PATENT DOCUMENT

Abstract:
The present invention is related to the obtaining of chimeric chains coding for proteins capable of inducing, in the recipient, a serotype-specific and protective humoral immune response against the infection by the Dengue virus, thus eliminating the effects of the serotype-nonespecific viral immunoenhancement that causes hemorrhagies and clinical complications described for this kind of pathology. These chimeric chains of nucleic acids are composed by the specific combination of fragments belonging to the gene of a mutated protein from  Neisseria meningitidis  with dehydrogenase activity and fragments that codify for a region of the envelope (E) protein from the Dengue virus which, when inserted to an expression vector, give rise to chimeric proteins with particular properties. The resultant chimeric molecules from this invention are applicable to the pharmaceutical industry for the obtaining of vaccine preparations and diagnostic means of high serotype-specificity to be used in humans.

Full Description:
FIELD OF THE INVENTION  
         [0001]    The present invention is related with the field of the biotechnology and the pharmaceutical industry, in particular with the obtaining of chimeric nucleotide chains which, when introduced into an expression vector, give rise to proteins able to elicit a serotype-specific humoral immune response and protection against the Dengue virus infection; quoted from now on as DEN, avoiding the effects of the serotype-specific viral immunoamplification, causing hemorrhages and clinical complications described in this type of pathology.  
         PREVIOUS TECHNIQUE  
         [0002]    The Dengue virus (DEN), is a coated virus whose lipid membrane contains two of its three structural proteins: the envelope protein (E) and the membrane protein (M). The E protein covers an icosaedric nucleocapsid composed by the third of its structural proteins, the core protein. This virus belongs to the  Flaviviridae  family and four different serotypes exist. Its transmission to the man is carried out through the mosquito  Aedes aegypti  that belongs to the  Stegomia  family. The disease produced in the human for this virus was considered as benign and was described as Dengue Fever or Classical Dengue (DF) until the appearance of a more serious modality and sometimes lethal, characterized by hemorrhagic fever and shock, denominated: Hemorrhagic Dengue Fever and Dengue Shock Syndrome (HDF/DSS) (Hammon WMc. New hemorrhagic fever in children in the Philippines and Thailand. Trans Assoc Physicians 1960; 73: 140-155). Several epidemiological studies have been carried out evidencing as a risk factor the sequential infection of two different viral serotypes (Kouri G P, Guzman M G, Brave J R. Why dengue hemorrhagic fever in Cuba) 2. An integral analysis. Trans Roy Soc Trop Med Hyg 1987; 72: 821-823). This phenomenon is explained by the immuno-enhancement, which is based on an increase of the viral infection by increasing the entrance of the virus-antibody complex to the cell through the Fc receptors of the target cell (monocytes) (haistead SB. Pathogenesis of dengue: challenges to molecular biology. Science 1988; 239: 476-481).  
           [0003]    Different technologies have been developed to produce live attenuated vaccines, but at present there exist multiple unsolved issues on the possible benefits of these vaccines, since they could revert to the virulence, viral interference and inter-genomic recombination. Alternately, recombinant antigens can be obtained as possible components of a subunit vaccine (Feighny, R., Borrous, J. and Putnak R. Dengue type-2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge. Am. J. Trop. Med. Hyg. 1994. 50(3). 322-328; Deubel, V., Staropoli, I., Megret, F., et al. Affinity-purified dengue-2 virus envelope glycoprotein induces neutralizing antibodies and protective immunity in mice. Vaccine. 1997. 15, 1946-1954).  
           [0004]    The main antigen of the virus is the envelope protein DENe. This protein is the major component of the viral surface and is thought to mediate the binding of the virus to the cellular receptor (A Heinz F X, Berge R, Tuma W et al. A topological and functional model of epitopes on the structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies. Virology. 1983; 126: 525). This protein has structural homology with that of the tick borne encephalitis virus (TBE) (Rey, F. A., Heinz, F. X., Mandl, C., et al. The envelope glycoprotein from tick borne encephalitis virus at 2 A° resolution. Nature 1995; 375: 291-298) and it is also structurally conserved among serotypes.  
           [0005]    The insect cells constitute one of the systems most used for the expression of diverse heterologous genes that employ the baculovirus system as vectors. These vectors have been used for the expression of several combinations of structural and nonstructural proteins of the Encephalitis Japanese virus (JEV), DEN-1, DEN-2 and DEN4, (Matsuura Y, Miyamoto M, Soto T et al. Characterization of japanese encephalitis virus envelope protein expressed by recombinant baculoviruses. Virology 1989; 173: 677-682; Deubel V, Bordier M, Megret F et al. Processing, secretion and immunoreactivity of carboxy terminally truncated dengue-2 envelope proteins expressed in insect cell by recombinant baculoviruses. Virology 1991; 180: 440-447; Putnak R, Feighny R, Burrous J et al. Dengue 1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicit virus neutralization antibodies in mice and protects them from virus challenge. Am J Trop Med Hyg 1991; 45: 159-167; Feighny R, Burrous J, Putnak R. Dengue type 2 virus envelope protein made using recombinant baculovirus protects mice against virus challenge. Am J Trop Med Hyg 1994; 50: 322-328). Another system used has been the cells of  Drosophila melanogaster  expressing different variants of the E protein (PCT/US96/07627). In spite of obtaining an appropriate functional response, upon using the proteins expressed in these systems, they imply a high cost for the development of scale-up production processes; therefore, the expression in yeast has been an alternative to produce recombinant structural proteins of flavivirus. However, in the case of the DENe protein, expressed in  Pichia pastoris  (PCT/US96/07627; Sugrue R. J., Fu H., Howe J., Chan Y. Expression of the Dengue virus structural proteins in  Pichia pastoris  leads to the generation of virus-like particles. J. Virol. 1997. 78, 1861-1866), the levels of expression are low, either secreted or intracellularly, hindering the purification process.  
           [0006]    In parallel, several variants of the DENe protein have been obtained in bacteria. One of them was the C-terminal portion of the E protein of the JEV fused to a protein of the Tryptophan metabolism (TrpE) of  E. coli . This protein was produced as inclusion bodies and was recognized by neutralizing monoclonal antibodies (Mabs) using immunodetection techniques. However, pure preparations of this protein were unable to develop neutralizing antibodies and to protect against viral challenge (Mason P. W., Zogel M. V., Semproni A. R., et al. The antigenic structure of dengue type I virus envelope and NS1 protein expressed in  E. coli . J Gen Virol. 1990. 71: 2107-2114). In addition, another construction was made (Srivastava A. K., Morita K., Matsuo S., et al. Japanese encephalitis virus fusion protein with protein A expressed in  E. coli  confers protection in mice. Microbiol Immunol. 1991. 35: 863-870), that contains the protein A of  Staphylococcus aurius  fused to the C-terminal fragment of the E protein, followed by the N-terminal segment of the nonstructural protein of the JEV, the NS1. In this case the fused protein was soluble, facilitating its purification by affinity chromatography. Upon immunizing mice with this pure protein high neutralizing antibodies titers were obtained, which also inhibited haemagglutination and protected against the viral challenge with the JEV. Similar results were obtained using the DENe region of the DEN-2 fused to the protein A of  S. aureus  (Srivastava A. K., Putnak R. J., Warren R. L., Hoke C. H. Mice immunized with a dengue type 2 virus E and NS1 fusion protein made in  Escherichia coli  are protected against lethal dengue virus infection. Vaccine. 1995. 13: 1251-1258); however, it is not possible to use these preparations in humans due to the presence of the protein A, which has shown a high affinity for the human immunoglobulin G (IgG).  
           [0007]    Finally, it has been reported a fusion protein that contains the B domain of the DENe protein of DEN-2 and the maltose binding protein (MBP) of  E. coli  (Simmons M., Nelson W. M., Wu S. J., Hayes C. G. Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice. Am J Trop Med Hyg. 1998. 58: 655-662) denominated MBP-DomB. This protein variant was immunogenic in mice and elicited neutralizing antibodies.  
           [0008]    In our case, the subject of this invention relies on the obtaining of chimeric sequences, as for instance, in the first case, the sequence coding for a region of the DENe protein linked to the N-terminal fragment of a mutated protein with dehydrogenase activity (MDH) from  Neisseria meningitidis ; in the second case, the sequence coding for a region of the DENe protein linked to the entire gene of the MDH protein in two different positions, and in the third case, the chimeric sequences are formed by two fragments of the DENe protein from two different viral serotypes fused to the same gene coding for the MDH protein. These chimeric chains when inserted into a suitable vector, give rise to insoluble chimeric proteins within the bacterium&#39;s cytoplasm. These proteins are then capable to elicit high levels of neutralizing antibodies against DEN, inhibitors of the viral hemagglutination and to protect immunized mice against viral challenge.  
           [0009]    With regards to the insolubility of the aforementioned proteins, an easy scale-up folding process was achieved in vitro, as well as the expression and purification processes which were superior to those used by Simmons et al, 1998. On the other hand, the serotype specificity of the antibodies is demonstrated, generated by immunization of mice with these proteins, at the level of neutralization, inhibition of hemaglutination and ELISA, using doses lower than those employed by Simmons et al, 1998. This fact constitutes the first report on the expression of insoluble DENe proteins in  E. coli  capable of stimulating a functionl immune response.  
           [0010]    In addition, considering the results obtained with the dimeric variant, it is possible to generate serotype-specific antibodies with the same molecule for two different viral serotypes, capable of neutralizing viral infection and protect mice against viral challenge. Concerning the MDH protein, a search for homology with other sequences was done in the EMBL data base, revealing that the first 110 amino acids are highly similar to the lipoic binding domain region and the flexible hinge of the dihydrolipoamide acetyltransferase (E2 enzyme of the pyruvate dehydrogenase complex and α-cetoglutarate dehydrogenase), and the rest of the protein is highly similar to the lipoamide dehydrogenase (LPDH), enzyme E3 of said complexes (Stephens, P. E; H. M. Darlinson, and J. R. Guest., 1983. The Pyruvate dehydrogenase complex of  E. coli . Eur. J. Biochem. 133:155-162.  
           [0011]    On the other hand, it was also found that patients with Primary Biliary Cirrhosis (PBC) produced anti-mitochondrial autoantibodies, specific for the lipoic acid binding site, common among these proteins (Gershwin M E, Mackay I R, Sturgess A, Coppel R L. Identification and specificity of a cDNA encoding the 70 KDa mitochondrial antigen recognized in primary biliary cirrhosis. J Immunol 1987;138:3525-31). Therefore, we decided to mutate this region within the protein to avoid any autoimmune response when immunized in humans as chimeric proteins. The mutated MDH protein of our invention was used in a Phase I clinical trial and showed to be safe and immunogenic in humans, and also was not recognized by sera of patients with PBC (Perez, A., F. Dickinson, Z. Cinza, A. Ruiz, T. Serrano, J. Sosa, S. González, Y. Gutiérrez, C. Nazábal, O. Gutiérrez, D. Guzmán, M. Diaz, M. Delgado, E. Caballero, G. Sardiñas, A. Alvarez, A. Martin, G. Guillén, R. Silva. Safety and preliminary immunogenicity of the recombinant outer membrane protein of  Neisseria meningitidis  in human volunteers. Biotech. Appl. Biochem. 34: 121-125). However, the possible use of the MBP in humans has not been demonstrated yet (Simmons M., Nelson W. M., Wu S. J., Hayes C. G. Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice. Am J Trop Med Hyg. 1998. 58: 655-662).  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0012]    In this invention is described the obtaining of chimeric nucleotide chains that when introduced into an expression vector, give rise to chimeric proteins capable of inducing a serotype-specific humoral immune response and protecting against the infection by Dengue virus, as for instance, the sequence coding for a region of the DENe protein from each one of the viral serotypes of the Dengue virus, linked to the N-terminal fragment of a mutated protein with dehydrogenase activity (MDH) from  Neisseria meningitidis ; in the second case, the sequence coding for a region of the DENe protein linked to the entire gene of the MDH protein in two different positions: within one site of the sequence coding for the structural domain of the MDH protein (lipoic acid binding domain and the 3′ end of the gen), and in the third case, the chimeric sequences are formed by two fragments of the DENe protein from two different viral serotypes, DEN-2 and DEN-4, in two different positions of the MDH gen: one within a particular site of the sequence coding for the lipoic acid binding domain (serotype 4) and the other in the 3′ end of the MDH gen (serotype 2). This was called a dimeric construct.  
           [0013]    This chimeric proteins were obtained insoluble within the bacterium&#39;s cytoplasm. A purification process by immobilazed metal affinity chromatography (IMAC) was done which led to obtain pure proteins for immunogenicity studies.  
           [0014]    When antigenicity results were analyzed a strong recognition of all the recombinant chimeric proteins for the hyperimmune ascitic liquids (HMAF) anti-DEN was demonstrated, being higher for the case of the fusion to the entire MDH gene, which evidences a positive effect on the folding of the region from the DENe protein given by the MDH. In the cases where the serotype 2 was used, all recombinant proteins obtained were recognized by a serotype-specific neutralizing antibody (3H5), being also higher for the case of the fusion to the entire MDH gene, as well as in the dimeric protein. It was also observed that the recognition for the HMAF from the homologous serotype in each case was significantly higher than the recognition for the HMAF from the heterologous serotypes, evidencing the exposure of serotype-specific epitopes and permitting thus its use as a diagnostic mean for Dengue and serotyping.  
           [0015]    When all the recombinant chimeric proteins were immunized in mice a neutralizing and protective response was obtained. Highest neutralizing titers were obtained with the sequences fused to the entire gene of the MDH and with the dimeric protein, independently of the position of the fragment from the DENe protein. This showed an immunopotentiator effect of the immune response mediated by the MDH that can be explained by the influence in the folding of the DENe protein reflected in the antigenicity results obtained. It was also demonstrated for the first time, and contrary to the previous state of the art, that the insolubility of these proteins do not affect the capacity of generating a suitable immune response.  
           [0016]    The immune response produced in all the cases was serotype-specific (antibodies against the immunized homologous serotype) in the viral neutralization, the hemoagglutination inhibition and ELISA. The generation of serotype-specific antibodies means that they are not capable to recognize antigenic determinants from virus of heterologous serotypes that favour the immunoenhacement phenomenum. This characteristic is of great importance for the development of a vaccine candidate against the Dengue virus since the recognition of antibodies to heterologous serotypes could be one of the causes for the Hemorrhagic Dengue Fever (HDF).  
           [0017]    Besides it was showed the induction of antibodies against two viral serotypes after immunization with just one of the chimeric proteins, which permits the formulation of a vaccine candidate against the four serotypes, using only two of our available recombinant chimeric proteins.  
           [0018]    The obtaining of the mutant MDH protein consisted of the elimination of the lipoic acid binding site in the sequence ETDKAT, based on the covalent binding of this fatty acid with the epsilon-amine groups of lysine (K) (Tuaillon N, Andre C, Briand J P et al. A lipoyl synthetic octadecapeptide of dihydrolipoamide acetyltransferase specifically recognized by anti-M2 autoantibodies in Primary Biliary Cirrhosis. J Immunol 1992; 148:445-50).  
           [0019]    The mutagenesis was done by using PCR with a pair of primers to amplify the N-terminal region (from the start codon of the lpdA gene until the lipoic acid binding site, 135 bp) and the C-terminal of the protein (from the lipoic acid binding site until the 3′ end of the gene); thus, being eliminated the possibility to generate autoimmune reactions, as demonstrated in the human clinical trials.  
           [0020]    Deposit of the Biological Material  
           [0021]    Plasmids PLL1, PLL2, PLL3, PLH1, PLH2, PLH3, PAZ1, PAZ2, PAZ3, PID1, PID2 and PID3 were deposited according to the Budapest Treaty in the Belgian Coordinated collection of Microorganism—BCCM™, LMBP-COLLECTION, and with the date and under the access numbers 
       
