Title of Invention

A PHARMACEUTICAL PREPARATION AGAINST DENGUE VIRUSES

Abstract Pharmaceutical compound capable of induce immune protective response against Dengue virus having the capsid protein of the Dengue virus. This invention describe the a pharmaceutical compound having the capsid protein of Dengue virus capable of induce in the receptor a protective immune response before the viral challenge without inducing the Ab-dependent enhancement phenomenon.
Full Text

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, avoiding the antibody-dependent enhancement phenomenon described in persons re-infected with this virus.
Dengue fever (DF) and dengue hemorrhagic fever (DHF) acquire every time more 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. (Guzman 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. SchelesingerR.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 DJ. 1998. Dengue and Dengue Hembrrhagic 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 EJ., 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 DM,
Halstead SB, Marchette NJ. 1987. Profiles of antibody-dependent enhancement of dengue virus type 2
infection. Microb Pathog. Oct;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 Fcy receptors in the membranes, like monocytes and macrophages. This mechanism, known as antibodies-dependent enhancement (ADE) occurs during secondary infections. (Morens DM, Halstead SB, Marchette NJ. 1987. Profiles of antibody-dependent enhancement of dengue virus type 2 infection. Microb Pathog. Oct;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 rnaternal dengue antibodies are ImporlatU 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).
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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 vaccinees, 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, Jagsudee A, Saluzzo JF, 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 JF, 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 l/ll, 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 JF, 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 MT, van Amerongen G, Copra C, Osterhaus AD, Deubel V. 1999. Induction of

protective immunity against Dengue virus type 2: comparison of candidate live attenuated and recombinant vaccines. Vaccine. Mar 17;17(11-12):1312-20). On the other hand, these candidates friiquire 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 MG, 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 Jan;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 DW, Kalayanarooj S, Nimmannitya S, Nisalak A, Kurane I, Rothman AL, Ennis FA. 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 MA, Ennis FA. 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 CDS human cytotoxic T lymphocytes. J. Virol. 63:5086-5091; Gagnon SJ., 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 SF, Liao CL, Lin YL, Yeh CT, Chen LK, Huang YF, Chou HY, Huang JL, Shaio MF, Sytwu HK. 2003. Evaluation of protective efficacy and Immune mechanisms of using a non-structural protein NS1 In DNA vaccine against dengue 7 virus in mice. Vaccine. Sep 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 Call 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 bene however, it was demonstrated that this protection was mediated by the raised antibodies since the same protection using passive immunization was obtained (Tan CH, Yap EH, Singh M, Deubel V, Chan YC. 1990. Passive protection studies in mice with monoclonal antibodies directed against the non-structural protein NS3 of dengue 1 virus. J Gen Virol. 1990 Mar;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 FA, Rothman AL. 1999. Partial agonist effect influences the CTL response to a heterologous dengue virusserotype. J Immunol. Sep1; 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 PW, 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. Sep 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, Alvarez-Obregon JC, Herrera A, Lorenzo LJ, 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. Nov 22;19(7-8):992-7; Suzich JA, Ghin SJ, Palmer-Hill FJ, etal. 1995. Systemic immunization with papillomavirus LI 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 arGlnine and lysine. The presence ot these amino adds 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-I -arGlnine 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 RJ, Zhang W, Rossmann MG, Pletnev SV. Corver J, cenches E, Jones CT, Mukhopadhyay S, Chipman PR, Strauss EG, Baker TS, Strauss JH. 2002. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell. Mar 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, p7143-7149, 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 SJ. Zeng W, Kurane I, Ennis FA. 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 CP, Dong T, Chau NV, Dung NT, Chau TN, Thao le TT, Dung NT. Hien TT, Rowland-Jones S, Farrar J. 2005. Eariy 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 MG. 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 Jan;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 lyrnphoproliferative 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,

If , '■„~
phenomenon described for other recombinant capsids from other viruses like the hepatitis B virus (Alvarez JC, Guillen G. Formulations containing virus like particles as immunoenhancers by mucosal pute. 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 semipurifled preparation was detected a fraction with lower
retention times, while in the purified sample a retention time corresponding lo 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 dissapear. 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 MG, 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 Jan;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 Ralb/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.
lle. Manuel Selman-Housein Sosa.
Legal Representative.
CIGB.

FIGURES DESCRIPTION
1-Fgure 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.
Figure 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.
Figure 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 NaCI), 4. Eluted
fraction (750 mM NaCI), 5. Fraction in Tris 10 mM, EDTA 1 mM.
Figure 4, Chromatographic profile in Superdex 200 of the semipurified (A) and pure (B) preparations of
PDC-2.
Figure 5. Electronic microscopy pictures of the pure PDC-2 preparation before (A) and after (B) the
treatment with oligonucleotides.
Figure 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.
Figure 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/Hindlll, which contains the phage T5 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 1mM 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 10mM 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.


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.

* 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 aliiminum 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.



* 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).
EXAMPLES. 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 10mM, Tween 0.5%, urea 7M, pH 8. The column was washed with buffer diethanolamine 30mM, NaCI 350 mM, pH 10.3. The elution of the protein of interest was done with buffer diethanolamine 30mM, NaCI 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 (Figure 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 ranGlng from 15 to 20 minutes, evidencing the presence of high molecular weight species (Figure 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 (Figure 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, whilo in the control sample no particles were observed (Figure 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 partlculated capsid protein adjuvanted with aluminum hydroxide. The soluble fraction from the rupture of XL-7 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).

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.


