# Patent

## Title
Pharmaceutical compound capable of induce immune protective response against Dengue virus having the capsid protein of the Dengue virus

## Abstract

This invention describes a pharmaceutical compound which has the capsid protein of Dengue virus and which is capable of inducing, in the receptor, a protective immune response before the viral challenge, without inducing the Ab-dependent enhancement phenomenon.

## Background
This application is the U.S. National Phase of, and Applicants claim priority from, International Application Number PCT/CU2006/000008 filed 18 Sep. 2006 and Cuban Application bearing Serial No. CU2005-0168 filed 16 Sep. 2005, which are incorporated herein by reference.
The present invention is related to the field of biotechnology and the pharmaceutical industry, in particular to the obtaining of proteins capable of inducing an immune response against the infection with Dengue virus, quoted from now on as DEN, while avoiding the antibody-dependent enhancement phenomenon that has been described in persons re-infected with this virus.
Dengue fever (DF) and dengue hemorrhagic fever (DHF) acquire increasing importance as health problems, affecting several countries of the tropical and subtropical zones of the planet. Dengue virus has been recognized in more than 100 countries and 2 500 million people living in risk areas are estimated. Between 50 and 100 million cases from DF and 250 000 to 500 000 of DHF are reported each year. (Guzmán M. G. and Kouri G. 2002. Dengue: an update. Lancet Infect. Dis. 2: 33-42).
The causal agent of this disease is the Dengue virus of the genus Flavivirus, family Flaviviridae, which is transmitted by the mosquito Aedes aegypti (Leyssen P., De Clerco E., Neyts J. 2000. Perspectives for the treatment of infections with Flaviviridae. Clin. Microbiol. Rev. 13: 67-82).
Until now four serotypes have been reported that can circulate in a same region. Dengue virus is an RNA positive coated virus, whose genome contains only one reading frame. This RNA is translated in a polyprotein that is processed in three structural proteins and seven non-structural proteins. (Russell P. K., Brandt W. E., Dalrymple J. M. 1980. Chemical and antigenic structure of flaviviruses. The togaviruses: biology, structure, replication. Schelesinger R. W. (ed.). 503-529).
Multiple epidemiological studies have been made to determine the risk factors that entail to the most severe form of Dengue disease. This is characterized by high fever, extrusion of liquids, hemorrhages and finally the Dengue shock. (Gubler D. J. 1998. Dengue and Dengue Hemorrhagic Fever. Clin. Microbiol. Rev. 11: 480-496). One of the most important risk factors is the secondary infection by a heterologous serotype. Cross-protection among the infections of the different serotypes does not exist. (Kouri G., Guzman M. G., Bravo J., Trina C. 1989. Dengue hemorrhagic fever/dengue shock syndrome: lessons from the Cuban epidemic. WHO Bulletin OMS. 67: 375-380).
Several hypotheses exist to explain this phenomenon. One of the most important is the antibody depend enhancement. (Halstead S. B., Scanlon J. E., Umpaivit P., Udomsakdi S. 1969. Dengue and Chikungunya virus infection in man in Thailand, 1962-1964. IV. Epidemiologic studies in the Bangkok metropolitan area. Am. J. Trop. Med. Hyg. 18: 997-1021).
From the first studies, it was raised that DEN virus replicates in greater measurement in peripheral mononuclear cells from the blood of patients who had undergone a previous infection with the virus (Halstead S. B., O'Rourke E. J., Allison A. C. 1977. Dengue viruses and mononuclear phagocytes. II. Identity of blood and tissue leukocytes supporting in vitro infection. J. Exp. Med. 146: 218-229). Later, it was demonstrated that the residual antibodies were the responsables of this effect (Morens D M, Halstead S B, Marchette N J. 1987. Profiles of antibody-dependent enhancement of dengue virus type 2 infection. Microb Pathog. October; 3(4):231-7).
In conditions of specificity or concentration of antibodies in which there is not neutralization, the antibody-virus complexes can be internalized by cells presenting Fey receptors in the membranes, like monocytes and macrophages. This mechanism, known as antibodies-dependent enhancement (ADE) occurs during secondary infections. (Morens D M, Halstead S B, Marchette N J. 1987. Profiles of antibody-dependent enhancement of dengue virus type 2 infection. Microb Pathog. October; 3(4):231-7; Kliks S. C., Nimmannitya S., Nisalak A., Burke D. S. 1988. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am. J. Trop. Med. Hyg. 38: 411-419).
Halstead et al. (Halstead S. B., Scanlon J. E., Umpaivit P., Udomsakdi S. 1969. Dengue and Chikungunya virus infection in man in Thailand, 1962-1964. IV. Epidemiologic studies in the Bangkok metropolitan area. Am. J. Trop. Med. Hyg. 18: 997-1021.), in a 3-year study in Bangkok, Thailand, reported that the hospitalization indices by DEN infection among children, reached a maximum in those between 7 and 8 months old. These indices were four to eight times greater than the observed between children of 1-3 months and twice than that in children of 3 years. Kliks et al. (Kliks S. C., Nimmannitya S., Nisalak A., Burke D. S. 1988. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am. J. Trop. Med. Hyg. 38: 411-419), determined the relation between the maternal neutralizing antibody titers against DEN-2 and the ages of thirteen children with FHD caused by the infection with the homologous virus. The results showed that the infection serious cases with the virus occurred when the maternal antibody levels diminished to sub-neutralizing levels. These data are consistent with the hypothesis in which the maternal antibodies play the double role of protecting first and stimulate the development of DHF later on.
Despite this immunological phenomenon, nowadays the most advanced vaccine candidates worldwide are based in attenuated virus of the different four serotypes, containing the envelope protein. These candidates are able to induce potential amplifying antibodies against the exposed proteins (PrM/M and Envelope) and protector neutralizing antibodies against the four viral serotypes in human volunteers. (Kanesa-thasan N., Sun W., Kim-Ahn G., Van Albert S., Putnak J. R., King A., Raengsakulsrach B., Christ-Schmidt H., Gilson K., Zahradnik J. M., Vaughn D. W., Innis B. L., Saluzzo J. F. y Hoke C. H. 2001. Safety and immunogenicity of attenuated dengue virus vaccines (Aventis Pasteur) in human volunteers. Vaccine. 19: 3179-3188).
High levels of neutralizing antibodies after immunization could prevent the viral replication despite the induction of enhancing antibodies. The problem can take place when the total seroconversion to the four serotypes in the vaccines, in terms of neutralizing Abs, is not obtained or is diminished to low levels in blood and the individuals would become then susceptible to a severe secondary infection with a viral serotype whose protective antibodies are not present. In fact, several tests in monkeys and humans have been made to define the viral amounts in the vaccine formulations. (Guirakhoo F., Arroyo J., Pugachev K. V., Miller C., Zhang Z.-X., Weltzin R., Georgakopoulos K., Catalan J., Ocran S., Soike K., Raterree M., Monath T. P. 2001. Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J. Virol. 75: 7290-7304).
In some cases the balance of seroconversion has not been obtained to the four serotypes (Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C, Chokejindachai W, Jagsudec A, Saluzzo J F, Bhamarapravati N. 2002. Safety and immunogenicity of tetravalent live-attenuated dengue vaccines in Thai adult volunteers: role of serotype concentration, ratio, and multiple doses. Am J Trop Med Hyg. 66(3): 264-72). In addition, it has been necessary to administer up to three doses of attenuated vaccines in children for a total seroconversion, in terms of neutralizing antibodies and still not known whether these will last in the time (Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C, Chambonneau L, Saluzzo J F, Bhamarapravati N. 2004. Safety and immunogenicity of a three dose regimen of two tetravalent live-attenuated dengue vaccines in five- to twelve-year-old Thai children. Pediatr Infect Dis J.; 23(2):99-109). This is one of the most questionable concerns in the vaccine formulations that include the envelope protein of Dengue virus, consequently of the vaccines candidates in development.
Another drawback of the attenuated vaccines, currently in phase I/II, is the safety. Upon the first dose the existence of adverse effects in adults and children like fever, myalgia, petequias and headache, has been demonstrated in several studies (Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C, Chambonneau L, Saluzzo J F, Bhamarapravati N. 2004. Safety and immunogenicity of a three dose regimen of two tetravalent live-attenuated dengue vaccines in five- to twelve-year-old Thai children. Pediatr Infect Dis J.; 23(2):99-109). In general the phenomenon of reversion to the virulence potentially associated to live vaccines may be presented.
In the search of new alternatives, it has been developed different variants of vaccine candidates based on the envelope protein or fragments of this one, obtained through a recombinant way. These recombinant candidates avoid the safety problems related to the inoculation of live virus, and are able to sensitize the individual if a balanced response against the four serotypes is not induced (Velzing J, Groen J, Drouet M T, van Amerongen G, Copra C, Osterhaus A D, Deubel V. 1999. Induction of protective immunity against Dengue virus type 2: comparison of candidate live attenuated and recombinant vaccines. Vaccine. March 17; 17(11-12):1312-20). On the other hand, these candidates require powerful adjuvants—not approved for their use in humans yet—to stimulate a serotype-specific suitable protective immune response (Hermida L, Rodriguez R, Lazo L, Silva R, Zulueta A, Chinea G, Lopez C, Guzman M G, Guillen G. 2004. 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 January; 115(1):41-9).
The humoral response of neutralizing antibodies has been extensively studied in animals and its protector effect has been demonstrated. The cytotoxic cellular immune response as a protector mechanism in Dengue has not deeply been explored. On the contrary, there are several reports in which the correlation between the induction of a cellular response and the most severe form of the disease is demonstrated (Rothman A. L. y Ennis F. A. 1999. Immunopathogenesis of Dengue Hemorragic Fever. Virology. 257: 1-6). These studies are based on the presence of high levels of activated T-cells in those individuals that exhibit DHF (Green S, Pichyangkul S, Vaughn D W, Kalayanarooj S, Nimmannitya S, Nisalak A, Kurane I. Rothman A L, Ennis F A. 1999. Early CD69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever. J Infect Dis. 180(5):1429-35).
T-cells epitopes have been reported mainly in nonstructural proteins (Kurane I, Zeng L, Brinton M A, Ennis F A. 1998. Definition of an epitope on NS3 recognized by human CD4+ cytotoxic T lymphocyte clones cross-reactive for dengue virus types 2, 3, and 4. Virology. 1998 Jan. 20; 240(2):169-74), but also are present in the envelope and in the capsid proteins (Bukowski, J. F., I. Kurane, C.-J. Lai, M. Bray, B. Falgout, and F. A. Ennis. 1989. Dengue virus-specific cross-reactive CD8 human cytotoxic T lymphocytes. J. Virol. 63:5086-5091; Gagnon S. J., Zeng W., Kurane I., Ennis F. A. 1996. Identification of two epitopes on the dengue 4 virus capsid protein recognized by a serotype-specific and a panel of serotype-cross-reactive human CD4+ cytotoxic T-lymphocyte clones. J Virol. 70: 141-147). Nevertheless, the protective character of some of these proteins based only on the induction of a cellular immune response has not been demonstrated.
In the search of vaccine candidates that avoid the immunoenhancement phenomenon. studies using the non-structural proteins NS1 and NS3 have been made. In the case of NS1, some level of protection in mice immunized with the recombinant protein has been reached.