    
    
     DESCRIPTION OF FIGURES  
       [0022]    [0022]FIG. 1. Cloning strategy of the E2 fragment to obtain PLL1.  
         [0023]    DENe2: Fragment of the envelope protein of DEN-2.  
         [0024]    N-term: Nucleotide sequence that codifies for the first 45 amino acids of the MDH protein.  
         [0025]    [0025]FIG. 2. Cloning strategy of the E2 fragment to obtain PLL2.  
         [0026]    DENe2: Fragment of the envelope protein of DEN-2.  
         [0027]    MDH: dehydrogenase mutant.  
         [0028]    [0028]FIG. 3. Cloning strategy of the E2 fragment to obtain PLL3.  
         [0029]    DENe2: Fragment of the envelope protein of DEN-2.  
         [0030]    MDH: dehydrogenase mutant.  
         [0031]    [0031]FIG. 4. Cloning strategy of the E1 fragment to obtain PLH1.  
         [0032]    DENe1: Fragment of the envelope protein of DEN-1.  
         [0033]    N-term: Nucleotide sequence that codifies for the first 45 amino acids of the MDH protein.  
         [0034]    [0034]FIG. 5. Cloning strategy of the E1 fragment to obtain PLH2.  
         [0035]    DENe1: Fragment of the envelope protein of DEN-1.  
         [0036]    N-term: Nucleotide sequence that codifies for the first 45 amino acids of the MDH protein.  
         [0037]    [0037]FIG. 6. Cloning strategy of the E1 fragment to obtain PLH3.  
         [0038]    DENe1: Fragment of the envelope protein of DEN-1.  
         [0039]    MDH: dehydrogenase mutant.  
         [0040]    [0040]FIG. 7. Cloning strategy of the E3 fragment to obtain PAZ1.  
         [0041]    DENe3: Fragment of the envelope protein of DEN-3.  
         [0042]    N-term: Nucleotide sequence that codifies for the first 45 amino acids of the MDH protein.  
         [0043]    [0043]FIG. 8. Cloning strategy of the E3 fragment to obtain PAZ2.  
         [0044]    DENe3: Fragment of the envelope protein of DEN-3.  
         [0045]    MDH: dehydrogenase mutant.  
         [0046]    [0046]FIG. 9. Cloning strategy of the E3 fragment to obtain PAZ3.  
         [0047]    DENe3: Fragment of the envelope protein of DEN-3.  
         [0048]    MDH: dehydrogenase mutant.  
         [0049]    [0049]FIG. 10. Cloning strategy of the E4 fragment to obtain PID1.  
         [0050]    DENe4: Fragment of the envelope protein of DEN-4.  
         [0051]    N-term: Nucleotide sequence that codifies for the first 45 amino acids of the MDH protein.  
         [0052]    [0052]FIG. 11. Cloning strategy of the E4 fragment to obtain PID2.  
         [0053]    DENe4: Fragment of the envelope protein of DEN4.  
         [0054]    MDH: dehydrogenase mutant.  
         [0055]    [0055]FIG. 12. Cloning strategy of the E4 fragment to obtain PID3.  
         [0056]    DENe4: Fragment of the envelope protein of DEN4.  
         [0057]    MDH: dehydrogenase mutant.  
         [0058]    [0058]FIG. 13. Cloning strategy to obtain PD4D2.  
         [0059]    DENe4: Fragment of the envelope protein of DEN4.  
         [0060]    DENe2: Fragment of the envelope protein of DEN-2.  
         [0061]    MDH: dehydrogenase mutant.  
     
    
     EXAMPLES OF PERFORMANCE  
     Example 1  
     Obtaining of PLL1  
       [0062]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-2 virus (Sec. Id. No. 22) was amplified with the oligonucleotides identified in the list of sequences 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).  
         [0063]    The vector was created by digestion of the pM108 His plasmid with Xba I/Bam HI, which contains the nucleotide sequence that codifies for the N-terminal region of the MDH and for a sequence of 6 histidines (Sequence No. 23). Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 (Hill C. W., Harnish B. W. 1982. Transposition of a chromosomal segment bounded by redundant rRNA genes in  Escherichia coli . J Bacteriology. 149:449-457) were transformed with the selected clone, called pLL1 (FIG. 1 and Sequence No. 24). Upon growing the colony in Luria Bertani (LB) medium, a SDS-PAGE of the cellular lysate was done. As a result a 25 kDA band was obtained, which accounted for 10% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the N-terminal region from the MDH protein and the DENe protein fragment from the DEN-2 virus. The protein was recognized in Immunoblotting by polyclonal antibodies (PA) anti-DEN-2 contained in the HMAF. This protein was denominated PLL1 (Sequence No. 25).  
       Example 2  
     Purification of the PLL1 Protein  
       [0064]    The biomass obtained from the  E. coli  strain transformed with pLL1 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained preponderantly as insoluble form associated to the pellet of the cellular disruption. From the pellet the protein was extracted with urea 6 M and the supernatant, containing the PLL1 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The protein was eluted with Imidazole 50 mM and the obtained volume was loaded onto a G-25 column to finally obtain the protein in the formulation buffer NaCl 100 mM, KCl 2  2 mM, Na 2 HPO 4  10 mM, pH 7.2, KH 2 PO 4  1 mM (PBS). This preparation was used for immunological studies.  
       Example 3  
     Antigenic characterization of PLL1  
       [0065]    The purified fraction of PLL1 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 1).  
                                 TABLE 1                           Reactivity of PLL1 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PLL1*                       HMAF   DEN-1   +           HMAF   DEN-2   ++           HMAF   DEN-3   −           HMAF   DEN-4   −           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   +                                                          
 
         [0066]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-2. This result coincides with the fact that the cloned region belongs to serotype 2. The recognition by HMAF against the other serotypes was less than in the case of serotype 2, in decreasing order: DEN-1, DEN-3 and DEN4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. On the other hand, the Mab 3H5 had reactivity indeed. This recognition by Western blotting relied on disulphide bond since when the sample was reduced the signal was lost. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-2 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting.  
       Example 4  
     Characterization of the Antibody Response Generated by PLL1  
       [0067]    A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PLL1 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-2 were obtained while, no reactivity was obtained against the rest of the serotypes (table 2 and table 5). In addition, the hemagglutination inhibition assay (HIA) was done and only positive titers were found against DEN-2 (table 3 and table 5). Finally, the in vitro neutralization assay was done and neutralization titers of 1:320 against DEN-2 were obtained. However, no neutralization of the viral infection was found against the rest of the serotypes (table 4 and table 5). These results indicate the high serotype-specificity of the antibodies elicited by PLL1.  
                                           TABLE 2                           Antibody titers against DEN-2 from the sera obtained       upon immunization of mice with PLL1.                Titers   Titers           anti-DEN-2   anti-DEN-2       Mouse   PLL1   PBSControl (−)                    1   1/128 000   &lt;1:100       2   1/64 000   &lt;1:100       3   1/64 000   &lt;1:100       4   1/128 000   &lt;1:100       5   1/32 000   &lt;1:100       6   1/32 000   &lt;1:100       7   1/64 000   &lt;1:100       8   1/32 000   &lt;1:100       9   1/128 000   &lt;1:100       10   1/512 000   &lt;1:100                  
 
         [0068]    [0068]                                           TABLE 3                           Titers by HI of the sera from the animals immunized with PLL1.                Titers by   Titers by           HI* anti-DEN-2   HI anti-DEN-2       Mouse   PLL1   PBS C(−)                    1   &lt;1:5   &lt;1:5       2   &gt;1:640   &lt;1:5       3    1:320   &lt;1:5       4    1:320   &lt;1:5       5   &gt;1:640   &lt;1:5       6   &gt;1:640   &lt;1:5       7   &gt;1:640   &lt;1:5       8    1:320   &lt;1:5       9   &gt;1:640   &lt;1:5       10   &lt;1:5   &lt;1:5                            
         [0069]    [0069]                                           TABLE 4                           Viral neutralization assay with the       sera of animals immunized with PLL1.                Neutralizing   Neutralizing           titers*   titers           anti-DEN-2   anti-DEN-2       Mouse   PLL1   PBS C(−)                    1   1:320   &lt;1:5       2   1:320   &lt;1:5       3   1:320   &lt;1:5       4   1:320   &lt;1:5       5   1:80   &lt;1:5       6   1:160   &lt;1:5       7   1:320   &lt;1:5       8   1:40   &lt;1:5       9   1:160   &lt;1:5       10   1:320   &lt;1:5                            
         [0070]    [0070]                                                                                 TABLE 5                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PLL1.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1 (PLL1)   &lt;1/100   &gt;1:128 000   &lt;1/100   &lt;1/100       2 (PLL1)   &lt;1/100    1:128 000   &lt;1/100   &lt;1/100                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PLL1   &lt;1/5   &gt;1/320   &lt;1/5   &lt;1/5                        Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers   titers   titers       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1 (PLL1)   &lt;1:5   1:320   &lt;1:5   &lt;1:5       2 (PLL1)   &lt;1:5   1:160   &lt;1:5   &lt;1:5                                            
       Example 5  
     Obtaining of PLL2  
       [0071]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-2 virus (Sec. Id. No. 22) was amplified with the oligonucleotides identified in the list of sequences as Sequence No. 1 and Sequence No. 3 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).  
         [0072]    The vector was created by digestion of the pM84 His plasmid with Xba I/Eco RI, which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines (Sequence No. 26). This digestion permits the insertion of the amplified fragment by PCR within the coding region for a structural domain of the MDH protein.  
         [0073]    Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells MM294 (Hanahan D. 1983. Studies on transformation of  Escherichia coli  with plasmids. J. Mol. Biol. 166:557-580) were transformed with the selected clone, called pLL2 (FIG. 2 and Sequence No. 27). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 10% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-2 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-2 and was denominated PLL2 (Sequence No. 28).  
       Example 6  
     Purification of the PLL2 Protein  
       [0074]    The biomass obtained from the  E. coli  strain transformed with pLL2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. From the soluble fraction a metal ions-affinity chromatography was done using the Chelating-sepharose FF column previously coupled with Cu ++  ions. The column was washed using Imidazolee 15 mM and the protein was eluted with Imidazolee 100 mM. On the other hand, the protein associated to the insoluble fraction was extracted using Urea 8 M, and the supernatant, containing the PLL2 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The protein was eluted with Imidazolee 100 mM. Finally, the pure fraction of each form of the protein were loaded onto a G-25 column to obtain the protein in the formulation buffer NaCl 100 mM, KCl 2  2 mM, Na 2 HPO 4  10 mM, pH 7.2, KH 2 PO 4  1 mM (PBS). This preparations were used for immunological studies.  
       Example 7  
     Antigenic characterization of PLL2  
       [0075]    The purified fraction of PLL2 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 6).  
         [0076]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-2. The recognition by HMAF against the other serotypes was less than in the case of serotype 2, in decreasing order: DEN-1, DEN-3 and DEN4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Nevertheless, related to the Mab 3H5, a great reactivity was observed (even higher than those obtained with the PLL1) either by Dot blot and Western blot. Contrariously to the PLL1 results, the recognition with the Mab 3H5 was the same when the reducing agents are present in the sample, indicating a possible conformational difference between both proteins. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-2 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting.  
                                 TABLE 6                           Reactivity of PLL2 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PLL2 sol, ins*                       HMAF   DEN-1   ++           HMAF   DEN-2   +++           HMAF   DEN-3   −           HMAF   DEN-4   −           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   +++                                                          
 