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 wore dctcctod. 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.



stimulation index: quotient average of counts / minutes of samples between counts / minutes of the ADN spontaneous .ynthesis control.
** Preparation of DEN-2 infected mice brain. *** Preparation of not infected mice brain. **** Phytohemaglutinina Mitogen (Positive Control).
EXAMPLE 12. CDS 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 transfonned 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 (20ug) were administered to groups of 20 animals, using aluminum hydroxide as adjuvant. One month after the last dose, 1mg of a rat anti-mouse CDS 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


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/Hindlll 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 co//with plasmids. J. Mol. Biol. 166:557-580) were transformed with the selected clone called pDC-1 (Figure 6 y Sequence No. 10). The E. colt strains transformed were cultivated in LB supplemented with Ampicilline 50 µg/mL for 10 h at 37°C. Isopropyl-S-D-thiogalactopyranoside (IPTG) to a final concentration of ImM 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 £ 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 lOmM 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

w
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.

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.


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.

* 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 Domlll.
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 III 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/Bamtil, 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 Domlll. The E. coli strains transformed were cultivated in LB supplemented with Ampicilline 50 ^g/mL for 10 h at 37°C. Isopropyl-B-D-thiogalactopyranoside (IPTG) to a final concentration of ImM 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 Domlll 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 butter Tris lOmM 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 semlpurlfled 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 ressilting 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 esponse 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 lll-capsid protein.

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.

r V
Table 15. Stimulation indexes against the homologous serotype of the lymphocytes from immunized
mire

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).
lle. Manuel Selman-Housein Sosa
Legal Representative
CIGB

SEQUENCE LISTING Center for Genetic EnGlneering and Biotecnology
The capsid protein of Dengue virus inducer of a protective immune response and pharmaceutical composition,
Dengue
24
Patentin version 3.2
1
24
DNA
Dengue virus

primer_bind
(1)..(24)
Sequence of the oligonucleotide BamH-l to the amplification of the
capsid protein of dengue 2 virus
1
cgggatccaa taaccaacga aaaa 24
2
23
DNA
Dengue virus

primer_bind (1).-(23)
Sequence of the oligonucleotide Hind-Ill to the amplification of the capsid protein of DEN-2 virus
2 acaagctttt acctgcgtct cct
3 339 DNA Dengue virus
gene

(1).-(339)
Nucleotide sequence that codes for amino acids 1 to
113 of the capsid protein of dengue 2 virus
3
aataaccaac gaaaaaaggc gagaagtacg cctttcaata tgctgaaacg cgagagaaac 60
cgcgtgtcaa ctgtgcaaca gctgacaaag agattctcac ttggaatgct gcaaggacga 120
ggaccattaa aactgttcat ggcccttgtg gcgttccttc gtttcctaac aatcccacca 180
acagcaggga tactgaaaag atggggaacg atcaaaaaat caaaagctat caatgttttg 240
agagggttca ggaaagagat tggaaggatg ctgaacatct tgaacaggag acgcaggaca 300
gcaggcgtga ttattatgtt gattccaaca gcgatggcg 339
4 339 DNA Dengue virus

gene {1)-(339)
Nucleotide sequence that codes for the quimeric protein PDC-2 in the plasmid pQE-30
4
atgagaggat cgcatcacca tcaccatcac ggatccaata accaacgaaa aaaggcgaga 60
agtacgcctt tcaatatgct gaaacgcgag agaaaccgcg tgtcaactgt gcaacagctg 120
acaaagagat tctcacttgg aatgctgcaa ggacgaggac cattaaaact gttcatggcc 180
cttgtggcgt tccttcgttt cctaacaatc ccaccaacag cagggatact gaaaagatgg 240
ggaacgatca aaaaatcaaa agctatcaat gttttgagag ggttcaggaa agagattgga 300
aggatgctga acatcttgaa caggagacgc taaaagctt 339
5 111 PRT Dengue virus

CHAIN
(1)..(111)
Aminoacidic sequence coresponding to the protein PDC-2.
5
Met Arg Gly Ser His His His His His His Gly Ser Asn Asn Gln Arg
15 10 15

ys Lys Ala Arg Ser Thr Pro Phe Asn Met Leu Lys Arg Glu Arg Asn
20 25 30
Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg Phe Ser Leu Gly Met
35 40 45
Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met Ala Leu Val Ala Phe
50 55 60
Leu Arg Phe Leu Thr lle Pro Pro Thr Ala Gly lle Leu Lys Arg Trp
65 70 75 80
Gly Thr lle Lys Lys Ser Lys Ala lle Asn Val Leu Arg Gly Phe Arg
85 90 95
Lys Glu lle Gly Arg Met Leu Asn lle Leu Asn Arg Arg Arg Arg
100 105 110
6 3461 DNA Artificial

plasmid pQE-30

misc_feature
Nucleotide sequence of the plasmid pQE-30. In italic the restriction
sites BamHI/Hind III are underlined.
6
ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60
attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga 120
ggatcgcatc accatcacca tcacggatcc gcatgcgagc tcggtacccc gggtcgacct 180
gcagccaagc ttaattagct gagcttggac tcctgttgat agatccagta atgacctcag 240
aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga 300
atccaagcta gcttggcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc 360
actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt 420
cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta 480
aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc 540
ctgatgaatg ctcatccgga atttcgtatg gcaatgaaag acggtgagct ggtgatatgg 600
gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc 660
tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg 720

tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc 780 cagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac 840 ttcttcgccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc 900 cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta 960 atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt 1020 attggtgccc ttaaacgcct ggggtaatga ctctctagct tgaggcatca aataaaacga 1080 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 1140 ctgagtagga caaatccgcc gctctagagc tgcctcgcgc gtttcggtga tgacggtgaa 1200 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 1260 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg 1320 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga 1380 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 1440 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtctgtcggc 1500 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 1560 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 1620 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 1680 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 1740 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 1800 ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagllcgg 1860 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 1920 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 1980 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 2040 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 2100 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 2160 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 2220 ctcaagoago tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 2280 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 2340 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 2400 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagctg 2460

cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 2520
tgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 2580
cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 2640
ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 2700
ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 2760
ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 2820
gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 2880
ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 2940
ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 3000
gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 3060
ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 3120
cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 3180
ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 3240
aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 3300
gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 3360
gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 3420
cctataaaaa taggcgtatc acgaggccct ttcgtcttca c 3461
7 300 DNA Dengue virus
gene (1)-.(300)
Nucleotide sequence that codes for amino acids 11 to 100 of the capsid protein of Dengue 1 virus.
7
atgaacaacc aacggaaaaa gacggctcga ccgtctttca atatgctgaa acgcgcgaga 60
aaccgcgtgt caactgtttc acagttggcg aagagattct caaaaggatt gctctcaggc 120
caaggaccca tgaaattggt gatggccttc atagcattcc taagatttct agccataccc 180
ccaacagcag gaattttggc tagatggggc tcattcaaga agaatggagc gatcaaagtg 240
ctacggggtt tcaagaaaga aatctcaaac atgtfgaata taatgaatag aaggaaaaga 300

8 26 DNA Dengue virus

primer_bind
(1)-.(26)
Sequence of the oligonucleotide BamH-l to the amplification of the
capsid protein of DEN-1 virus.
8
ggatccatga acaaccaacg gaaaaa 26
9
24
DNA
Dengue virus

primer_bind
(1)..(24)
Sequence of the oligonucleotide Hind-Ill to the amplification of the
capsid protein of DEN-1 virus
9
aagctttctt ttccttctat tcat 24
10 345 DNA Dengue virus

gene (1)..(345)
Nucleotide sequence that codes to the quimeric protein PDC-1 in the plasmid pQE-30
10
atgagaggat cgcatcacca tcaccatcac ggatccatga acaaccaacg gaaaaagacg 60
gctcgaccgt ctttcaatat gctgaaacgc gcgagaaacc gcgtgtcaac tgtttcacag 120
ttggcgaaga gattctcaaa aggattgctc tcaggccaag gacccatgaa attggtgatg 180
gccttcatag cattcctaag atttctagcc atacccccaa cagcaggaat tttggctaga 240
tggggctcat tcaagaagaa tggagcgatc aaagtgctac ggggtttcaa gaaagaaatc 300
tcaaacatgt tgaatataat gaatagaagg aaaagataaa agctt 345

11
112
PRT
Dengue virus

CHAIN
(1)..(112)
Aminoacidic sequence corresponding to the protein PDC-1
11
Met Arg Gly Ser His His His His His His Gly Ser Met Asn Asn Gln
15 10 15
Arg Lys Lys Thr Ala Arg Pro Ser Phe Asn Met Leu Lys Arg Ala Arg
20 25 30
Asn Arg Val Ser Thr Val Ser Gln Leu Ala Lys Arg Phe Ser Lys Gly
35 40 45
Leu Leu Ser Gly Gln Gly Pro Met Lys Leu Val Met Ala Phe lle Ala
50 55 60
Phe Leu Arg Phe Leu Ala lle Pro Pro Thr Ala Gly lle Leu Ala Arg
65 70 75 80
Trp Gly Ser Phe Lys Lys Asn Gly Ala lle Lys Val Leu Arg Gly Phe
85 90 95
Lys Lys Glu He Ser Asn Met Leu Asn lle Met Asn Arg Arg Lys Arg
100 105 110
12 426 DNA Dengue virus

gene (1)..(426)
Nucleotide sequence that codes for DomB of the envelope protein of DEN-2 virus
12
aggctgagaa tggacaaact acagctcaaa ggaatgtcat actctatgtg tacaggaaag 60
tttaaaattg tgaaggaaat agcagaaaca caacatggaa caatagttat cagagtacaa 120
tatgaagggg acggctctcc atgtaagatc ccttttgaga taatggattt ggaaaaaaga 180
cacgtcttag gtcgcctgat tacagttaac ccgatcgtaa cagaaaaaga tagcccagtc 240

aacatagaag cagaacctcc attcggagac agctacatca tcataggagt agagccggga 300 caattgaaac tcaactggtt taagaaagga agttccatcg gccaaatgtt tgagacaaca 360
tgagaggag cgaagagaat ggccatttta ggtgacacag cctgggattt tggaagcctg 420
ggaggg 426
13
21
DNA
Dengue virus

primer_bind
(1)..(21)
Sequence of the oligonucleotide 5' for the amplifcation of
the region that codes for the amino acids 286 to 426 of the envelope
protein of DEN-2 virus
13
cttggatcca ttctgagaat g 21
14 33 DNA Dengue virus

primer_bind
(1)..(33)
Sequence of the oligonucleotide 3' for the amplification of
the region that codes for the amino acids 286 to 426 of the envelope
protein of DEN-2 virus
14
tgtggatcct cctcctaggc ttccaaaatc cca 33
15
753
DNA
Dengue virus

gene
(1)..(753)
Nucleotide sequence that codes for the quimeric protein pDC-2 Domlll
15
catcaccatc accatcacgg atccaggctg agaatggaca aactacagct caaaggaatg 60
tcatactcta tgtgtacagg aaagtttaaa attgtgaagg aaatagcaga aacacaacat 120