Similar results have been obtained using a naked DNA containing the NS1 gene, through the ADCC mechanism (Wu S F. Liao C L, Lin Y L, Yeh C T, Chen L K, Huang Y F, Chou H Y, Huang J L, Shaio M F, Sytwu H K. 2003. Evaluation of protective efficacy and immune mechanisms of using a non-structural protein NS1 in DNA vaccine against dengue 2 virus in mice. Vaccine. September 8; 21(25-26):3919-29). Nevertheless, reports of their possible roll in phenomena of autoimmunity due to the induction of antibodies that recognize human endothelial cells exist (Chiou-Feng Lin, Huan-Yao Lei, Ai-Li Shiau, Hsiao-Sheng Liu, Trai-Ming Yeh, Shun-Hua Chen, Ching-Chuan Liu, Shu-Chen Chiu, and Yee-Shin Lin. 2002. Endothelial Cell Apoptosis Induced by Antibodies against Dengue Virus nonstructural Protein 1 via Production of Nitric Oxide. J. Immunol. 657-664). Additionally, there is a report of protection using a naked DNA formulation containing the NS3 gene; however, it was demonstrated that this protection was mediated by the raised antibodies since the same protection using passive immunization was obtained (Tan C H, Yap E H, Singh M, Deubel V, Chan Y C. 1990. Passive protection studies in mice with monoclonal antibodies directed against the non-structural protein NS3 of dengue 1 virus. J Gen Virol. 1990 March; 71 (Pt 3):745-9). In addition, it is worth noting the hypothesis, that the cellular response can be potentially harmful facing an infection with heterologous virus based on studies of NS3 protein epitopes (Zivny J, DeFronzo M, Jarry W, Jameson J, Cruz J, Ennis F A, Rothman A L. 1999. Partial agonist effect influences the CTL response to a heterologous dengue virus serotype. J Immunol. September 1; 163(5):2754-60).
In the case of the capsid protein of dengue virus, no evidences of protection in the challenge with a lethal dengue virus are reported. Concerning related flaviviruses, a report was published where authors inoculated mice with a naked DNA formulation containing the gene of the Japanese Encephalitis (JE) capsid protein. This formulation did not induce a protective response against the challenge with lethal JE in mice, despite the demonstration of a cytotoxic response (Konishi E, Ajiro N, Nukuzuma C, Mason P W, Kurane I. 2003. Comparison of protective efficacies of plasmid DNAs encoding Japanese encephalitis virus proteins that induce neutralizing antibody or cytotoxic T lymphocytes in mice. Vaccine. September 8; 21(25-26):3675-83).
Protection using the recombinant protein capsid has been demonstrated only in the case of human papilloma virus. However it has been suggested its protector role with other virus like Hepatitis C virus. Nevertheless, in all cases, they are chronic infections or tumors, in which the cellular cytotoxic response is the only mean of the immune system to clear viral infection (Duenas-Carrera S, Alvarez-Lajonchere L, Alvarcz-Obregon J C, Herrera A, Lorenzo L J, Pichardo D, Morales J. 2000. A truncated variant of the hepatitis C virus core induces a slow but potent immune response in mice following DNA immunization. Vaccine. November 22; 19(7-8):992-7; Suzich J A, Chin S J, Palmer-Hill F J, et al. 1995. Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas. Proc Natl Acad Sci USA; 92: 11553-57). These diseases do not correspond with the acute profile that is exhibited in the infection by Dengue in humans (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. y Nisalak A. 2000. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis. 181: 2-9).
The capsid protein of Dengue virus has a molecular weight of 9 to 12 kDa (112-127 amino acids) and has a marked basic character because the 25% of its amino acids are arginine and lysine. The presence of these amino acids could favor antigenic presentations to the immune system due to the capacity of polycationic peptides to do so. (Lingnau K., Egyed A., Schellack C., Mattner F, Buschle M., Schmidt W. 2002. Poly-1-arginine synergizes with oligodeoxynucleotides containing CpG-motifs (CpG-ODN) for enhanced and prolonged immune responses and prevents the CpG-ODN-induced systemic release of pro-inflammatory cytokines. Vaccine. 20: 3498-3508). The protein is located totally within the virion structure without any exposed region (Kuhn R J, Zhang W, Rossmann M G, Pletnev S V, Corver J, Lenches E, Jones C T, Mukhopadhyay S, Chipman P R, Strauss E G, Baker T S, Strauss J H. 2002. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell. March 8: 108(5):717-25).
Jones y cols. (Christopher T. Jones, Lixin Ma, John W. Burgner, Teresa D. Groesch, Carol B. Post, and Richard J. Kuhn. 2003. Flavivirus Capsid Is a Dimeric Alpha-Helical Protein. Journal of Virology, p 7143-7149, Vol. 77, No. 12) purified the capsid protein of VD2 obtained by the recombinant way in Escherichia coli (E. coli) and demonstrated that this protein behaves like a dimmer in solution without nucleic acids. Its secondary structure is mainly in form of alpha-helices and is composed by four of these helices, being one of those of greater length in the C-terminal end. The N-terminal end does not present a defined structure and its deletion does not affect the structural integrity of the protein.
This invention describes for the first time that the capsid of DEN-2 virus, obtained by a recombinant way in E. coli and with only a 40% of purity, is able to induce a protective immune response against the challenge with lethal DEN-2 virus in mice. It was demonstrated that this highly purified protein, retained its protective capacity, which was surpassed in the immunization of mice with the particulated form of the molecule. Moreover, it was demonstrated that the reached protection was mediated by CD8+ T-cells, a novel element considering that the reported T-cells epitopes for the capsid so far, are recognized by CD4+ T cells (Gagnon S J, Zeng W, Kurane I, Ennis F A. 1996. Identification of two epitopes on the dengue 4 virus capsid protein recognized by a serotype-specific and a panel of serotype-cross-reactive human CD4+ cytotoxic T-lymphocyte clones. J Virol. 70(1): 141-7; Simmons C P, Dong T, Chau N V, Dung N T, Chau T N, Thao le T T, Dung N T, Hien T T, Rowland-Jones S, Farrar J. 2005. Early T-cell responses to dengue virus epitopes in Vietnamese adults with secondary dengue virus infections. J Virol. 79(9):5665-75). Additionally, this recombinant molecule was mixed with the PD5 protein, which is formed by the P64k protein of Neisseria meningitidis and the III domain of the envelope protein of the Dengue-2 virus. This fusion protein is able to generate a highly serotype specific, protective and neutralizing immune response, with a low probability of generating the phenomenon of antibodies dependent enhancement (Hermida L, Rodriguez R, Lazo L, Silva R, Zulueta A, Chinea G, Lopez C, Guzman M G, Guillen G. 2004. 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 January; 115(1):41-9).
The obtaining of a genetic construction formed by the fusion of the capsid protein and the III domain of the envelope protein is also described to reach the same objective. As a result, the two formulations where the capsid is combined with the III domain of DEN-2, generated a lymphoproliferative response in mice higher than that generated by the capsid only, and in addition a serotype specific antibodies response higher than the generated by PD5 only. This last result demonstrates the immunoenhancing capacity of the capsid protein of dengue virus in the generation of Abs by a heterologous antigen, phenomenon described for other recombinant capsids from other viruses like the hepatitis B virus (Alvarez J C, Guillén G. Formulations containing virus like particles as immunoenhancers by mucosal route. Cuban office of the Industrial property. CU 1998/183).
DETAILED DESCRIPTION OF THE INVENTION
The objective of this invention is to obtain a recombinant protein corresponding to the capsid protein of Dengue virus, which generates a protective response against the infection with the lethal virus when is inoculated in mice.
The gene codifying for the capsid protein of Dengue virus was inserted into a plasmid containing the phage T5 promoter. The cells XL-1Blue, transformed with the recombinant plasmid, expressed high levels of the resulting protein.
This protein was purified approximately till a 40% of purity, and was adjuvated in aluminum hydroxide to be inoculated in Balb/c mice. A month upon the last dose the antiviral antibody response was measured. At the same time the lymphoproliferative response in spleens stimulated in vitro with the dengue virus was determined. As a result no antiviral antibodies were induced while a significant lymphoproliferative response was detected. In parallel, in not bleeding mice, the protection assay was done. A lethal doses corresponding to 100 LD50 of Dengue virus was inoculated, the disease symptoms and death were observed during 21 days. As a result a 44% of survival-immunized mice were obtained while in the negative control group all mice died. This is the first evidence of a protective response against Dengue virus by the immunization only with the capsid protein.
Later, a high-resolution purification process was conducted, obtaining a 95% of purity of the recombinant protein.
Both preparations, the semi- and purified ones, were analyzed by HPLC to know the aggregation state of the protein in each sample. In the semipurified preparation was detected a fraction with lower retention times, while in the purified sample a retention time corresponding to the dimeric form of the molecule was detected.
To obtain an aggregation state in the purified variant, an in vitro particulation process employing low quantities of oligonucleotides was done. As a result of the process, particles of 21 nm of diameter were obtained.
The dimeric and particulated preparations, both with more than 95% of purity, were inoculated in mice. The dimeric preparation was adjuvated with Freund Adjuvant and aluminum hydroxide, while the particulated variant was adjuvated only with aluminum hydroxide.
Similar to the semipurified preparation, high levels of lymphoproliferation were detected. In the protection assay a 40 and 20% of survival were obtained with the dimeric preparation adjuvated with Freund and aluminum, respectively; however, the particulated protein adjuvated with aluminum induced a higher protection percentage.
These results together with those obtained with the semipurified protein showed the capacity of the capsid protein of inducing a protective response in Balb/c mice and demonstrated the superiority of the particulated form of the protein, letting it to be used to humans in the future together to the aluminum hydroxide as adjuvant. Additionally, not inducing an antiviral response would eliminate the phenomenon of antibodies dependent enhancement as a risk factor for the occurrence of the most severe form of the disease: the dengue hemorrhagic fever.
With the aim to determine the possible mechanism of protection, which it is not related to the induction of Abs due to its demonstrated absence, a study of CD8+ cells depletion was made. As a result, the protection reached with pure proteins of each variant was dependent of the presence of the cells that present this marker, since eliminating them the induced protective effect disappear.
Similarly, a study was made to know if the combination of the particulated recombinant capsid with antigens inducing humoral response does not affect the generation of the lymphoproliferative response and to count with a mixture of immunogens able to contribute to both branches of the immune response. To this end, the purified particulated variant of the capsid and a fusion protein containing the III domain of the envelope protein of the dengue-2 virus was inoculated in mice, which is able to generate a serotype-specific immune response diminishing the phenomenon of ADE (Hermida L, Rodriguez R, Lazo L, Silva R, Zulueta A, Chinea G, Lopez C, Guzman M G, Guillen G. 2004. 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 January; 115(1):41-9). When administering three doses and analyzing the raised Abs, it was demonstrated a higher induction of antiviral serotype specifics Abs. As well, a lymphoproliferative response higher than that induced only by the capsid and significantly higher than that induced by the fusion protein was detected.
In parallel, to know whether it is possible to obtain the combination effect using a genetic fusion of both antigens, a plasmid containing the III domain of the envelope protein of DEN-2 virus fused to the N-terminal of the capsid protein gene was constructed. The resulting protein, with a 40% of purity, generated in Balb/c mice a lymphoproliferative response higher than that induced by the capsid alone and a serotype specific antibodies response higher than that induced by PD5.