       Example 8  
     Characterization of the Antibody Response Generated by PLL2  
       [0077]    A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PLL2 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-2 were obtained while, no reactivity was obtained against the rest of the serotypes (table 7 and table 10). In addition, the hemagglutination inhibition assay (HI) was done and only positive titers were found against DEN-2 (table 8 and table 10). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-2 were obtained. Similarly to the results obtained with PLL1, no neutralization of the viral infection was found against the rest of the serotypes (table 9 and table 10). On the other hand, the results obtained with both variants of PLL2 were similar, indicating that the solubility status of the protein do not influece in the capacity of generating functional antibodies.  
                                                                     TABLE 7                           Antibody titers against DEN-2 from the sera obtained upon       immunization of mice with PLL2 soluble and insoluble.                        Titers           Titers anti-DEN-2 (PLL2)       anti-DEN-2            Mouse   PLL2 s   PLL2 ins   PBS C(−)                    1   &gt;1:128 000    1:64000   &lt;1:100       2    1:128 000   &gt;1:128 000   &lt;1:100       3   &gt;1:128 000    1:128 000   &lt;1:100       4   &gt;1:128 000   &gt;1:128 000   &lt;1:100       5    1:64 000   &gt;1:128 000   &lt;1:100       6   &gt;1:128 000    1:128 000   &lt;1:100       7    1:64000   &gt;1:128 000   &lt;1:100       8   &gt;1:128 000    1:64000   &lt;1:100       9   &gt;1:128 000    1:64000   &lt;1:100       10    1:128 000   &gt;1:128 000   &lt;1:100                  
 
         [0078]    [0078]                                                                     TABLE 8                           Titers by HI of the sera from the animals       immunized with PLL2 soluble and insoluble.                Titers by HI*       Titers by HI           anti-DEN-2 (PLL2)       anti-DEN-2            Mouse   PLL2 s   PLL2 ins   PBS C(−)                    1   &gt;1:640   &gt;1:640   &lt;1:5       2   &gt;1:640   &gt;1:640   &lt;1:5       3    1:320   &gt;1:640   &lt;1:5       4   &gt;1:640    1:320   &lt;1:5       5    1:320   &lt;1:5   &lt;1:5       6   &gt;1:640   &gt;1:640   &lt;1:5       7   &gt;1:640    1:320   &lt;1:5       8   &lt;1:5    1:320   &lt;1:5       9    1:320   &gt;1:640   &lt;1:5       10   &gt;1:640   &gt;1:640   &lt;1:5                            
         [0079]    [0079]                                                                     TABLE 9                           Viral neutralization assay with the sera of animals       immunized with PLL2 soluble and insoluble.                        Neutralizing           Neutralizing titers*       titers           anti-DEN-2 (PLL2)       anti-DEN-2            Mouse   PLL2 s   PLL2 ins   PBS C(−)                    1   &gt;1:1280   &gt;1:1280   &gt;1:1280       2   &gt;1:1280   &gt;1:1280   &lt;1:5       3   &gt;1:1280    1:640   &lt;1:5       4    1:640   &gt;1:1280   &lt;1:5       5    1:640    1:640   &lt;1:5       6   &gt;1:1280   &gt;1:1280   &lt;1:5       7   &gt;1:1280   &gt;1:1280   &lt;1:5       8   &gt;1:1280   &gt;1:1280   &lt;1:5       9   &gt;1:1280   &gt;1:1280   &lt;1:5       10   &gt;1:1280   &gt;1:1280   &lt;1:5                            
         [0080]    [0080]                                                           TABLE 10                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the sera of       animals immunized with PLL2 soluble and insoluble.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1 (PLL2 sol.)   &lt;1/100   &gt;1:128 000   &lt;1/100   &lt;1/100       2 (PLL2 sol.)   &lt;1/100    1:64000   &lt;1/100   &lt;1/100       1 (PLL2 ins.)   &lt;1/100    1:64000   &lt;1/100   &lt;1/100       2 (PLL2 ins.)   &lt;1/100   &gt;1:128 000   &lt;1/100   &lt;1/100                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PLL2 sol.   &lt;1/5   &gt;1/320   &lt;1/5   &lt;1/5       PLL2 ins.   &lt;1/5   &gt;1/320   &lt;1/5   &lt;1/5                   Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers   titers   titers       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1 (PLL2)   &lt;1:5   1:320   &lt;1:5   &lt;1:5       2 (PLL2)   &lt;1:5   1:160   &lt;1:5   &lt;1:5                                            
       Example 9  
     Obtaining of pLL3  
       [0081]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-2 virus (Sec. Id. No. 22) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.4 and Sequence No.5 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).  
         [0082]    The vector was created by digestion of the pD4 plasmid with Bam HI/Bam HI which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines without stop codon (Sequence No. 29). This digestion permits to the fusion of the amplified fragment by PCR after the C-terminal region for the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pLL3 (FIG. 3 and Sequence No. 30). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-2 virus. The protein was recognized in Immunoblotting by a HMAFI anti-DEN-2 and was denominated PLL3 (Sequence No. 31).  
       Example 10  
     Purification of the PLL3 Protein  
       [0083]    The biomass obtained from the  E. coli  strain transformed with pLL2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. From the insoluble fraction, the protein was extracted using Urea 6 M, and the supernatant, containing the PLL3 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-Sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The colum was washed with Imidazolee 30 mM and the protein was eluted with Imidazole 100 mM. Finally, the pure fraction of the protein was loaded onto a G-25 column to obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.  
       Example 11  
     Antigenic Characterization of PLL3  
       [0084]    The purified fraction of PLL3 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 11).  
         [0085]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-2. The recognition by HMAF against the other serotypes was lower than in the case of serotype 2, in decreasing order: DEN-1, DEN-3 and DEN-4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Nevertheless, related to the Mab 3H5, a great reactivity was observed (similar to those obtained with the PLL2) either by Dot blot and Western blot. Contrariously to the PLL1 results, the recognition with the Mab 3H5 was the same when the reducing agents are present in the sample, indicating a possible conformational difference between both proteins. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-2 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting. These results were similar to those obtained with PLL2.  
                                 TABLE 11                           Reactivity of PLL3 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PLL3*                       HMAF   DEN-1   ++           HMAF   DEN-2   +++           HMAF   DEN-3   −           HMAF   DEN-4   −           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   +++                                                          
 
       Example 12  
     Characterization of the Antibody Response Generated by PLL3  
       [0086]    A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PLL3 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-2 were obtained while, no reactivity was obtained against the rest of the serotypes (table 12 and table 15). In addition, the hemagglutination inhibition (HI) assay was done and only positive titers were found against DEN-2 (table 13 and table 15). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-2 were obtained (table 14). No neutralization of the viral infection was found against the rest of the serotypes (table 15). Using the three tests, high levels of serotype-specific antibodies were detected, similar of those obtained after the immunization with the PLL2 protein.  
                                           TABLE 12                           Antibody titers against DEN-2 from the sera obtained       upon immunization of mice with PLL3.                Titers   Titers           anti-DEN-2   anti-DEN-2       Mouse   (PLL3)   PBS C(−)                    1    1:128 000   &lt;1:100       2   &gt;1:128 000   &lt;1:100       3   &gt;1:128 000   &lt;1:100       4    1:64 000   &lt;1:100       5   &gt;1:128 000   &lt;1:100       6    1:64 000   &lt;1:100       7   &gt;1:128 000   &lt;1:100       8   &gt;1:128 000   &lt;1:100       9    1:128 000   &lt;1:100       10   &gt;1:128 000   &lt;1:100                  
 
         [0087]    [0087]                                           TABLE 13                           Titers by HI of the sera from the animals immunized with PLL3.                Titers by HI*   Titers by HI           anti-DEN-2   anti-DEN-2       Mouse   (PLL3)   PBS C(−)                    1   &gt;1:640   &lt;1:5       2    1:320   &lt;1:5       3    1:320   &lt;1:5       4   &gt;1:640   &lt;1:5       5    1:320   &lt;1:5       6   &gt;1:640   &lt;1:5       7   &gt;1:640   &lt;1:5       8   &gt;1:640   &lt;1:5       9    1:320   &lt;1:5       10   &gt;1:640   &lt;1:5                            
         [0088]    [0088]                                           TABLE 14                           Viral neutralization assay with the       sera of animals immunized with PLL3.                Neutralizing   Neutralizing           titers   titers           anti-DEN-2   anti-DEN-2       Mouse   PLL3   PBS C(−)                    1   &gt;1:1280   &lt;1:5       2   &gt;1:1280   &lt;1:5       3   &gt;1:1280   &lt;1:5       4    1:640   &lt;1:5       5   &gt;1:1280   &lt;1:5       6   &gt;1:1280   &lt;1:5       7   &gt;1:1280   &lt;1:5       8   &gt;1:1280   &lt;1:5       9    1:640   &lt;1:5       10   &gt;1:1280   &lt;1:5                            
         [0089]    [0089]                                                                                 TABLE 15                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PLL3.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1(PLL3)   &lt;1/100   &gt;1:128000   &lt;1/100   &lt;1/100       2(PLL3)   &lt;1/100   &gt;1:128000   &lt;1/100   &lt;1/100                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PLL3   &lt;1/5   &gt;1/320   &lt;1/5   &lt;1/5                        Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers***   titers***   titers***       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PLL3)   &lt;1:5   &gt;1:1280   &lt;1:5   &lt;1:5       2(PLL3)   &lt;1:5   &gt;1:1280   &lt;1:5   &lt;1:5                                            
       Example 13  
     Obtaining of pLH1  
       [0090]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-1 virus (Sec. Id. No. 32) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.6 and Sequence No.7 from the DEN-1 virus strain genotype (Chu M. C., O&#39;Rourke E. J., Trent D. W. Genetic relatedness among structural protein genes of dengue 1 virus strains. J. Gen. Virol. 1989. 70:1701-1712).  
         [0091]    The vector was created by digestion of the pM108 His plasmid with Xba I/Bam HI, which contains the nucleotide sequence that codifies for the N-terminal region of the MDH and for a sequence of 6 histidines (Sequence No. 23). Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pLH1 (FIG. 4 and Sequence No.33). Upon growing the colony in Luria Bertani (LB) medium, a SDS-PAGE of the cellular lysate was done. As a result a 25 kDA band was obtained, which accounted for 10% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the N-terminal region from the MDH protein and the DENe protein fragment from the DEN-1 virus. The protein was recognized in Immunoblotting by polyclonal antibodies (PA) anti-DEN-1 contained in the HMAF. This protein was denominated PLH1 (Sequence No.34).  
       Example 14  
     Purification of the Protein PLH1  
       [0092]    The biomass obtained from the  E. coli  strain transformed with pLH1 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained preponderantly as insoluble form associated to the pellet of the cellular disruption. From the pellet, the protein was extracted with urea 7 M and the supernatant, containing the PLH1 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The protein was eluted with Imidazolee 60 mM and the obtained volume was loaded onto a G-25 column to finally obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.  
       Example 15  
     Antigenic Characterization of PLH1  
       [0093]    The purified fraction of PLH1 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 16).  
         [0094]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-1. The recognition by HMAF against the other serotypes was lower than in the case of serotype 1. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-1 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting.  
                                 TABLE 16                           Reactivity of PLH1 protein to monoclonal       and polyclonal antibodies                Abs**   Specificity***   PLH1                       HMAF   DEN-1   ++           HMAF   DEN-2   +           HMAF   DEN-3   −           HMAF   DEN-4   −           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   −                                                          
 