ggaacaatag ttatcagagt acaatatgaa ggggacggct ctccatgtaa gatccctttt 180
gagataatgg atttggaaaa aagacacgtc ttaggtcgcc tgattacagt taacccgatc 240
gtaacagaaa aagatagccc agtcaacata gaagcagaac ctccattcgg agacagctac 300
atcatcatag gagtagagcc gggacaattg aaactcaact ggtttaagaa aggaagttcc 360
atcggccaaa tgtttgagac aacaatgaga ggagcgaaga gaatggccat tttaggtgac 420
acagcctggg attttggatc cctgggagga ggatccaata accaacgaaa aaaggcgaga 480
agtacgcctt tcaatatgct gaaacgcgag agaaaccgcg tgtcaactgt gcaacagctg 540
acaaagagat tctcacttgg aatgctgcaa ggacgaggac cattaaaact gttcatggcc 600
cttgtggcgt tccttcgttt cctaacaatc ccaccaacag cagggatact gaaaagatgg 660
ggaacgatca aaaaatcaaa agctatcaat gttttgagag ggttcaggaa agagattgga 720
aggatgctga acatcttgaa caggagacgc taa 753
16 250 PRT Dengue virus

CHAIN
(1)..(250)
Aminoacidic sequence of PDC-2 Dom III
16
His His His His His His Gly Ser Arg Leu Arg Met Asp Lys Leu Gln
15 10 15
Leu Lys Gly Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys lle Val
20 25 30
Lys Glu He Ala Glu Thr Gln His Gly Thr lle Val lle Arg Val Gln
35 40 45
Tyr Glu Gly Asp Gly Ser Pro Cys Lys He Pro Phe Glu lle Met Asp
50 55 60
Leu Glu Lys Arg His Val Leu Gly Arg Leu He Thr Val Asn Pro He
65 70 75 80
Val Tlif Glu Lys Asp Ser Pro Val Asn He Glu Ala Glu Pro Pro Phe
85 90 95
Gly Asp Ser Tyr He He He Gly Val Glu Pro Gly Gln Leu Lys Leu
100 105 110

Asn Trp Phe Lys Lys Gly Ser Ser He Gly Gln Met Phe Glu Thr Thr
115 120 125
Met Arg Gly Ala Lys Arg Met Ala lle Leu Gly Asp Thr Ala Trp Asp
130 135 140
Phe Gly Ser Leu Gly Gly Gly Ser Asn Asn Gln Arg Lys Lys Ala Arg
145 150 155 160
Ser Thr Pro Phe Asn Met Leu Lys Arg Glu Arg Asn Arg Val Ser Thr
165 170 175
Val Gln Gln Leu Thr Lys Arg Phe Ser Leu Gly Met Leu Gln Gly Arg
180 185 190
Gly Pro Leu Lys Leu Phe Met Ala Leu Val Ala Phe Leu Arg Phe Leu
195 200 205
Thr lle Pro Pro Thr Ala Gly lle Leu Lys Arg Trp Gly Thr lle Lys
210 215 220
Lys Ser Lys Ala lle Asn Val Leu Arg Gly Phe Arg Lys Glu lle Gly
225 230 235 240
Arg Met Leu Asn lle Leu Asn Arg Arg Arg
245 250
17
142
PRT
Dengue virus type 1

CHAIN
(1)..{142)
Aminoacidic sequence corresponding to DomB of the envelope
protein of DEN-1 virus
17
Arg Leu Lys Met Asp Lys Leu Thr Leu Lys Gly Val Ser Tyr Val Met
1 5 10 15
Cys Thr Gly Ser Phe Lys Leu Glu Lys Glu Val Ala Glu Thr Gln His
20 25 30
Gly Thr Val Leu Val Gln Val Lys Tyr Glu Gly Thr Asp Ala Pro Cys
35 40 45
Lys lle Pro Phe Ser Ser Gln Asp Glu Lys Gly Val Thr Gln Asn Gly
50 55 60
Arg Leu lle Thr Ala Asn Pro lle Val lle Asp Lys Glu Lys Pro Val
65 70 75 80
Asn lle Glu Ala Glu Pro Pro Phe Gly Glu Ser Tyr lle Val Val Gly

85 90 95
Ala Gly Glu Lys Ala Leu Lys Leu Ser Trp Phe Lys Lys Gly Ser Ser
100 105 110
lle Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg Arg Met Ala
115 120 125
lle Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser lle Gly Gly
130 135 140
18
142
PRT
Dengue virus type 3

CHAIN (1)..(142)
Aminoacidic sequence corresponding to DomB of the envelope protein of DEN-3 virus
18
Arg Leu Lys Met Asp Lys Leu Glu Leu Lys Gly Met Ser Tyr Ala Met
15 10 15
Cys Thr Asn Thr Phe Val Leu Lys Lys Glu Val Ser Glu Thr Gln His
20 25 30
Gly Thr lle Leu lle Lys Val Glu Tyr Lys Gly Glu Asp Val Pro Cys
35 40 45
Lys lle Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys Ala His Asn Gly
50 55 60
Arg Leu He Thr Ala Asn Pro Val Val Thr Lys Lys Glu Glu Pro Val
65 70 75 80
Asn lle Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn lle Val lle Gly
85 90 95
lle Gly Asp Asn Ala Leu Lys lle Asn Trp Tyr Lys Lys Gly Ser Ser
100 105 110
lle Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg Arg Met Ala
115 120 125
lle Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly Gly
130 135 140
19 142