FIGURES DESCRIPTION
FIG. 1. Cloning strategy of the capsid protein of DEN-2 virus to generate PDC-2. DEN2 C: Fragment of the capsid protein of DEN-2.
FIG. 2. Analysis by SDS-PAGE at 15% of the PDC-2 semipurification process. 1. Rupture supernatant. 2 and 3. Fraction not adsorbed to Q Sepharose FF. 4. Fraction eluted with NaCl 1M.
FIG. 3. Analysis by SDS-PAGE at 15% of the PDC-2 purification process. 1. Rupture supernatant, 2. Fraction not absorbed to the gel, 3. Washed (350 mM NaCl), 4. Eluted fraction (750 mM NaCl), 5. Fraction in Tris 10 mM, EDTA 1 mM.
FIG. 4. Chromatographic profile in Superdex 200 of the semipurified (A) and pure (B) preparations of PDC-2.
FIG. 5. Electronic microscopy pictures of the pure PDC-2 preparation before (A) and after (B) the treatment with oligonucleotides.
FIG. 6. Cloning strategy of the capsid protein of DEN-1 virus to generate PDC-1. DEN1 C: Fragment of the capsid protein of DEN-1.
FIG. 7. Analysis by SDS-PAGE at 15% of the PDC-1 semipurification process. 1. Molecular weight marker. 2. Rupture supernatant 3. Fraction not adsorbed to Q Sepharose FF.