       Example 16  
     Characterization of the Antibody Response Generated by PLH1  
       [0095]    A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PLH1 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-1 were obtained while, no reactivity was obtained against the rest of the serotypes (table 17 and table 20). In addition, the HI assay was done and only positive titers were found against DEN-1 (table 18 and table  20 ). Finally, the in vitro neutralization assay was done and neutralization titers of 1:320 against DEN-1 were obtained. However, no neutralization of the viral infection was found against the rest of the serotypes (table 19 and table 20). These results indicate the high serotype-specificity of the antibodies elicited by PLH1.  
                                           TABLE 17                           Antibody titers against DEN-1 from the sera obtained       upon immunization of mice with PLH1.                Titers   Titers           anti-DEN-1   anti-DEN-1       Mouse   (PLH1)   PBS C(−)                    1   1/64 000   &lt;1:100       2   1/128 000   &lt;1:100       3   1/64 000   &lt;1:100       4   1/32 000   &lt;1:100       5   1/32 000   &lt;1:100       6   1/64 000   &lt;1:100       7   1/128 000   &lt;1:100       8   1/64 000   &lt;1:100       9   1/128 000   &lt;1:100       10   1/128 000   &lt;1:100                  
 
         [0096]    [0096]                                           TABLE 18                           Titers by HI of the sera from the animals immunized with PLH1                Titers by HI*   Titers by HI*           anti-DEN-1   anti-DEN-1       Mouse   (PLH1)   PBS C(−)                    1   &gt;1:640   &lt;1:5       2    1:320   &lt;1:5       3   &gt;1:640   &lt;1:5       4   &gt;1:640   &lt;1:5       5   &gt;1:640   &lt;1:5       6    1:320   &lt;1:5       7    1:40   &lt;1:5       8    1:320   &lt;1:5       9   &gt;1:640   &lt;1:5       10   &lt;1:5   &lt;1:5                            
         [0097]    [0097]                                           TABLE 19                           Viral neutralization assay with the       sera of animals immunized with PLH1.                Neutralizing   Neutralizing           titers*   titers*           anti-DEN-1   anti-DEN-1       Mouse   (PLH1)   PBS C(−)                    1   1:80   &lt;1:5       2   1:320   &lt;1:5       3   1:40   &lt;1:5       4   1:320   &lt;1:5       5   1:80   &lt;1:5       6   1:160   &lt;1:5       7   1:320   &lt;1:5       8   1:320   &lt;1:5       9   1:320   &lt;1:5       10   1:320   &lt;1:5                            
         [0098]    [0098]                                                                                 TABLE 20                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PLH1.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1 (PLH1)    1:128 000   &lt;1/100   &lt;1/100   &lt;1/100       2 (PLH1)   &gt;1:128 000   &lt;1/100   &lt;1/100   &lt;1/100                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PLH1   &gt;1:320   &lt;1/5   &lt;1/5   &lt;1/5                        Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers***   titers***   titers***       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PLH1)   1:160   &lt;1:5   &lt;1:5   &lt;1:5       2(PLH1)   1:320   &lt;1:5   &lt;1:5   &lt;1:5                                            
       Example 17  
     Obtaining of pLH2  
       [0099]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-1 virus (Sequence No. 32) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.6 and Sequence No.8 from a DEN-1 viral strain (Chu M. C., O&#39;Rourke E. J., Trent D. W. Genetic relatedness among structural protein genes of dengue 1 virus strains. J. Gen. Virol. 1989. 70:1701-1712).  
         [0100]    The vector was created by digestion of the pM84 His plasmid with Xba I/Eco RI, which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines (Sequence No. 26). This digestion permits the insertion of the amplified fragment by PCR within the coding region for a structural domain of the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells MM294 were transformed with the selected clone, called pLH2 (FIG. 5 and Sequence No.35). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-1 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-1 and was denominated PLH2 (Sequence No.36).  
       Example 18  
     Purification of the PLH2 Protein  
       [0101]    The biomass obtained from the  E. coli  strain transformed with pLH2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. The protein associated to the insoluble fraction was extracted using Urea 7 M, and the supernatant, containing the PLH2 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The column was washed with Imidazolee 40 mM and the protein was eluted with Imidazole 100 mM. Finally, the pure fraction was loaded onto a G-25 column to obtain the protein in the formulation (PBS). This preparation was used for immunological studies.  
       Example 19  
     Antigenic Characterization of PLH2  
       [0102]    The purified fraction of PLH2 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 21).  
         [0103]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-1 (higher than those obtained with PLH1). The recognition by HMAF against the other serotypes was lower than in the case of serotype 1. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against five human sera of high titers and three of low titers against DEN-1 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting.  
                                 TABLE 21                           Reactivity of PLH2 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PLH2                       HMAF   DEN-1   +++           HMAF   DEN-2   +           HMAF   DEN-3   −           HMAF   DEN-4   −           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   −                                                          
 
       Example 20  
     Characterization of the Antibody Response Generated by PLH2  
       [0104]    A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PLH2 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-1 were obtained while, no reactivity was obtained against the rest of the serotypes (table 22 and table 25). In addition, the HI assay was done and only positive titers were found against DEN-1 (table 23 and table 25). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-1 were obtained (table 24). No neutralization of the viral infection was found against the rest of the serotypes. (table 25).  
                                           TABLE 22                           Antibody titers against DEN-1 from the sera obtained       upon immunization of mice with PLH2.                Titers   Titers           anti-DEN-1   anti-DEN-1       Mouse   (PLH2)   PBS C(−)                    1    1:128 000   &lt;1:100       2   &gt;1:128 000   &lt;1:100       3    1:64000   &lt;1:100       4   &gt;1:128 000   &lt;1:100       5    1:128 000   &lt;1:100       6    1:64000   &lt;1:100       7   &gt;1:128 000   &lt;1:100       8    1:128 000   &lt;1:100       9   &gt;1:128 000   &lt;1:100       10   &gt;1:128 000   &lt;1:100                  
 
         [0105]    [0105]                                           TABLE 23                           Titers by HI of the sera from the animals immunized with PLH2.                Titers by HI*   Titers by HI           anti-DEN-1   anti-DEN-1       Mouse   (PLH2)   PBS C(−)                    1   &lt;1:5   &lt;1:5       2   &gt;1:640   &lt;1:5       3    1:320   &lt;1:5       4    1:320   &lt;1:5       5   &gt;1:640   &lt;1:5       6   &gt;1:640   &lt;1:5       7   &gt;1:640   &lt;1:5       8    1:320   &lt;1:5       9   &gt;1:640   &lt;1:5       10   &lt;1:5   &lt;1:5                            
         [0106]    [0106]                                           TABLE 24                           Viral neutralization assay with the       sera of animals immunized with PLH2.                Neutralizing   Neutralizing           titers*   titers*           anti-DEN-1   anti-DEN-1.       Mouse   (PLH2)   C(−)                    1   &gt;1:1280   &lt;1:5       2   &gt;1:1280   &lt;1:5       3   &gt;1:1280   &lt;1:5       4   &gt;1:1280   &lt;1:5       5   &gt;1:1280   &lt;1:5       6   &gt;1:1280   &lt;1:5       7   &gt;1:1280   &lt;1:5       8   &gt;1:1280   &lt;1:5       9    1:640   &lt;1:5       10   &gt;1:1280   &lt;1:5                            
         [0107]    [0107]                                                                                 TABLE 25                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PLH2.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1 (PLH2)    1:64 000   &lt;1/100   &lt;1/100   &lt;1/100       2 (PLH2)   &gt;1:128 000   &lt;1/100   &lt;1/100   &lt;1/100                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PLH2   &gt;1/320   &lt;1/5   &lt;1/5   &lt;1/5                        Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers   titers   titers       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PLH2)   &gt;1:1280   &lt;1:5   &lt;1:5   &lt;1:5       2(PLH2)   &gt;1:1280   &lt;1:5   &lt;1:5   &lt;1:5                                            
       Example 21  
     Obtaining of pLH3  
       [0108]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-1 virus (Sec. Id. No. 32) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.9 and Sequence No.10 from the DEN-1 virus strain (Chu M. C., O&#39;Rourke E. J., Trent D. W. Genetic relatedness among structural protein genes of dengue 1 virus strain. J. Gen. Virol.1989. 70:1701-1712).  
         [0109]    The vector was created by digestion of the pD4 plasmid with Bam HI/Bam HI which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines without stop codon (Sequence No.29). This digestion permits the fusion of the amplified fragment by PCR after the C-terminal region for the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pLH3 (FIG. 6 and Sequence No.37). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-1 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-1 and was denominated PLH3 (Sequence No.38).  
       Example 22  
     Purification of the PLH3 Protein  
       [0110]    The biomass obtained from the  E. coli  strain transformed with pLH3 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. From the insoluble fraction, the protein was extracted using Urea 6 M, and the supernatant, containing the PLH3 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-Sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The colum was washed with Imidazolee 30 mM and the protein was eluted with Imidazole 250 mM. Finally, the pure fraction of the protein was loaded onto a G-25 column to obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.  
       Example 23  
     Antigenic characterization of PLH3  
       [0111]    The purified fraction of PLH3 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 26).  
         [0112]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-1. The recognition by HMAF against the other serotypes was lower than in the case of serotype 1. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-1 was measured, achieving a substantial signal in both cases by Dot blotting and Western blotting.  
                                 TABLE 26                           Reactivity of PLH3 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PLH3                       HMAF   DEN-1   +++           HMAF   DEN-2   +           HMAF   DEN-3   −           HMAF   DEN-4   −           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   −                                                          
 
       Example 24  
     Characterization of the Antibody Response Generated by PLH3  
       [0113]    A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PLH3 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-1 were obtained while, no reactivity was obtained against the rest of the serotypes (table 27 and table 30). In addition, the HI assay was done and only positive titers were found against DEN-1 (table 28 and table 30). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-1 were obtained (table 29). No neutralization of the viral infection was found against the rest of the serotypes (table 30).  
                                           TABLE 27                           Antibody titers against DEN-1 from the sera obtained       upon immunization of mice with PLH3.                Titers   Titers           anti-DEN-1   anti-DEN-1       Mouse   PLH3   PBSControl (−)                    1    1:64 000   &lt;1:100       2   &gt;1:128 000   &lt;1:100       3    1:64 000   &lt;1:100       4   &gt;1:128 000   &lt;1:100       5    1:128 000   &lt;1:100       6    1:128 000   &lt;1:100       7    1:128 000   &lt;1:100       8   &gt;1:128 000   &lt;1:100       9    1:64 000   &lt;1:100       10   &gt;1:128 000   &lt;1:100                  
 
         [0114]    [0114]                                           TABLE 28                           Titers by HI of the sera from the animals immunized with PLH3.                Titers by HI*   Titers by HI           anti-DEN-1   anti-DEN-1       Mouse   PLH3   PBS C(−)                    1    1:320   &lt;1:5       2   &gt;1:640   &lt;1:5       3   &gt;1:640   &lt;1:5       4    1:320   &lt;1:5       5    1:320   &lt;1:5       6   &gt;1:640   &lt;1:5       7    1:320   &lt;1:5       8   &lt;1:5   &lt;1:5       9   &gt;1:640   &lt;1:5       10   &gt;1:640   &lt;1:5                            
         [0115]    [0115]                                           TABLE 29                           Viral neutralization assay with the       sera of animals immunized with PLH3.                Neutralizing   Neutralizing           titers*   titers*           anti-DEN-1   anti-DEN-1       Mouse   PLH3   PBS C(−)                    1   &gt;1:1280   &lt;1:5       2   &gt;1:1280   &lt;1:5       3   &gt;1:1280   &lt;1:5       4    1:640   &lt;1:5       5    1:640   &lt;1:5       6   &gt;1:1280   &lt;1:5       7   &gt;1:1280   &lt;1:5       8   &gt;1:1280   &lt;1:5       9   &gt;1:1280   &lt;1:5       10   &gt;1:1280   &lt;1:5                            
         [0116]    [0116]                                                                                 TABLE 30                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PLH3.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   anti DEN-2)   anti-DEN-3)   (anti-DEN- 4)               1(PLH3)    1:128 000   &lt;1/100   &lt;1/100   &lt;1/100       2(PLH3)   &gt;1:128 000   &lt;1/100   &lt;1/100   &lt;1/100                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PLH3   &gt;1/320   &lt;1/5   &lt;1/5   &lt;1/5                        Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers   titers   titers       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PLH3)   &gt;1:1280   &lt;1:5   &lt;1:5   &lt;1:5       2(PLH3)   &gt;1:1280   &lt;1:5   &lt;1:5   &lt;1:5                                            
       Example 25  
     Obtaining of pAZ1  
       [0117]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-3 virus (Seq. 39) was amplified with the oligonucleotides identified in the list of sequences as Sequence No. 11 and Sequence No.12 from the DEN-3 virus strain genotype (Osatomi K., Sumiyoshi H. Complete nucleotide sequence of dengue type 3 virus genome RNA. Virology.1990. 176(2):643-647).  
         [0118]    The vector was created by digestion of the pM108 His plasmid with Xba I/Bam HI, which contains the nucleotide sequence that codifies for the N-terminal region of the MDH and for a sequence of 6 histidines (Sequence No. 23). Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pAZ1 (FIG. 7 and Sequence No.40). Upon growing the colony in Luria Bertani (LB) medium, a SDS-PAGE of the cellular lysate was done. As a result a 25 kDA band was obtained, which accounted for 10% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the N-terminal region from the MDH protein and the DENe protein fragment from the DEN-3 virus. The protein was recognized in Immunoblotting by polyclonal antibodies (PA) anti-DEN-3 contained in the HMAF. This protein was denominated PAZ1 (Sequence No.41).  
       Example 26  
     Purification of the Protein PAZ1  
       [0119]    The biomass obtained from the  E. coli  strain transformed with pAZ1 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained preponderantly as insoluble form associated to the pellet of the cellular disruption. From the pellet, the protein was extracted with urea 7 M and the supernatant, containing the PLH1 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The protein was eluted with Imidazolee 60 mM and the obtained volume was loaded onto a G-25 column to finally obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.  
       Example 27  
     Antigenic Characterization of PAZ1  
       [0120]    The purified fraction of PAZ1 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 31).  
         [0121]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-3. The recognition by HMAF against the other serotypes was lower than in the case of serotype 3. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-3 was measured, achieving a substantial signal in both cases by Dot blotting Western blotting.  
                                 TABLE 31                           Reactivity of PAZ1 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PAZ1                       HMAF   DEN-1   −           HMAF   DEN-2   +           HMAF   DEN-3   ++           HMAF   DEN-4   +           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   −                                                                                              
 