PRT
Dengue virus type 4


CHAIN
(1)..(142)
Aminoacidic sequence corresponding to Dom B of the envelope
protein of DEN-4 virus
19
Lys Val Arg Met Glu Lys Leu Arg lle Lys Gly Met Ser Tyr Thr Met
15 10 15
Cys Ser Gly Lys Phe Ser lle Asp Lys Glu Met Ala Glu Thr Gln His
20 25 30
Gly Tlir Thr Val Val Lys Val Lys Tyr Glu Gly Ala Gly Ala Pro Cys
35 40 45
Lys Val Pro lle Glu lle Arg Asp Val Asn Lys Glu Lys Val Val Gly
50 55 60
Arg He lle Ser Ser Thr Pro Leu Ala Glu Asn Thr Asn Ser Val Thr
65 70 75 80
Asn lle Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr He Val He Gly
85 90 95
Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg Lys Gly Ser Ser
100 105 110
lle Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly Ala Lys Arg Met Ala
115 120 125
He Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser Val Gly Gly
130 135 140
20
749
PRT
Dengue virus type 1

CHAIN
(1)..(749)
Aminoacidic sequence corresponding to the protein PLH3.
20
Met Gly His His His His His His Ala Met Val Asp Lys Arg Met Ala
15 10 15
Leu Val Glu Leu Lys Val Pro Asp He Gly Gly His Glu Asn Val Asp
20 25 30

He lle Ala Val Glu Val Asn Val Gly Asp Thr He Ala Val Asp Asp
35 40 45
Thr Leu He Thr Leu Asp Leu Glu Met Asp Val Pro Ala Glu Val Ala
50 55 60
Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys He Ser Glu
65 70 75 80
Gly Gly Leu lle Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro
85 90 95
Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala
100 105 110
Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu
115 120 125
Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala
130 135 140
Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala He Val Glu Arg Tyr
145 150 155 160
Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys He Pro Ser Lys
165 170 175
Ala Leu Leu His Asn Ala Ala Val He Asp Glu Val Arg His Leu Ala
180 185 190
Ala Asn Gly He Lys Tyr Pro Glu Pro Glu Leu Asp lle Asp Met Leu
195 200 205
Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala
210 215 220
Gly Met Ala Lys Ser Arg Lys Val Asp Val He Gln Gly Asp Gly Gln
225 230 235 240
Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala
245 250 255
Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys lle Val Ala Phe Lys
260 265 270
Asn Cys He He Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe He
275 280 285
Pro Glu Asp Pro Arg He lle Asp Ser Ser Gly Ala Leu Ala Leu Lys
290 295 300
Glu Val Pro Gly Lys Leu Leu He He Gly Gly Gly lle He Gly Leu
305 310 315 320
Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val
325 330 335
Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys

340 345 350
Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn lle Met Val Asn
355 360 365
Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr
370 375 380
Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val
385 390 395 400
Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu lle Ser Ala Glu
405 410 415
Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe lle Glu Val Asp Lys
420 425 430
Gln Met Arg Thr Asn Val Pro His lle Tyr Ala He Gly Asp lle Val
435 440 445
Gly Gln Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala
450 455 460
Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val lle
465 470 475 480
Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr
485 490 495
Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys lle Thr Lys Ala Asn Phe
500 505 510
Pro Trp Ala Ala Ser Gly Arg Ala lle Ala Asn Gly Cys Asp Lys Pro
515 520 525
Phe Thr Lys Leu lle Phe Asp Ala Glu Thr Gly Arg lle lle Gly Gly
530 535 540
Gly lle Val Gly Pro Asn Gly Gly Asp Met lle Gly Glu Val Cys Leu
545 550 555 560
Ala lle Glu Met Gly Cys Asp Ala Ala Asp lle Gly Lys Thr lle His
565 570 575
Pro His Pro Thr Leu Gly Glu Ser lle Gly Met Ala Ala Glu Val Ala
580 585 590
Leu Gly Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Arg
595 600 605
Leu Lys Met Asp Lys Leu Thr Leu Lys Gly Val Ser Tyr Val Met Cys
610 615 620
Thr Gly Ser Phe Lys Leu Glu Lys Glu Val Ala Glu Thr Gln His Gly
625 630 635 640
Thr Val Leu Val Gln Val Lys Tyr Glu Gly Thr Asp Ala Pro Cys Lys
645 650 655

lle Pro Phe Ser Ser Gln Asp Glu Lys Gly Val Thr Gln Asn Gly Arg
660 665 670
Leu lle Thr Ala Asn Pro lle Val lle Asp Lys Glu Lys Pro Val Asn
675 680 685
He Glu Ala Glu Pro Pro Phe Gly Glu Ser Tyr lle Val Val Gly Ala
690 695 700
Gly Glu Lys Ala Leu Lys Leu Ser Trp Phe Lys Lys Gly Ser Ser He
705 710 715 720
Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg Arg Met Ala He
725 730 735
Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser He Gly Gly
740 745
21
749
PRT
Dengue virus type 3

CHAIN
(1)..(749)
Aminoacidic sequence corresponding to the protein PAZ3.
21
Met Gly His His His His His His Ala Met Val Asp Lys Arg Met Ala
1 5 10 15
Leu Val Glu Leu Lys Val Pro Asp He Gly Gly His Glu Asn Val Asp
20 25 30
He lle Ala Val Glu Val Asn Val Gly Asp Thr He Ala Val Asp Asp
35 40 45
Thr Leu He Thr Leu Asp Leu Glu Met Asp Val Pro Ala Glu Val Ala
50 55 60
Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys lle Ser Glu
65 70 75 80
Gly Gly Leu lle Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro
85 90 95
Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala
100 105 110
Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu
115 120 125
Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala
130 135 140

Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala lle Val Glu Arg Tyr
145 150 155 160
Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys lle Pro Ser Lys
165 170 175
Ala Leu Leu His Asn Ala Ala Val lle Asp Glu Val Arg His Leu Ala
180 185 190
Ala Asn Gly lle Lys Tyr Pro Glu Pro Glu Leu Asp lle Asp Met Leu
195 200 205
Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala
210 215 220
Gly Met Ala Lys Ser Arg Lys Val Asp Val lle Gln Gly Asp Gly Gln
225 230 235 240
Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala
245 250 255
Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys lle Val Ala Phe Lys
260 265 270
Asn Cys lle lle Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe lle
275 280 285
Pro Glu Asp Pro Arg He lle Asp Ser Ser Gly Ala Leu Ala Leu Lys
290 295 300
Glu Val Pro Gly Lys Leu Leu He lle Gly Gly Gly He lle Gly Leu
305 310 315 320
Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val
325 330 335
Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys
340 345 350
Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn He Met Val Asn
355 360 365
Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr
370 375 380
Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val
385 390 395 400
Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu He Ser Ala Glu
405 410 415
Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe He Glu Val Asp Lys
420 425 430
Gln Met Arg Thr Asn Val Pro His He Tyr Ala He Gly Asp He Val
435 440 445

Gly Gln Pro Met Leu Ala Hls Lys Ala Val His Glu Gly His Val Ala
450 455 460
Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val lle
465 470 475 480
Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr
485 490 495
Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys lle Thr Lys Ala Asn Phe
500 505 510
Pro Trp Ala Ala Ser Gly Arg Ala lle Ala Asn Gly Cys Asp Lys Pro
515 520 525
Phe Thr Lys Leu lle Phe Asp Ala Glu Thr Gly Arg lle lle Gly Gly
530 535 540
Gly lle Val Giy Pro Asn Gly Gly Asp Met lle Gly Glu Val Cys Leu
545 550 555 560
Ala lle Glu Met Gly Cys Asp Ala Ala Asp lle Gly Lys Thr lle His
565 570 575
Pro His Pro Thr Leu Gly Glu Ser lle Gly Met Ala Ala Glu Val Ala
580 585 590
Leu Gly Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Arg
595 600 605
Leu Lys Met Asp Lys Leu Lys Leu Lys Gly Met Ser Tyr Ala Met Cys
610 615 620
Leu Asn Thr Phe Val Leu Lys Lys Glu Val Ser Glu Thr Gln His Gly
625 630 635 640
Thr lle Leu lle Lys Val Glu Tyr Lys Gly Glu Asp Ala Pro Cys Lys
645 650 655
lle Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys Ala His Asn Gly Arg
660 665 670
Leu lle Thr Ala Asn Pro Val Val Thr Lys Lys Glu Glu Pro Val Asn
675 680 685
He Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn lle Val lle Gly He
690 695 700
Gly Asp Lys Ala Leu Lys He Asn Trp Tyr Arg Lys Gly Ser Ser He
705 710 715 720
Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg Arg Met Ala II&
725 730 735
Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly Gly
740 745

22
749
PRT
Dengue virus type 4

CHAIN
(1)..{749)
Aminoacidic sequence corresponding to PID3.
22
Met Gly His His His His His His Ala iVIet Val Asp Lys Arg Met Ala
15 10 15
Leu Val Glu Leu Lys Val Pro Asp lle Gly Gly His Glu Asn Val Asp
20 25 30
lle lle Ala Val Glu Val Asn Val Gly Asp Thr lle Ala Val Asp Asp
35 40 45
Thr Leu lle Thr Leu Asp Leu Glu Met Asp Val Pro Ala Glu Val Ala
50 55 60
Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys lle Ser Glu
65 70 75 80
Gly Gly Leu lle Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro
85 90 95
Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala
100 105 110
Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu
115 120 125
Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala
130 135 140
Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala lle Val Glu Arg Tyr
145 150 165 160
Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys lle Pro Ser Lys
165 170 175
Ala Leu Leu His Asn Ala Ala Val He Asp Glu Val Arg His Leu Ala
180 185 190
Ala Asn Gly lle Lys Tyr Pro Glu Pro Glu Leu Asp lle Asp Met Leu
195 200 205
Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala
210 215 220
Gly Met Ala Lys Ser Arg Lys Val Asp Val lle Gln Gly Asp Gly Gln
225 230 235 240

Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala
245 250 255
lyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys lle Val Ala Phe Lys
260 265 270
Asn Cys lle lle Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe lle
275 280 285
Pro Glu Asp Pro Arg lle lle Asp Ser Ser Gly Ala Leu Ala Leu Lys
290 295 300
Glu Val Pro Gly Lys Leu Leu lle lle Gly Gly Gly lle lle Gly Leu
305 310 315 320
Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val
325 330 335
Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys
340 345 350
Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn lle Met Val Asn
355 360 365
Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr
370 375 380
Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val
385 390 395 400
Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu lle Ser Ala Glu
405 410 415
Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe lle Glu Val Asp Lys
420 425 430
Gln Met Arg Thr Asn Val Pro His lle Tyr Ala lle Gly Asp lle Val
435 440 445
Gly Gln Pro Mel Leu Ala His Lys Ala Val His Glu Gly His Val Ala
450 455 460
Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val lle
465 470 475 480
Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr
485 490 495
Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys lle Thr Lys Ala Asn Phe
500 505 510
Pro Trp Ala Ala Ser Gly Arg Ala lle Ala Asn Gly Cys Asp Lys Pro
515 520 525
Phe Thr Lys Leu He Phe Asp Ala Glu Thr Gly Arg lle lle Gly Gly
530 535 540
Gly lle Val Gly Pro Asn Gly Gly Asp Met lle Gly Glu Val Cys Leu