EXAMPLES
Example 1
Cloning and Expression of PDC-2
The nucleotide sequence that codes for amino acids 1 to 99 of the capsid protein from DEN-2 virus (Sequence No. 3), was amplified with the oligonucleotides identified in the sequence list as Sequence No. 1 and Sequence No. 2 from the DEN-2 virus strain genotype Jamaica (Deubel V., Kinney R. M., Trent D. W. Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of Dengue type 2 virus, Jamaica genotype: Comparative analysis of the full-length genome. Virology 1988.165:234-244).
The vector was created by digestion of the plasmid pQE-30 with BamHI/HindIII, which contains the phage 15 promoter and a 6-histidine tail in the N-terminal region (Sequence No. 6). Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions.
Competent cells XL-1 Blue (Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580) were transformed with the selected clone called pDC-2 (FIG. 1 and Sequence No. 4). The transformed E. coli strains were cultivated in Luria Bertani medium (LB) supplemented with Ampicilline 50 μg/mL for 10 h at 37° C. Isopropyl-B-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM was used for the induction of the promoter. Upon growing the colony, an SDS-PAGE of the cellular lysate was done. As a result, a 15-kDA band was obtained. The protein was recognized by an anti-DEN-2 hyperimmune ascitic fluid (HMAF). This protein was denominated PDC-2 (Sequence No. 5).
Example 2
Semipurification and Characterization of PDC-2
The biomass obtained from the E. coli strain transformed with pDC-2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained equally distributed between the soluble and insoluble fractions. The soluble fraction was subjected to an anionic interchange chromatography, using a Q Sepharose FF column and the buffer Tris 10 mM pH 8. The protein in the non-absorbed fraction was obtained with 40% purity and was used for the immunological studies (FIG. 2).
Example 3
Immunological Evaluation of Semipurified PDC-2
Three groups of 30 Balb/c mice were used. Two of them were immunized with 10 ug of the recombinant protein by intraperitoneal route, using Freund's Adjuvant (FA) in one of the groups and aluminum hydroxide in the other. The soluble fraction resulting from the rupture of the pQE-30-transformed cells was used as negative control adjuvanted with FA; 10 animals were bled 15 days after the third dose and the antibody titers against DEN-2 were determined by ELISA. After the immunization with the recombinant protein, formulated in either adjuvant, no antibody titers were obtained.
Table 1. Antibody titers against DEN-2 from the sera obtained upon immunization of mice with semipurified PDC-2.