       Example 28  
     Characterization of the Antibody Response Generated by PAZ1  
       [0122]    A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PAZ1 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-1 were obtained while, no reactivity was obtained against the rest of the serotypes (table 32 and table 35). In addition, the HI assay was done and only positive titers were found against DEN-3 (table 33 and table 35). Finally, the in vitro neutralization assay was done and neutralization titers of 1:320 against DEN-3 were obtained. However, no neutralization of the viral infection was found against the rest of the serotypes (table 34 and table 35). These results indicate the high serotype-specificity of the antibodies elicited by PAZ1.  
                                           TABLE 32                           Antibody titers against DEN-3 from the sera obtained       upon immunization of mice with PAZ1.                Titers   Titers           anti-DEN-3   anti-DEN-3       Mouse   PAZ1   PBSControl (−)                    1   1/64 000   &lt;1:100       2   1/128 000   &lt;1:100       3   1/32 000   &lt;1:100       4   1/64 000   &lt;1:100       5   1/64 000   &lt;1:100       6   1/128 000   &lt;1:100       7   1/64 000   &lt;1:100       8   1/64 000   &lt;1:100       9   1/128 000   &lt;1:100       10   1/128 000   &lt;1:100                  
 
         [0123]    [0123]                                           TABLE 33                           Titers by HI of the sera from the animals immunized with PAZ1.                Titers by HI*   Titers by HI           anti-DEN-3   anti-DEN-3       Mouse   PAZ1   PBS C(−)                    1   &gt;1:640   &lt;1:5       2    1:320   &lt;1:5       3    1:320   &lt;1:5       4    1:640   &lt;1:5       5   &lt;1/5   &lt;1:5       6    1:320   &lt;1:5       7   &lt;1/5   &lt;1:5       8    1:320   &lt;1:5       9   &gt;1:640   &lt;1:5       10   &gt;1:640   &lt;1:5                            
         [0124]    [0124]                                           TABLE 34                           Viral neutralization assay with the       sera of animals immunized with PAZ1.                Neutralizing   Neutralizing           titers   titers           anti-DEN-3   anti-DEN-3       Mouse   PAZ1   PBS C(−)                    1   1:160   &lt;1:5       2   1:320   &lt;1:5       3   1:320   &lt;1:5       4   1:320   &lt;1:5       5   1:40   &lt;1:5       6   1:40   &lt;1:5       7   1:320   &lt;1:5       8   1:320   &lt;1:5       9   1:160   &lt;1:5       10   1:320   &lt;1:5                            
         [0125]    [0125]                                                                                 TABLE 35                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PAZ1.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti-DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1 (PAZ1)   &lt;1/100   &lt;1/100    1:64 000   &lt;1/100       2 (PAZ1)   &lt;1/100   &lt;1/100   &gt;1:128000   &lt;1/100                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PAZ1   &lt;1/5   &lt;1/5   &gt;1:320   &lt;1/5                        Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers   titers   titers       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PAZ1)   &lt;1:5   &lt;1:5   1:320   &lt;1:5       2(PAZ1)   &lt;1:5   &lt;1:5   1:320   &lt;1:5                                            
       Example 29  
     Obtaining of pAZ2  
       [0126]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-3 virus (Sequence No.39) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.11 and Sequence No.13 from a DEN-3 viral strain (Osatomi K., Sumiyoshi H. Complete nucleotide sequence of dengue type 3 virus genome RNA. Virology. 1990.176(2):643-647).  
         [0127]    The vector was created by digestion of the pM84 His plasmid with Xba I/Eco RI, which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines (Sequence No. 26). This digestion permits the insertion of the amplified fragment by PCR within the coding region for a structural domain of the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells MM294 were transformed with the selected clone, called pAZ2 (FIG. 8 and Sequence No.42). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-3 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-3 and was denominated PAZ2 (Sequence No.43).  
       Example 30  
     Purification of the PAZ2 Protein  
       [0128]    The biomass obtained from the  E. coli  strain transformed with pAZ2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. The protein associated to the insoluble fraction was extracted using Urea 7 M, and the supernatant, containing the PAZ2 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The column was washed with Imidazolee 40 mM and the protein was eluted with Imidazole 100 mM. Finally, the pure fraction was loaded onto a G-25 column to obtain the protein in the formulation (PBS). This preparation was used for immunological studies.  
       Example 31  
     Antigenic Characterization of PAZ2  
       [0129]    The purified fraction of PAZ2 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 36).  
         [0130]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-3 (higher than those obtained with PAZ1). The recognition by HMAF against the other serotypes was lower than in the case of serotype 3. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against five human sera of high titers and three of low titers against DEN-3 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting.  
                                 TABLE 36                           Reactivity of PAZ2 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PAZ2                       HMAF   DEN-1   −           HMAF   DEN-2   −           HMAF   DEN-3   +++           HMAF   DEN-4   +           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   −                                                          
 
       Example 32  
     Characterization of the Antibody Response Generated by PAZ2  
       [0131]    A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PAZ2 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-3 were obtained while, no reactivity was obtained against the rest of the serotypes (table 37 and table 40). In addition, the HI assay was done and only positive titers were found against DEN-3 (table 38 and table 40). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-3 were obtained (table 39). No neutralization of the viral infection was found against the rest of the serotypes (table 40).  
                                           TABLE 37                           Antibody titers against DEN-3 from the sera obtained       upon immunization of mice with PAZ2.                Titers   Titers           anti-DEN-3   anti-DEN-3       Mouse   PAZ2   PBS Control (−)                    1   &gt;1:128 000   &lt;1:100       2    1:128 000   &lt;1:100       3   &gt;1:128 000   &lt;1:100       4   &gt;1:128 000   &lt;1:100       5    1:128 000   &lt;1:100       6   &gt;1:128 000   &lt;1:100       7    1:64000   &lt;1:100       8   &gt;1:128 000   &lt;1:100       9    1:64000   &lt;1:100       10   &gt;1:128 000   &lt;1:100                  
 
         [0132]    [0132]                                           TABLE 38                           Titers by HI of the sera from the animals immunized with PAZ2.                Titers   Titers           anti-DEN-3   anti-DEN-3       Mouse   PAZ2   PBS                    1   &gt;1:640   &lt;1:5       2    1:320   &lt;1:5       3   &gt;1:640   &lt;1:5       4   &gt;1:640   &lt;1:5       5   &gt;1:640   &lt;1:5       6    1:320   &lt;1:5       7   &lt;1:5   &lt;1:5       8    1:320   &lt;1:5       9   &gt;1:640   &lt;1:5       10   &gt;1:640   &lt;1:5                            
         [0133]    [0133]                                           TABLE 39                           Viral neutralization assay with the       sera of animals immunized with PAZ2.                Neutralizing   Neutralizing           titers   titers           anti-DEN-3   anti-DEN-3       Mouse   PAZ2   PBS C(−)                    1   &gt;1:1280   &lt;1:5       2   &gt;1:1280   &lt;1:5       3   &gt;1:1280   &lt;1:5       4   &gt;1:1280   &lt;1:5       5   &gt;1:1280   &lt;1:5       6    1:640   &lt;1:5       7   &gt;1:1280   &lt;1:5       8    1:640   &lt;1:5       9   &gt;1:1280   &lt;1:5       10    1:640   &lt;1:5                            
         [0134]    [0134]                                                                                 TABLE 40                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PAZ2.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1(PAZ2)   &lt;1/100   &lt;1/100   &gt;1:128 000   &lt;1/100       2(PAZ2)   &lt;1/100   &lt;1/100   &gt;1:128 000   &lt;1/100                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PAZ2   &lt;1/5   &lt;1/5   &gt;1/320   &lt;1/5                        Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers   titers   titers       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PAZ2)   &lt;1:5   &lt;1:5   &gt;1:1280   &lt;1:5       2(PAZ2)   &lt;1:5   &lt;1:5   &gt;1:1280   &lt;1:5                                            
       Example 33  
     Obtaining of pAZ3  
       [0135]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-3 virus (Seq. 39) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.14 and Sequence No.15 from the DEN-3 viral strain (Osatomi K., Sumiyoshi H. Complete nucleotide sequence of dengue type 3 virus genome RNA. Virology.1990. 176(2):643-647).  
         [0136]    The vector was created by digestion of the pD4 plasmid with Bam HI/Bam HI which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines without stop codon (Sequence No.29). This digestion permits the fusion of the amplified fragment by PCR after the C-terminal region for the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pAZ3 (FIG. 9 and Sequence No.44). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-3 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-3 and was denominated PAZ3 (Sequence No.45).  
       Example 34  
     Purification of the PAZ3 Protein  
       [0137]    The biomass obtained from the  E. coli  strain transformed with pAZ3 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. From the insoluble fraction, the protein was extracted using Urea 7 M, and the supernatant, containing the PAZ3 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-Sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The colum was washed with Imidazolee 45 mM and the protein was eluted with Imidazole 230 mM. Finally, the pure fraction of the protein was loaded onto a G-25 column to obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.  
       Example 35  
     Antigenic Characterization of PAZ3  
       [0138]    The purified fraction of PAZ3 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 26).  
         [0139]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-3. The recognition by HMAF against the other serotypes was lower than in the case of serotype 3. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN-3 was measured, achieving a substantial signal in both cases by Dot blotting and Western blotting.  
                                 TABLE 41                           Reactivity of PAZ3 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PAZ3                       HMAF   DEN-1   −           HMAF   DEN-2   −           HMAF   DEN-3   +++           HMAF   DEN-4   +           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   −                                                          
 
       Example 36  
     Characterization of the Antibody Response Generated by PAZ3  
       [0140]    A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PAZ3 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-3 were obtained while, no reactivity was obtained against the rest of the serotypes (table 42 and table 45). In addition, the HI assay was done and only positive titers were found against DEN-3 (table 43 and table 45). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-3 were obtained (table 44). No neutralization of the viral infection was found against the rest of the serotypes (table 45).  
                                           TABLE 42                           Antibody titers against DEN-3 from the sera obtained       upon immunization of mice with PAZ3.                Titers   Titers           anti-DEN-3   anti-DEN-3       Mouse   PAZ3   PBSControl (−)                    1   &gt;1:128 000   &lt;1:100       2    1:128 000   &lt;1:100       3   &gt;1:128 000   &lt;1:100       4    1:128000   &lt;1:100       5    1:128 000   &lt;1:100       6   &gt;1:128 000   &lt;1:100       7   &gt;1:128 000   &lt;1:100       8    1:128 000   &lt;1:100       9    1:128 000   &lt;1:100       10   &gt;1:128 000   &lt;1:100                  
 