545 550 555 560
Ala lle Glu Met Gly Cys Asp Ala Ala Asp lle Gly Lys Thr lle His
565 570 575
Pro His Pro Thr Leu Gly Glu Ser lle Gly Met Ala Ala Glu Val Ala
580 585 590
Leu Gly Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Lys
595 600 605
Val Arg Met Glu Lys Leu Arg He Lys Gly Met Ser Tyr Thr Met Cys
610 615 620
Ser Gly Lys Phe Ser lle Asp Lys Glu Met Ala Glu Thr Gln His Gly
625 630 635 640
Thr Thr Val Val Lys Val Lys Tyr Glu Gly Ala Gly Ala Pro Cys Lys
645 650 655
Val Pro lle Glu He Arg Asp Val Asn Lys Glu Lys Val Val Gly Arg
660 665 670
He lle Ser Ser Thr Pro Leu Ala Glu Asn Thr Asn Ser Val Thr Asn
675 680 685
lle Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr lle Val lle Gly Val
690 695 700
Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg Lys Gly Ser Ser lle
705 710 715 720
Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly Ala Lys Arg Met Ala lle
725 730 735
Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser Val Gly Gly
740 745
23
750
PRT
Dengue virus type 2

CHAIN
(1)..(750)
Aminoacidic sequence corresponding to PLL3.
23
Met Gly His His His His His His Ala Met Val Asp Lys Arg Met Ala
15 10 15
Leu Val Glu Leu Lys Val Pro Asp lle Gly Gly His Glu Asn Val Asp
20 25 30

lle lle Ala Val Glu Val Asn Val Gly Asp Thr lle Ala Val Asp Asp
35 40 45
Thr Leu lle Thr Leu Asp Leu Glu Met Asp Val Pro Ala Glu Val Ala
50 55 60
Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys lle Ser Glu
5 70 75 80
Gly Gly Leu lle Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro
85 90 95
Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala
100 105 110
Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu
115 120 125
Tyr Asp Val Vai Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala
130 135 140
Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala lle Val Glu Arg Tyr
145 150 155 160
Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys lle Pro Ser Lys
165 170 175
Ala Leu Leu His Asn Ala Ala Val lle Asp Glu Val Arg His Leu Ala
180 185 190
Ala Asn Gly lle Lys Tyr Pro Glu Pro Glu Leu Asp lle Asp Met Leu
195 200 205
Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala
210 215 220
Gly Met Ala Lys Ser Arg Lys Val Asp Val lle Gln Gly Asp Gly Gln
225 230 235 240
Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala
245 250 255
Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys He Val Ala Phe Lys
260 265 270
Asn Cys lle lle Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe He
275 280 285
Pro Glu Asp Pro Arg lle He Asp Ser Ser Gly Ala Leu Ala Leu Lys
290 295 300
Glu Val Pro Gly Lys Leu Leu He He Gly Gly Gly He He Gly Leu
305 310 315 320
Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val
325 330 335

Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys
340 345 350
al Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn He Met Val Asn
355 360 365
Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr
370 375 380
Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val
385 390 395 400
Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu lle Ser Ala Glu
405 410 415
Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe lle Glu Val Asp Lys
420 425 430
Gln Met Arg Thr Asn Val Pro His lle Tyr Ala lle Gly Asp lle Val
435 440 445
Gly Gln Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala
450 455 460
Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val lle
465 470 475 480
Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr
485 490 495
Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys lle Thr Lys Ala Asn Phe
500 505 510
Pro Trp Ala Ala Ser Gly Arg Ala lle Ala Asn Gly Cys Asp Lys Pro
515 520 525
Phe Thr Lys Leu lle Phe Asp Ala Glu Thr Gly Arg lle lle Gly Gly
530 535 540
Gly lle Val Gly Pro Asn Gly Gly Asp Met lle Gly Glu Val Cys Leu
545 550 555 560
Ala He Glu Met Gly Cys Asp Ala Ala Asp lle Gly Lys Thr lle His
565 570 575
Pro His Pro Thr Leu Gly Glu Ser He Gly Met Ala Ala Glu Val Ala
580 585 590
Leu Gly Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Asp
595 600 605
Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met
610 615 620
Cys Thr Gly Lys Phe Lys He Val Lys Glu He Ala Glu Thr Gln His
625 630 635 640
Gly Thr He Val He Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys

645 650 655
Lys He Pro Phe Glu He Met Asp Leu Glu Lys Arg His Val Leu Gly
660 665 670
Arg Leu He Thr Val Asn Pro He Val Thr Glu Lys Asp Ser Pro Val
675 680 685
Asn lle Glu Ala Glu Pro Pro Phe Gly Asp Ser Tyr lle lle lle Gly
690 695 700
Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser
705 710 715 720
lle Gly Gln Met Phe Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala
725 730 735
He Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Leu Gly Gly
740 745 750
24
142
PRT
Dengue virus type 2

CHAIN
(1)..(142)
Aminoacidic sequence corresponding to the DomB of the envelope
protein of DEN-2 virus.
24
Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met
15 10 15
Cys Thr Gly Lys Phe Lys He Val Lys Glu lle Ala Glu Thr Gln His
20 25 30
Gly Thr He Val lle Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys
35 40 45
Lys He Pro Phe Glu He Met Asp Leu Glu Lys Arg His Val Leu Gly
50 55 60
Arg Leu lle Thr Val Asn Pro lle Val Thr Glu Lys Asp Ser Pro Vai
65 70 75 80
Asn He Glu Ala Glu Pro Pro Phe Gly Asp Ser Tyr lle lle lle Gly
85 90 95
Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser
100 105 110
lle Gly Gln Met Phe Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala
115 120 125

lie Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Leu Gly Gly
130 135 140
Lic. Manuel Selman-Housein Sosa.
Legal Representative.
CIGB.