TABLE 1







Antibody titers against DEN-2 from the sera obtained


upon immunization of mice with semipurified PDC-2.









ELISA Titers against DEN-2











PDC-2
PDC-2
XL-1 Blue


Mouse
Freund A.
Aluminum hydroxide
Freund (Neg. Control)













1
<1:100
<1:100
<1:100


2
<1:100
<1:100
<1:100


3
<1:100
<1:100
<1:100


4
<1:100
<1:100
<1:100


5
<1:100
<1:100
<1:100


6
<1:100
<1:100
<1:100


7
<1:100
<1:100
<1:100


8
<1:100
<1:100
<1:100


9
<1:100
<1:100
<1:100


10
<1:100
<1:100
<1:100









Example 4
Protection Assay
For the evaluation of the protection conferred to mice against challenge with lethal homologous DEN virus by the immunization with the described variants, 10 mice were used from each of the groups immunized with the recombinant protein adsorbed in aluminum hydroxide and with the control preparation. Each animal received a dose of 100 LD50 of lethal DEN-2 virus by intracranial inoculation and was observed for 21 days to obtain the percentages of lethality in terms of death by viral encephalitis. As a positive control, a group of 10 mice immunized with infective DEN-2 virus (104 pfu) was used. All mice in the positive control group survived, while in the negative control group all mice were sick by day 7-11 after challenge and 100% mortality was obtained by day 21. Finally, the group immunized with the recombinant protein PDC-2 presented 44.4% protection (table 2).







TABLE 2







Percentage of survival in PDC-2 immunized mice upon


challenge with the homologous lethal Dengue virus.











Survival



Immunogen
percentage














XL-1 blue
0



DEN-2
100



PDC-2 alum.
44.4







* It was calculated: (# de survivors)/(total # of mice). Data of survivors were taken 21 days after challenge.






Example 5
Lymphoproliferative Response
The rest of the animals from the group immunized with the capsid protein adjuvanted with aluminum hydroxide were sacrificed 30 days after the last dose. Then, their spleens were extracted and the lymphoproliferative response to DEN-2 was studied. The results in table 3 show the stimulation indexes obtained.







TABLE 3







Stimulation indexes against the homologous serotype


of the lymphocytes from immunized mice.









PDC-2



Aluminum



hydroxide














DEN-2**
8*



Control
1.5



Antigen***



PHA****
7







*Stimulation index: quotient average of counts/minutes of samples between counts/minutes of the ADN spontaneous synthesis control.



**Preparation of DEN-2 infected mice brain.



***Preparation of not infected mice brain.



****Phytohemaglutinina Mitogen (Positive Control).






Example 6
Purification of PDC-2
The biomass obtained from the E. coli strain transformed with pDC-2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained equally distributed between the soluble and insoluble fractions. The soluble fraction was subjected to a cationic interchange chromatography, using an SP-Sepharose FF column and the buffer Tris 10 mM, Tween 0.5%, urea 7M, pH 8. The column was washed with buffer diethanolamine 30 mM, NaCl 350 mM, pH 10.3. The elution of the protein of interest was done with buffer diethanolamine 30 mM, NaCl 750 mM, pH 10.3. Once eluted the protein, the buffer was exchanged using G-25 columns. Finally, the protein was obtained with 96% purity in buffer Tris 10 mM, EDTA 1 mM (FIG. 3).
Example 7
Characterization of the Semipurified and Purified Variants
With the aim of characterizing the state of aggregation of the semipurified and the purified preparations, gel filtration chromatographies were done using the TSK-5000 column (Tosoh bioscience, Japan). After applying the semipurified sample, a homogeneous and major peak was obtained, with a retention time ranging from 15 to 20 minutes, evidencing the presence of high molecular weight species (FIG. 4A). Contrarily, in the sample from the highly purified fraction of the capsid protein, retention times of 30 minutes were detected, corresponding to the dimeric form of the molecule (FIG. 4B).
Example 8
Studies of Reparticulation “In Vitro”
In order to reparticulate the pure capsid protein in a dimeric form, the buffer was exchanged to Hepes 25 mM, KAc 100 mM, MgAc2 1.7 mM, pH 7.4. After heating the protein and the mixture of oligonucleotides for 1 min at 37° C., they were incubated in an equal volume for 30 min at 30° C. As a negative control of the experiment, the protein was incubated without the oligonucleotides. When both preparations were observed with an electron microscope, a large quantity of particles of approximately 21 nm diameter, were observed in the sample of protein previously incubated with the mixture of oligonucleotides, while in the control sample no particles were observed (FIG. 5).
Example 9
Immunological Evaluation in Mice of the Purified Capsid
Five groups of 20 Balb/c mice were used. Two of them were immunized with 10 ug of the dimeric purified recombinant protein by intraperitoneal route, using aluminum hydroxide and Freund's adjuvant. Another group was immunized with 10 ug of the purified and particulated capsid protein adjuvanted with aluminum hydroxide. The soluble fraction from the rupture of XL-1 blue cells transformed with the plasmid pQE-30 and subjected to the same purification steps than PDC-2 was used as negative control, adjuvanted with Freund's adjuvant. The fifth group was immunized with DEN-2 virus as positive control. One month after the last dose 10 animals from each group received a dose of 100 LD50 of lethal DEN-2 by intracranial inoculation and were observed for 21 days to obtain the percentages of survival. All mice in the positive control group survived, while in the negative control group all mice were sick by day 7-11 after challenge and 0% mortality was obtained. Finally, from the groups immunized with the recombinant protein, the group immunized with pure dimeric PDC-2 presented a 20% protection when immunized with aluminum hydroxide and a 40% protection when Freund's adjuvant was used. Additionally, in the group that received the reparticulated pure protein adjuvanted with aluminum hydroxide. 90% of mice were protected (Table 4).