         [0141]    [0141]                                           TABLE 43                           Titers by HI of the sera from the animals immunized with PAZ3.                Titers by HI*   Titers by HI           anti-DEN-3   anti-DEN-3       Mouse   PAZ3   PBS C(−)                    1   &gt;1:640   &lt;1:5       2   &gt;1:640   &lt;1:5       3    1:320   &lt;1:5       4   &lt;1:5   &lt;1:5       5   &gt;1:640   &lt;1:5       6   &lt;1:5   &lt;1:5       7    1:320   &lt;1:5       8   &gt;1:640   &lt;1:5       9   &gt;1:640   &lt;1:5       10    1:320   &lt;1:5                            
         [0142]    [0142]                                           TABLE 44                           Viral neutralization assay with the       sera of animals immunized with PAZ3.                Neutralizing   Neutralizing           titers   titers           anti-DEN-3   anti-DEN-3       Mouse   PAZ3   PBS C(−)                    1   &gt;1:1280   &lt;1:5       2    1:640   &lt;1:5       3   &gt;1:1280   &lt;1:5       4   &gt;1:1280   &lt;1:5       5   &gt;1:1280   &lt;1:5       6   &gt;1:1280   &lt;1:5       7   &gt;1:1280   &lt;1:5       8   &gt;1:1280   &lt;1:5       9   &gt;1:1280   &lt;1:5       10   &gt;1:1280   &lt;1:5                            
         [0143]    [0143]                                                                                 TABLE 45                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PAZ3.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1(PAZ3)   &lt;1/100   &lt;1/100   &gt;1:128 000   &lt;1/100       2(PAZ3)   &lt;1/100   &lt;1/100    1:128 000   &lt;1/100                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PAZ3   &lt;1/5   &lt;1/5   &gt;1/320   &lt;1/5                        Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers   titers   titers       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PAZ3)   &lt;1:5   &lt;1:5   &gt;1:1280   &lt;1:5       2(PAZ3)   &lt;1:5   &lt;1:5   &gt;1:1280   &lt;1:5                                            
       Example 37  
     Obtaining of pID1  
       [0144]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-4 virus (Sequence No.46) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.17 and Sequence No.18 from the DEN4 virus strain genotype (Zhao B., Mackow E. R., Buckler-White A. J., Markoff L., Chancock R. M., Lai C. -J., Makino Y. Cloning full-length Dengue type 4 viral DNA sequences: Analysis of genes coding for structural proteins. Virology 1986. 155:77-88).  
         [0145]    The vector was created by digestion of the pM108 His plasmid with Xba I/Bam HI, which contains the nucleotide sequence that codifies for the N-terminal region of the MDH and for a sequence of 6 histidines (Sequence No. 23). Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pID1 (FIG. 10 and Sequence No.47). Upon growing the colony in Luria Bertani (LB) medium, a SDS-PAGE of the cellular lysate was done. As a result a 25 kDA band was obtained, which accounted for 10% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the N-terminal region from the MDH protein and the DENe protein fragment from the DEN-4 virus. The protein was recognized in Immunoblotting by polyclonal antibodies (PA) anti-DEN4 contained in the HMAF. This protein was denominated PID1 (Sequence No.48).  
       Example 38  
     Purification of the Protein PID1  
       [0146]    The biomass obtained from the  E. coli  strain transformed with pID1 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained preponderantly as insoluble form associated to the pellet of the cellular disruption. From the pellet, the protein was extracted with urea 6 M and the supernatant, containing the PID1 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The protein was eluted with Imidazolee 60 mM and the obtained volume was loaded onto a G-25 column to finally obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.  
       Example 39  
     Antigenic Characterization of PID1  
       [0147]    The purified fraction of PID1 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 46).  
         [0148]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN4. The recognition by HMAF against the other serotypes was lower than in the case of serotype 4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN4 was measured, achieving a substantial signal in both cases by Dot blotting Western blotting.  
                                 TABLE 46                           Reactivity of PID1 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PID1                       HMAF   DEN-1   −           HMAF   DEN-2   −           HMAF   DEN-3   +           HMAF   DEN-4   ++           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   −                                                          
 
       Example 40  
     Characterization of the Antibody Response Generated by PID1  
       [0149]    A total of 25 Balb/c mice were i.p immunized with 35 ug of purified PID1 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-1 were obtained while, no reactivity was obtained against the rest of the serotypes (table 47 and table 50). In addition, the HI assay was done and only positive titers were found against DEN-4 (table 48 and table 50). Finally, the in vitro neutralization assay was done and neutralization titers of 1:320 against DEN-4 were obtained. However, no neutralization of the viral infection was found against the rest of the serotypes (table 49 and table 50). These results indicate the high serotype-specificity of the antibodies elicited by PID1.  
                                           TABLE 47                           Antibody titers against DEN-4 from the sera obtained       upon immunization of mice with PID1.                Titers   Titers           anti-DEN-4   anti-DEN-4       Mouse   PID1   PBS Control (−)                    1   1/128 000   &lt;1:100       2   1/128 000   &lt;1:100       3   1/64 000   &lt;1:100       4   1/64 000   &lt;1:100       5   1/128 000   &lt;1:100       6   1/32 000   &lt;1:100       7   1/128 000   &lt;1:100       8   1/32 000   &lt;1:100       9   1/128 000   &lt;1:100       10   1/128 000   &lt;1:100                  
 
         [0150]    [0150]                                           TABLE 48                           Titers by HI of the sera from the animals immunized with PID1.                Titers by HI*   Titers by HI           anti-DEN-4   anti-DEN-4       Mouse   PID1   PBS C(−)                    1   1:320   &lt;1:5       2   1:320   &lt;1:5       3   1:640   &lt;1:5       4   1:40   &lt;1:5       5   &lt;1/5   &lt;1:5       6   1:320   &lt;1:5       7   1:640   &lt;1:5       8   1:640   &lt;1:5       9   1:40   &lt;1:5       10   1:320   &lt;1:5                            
         [0151]    [0151]                                           TABLE 49                           Viral neutralization assay with the       sera of animals immunized with PID1.                Neutralizing   Neutralizing           titers   titers           anti-DEN-4   anti-DEN-4       Mouse   PID1   PBS C(−)                    1   1:320   &lt;1:5       2   1:80   &lt;1:5       3   1:320   &lt;1:5       4   1:320   &lt;1:5       5   1:160   &lt;1:5       6   1:320   &lt;1:5       7   1:320   &lt;1:5       8   1:320   &lt;1:5       9   1:160   &lt;1:5       10   1:40   &lt;1:5                            
         [0152]    [0152]                                                           TABLE 50                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PID1.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1 (PID1)   &lt;1/100   &lt;1/100   &lt;1/100    1:64 000       2(PID1)   &lt;1/100   &lt;1/100   &lt;1/100   &gt;1:128 000                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PID1   &lt;1/5   &lt;1/5   &lt;1/5   &gt;1:320                   Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers   titers   titers       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PID1)   &lt;1:5   &lt;1:5   &lt;1:5   1:160       2(PID1)   &lt;1:5   &lt;1:5   &lt;1:5   1:320                                            
       Example 41  
     Obtención de pID2  
       [0153]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN-4 virus (Sequence No.46) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.16 and Sequence No.18 from a DEN-4 viral strain (Zhao B., Mackow E. R., Buckler-White A. J., Markoff L., Chancock R. M., Lai C. -J., Makino Y. Cloning full-length Dengue type 4 viral DNA sequences: Analysis of genes coding for structural proteins. Virology 1986.155:77-88. The vector was created by digestion of the pM84 His plasmid with Xba I/Eco RI, which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines (Sequence No. 26). This digestion permits the insertion of the amplified fragment by PCR within the coding region for a structural domain of the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells MM294 were transformed with the selected clone, called pID2 (FIG. 11 and Sequence No.49). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN-4 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN4 and was denominated PID2 (Sequence No.50).  
       Example 42  
     Purification of the PID2 Protein  
       [0154]    The biomass obtained from the  E. coli  strain transformed with pID2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. The protein associated to the insoluble fraction was extracted using Urea 6 M, and the supernatant, containing the PID2 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The column was washed with Imidazolee 30 mM and the protein was eluted with Imidazole 250 mM. Finally, the pure fraction was loaded onto a G-25 column to obtain the protein in the formulation (PBS). This preparation was used for immunological studies.  
       Example 43  
     Caracterización antigénica de PID2  
       [0155]    The purified fraction of PID2 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 51).  
         [0156]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN4 (higher than those obtained with PID1). The recognition by HMAF against the other serotypes was lower than in the case of serotype 4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against five human sera of high titers and three of low titers against DEN-3 was measured, achieving a substantial signal in both cases by Western blotting and Dot blotting.  
                                 TABLE 51                           Reactivity of PID2 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PID2                       HMAF   DEN-1   −           HMAF   DEN-2   −           HMAF   DEN-3   +           HMAF   DEN-4   +++           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   −                                                          
 
       Example 44  
     Characterization of the Antibody Response Generated by PID2  
       [0157]    A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PID2 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-4 were obtained while, no reactivity was obtained against the rest of the serotypes (table 52 and table 55). In addition, the HI assay was done and only positive titers were found against DEN-4 (table 53 and table 55). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN-4 were obtained (table 54). No neutralization of the viral infection was found against the rest of the serotypes (table 55).  
                                           TABLE 52                           Antibody titers against DEN-4 from the sera obtained       upon immunization of mice with PID2.                Titers   Titers           anti-DEN-4   anti-DEN-4       Mouse   PID2   PBSControl (−)                    1    1:64000   &lt;1:100       2   &gt;1:128 000   &lt;1:100       3    1:128 000   &lt;1:100       4   &gt;1:128 000   &lt;1:100       5    1:64000   &lt;1:100       6   &gt;1:128 000   &lt;1:100       7   &gt;1:128 000   &lt;1:100       8   &gt;1:128 000   &lt;1:100       9   &gt;1:128 000   &lt;1:100       10    1:128 000   &lt;1:100                  
 
         [0158]    [0158]                                           TABLE 53                           Titers by HI of the sera from the animals immunized with PID2.                Titers by HI*   Titers by HI           anti-DEN-4   anti-DEN-4       Mouse   (PID2)   PBS C(−)                    1   1:320   &lt;1:5       2   1:320   &lt;1:5       3   1:640   &lt;1:5       4   1:40   &lt;1:5       5   &lt;1/5   &lt;1:5       6   1:320   &lt;1:5       7   1:640   &lt;1:5       8   1:640   &lt;1:5       9   1:40   &lt;1:5       10   1:320   &lt;1:5                            
         [0159]    [0159]                                           TABLE 54                           Viral neutralization assay with the       sera of animals immunized with PID2.                Neutralizing   Neutralizing           titers   titers           anti-DEN-4   anti-DEN-4       Mouse   PID2   PBS C(−)                    1   &gt;1:1280   &lt;1:5       2   &gt;1:1280   &lt;1:5       3   &gt;1:1280   &lt;1:5       4   &gt;1:1280   &lt;1:5       5    1:640   &lt;1:5       6   &gt;1:1280   &lt;1:5       7   &gt;1:1280   &lt;1:5       8   &gt;1:1280   &lt;1:5       9    1:640   &lt;1:5       10   &gt;1:1280   &lt;1:5                            
         [0160]    [0160]                                                           TABLE 55                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PID2.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1(PID2)   &lt;1/100   &lt;1/100   &lt;1/100   &gt;1:128 000       2(PID2)   &lt;1/100   &lt;1/100   &lt;1/100    1:64 000                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PID2   &lt;1/5   &lt;1/5   &lt;1/5   &gt;1/320                   Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers   titers   titers       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PID2)   &lt;1:5   &lt;1:5   &lt;1:5   &gt;1:1280       2(PID2)   &lt;1:5   &lt;1:5   &lt;1:5   &gt;1:1280                                            
       Example 45  
     Obtaining of pID3  
       [0161]    The nucleotide sequence that codifies for the amino acids from 286 to 426 of the envelope protein from the DEN4 virus (Sequence No.46) was amplified with the oligonucleotides identified in the list of sequences as Sequence No.19 and Sequence No.20 from the DEN4 viral strain (Zhao B., Mackow E. R., Buckler-White A. J., Markoff L., Chancock R. M., Lai C. -J., Makino Y. Cloning full-length Dengue type 4 viral DNA sequences: Analysis of genes coding for structural proteins. Virology 1986.155:77-88).  
         [0162]    The vector was created by digestion of the pD4 plasmid with Bam HI/Bam HI which contains the nucleotide sequence that codifies for the MDH protein and for a sequence of 6 histidines without stop codon (Sequence No.29). This digestion permits the fusion of the amplified fragment by PCR after the C-terminal region for the MDH protein. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells W3110 were transformed with the selected clone, called pID3 (FIG. 12 and Sequence No.51). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 80 kDA band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the size of the MDH protein and the DENe protein fragment from the DEN4 virus. The protein was recognized in Immunoblotting by a HMAF anti-DEN-4 and was denominated PID3 (Sequence No.52).  
       Example 46  
     Purificación de la proteina PID3  
       [0163]    The biomass obtained from the  E. coli  strain transformed with pID3 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained in both forms: soluble and insoluble. From the insoluble fraction, the protein was extracted using Urea 6 M, and the supernatant, containing the PID3 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-Sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. The colum was washed with Imidazolee 45 mM and the protein was eluted with Imidazole 200 mM. Finally, the pure fraction of the protein was loaded onto a G-25 column to obtain the protein in the formulation buffer (PBS). This preparation was used for immunological studies.  
       Example 47  
     Antigenic Characterization of PID3  
       [0164]    The purified fraction of PID3 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 56).  
         [0165]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN4. The recognition by HMAF against the other serotypes was lower than in the case of serotype 4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. Finally, the reactivity against three human sera of high titers and three of low titers against DEN4 was measured, achieving a substantial signal in both cases by Dot blotting and Western blotting.  
                                 TABLE 56                           Reactivity of PID3 protein to monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PID3                       HMAF   DEN-1   −           HMAF   DEN-2   −           HMAF   DEN-3   +           HMAF   DEN-4   +++           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   −                                                          
 