1 A pharmaceutical preparation characterized by being a vaccine capable to induce in the receptor organism an immune response against Dengue viruses and containing the capsid protein of one or several Dengue virus serotypes, alone or combined with other antigens.
2. A pharmaceutical preparation according to claim 1, characterized by containing the capsid
protein of Dengue virus 1, 2, 3 or 4,, alone, mixed or combined among them.
3. A pharmaceutical preparation according to claim 1, characterized by containing the capsid
protein mixed or combined with antigens capable of inducing a hunnoral and/or cellular
response.
4. A pharmaceutical preparation according to claims 1 and 3, characterized by containing the
capsid protein mixed with one or several proteins identified in the sequencing list as
Sequence 20, Sequence 21, Sequence 22 and Sequence 23,
5. A pharmaceutical preparation according to claims 1 and 3, characterized by containing the
capsid protein fused chemically or genetically with one or several sequences identified in the
sequencing list as Sequence 17, Sequence 18, Sequence 19 y Sequence 24.
6. A pharmaceutical preparation according to claims 1, 2, 3, 4 and 5 characterized by
containing the capsid protein in aggregated or particulate form.
7. A pharmaceutical preparation according to claims 1, 2, 3, 4, 5 and 6 characterized by
containing a pharmacologically acceptable vehicle and an oily or not oily adjuvant
8. A pharmaceutical preparation according to claims 1,2, 3, 4, 5, 6 and 7 characterized by
being a preventive or therapeutic agent against the Dengue viruses, for oral, intramuscular,
subcutaneous, mucosal or intravenous use,
9, A pharmaceutical preparation substantially such as herein described with reference to the accompanying drawings and as illustrated in the forgoing examples.


Documents:

1634-chenp-2008 form-1 26-08-2011.pdf

1634-chenp-2008 form-3 26-08-2011.pdf

1634-CHENP-2008 AMENDED PAGES OF SPECIFICATION 26-08-2011.pdf

1634-CHENP-2008 AMENDED CLAIMS 26-08-2011.pdf

1634-CHENP-2008 EXAMINATION REPORT REPLY RECEIVED 26-08-2011.pdf

1634-chenp-2008-abstract.pdf

1634-chenp-2008-claims.pdf

1634-chenp-2008-correspondnece-others.pdf

1634-chenp-2008-description(complete).pdf

1634-chenp-2008-drawings.pdf

1634-chenp-2008-form 1.pdf

1634-chenp-2008-form 3.pdf

1634-chenp-2008-form 5.pdf

1634-chenp-2008-pct.pdf


Patent Number 248882
Indian Patent Application Number 1634/CHENP/2008
PG Journal Number 36/2011
Publication Date 09-Sep-2011
Grant Date 05-Sep-2011
Date of Filing 01-Apr-2008
Name of Patentee CENTRO DE INGENIERIA GENETICA Y BIOTECNOLOGIA
Applicant Address AVE. 31 ENTRE 158Y 190 CUBANACAN, PLAYA C HABANA 12100
Inventors:
# Inventor's Name Inventor's Address
1 LAZO VAZQUEZ, LAURA ARELLANO 274E/C Y ALTARRIBA LAWTON 10 DE OCTUBRE CIUDAD DE LA HABANA 10700
2 HERMIDA CRUZ, LISSET CASTILLO 61E/1RA Y 2DA REPARTO CALIFORNIA SAN MIGUEL DEL PADRON CIUDAD DE LA HABANA 11000
3 LOPEZ ABARRATEGUI, CARLOS CISNERO BETANCOURT 453 LOS PINOS, ARROYO NARANJO CIUDAD DE LA HABANA 11800
4 SIERRA VAZQUEZ, BEATRIZ DE LA CARIDAD CALLE 312 NO.2925 E/29 Y 31 JUAN DE DIOS FRAGA, LA LISA, CIUDAD DE LA HABANA 17100
5 VAZQUEZ RAMUNDO, SUSANA AVE 31 NO 31607 E/ 316 Y 318 REPARTO FRAGA, LA LISA CIUDAD DE LA HABANA 17100
6 VALDEZ PRADO, IRIS SANTA FELICIA 427 E/ROSA ENRIQUE Y MELONES LUYANO, CIUDAD DE LA HABANA 10700
7 GUILLEN NIETO, GERARDO, ENRIQUE LINEA 6 E/ N Y O PISO 4 VEDADO PLAZA DE LA REVOLUCION CIUDAD DE LA HABANA 10400
8 GUZMAN TIRADO, MARIA, GUADALUPE CALLE 28 NO 116/E 1RA Y 3RA, MIRAMAR, PLAYA, CIUDAD DE LA HABANA 11300
9 ZULUETA MORALES, AIDA EDIFICIO 77 APTO 32 ZONA 21 ALAMAR, CIUDAD DE LA HABANA 17200
PCT International Classification Number A61K 39/12
PCT International Application Number PCT/CU2006/000008
PCT International Filing date 2006-09-18
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 CU2005-0168 2005-09-16 Cuba