TABLE 4







Percentage of survival in mice immunized with


the protein variants assayed upon challenge


with the homologous lethal Dengue virus.










Immunogen (adjuvant)
Survival percentage*














Xl-1 Blue (Freund)
0



Pure and dimeric PDC-2 (Aluminum)
20



Pure and dimeric PDC-2 (Freund)
40



Pure and reparticulated
90



PDC-2 (Aluminum)



DEN-2
100







*It was calculated: (# de survivors)/(total # of mice). Data of survivors were taken 21 days after challenge.






Example 10
Lymphoproliferative Response
The rest of the animals from the groups immunized with the capsid protein (10 animals), either dimeric or reparticulated, adjuvanted with aluminum hydroxide, were sacrificed 15 days after the last dose. Then, their spleens were extracted and the lymphoproliferative response to DEN-2 was studied. The results in table 5 show the stimulation indexes obtained.







TABLE 5







Stimulation indexes against the homologous serotype


of the lymphocytes from immunized mice.










Pure and




reparticulated
Pure



PDC-2
PDC-2















DEN-2**
10*
4



Antigen
1.5
1.2



Control***



PHA****
7
8







*Stimulation index: quotient average of counts/minutes of samples between counts/minutes of the ADN spontaneous synthesis control.



**Preparation of DEN-2 infected mice brain.



***Preparation of not infected mice brain.



****Phytohemaglutinina Mitogen (Positive Control).






Example 11
Immunological Evaluation of the Mixture Formed by PD5 and PDC-2
Twenty animals were inoculated with the mixture of 10 ug of the particulated pure capsid protein and 20 ug of protein PD5 (Sequence No. 23) in three doses spaced fifteen days apart. A group immunized with 10 ug of the pure capsid protein, a group immunized with 20 ug of protein PD5 mixed with the equivalent volume of PDC-2 but obtained from a negative control run, and a group immunized with protein P64k, the carrier protein present in the construction of PD5, were used as controls. In all cases, aluminum hydroxide was used as adjuvant.
Fifteen days after the last dose, the animals were bled and the sera tested for antiviral antibodies by ELISA. As shown in tables 6 and 7, the group immunized with the mixture developed serotype-specific antibodies with titers higher than those of the group immunized only with protein PD5 and, at the same time, titers in these two groups were higher than those in the group immunized with protein PDC-2, where no Abs against DEN-2 virus were detected. On the other hand, 10 additional animals were taken from each group for lymphoproliferation assays. The cells from the spleens of these animals were extracted and stimulated with the infective DEN-2 virus. As shown in table 8, in the group immunized with the mixture the stimulation indexes were higher than those in the group immunized with the capsid protein only. The lowest stimulation indexes were obtained in the group immunized with protein PD5.







TABLE 6







Antibody titers against DEN-2 virus in


sera obtained after the immunization.










Groups inoculated with:












Mouse
PDC-2
PDC-2/PD5
PD5
P64k














1
<1:100
<1:128000
<1:64000
<1:100


2
<1:100
<1:320000
<1:32000
<1:100


3
<1:100
<1:320000
<1:64000
<1:100


4
<1:100
<1:320000
<1:16000
<1:100


5
<1:100
<1:64000 
<1:64000
<1:100


6
<1:100
<1:128000
 <1:128000
<1:100


7
<1:100
<1:64000 
<1:64000
<1:100


8
<1:100
<1:128000
<1:32000
<1:100


9
<1:100
<1:320000
<1:64000
<1:100


10
<1:100
<1:320000
<1:32000
<1:100
















TABLE 7







Determination of the serotype-specificity of the antibodies contained


in the mixtures of the sera obtained from each group.








Viral
Groups inoculated with:











Antigen
PDC-2
PDC-2/PD5
PD5
P64k





DEN-1
<1:100
<1:200
<1:200
<1:100


DEN-2
<1:100
   1:320 000
  1:64000
<1:100


DEN-3
<1:100
<1:200
<1:200
<1:100


DEN-4
<1:100
<1:200
<1:200
<1:100
















TABLE 8







Stimulation indexes against the homologous serotype


of the lymphocytes from immunized mice.













PDC-





PDC-2
2/PD5
PD5
P64k

















DEN-2**
9*
11
2.1
1.1



Antigen***
1.3
1.6
1.5
1.2



Control (−)



PHA****
7.5
7.3
7.9
8







*Stimulation index: quotient average of counts/minutes of samples between counts/minutes of the ADN spontaneous synthesis control.



**Preparation of DEN-2 infected mice brain.



***Preparation of not infected mice brain.



****Phytohemaglutinina Mitogen (Positive Control).






Example 12
CD8 Depletion Studies
The reparticulated and the dimeric capsid proteins were inoculated in Balb/c mice to obtain some evidence of induction of cellular immune response. A preparation obtained from cells transformed with the plasmid used to generate pDC-2, and by a purification process similar to the one used for the protein PDC-2, was employed as a negative control.
Three doses of the protein (20 ug) were administered to groups of 20 animals, using aluminum hydroxide as adjuvant. One month after the last dose, 1 mg of a rat anti-mouse CD8 mAb, able to deplete the cells of the mouse immune system containing this marker was administered to half of the animals of each group. On the next day, all the animals were challenged with 100 LD50 (Median Lethal doses) of DEN-2 virus. They were observed for the onset of signs of disease and deaths were recorded.
In the case of the immunized non-treated groups, 20 and 80% protection was obtained in the groups immunized with the dimeric and the reparticulated capsid, respectively. Parallely, in the treated groups the percentage of protection was lower than in the non-treated groups: 0% protection for the dimeric PDC-2 and 10% protection for the reparticulated protein. In the case of the negative control group no protection was obtained in either the treated or the non-treated animals.







TABLE 9







Challenge assay with DEN-2 lethal virus in the animals


immunized with variants of the recombinant capsid










*Survival percentages
Survival percentages



in mice treated
in mice non



with the
treated with the


Groups
anti-CD8 mAb
anti-CD8 mAb












PCD12 reparticulated
10
80


PCD12
0
20


non-particulated


Control (−)
0
0





*It was calculated: (# de survivors)/(total # of mice). Dataa of survivors were taken 21 days after challenge.