       Example 48  
     Characterization of the Antibody Response Generated by PID3  
       [0166]    A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PAZ3 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-4 were obtained while, no reactivity was obtained against the rest of the serotypes (table 57 and table 60). In addition, the HI assay was done and only positive titers were found against DEN4 (table 58 and table 60). Finally, the in vitro neutralization assay was done and neutralization titers of 1: 1280 against DEN4 were obtained (table 59). No neutralization of the viral infection was found against the rest of the serotypes (table 60).  
                                           TABLE 57                           Antibody titers against DEN-4 from the sera obtained       upon immunization of mice with PID3.                Titers   Titers           anti-DEN-4   anti-DEN-4       Mouse   (PID3)   PBSControl (−)                    1   &gt;1:128 000   &lt;1:100       2   &gt;1:128 000   &lt;1:100       3   &gt;1:128 000   &lt;1:100       4    1:64000   &lt;1:100       5    1:64 000   &lt;1:100       6   &gt;1:128 000   &lt;1:100       7    1:128 000   &lt;1:100       8   &gt;1:128 000   &lt;1:100       9    1:128 000   &lt;1:100       10   &gt;1:128 000   &lt;1:100                  
 
         [0167]    [0167]                                           TABLE 58                           Titers by HI of the sera from the animals immunized with PID3.                Titers by HI*   Titers by HI           anti-DEN-4   anti-DEN-4       Mouse   PID3   PBS C(−)                    1   &gt;1:640   &lt;1:5       2    1:320   &lt;1:5       3   &gt;1:640   &lt;1:5       4   &gt;1:640   &lt;1:5       5   &lt;1:5   &lt;1:5       6   &gt;1:640   &lt;1:5       7    1:320   &lt;1:5       8   &gt;1:640   &lt;1:5       9    1:320   &lt;1:5       10   &gt;1:640   &lt;1:5                            
         [0168]    [0168]                                           TABLE 59                           Viral neutralization assay with the       sera of animals immunized with PID3.                Neutralizing   Neutralizing           titers*   titers           anti-DEN-4   anti-DEN-4       Mouse   PID3   PBS C(−)                    1   &gt;1:1280   &lt;1:5       2   &gt;1:1280   &lt;1:5       3   &gt;1:1280   &lt;1:5       4   &gt;1:1280   &lt;1:5       5   &gt;1:1280   &lt;1:5       6   &gt;1:1280   &lt;1:5       7   &gt;1:1280   &lt;1:5       8    1:640   &lt;1:5       9   &gt;1:1280   &lt;1:5       10   &gt;1:1280   &lt;1:5                            
         [0169]    [0169]                                                                                 TABLE 60                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PID3.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1(PID3)   &lt;1/100   &lt;1/100   &lt;1/100   &gt;1:128 000       2(PID3)   &lt;1/100   &lt;1/100   &lt;1/100    1:128 000                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PID3   &lt;1/5   &lt;1/5   &lt;1/5   &gt;1/320                        Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture of   titers***   titers   titers   titers       sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PID3)   &lt;1:5   &lt;1:5   &lt;1:5   &gt;1:1280       2(PID3)   &lt;1:5   &lt;1:5   &lt;1:5   &gt;1/1280                                            
       Example 49  
     Obtaining of pD4D2  
       [0170]    The nucleotide sequence that codifies for the amino acids 286 to 426 of the E protein from the DEN-4 virus (Sequence No.46) was amplified with the oligonuclotides identified in the sequencing list as Sequence No.16 and Sequence No.21 from the viral strain of DEN-4 (Zhao B., Mackow E. R., Buckler-White A. J., Markoff L., Chancock R. M., Lai C. -J., Makino Y. Cloning full-length Dengue type 4 viral DNA sequences: Analysis of genes coding for structural proteins. Virology 1986. 155:77-88).  
         [0171]    The vector was created by digestion Xba/Xba I of the pLL3 plasmid, which contains the MDH gene plus a sequence of 6 histidines in the 3′ region of the gene and the sequence of the E fragment from DEN-2 in the 3′ end. As result, two regions of the E protein from serotypes 2 and 4 were obtained, fused to the same MDH gene. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Competent cells MM294 were transformed with the selected clone, called pD4D2 (FIG. 13 and Sequence No.53). Upon growing the colony in LB medium, a SDS-PAGE of the cellular lysate was done. As a result a 110 kDa band was obtained, which accounted for 20% of the total cellular proteins. The size of the protein obtained corresponded to the sum of the MDH protein and the two fragments of the DENe protein from Dengue virus. This protein was recognized by polyclonal antibodies anti-DEN-2 and anti-DEN-4 contained in the HMAF. This protein was denominated PD4D2 (Sequence No.54).  
       Example 50  
     Purification of the PD4D2 Protein  
       [0172]    The biomass obtained from the  E. coli  strain transformed with pD4D2 and grown at 37° C. was disrupted by French press. The recombinant protein was obtained either soluble or insoluble. From the pellet the protein was extracted with urea 6 M and the supernatant, containing the PD4D2 protein, was loaded onto a G-25 column to eliminate the chaotropic agent. The fraction obtained was then loaded onto the Chelating-sepharose FF column (Pharmacia, UK), in the presence of Cu ++  ions. A washing step was done with Imidazolee 30 mM and the protein was eluted with Imidazole 250 mM. Finally, the pure preparation was loaded onto a G-25 column to obtain the protein in the formulation buffer and use it for immunological studies.  
       Example 51  
     Antigenic Characterization of PD4D2  
       [0173]    The purified fraction of PD4D2 was characterized either by its recognition by different polyclonal sera and/or murine monoclonal antibodies, as well as by positive human sera to Dengue (table 61).  
         [0174]    The highest recognitions in Dot blotting were obtained with the HMAF anti-DEN-2 and anti-DEN-4. The recognition of the two other serotypes was less than for the case of serotypes 2 and 4. The antibodies generated by other flavivirus like the Yellow Fever virus and the Saint Louis Encephalitis virus had not any recognition at all. On the other hand, the Mab 3H5 had reactivity indeed, similar to that obtained for PLL2 and PLL3.  
         [0175]    Finally, the reactivity against human sera of high and low titers against DEN-2 and DEN-4 was measured, achieving a substantial signal for both cases by Western blotting and Dot blotting.  
                                 TABLE 61                           Reactivity of PD4D2 protein against monoclonal       and polyclonal antibodies.                Abs**   Specificity***   PD4D2                       HMAF   DEN-1   −           HMAF   DEN-2   +++           HMAF   DEN-3   −           HMAF   DEN-4   +++           HMAF   EEE   −           HMAF   YFV   −           HMAF   SLV   −           Mab 3H5   NT   −                                                          
 
       Example 52  
     Characterization of the Antibody Response Generated by PD4D2  
       [0176]    A total of 25 Balb/c mice were i.p immunized with 20 ug of purified PD4D2 in Freund adjuvant; 10 animals were bled after four doses and the antibodies anti-DEN were evaluated by ELISA. High antibody titers against DEN-2 and DEN4 were obtained while, no reactivity was obtained against the rest of the serotypes (table 62 and table 65). In addition, the hemagglutination inhibition assay (HI) was done and only positive titers were found against DEN-2 and DEN-4 (table 63 and table 65). Finally, the in vitro neutralization assay was done and neutralization titers of &gt;1:1280 against DEN-2 and &gt;1:1280 against DEN-4 were obtained (table 64). No neutralization of the viral infection was found against the rest of the serotypes (table 65).  
                                                                         TABLE 62                           Antibody titers against DEN-2 and DEN-4 from the sera       obtained upon immunization of mice with PD4D2.                Titers by ELISA   Titers by ELISA           (PD4D2)   PBS C(−)            Mouse   Anti-DEN-4   Anti-DEN-2   Anti-DEN-4   Anti-DEN-2                    1   &gt;1:128 000   &gt;1:128 000   &lt;1:100   &lt;1:100       2    1:128 000    1:128 000   &lt;1:100   &lt;1:100       3   &gt;1:128 000   &gt;1:128 000   &lt;1:100   &lt;1:100       4   &gt;1:128 000   &gt;1:128 000   &lt;1:100   &lt;1:100       5    1:64 000   &gt;1:128 000   &lt;1:100   &lt;1:100       6   &gt;1:128 000    1:128 000   &lt;1:100   &lt;1:100       7    1:64 000   &gt;1:128 000   &lt;1:100   &lt;1:100       8   &gt;1:128 000   &gt;1:128 000   &lt;1:100   &lt;1:100       9   &gt;1:128 000    1:128 000   &lt;1:100   &lt;1:100       10    1:128 000   &gt;1:128 000   &lt;1:100   &lt;1:100                  
 
         [0177]    [0177]                                                                         TABLE 63                           Titers by HI of the sera from the animals immunized with PD4D2.                Titers by HI (PD4D2)   Titers by HI PBS C(−)            Mouse   Anti-DEN-4   Anti-DEN-2   Anti-DEN-4   Anti-DEN-2                    1   &gt;1:640   &gt;1:640   &lt;1:5   &lt;1:5       2   &gt;1:640   &gt;1:640   &lt;1:5   &lt;1:5       3   &gt;1:640    1:320   &lt;1:5   &lt;1:5       4   &gt;1:640   &gt;1:640   &lt;1:5   &lt;1:5       5    1:320    1:640   &lt;1:5   &lt;1:5       6   &gt;1:640   &gt;1:640   &lt;1:5   &lt;1:5       7   &gt;1:640   &gt;1:640   &lt;1:5   &lt;1:5       8    1:320    1:320   &lt;1:5   &lt;1:5       9    1:320    1:320   &lt;1:5   &lt;1:5       10   &gt;1:640   &gt;1:640   &lt;1:5   &lt;1:5                            
         [0178]    [0178]                                                                         TABLE 64                           Viral neutralization assay with the sera       of animals immunized with PD4D2.                Neutralizing titers*   Neutralizing titers           (PD4D2)   PBS C(−)            Mouse   Anti-DEN-4   Anti-DEN-2   Anti-DEN-4   Anti-DEN-2                    1   &gt;1:1280   &gt;1:1280   &lt;1:5   &lt;1:5       2   &gt;1:1280   &gt;1:1280   &lt;1:5   &lt;1:5       3    1:1280    1:1280   &lt;1:5   &lt;1:5       4   &gt;1:1280   &gt;1:1280   &lt;1:5   &lt;1:5       5    1:640    1:1280   &lt;1:5   &lt;1:5       6   &gt;1:1280   &gt;1:1280   &lt;1:5   &lt;1:5       7   &gt;1:1280   &gt;1:1280   &lt;1:5   &lt;1:5       8    1:640   &gt;1:1280   &lt;1:5   &lt;1:5       9   &gt;1:1280   &gt;1:1280   &lt;1:5   &lt;1:5       10   &gt;1:1280   &gt;1:1280   &lt;1:5   &lt;1:5                            
         [0179]    [0179]                                                                                 TABLE 65                       Cross-reactivity assay against all the viral serotypes       by ELISA, HI and viral neutralization with the       sera of animals immunized with PD4D2.                                Mixture   ELISA   ELISA   ELISA   ELISA       of sera*   (anti-DEN-1)   (anti DEN-2)   (anti-DEN-3)   (anti-DEN-4)               1(PD4D2)   &lt;1/100   &gt;1:128 000   &lt;1/100    1:64 000       2(PD4D2)   &lt;1/100   &gt;1:128 000   &lt;1/100   &gt;1:128 000                    Mixture   HI**   HI   HI   HI       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               PD4D2   &lt;1:5   &gt;1:320   &lt;1:5   &gt;1:320                        Neutralizing   Neutralizing   Neutralizing   Neutralizing       Mixture   titers***   titers   titers   titers       of sera*   anti-DEN-1   anti-DEN-2   anti-DEN-3   anti-DEN-4               1(PD4D2)   &lt;1:5   &gt;1:1280   &lt;1:5   &gt;1:1280       2(PD4D2)   &lt;1:5   &gt;1:1280   &lt;1:5   &gt;1:1280                                            
       Example 53  
     Protection Assay  
       [0180]    For the evaluation of the protection conferred to mice immunized with all the assayed variants upon challenge with homologous lethal DEN, 15 mice of each group were used. Each animal received one dose of 100 LD 50  of lethal DEN by intracraneal inoculation and they were observed during 21 days to study the percentage of lethality. As positive controls, groups of 15 mice immunized with the four viral preparations (DEN-1, DEN-2, DEN-3 and DEN-4) were used. All the mice of these control groups survived while mice from the negative control group become sick between 7-11 days after challenge; therefore, achieving a 100% of mortality. Finally, groups immunized with the fusion proteins under study had between an 80% and 100% of protection and in all the cases, significant differences with respect to the control group were found (table 66).  
                                           TABLE 66                           Percentage of survival in mice immunized with       the protein variants assayed upon challenge       with the homologous lethal Dengue virus.                    Percentage of           Immunogen   survival*                            PBS   0           DEN-1   100           DEN-2   100           DEN-3   100           DEN-4   100           PLL1   86           PLL2   100           PLL3   100           PLH1   80           PLH2   100           PLH3   100           PAZ1   80           PAZ2   100           PAZ3   100           PID1   86           PID2   100           PID3   100           PD4D2   100               (DEN-4)               100               (DEN-2)                                  
 