Example 13
Obtaining and Semipurification of the DEN-1 Protein
The nucleotide sequence that codes for amino acids 1 to 100 of the capsid protein of DEN-1 virus (Sequence No. 7), was amplified with the oligonucleotides identified in the sequence list as Sequence No. 8 and Sequence No. 10 from the DEN-1 viral strain. The vector was generated by digestion BamHI/HindIII of the plasmid pQE-30, which contains the phage T5 promoter and a 6 histidine tail in the N-terminal region (Sequence No. 6). Upon ligation, the recombinants were analyzed by restriction and the positives clones were sequenced to check the junctions. Competent cells XL-1 Blue (Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580) were transformed with the selected clone called pDC-1 (FIG. 6 y Sequence No. 10). The E. coli strains transformed were cultivated in LB supplemented with Ampicilline 50 μg/mL for 10 h at 37° C. Isopropyl-B-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM was used for the induction of the promoter. Upon growing the colony, an SDS-PAGE of the cellular lysate was done. As a result a 15-kDA band was obtained. The protein was recognized by an anti-DEN-1 HMAF. This protein was denominated PDC-1 (Sequence No. 11).
Example 14
Semipurification and Characterization of PDC-1
The biomass obtained from the E. coli strain transformed with pDC-1 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained equally distributed between the soluble and insoluble fractions. From the soluble fraction an anionic interchange chromatography was done, using a Q Sepharose FF column and the buffer Tris 10 mM pH 8. The protein in the non-absorbed fraction was obtained with 45% of purity, and was used to the immunological studies.
Example 15
Immunological Evaluation of Semipurified PDC-1
Two groups of 30 Balb/c mice were used. One of them was immunized with 10 ug of the recombinant protein by intraperitoneal route, using the aluminum hydroxide as adjuvant. The soluble fraction resulting from the rupture of the pQE-30-transformed cells adjuvanted with aluminum hydroxide was used as negative control. A part of the animals (10 mice) were bled 15 days after the third dose and the antibody titers against DEN-1 were determined by ELISA. After the immunization with the recombinant protein, no antiviral antibody titers were obtained.







TABLE 10







Antibodies titers against DEN-1 virus from sera obtained


after the immunization with the semipurified PDC-1.










Anti- DEN-1 ELISA titers











XL-1 blue



Mouse
Control (−)
PDC-1












1
<1:100
<1:100


2
<1:100
<1:100


3
<1:100
<1:100


4
<1:100
<1:100


5
<1:100
<1:100


6
<1:100
<1:100


7
<1:100
<1:100


8
<1:100
<1:100


9
<1:100
<1:100


10
<1:100
<1:100









Example 16
Protection Assay
For the evaluation of the protection conferred to mice against challenge with lethal homologous DEN virus by the immunization with the described variants, 10 mice were used from each of the groups immunized with the recombinant protein adsorbed in aluminum hydroxide and with the control preparation. Each animal received a dose of 100 LD50 of lethal DEN-1 by intracranial inoculation and was observed for 21 days to obtain the percentages of lethality in terms of death by viral encephalitis. As a positive control, a group of 10 mice immunized with infective DEN-1 virus (104 pfu) was used. All mice in the positive control group survived, while in the negative control group all mice were sick at day 7-11 after challenge and 100% mortality was obtained at day 21. Finally, the group immunized with the recombinant protein PDC-1 presented 50% of protection (Table 11).







TABLE 11







Percentage of survival in mice immunized with the protein variants


assayed upon challenge with the homologous lethal DEN virus.











Survival



Immunogen
percentages*














XL-1 blue
0



(Control −)



DEN-1
100



(Control +)



PDC-1
50







*It was calculated: (# de survivors)/(total # of mice). Data of survivors were taken 21 days after challenge.






Example 17
Lymphoproliferative Response
The rest of the animals of the group immunized with the protein PDC-1 were sacrificed 15 days after the last dose. Then, their spleens were extracted and the lymphoproliferative response to DEN-1 was studied. The results in table 12 show the stimulation indexes obtained.







TABLE 12







Stimulation indexes against the homologous serotype


of the lymphocytes from immunized mice.









PDC-1 aluminum



hydroxide














DEN1**
8*



Control
1.5



Antigen***



PHA****
7







*Stimulation index: quotient average of counts/minutes of samples between counts/minutes of the ADN spontaneous synthesis control.



**Preparation of DEN-2 infected mice brain.



***Preparation of not infected mice brain.



****Phytohemaglutinina Mitogen (Positive Control).






Example 18
Cloning and Expression of PDC-2 DomIII
The nucleotide sequence that codes for amino acids 286 to 426 of the envelope protein from DEN-2 (Sequence No. 12), corresponding to the region of the domain 111 of the protein, was amplified with the oligonucleotides identified in the sequence list as Sequence No. 13 and Sequence No. 14 from the DEN-2 virus strain genotype Jamaica (Deubel V., Kinney R. M., Trent D. W. Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of Dengue type 2 virus, Jamaica genotype: Comparative analysis of the full-length genome. Virology 1988.165:234-244).
The vector was created by digestion of the plasmid pDC-2 with BamHI/BamHI, which contains the phage T5 promoter, a 6-histidine tail in the N-terminal region and the region corresponding to 100 amino acids of the capsid protein of DEN-2 virus. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Finally the clone selected was named pDC-2 Dom III (Sequence No 15).
Competent cells XL-1 Blue (Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580) were transformed with the selected clone called pDC-2 DomIII. The E. coli strains transformed were cultivated in LB supplemented with Ampicilline 50 μg/mL for 10 h at 37° C. isopropyl-B-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM was used to the induction of the promoter. Upon growing the colony, an SDS-PAGE of the cellular lysate was done. As a result, a 30-kDA band was obtained. The protein was recognized by an anti-DEN-2 HMAF. This protein was denominated PDC-2 Dom III (Sequence No. 16).
Example 19
Semipurification and Characterization of PDC-2 Dom III
The biomass obtained from the E. coli strain transformed with pDC-2 DomIII and grown at 37° C. was disrupted by French press. The recombinant protein was obtained equally distributed between the soluble and insoluble fractions. From the soluble fraction an anionic interchange chromatography was done, using a Q Sepharose FF column and the buffer Tris 10 mM pH 8. The protein in the non-absorbed fraction was obtained with 40% of purity, and was used to the immunological studies (FIG. 2).
Example 20
Immunological Evaluation in Mice of the Semipurified PDC-2 Dom III
Five groups of 30 Balb/c mice were used. One of the groups was immunized with 10 ug of the recombinant protein by intraperitoneal route, using aluminum hydroxide as adjuvant. The soluble fraction resulting from the rupture of the XL-1 Blue cells transformed with the plasmid pQE-30 was used as negative control, adjuvanted with aluminum hydroxide. Another two groups were included as controls. One of them was immunized with the protein PDC-2 and the other with the protein PD5 (this protein contains the domain III region of the envelope protein of DEN-2 virus). Ten animals from each group were bled 15 days after the third dose and the antibody titers against DEN-2 were determined by ELISA. As shown in Tables 13 and 14, the group immunized with PDC-2 Dom III developed high titers of serotype-specific antibodies against DEN-2, higher than those induced by the protein PD5. These results demonstrate that the genetic combination with the capsid protein enhances the antiviral immune response elicited by the domain III of the envelope protein.







TABLE 13







Antibodies titers against DEN-2 virus from sera obtained


after the immunization with the Dom III-capsid protein.












Groups immunized with:














Mouse
PDC-2
PDC-2 Dom III
PD5
P64k














1
<1:100
<1:320000
<1:32000
<1:100


2
<1:100
<1:640000
<1:32000
<1:100


3
<1:100
<1:640000
<1:64000
<1:100


4
<1:100
<1:640000
<1:64000
<1:100


5
<1:100
<1:128000
<1:64000
<1:100


6
<1:100
<1:320000
<1:32000
<1:100


7
<1:100
<1:128000
<1:64000
<1:100


8
<1:100
<1:64000 
 <1:128000
<1:100


9
<1:100
<1:64000 
<1:32000
<1:100


10
<1:100
<1:128000
<1:64000
<1:100
















TABLE 14







Determination of the serotype-specificity of the antibodies contained


in the mixtures of the sera obtained from each group.








Viral
Groups inoculated with:











Antigen
PDC-2
PDC-2 Dom III
PD5
P64k





DEN-1
<1:100
<1:200
<1:200
<1:100


DEN-2
<1:100
   1:320 000
   1:64 000
<1:100


DEN-3
<1:100
<1:200
<1:200
<1:100


DEN-4
<1:100
<1:200
<1:200
<1:100









On the other hand, 10 additional animals were taken from each group for the lymphoproliferation assays. The cells from the spleens of these animals were extracted and stimulated with the infective DEN-2 virus. Table 15 shows that in the group immunized with the combination, the stimulation indexes were higher than those in the group immunized with the capsid protein only. The stimulation indexes in the group immunized with protein PD5 were the lowest.







TABLE 15







Stimulation indexes against the homologous serotype


of the lymphocytes from immunized mice.












PDC-2
PDC-2 Dom III
PD5
P64k

















DEN-2**
9.5*
11.6
2.2
1.2



Antigen***
1.2
1.1
1.2
1.6



Control (−)



PHA****
7.6
7.4
7.5
7.9







*Stimulation index: quotient average of counts/minutes of samples between counts/minutes of the ADN spontaneous synthesis control.



**Preparation of DEN-2 infected mice brain.



***Preparation of not infected mice brain.



****Phytohemaglutinina Mitogen (Positive Control).






Incorporation of Sequence Listing
Incorporated herein by reference in its entirety is the Sequence Listing for the application. The Sequence Listing is disclosed on a computer-readable ASCII text file titled, “sequence_listing.txt”, created on Jul. 23, 2008. The sequence_listing.txt file is 44 kb in size.

## Claims

1. A pharmaceutical composition capable of generating an immune response against Dengue virus, comprising a capsid protein of Dengue virus type 1, a capsid protein of Dengue virus type 2, a capsid protein of Dengue virus type 3, and a capsid protein of Dengue virus type 4, wherein the capsid protein of Dengue serotype 1, the capsid protein of Dengue serotype 2, the capsid protein of Dengue serotype 3, or the capsid protein of Dengue serotype 4 is fused with a capsid protein of Dengue virus type 1, a capsid protein of Dengue virus type 2, a capsid protein of Dengue virus type 3, or a capsid protein of Dengue virus type 4.

2. The pharmaceutical composition of claim 1, wherein the capsid protein of Dengue serotype 1, the capsid protein of Dengue serotype 2, the capsid protein of Dengue serotype 3, or the capsid protein of Dengue serotype 4 is fused to an immunogenic antigen.

3. The pharmaceutical composition of claim 2, wherein the immunogenic antigen is a peptide having the amino acid sequence comprising SEQ ID NO: 19.

4. The pharmaceutical composition of claim 1, further comprising a capsid protein of Dengue virus type 1, a capsid protein of Dengue virus type 2, a capsid protein of Dengue virus type 3, and a capsid protein of Dengue virus type 4, or a combination thereof.

5. The pharmaceutical composition of claim 1, further comprising an immunogenic antigen.

6. A pharmaceutical composition capable of generating an immune response against Dengue virus, comprising
a) a capsid protein of Dengue virus type 1, a capsid protein of Dengue virus type 2, a capsid protein of Dengue virus type 3, and a capsid protein of Dengue virus type 4: and
b) an immunogenic antigen
wherein the immunogenic antigen is a peptide having the amino acid sequence comprising SEQ ID NO: 22.

7. A pharmaceutical composition capable of generating an immune response against Dengue virus, comprising a capsid protein of Dengue virus type 1, a capsid protein of Dengue virus type 2, a capsid protein of Dengue virus type 3, and a capsid protein of Dengue virus type 4, wherein the capsid protein is in aggregated form or particulate form.

8. The pharmaceutical composition of claim 1, further comprising a pharmacologically acceptable vehicle and an adjuvant.

9. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is suitable for oral, intramuscular, subcutaneous, mucosal, or intravenous administration.

10. A pharmaceutical composition capable of generating an immune response against Dengue virus, comprising a capsid protein of Dengue virus type 1, a capsid protein of Dengue virus type 2, a capsid protein of Dengue virus type 3, and a capsid protein of Dengue virus type 4, wherein the capsid protein of Dengue serotype 1, the capsid protein of Dengue serotype 2, the capsid protein of Dengue serotype 3, or the capsid protein of Dengue serotype 4 is fused to a peptide having the amino acid sequence consisting of SEQ ID NO: 19.