       Example 54  
     Lymphoproliferative Response  
       [0181]    Animals from different groups immunized with the chimeric proteins containing the E fragment from DEN-2 (PLL1, PLL2 and PLL3), and a placebo group, were sacrified 15 days after the last dose. Then, the spleen of the animals was harvested and the lymphoproliferative response against the four serotypes of Dengue virus was studied. Table 67 shows the results of the stimulation indexes obtained, which demonstrate that a serotype specific response was achieved.  
                                                               TABLE 67                           Stimulation indexes, against the four Dengue viral serotypes,       of lymphocytes from mice immunized with PLL1, PLL2 and PLL3.                PLL1   PLL2   PLL3   C(−)                            DEN-1     1.3*   1.0   0.8   1.2           DEN-2   12.5   10.3   8.9   1.4           DEN-3   1.0   1.6   1.8   1.4           DEN-4   1.7   1.5   1.7   1.1           Control Antigen   1.1   1.0   1.3   0.9           PHA**   13.3   16.5   11.1   12.0                                              
 
         [0182]    [0182] 
     
       
       
         1 
         
           
             54  
           
           
             1  
             25  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(25)  
               Sequence of the Xba-I primer for the 
      amplification of DENe-2 fragment  
             
           
            1 

cttctagaca ggctgcgcat ggaca                                           25 

 
           
             2  
             29  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(29)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-2 fragment  
             
           
            2 

gtggatcctt accctcccag gcttccaaa                                       29 

 
           
             3  
             25  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(25)  
               Sequence of the Eco-RI primer for the 
      amplification of DENe-2 fragment  
             
           
            3 

atgaattcac gcctcccaga gatcc                                           25 

 
           
             4  
             21  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(21)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-2 fragment  
             
           
            4 

cttggatcca ggctgagaat g                                               21 

 
           
             5  
             31  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(31)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-2 fragment  
             
           
            5 

gaggatcctt aaccacccag agacccaaaa t                                    31 

 
           
             6  
             25  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(25)  
               Sequence of the Xba-I primer for the 
      amplification of DENe-1 fragment  
             
           
            6 

cttctagaca ggctcaaaat ggata                                           25 

 
           
             7  
             28  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(28)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-1 fragment  
             
           
            7 

gaggatcctt acccgccaat agaaccga                                        28 

 
           
             8  
             25  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(25)  
               Sequence of the Eco-RI primer for the 
      amplification of DENe-1 fragment  
             
           
            8 

acgaattcac ccctcctata gatcc                                           25 

 
           
             9  
             24  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(24)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-1 fragment  
             
           
            9 

acaccttgga tccagactaa aaat                                            24 

 
           
             10  
             26  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(26)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-1 fragment  
             
           
            10 

ccggatccgt gaattaccca cctata                                          26 

 
           
             11  
             29  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(29)  
               Sequence of the Xba-I primer for the 
      amplification of DENe-3 fragment  
             
           
            11 

tttctagata gactcaagat ggacaaatt                                       29 

 
           
             12  
             28  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(28)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-3 fragment  
             
           
            12 

gaggatcctt aaccacccac tgaaccaa                                        28 

 
           
             13  
             25  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(25)  
               Sequence of the Eco-RI primer for the 
      amplification of DENe-3 fragment  
             
           
            13 

aagaattcac accacccaca gatcc                                           25 

 
           
             14  
             23  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(23)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-3 fragment  
             
           
            14 

acttaggatc cagactcaag atg                                             23 

 
           
             15  
             26  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(26)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-3 fragment  
             
           
            15 

gaggatcctt aaccacccac tgaacc                                          26 

 
           
             16  
             30  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(30)  
               Sequence of the Xba-I primer for the 
      amplification of DENe-4 fragment  
             
           
            16 

cttctagaca aagtgcgtat ggagaaattg                                      30 

 
           
             17  
             28  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(28)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-4 fragment  
             
           
            17 

gaggatcctt aaccaccaac agaaccaa                                        28 

 
           
             18  
             25  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(25)  
               Sequence of the Eco-RI primer for the 
      amplification of DENe-4 fragment  
             
           
            18 

atgaattcag tccaccaacg ctacc                                           25 

 
           
             19  
             27  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(27)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-4 fragment  
             
           
            19 

ggccatctag gatccaaagt gcgtatg                                         27 

 
           
             20  
             28  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(28)  
               Sequence of the Bam-HI primer for the 
      amplification of DENe-4 fragment  
             
           
            20 

gaggatcctt agccaccaac cgaaccaa                                        28 

 
           
             21  
             26  
             DNA  
             Escherichia coli  
             
               primer_bind  
               (1)..(26)  
               Sequence of the primer Xba-I for the 
      amplification of DENe-4 fragment  
             
           
            21 

attctagaag accaccaacg gaacca                                          26 

 
           
             22  
             429  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(426)  
               Nucleotidic sequence coding for the aminoacids 
      286 to 426 of DEN-2 envelope protein  
             
           
            22 

gacaggctga gaatggacaa actacagctc aaaggaatgt catactctat gtgtacagga     60 

aagtttaaaa ttgtgaagga aatagcagaa acacaacatg gaacaatagt tatcagagta    120 

caatatgaag gggacggctc tccatgtaag atcccttttg agataatgga tttggaaaaa    180 

agacacgtct taggtcgcct gattacagtt aacccgatcg taacagaaaa agatagccca    240 

gtcaacatag aagcagaacc tccattcgga gacagctaca tcatcatagg agtagagccg    300 

ggacaattga aactcaactg gtttaagaaa ggaagttcca tcggccaaat gtttgagaca    360 

acaatgagag gagcgaagag aatggccatt ttaggtgaca cagcctggga ttttggatcc    420 

ctgggagga                                                            429 

 
           
             23  
             168  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(168)  
               Nucleotidic sequence coding for the first 45 
      aminoacids of the MDH.  
             
           
            23 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagac                 168 

 
           
             24  
             603  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(603)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pLL1  
             
           
            24 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagacag gctgcgcatg    180 

gacaaactac agctcaaagg aatgtcatac tctatgtgta caggaaagtt taaaattgtg    240 

aaggaaatag cagaaacaca acatggaaca atagttatca gagtacaata tgaaggggac    300 

ggctctccat gtaagatccc ttttgagata atggatttgg aaaaaagaca cgtcttaggt    360 

cgcctgatta cagttaaccc gatcgtaaca gaaaaagata gcccagtcaa catagaagca    420 

gaacctccat tcggagacag ctacatcatc ataggagtag agccgggaca attgaaactc    480 

aactggttta agaaaggaag ttccatcggc caaatgtttg agacaacaat gagaggagcg    540 

aagagaatgg ccattttagg tgacacagcc tgggattttg gaagcctggg agggtaagga    600 

tcc                                                                  603 

 
           
             25  
             195  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(195)  
               Aminoacidic sequence of the PLL1 protein  
             
           
            25 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Leu Asp Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly 
     50                  55                  60 

Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Ile Val Lys Glu Ile 
 65                  70                  75                  80 

Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly 
                 85                  90                  95 

Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys 
            100                 105                 110 

Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu 
        115                 120                 125 

Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser 
    130                 135                 140 

Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe 
145                 150                 155                 160 

Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly 
                165                 170                 175 

Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 
            180                 185                 190 

Leu Gly Gly 
        195 

 
           
             26  
             1851  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(1851)  
               Nucleotidic sequence of the MDH in the plasmid 
      pM84 His.  
             
           
            26 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagaa    180 

gaactagtgg atcccccggg ctgcaggaat tcgatgaatt cgatggacgt acctgctgaa    240 

gttgcaggcg tagtcaaaga agttaaagtt aaagtcggcg acaaaatctc tgaaggtggt    300 

ttgattgtcg tcgttgaagc tgaaggcacg gcagccgctc ctaaagccga agcggctgcc    360 

gccccggcgc aagaagcccc taaagctgcc gctcctgctc cgcaagccgc gcaattcggc    420 

ggttctgccg atgccgagta cgacgtggtc gtattgggtg gcggtcccgg cggttactcc    480 

gctgcatttg ccgctgccga tgaaggcttg aaagtcgcca tcgtcgaacg ttacaaaact    540 

ttgggcggcg tttgcctgaa cgtcggctgt atcccttcca aagccttgtt gcacaatgcc    600 

gccgttatcg acgaagtgcg ccacttggct gccaacggta tcaaataccc cgagccggaa    660 

ctcgacatcg atatgcttcg cgcctacaaa gacggcgtag tttcccgcct cacgggcggt    720 

ttggcaggta tggcgaaaag ccgtaaagtg gacgttatcc aaggcgacgg gcaattctta    780 

gatccgcacc acttggaagt gtcgctgact gccggcgacg cgtacgaaca ggcagcccct    840 

accggcgaga aaaaaatcgt tgccttcaaa aactgtatca ttgcagcagg cagccgcgta    900 

accaaactgc ctttcattcc tgaagatccg cgcatcatcg attccagcgg cgcattggct    960 

ctgaaagaag taccgggcaa actgctgatt atcggcggcg gcattatcgg cctcgagatg   1020 

ggtacggttt acagcacgct gggttcgcgt ttggatgtgg ttgaaatgat ggacggcctg   1080 

atgcaaggcg cagaccgcga tttggtaaaa gtatggcaaa aacaaaacga ataccgtttt   1140 

gacaacatta tggtcaacac caaaaccgtt gcagttgagc cgaaagaaga cggcgtttac   1200 

gttacctttg aaggcgcgaa cgcgcctaaa gagccgcaac gctacgatgc cgtattggtt   1260 

gccgccggcc gcgcgcccaa cggcaaactc atcagcgcgg aaaaagcagg cgttgccgta   1320 

accgatcgcg gcttcatcga agtggacaaa caaatgcgta ccaatgtgcc gcacatctac   1380 

gccatcggcg acatcgtcgg tcagccgatg ttggcgcaca aagccgttca cgaaggccac   1440 

gttgccgccg aaaactgcgc cggccacaaa gcctacttcg acgcacgcgt gattccgggc   1500 

gttgcctaca cttcccccga agtggcgtgg gtgggcgaaa ccgaactgtc cgccaaagcc   1560 

tccggccgca aaatcaccaa agccaacttc ccgtgggcgg cttccggccg tgcgattgcc   1620 

aacggttgcg acaagccgtt taccaagctg atttttgatg ccgaaaccgg ccgcatcatc   1680 

ggcggcggca ttgtcggtcc gaacggtggc gatatgatcg gcgaagtctg ccttgccatc   1740 

gaaatgggct gcgacgcggc agacatcggc aaaaccatcc acccgcaccc gggcgaatcc   1800 

atcggtatgg cggcggaagt ggcattgggt acttgtaccg acaaaaaaaa a            1851 

 
           
             27  
             2253  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(2253)  
               Nucleotidic sequence of the quimeric protein in 
      the plasmid pLL2.  
             
           
            27 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagac    180 

aggctgcgca tggacaaact acagctcaaa ggaatgtcat actctatgtg tacaggaaag    240 

tttaaaattg tgaaggaaat agcagaaaca caacatggaa caatagttat cagagtacaa    300 

tatgaagggg acggctctcc atgtaagatc ccttttgaga taatggattt ggaaaaaaga    360 

cacgtcttag gtcgcctgat tacagttaac ccgatcgtaa cagaaaaaga tagcccagtc    420 

aacatagaag cagaacctcc attcggagac agctacatca tcataggagt agagccggga    480 

caattgaaac tcaactggtt taagaaagga agttccatcg gccaaatgtt tgagacaaca    540 

atgagaggag cgaagagaat ggccatttta ggtgacacag cctgggattt tggatctctg    600 

ggaggcgtga attcgatgaa ttcgatggac gtacctgctg aagttgcagg cgtagtcaaa    660 

gaagttaaag ttaaagtcgg cgacaaaatc tctgaaggtg gtttgattgt cgtcgttgaa    720 

gctgaaggca cggcagccgc tcctaaagcc gaagcggctg ccgccccggc gcaagaagcc    780 

cctaaagctg ccgctcctgc tccgcaagcc gcgcaattcg gcggttctgc cgatgccgag    840 

tacgacgtgg tcgtattggg tggcggtccc ggcggttact ccgctgcatt tgccgctgcc    900 

gatgaaggct tgaaagtcgc catcgtcgaa cgttacaaaa ctttgggcgg cgtttgcctg    960 

aacgtcggct gtatcccttc caaagccttg ttgcacaatg ccgccgttat cgacgaagtg   1020 

cgccacttgg ctgccaacgg tatcaaatac cccgagccgg aactcgacat cgatatgctt   1080 

cgcgcctaca aagacggcgt agtttcccgc ctcacgggcg gtttggcagg tatggcgaaa   1140 

agccgtaaag tggacgttat ccaaggcgac gggcaattct tagatccgca ccacttggaa   1200 

gtgtcgctga ctgccggcga cgcgtacgaa caggcagccc ctaccggcga gaaaaaaatc   1260 

gttgccttca aaaactgtat cattgcagca ggcagccgcg taaccaaact gcctttcatt   1320 

cctgaagatc cgcgcatcat cgattccagc ggcgcattgg ctctgaaaga agtaccgggc   1380 

aaactgctga ttatcggcgg cggcattatc ggcctcgaga tgggtacggt ttacagcacg   1440 

ctgggttcgc gtttggatgt ggttgaaatg atggacggcc tgatgcaagg cgcagaccgc   1500 

gatttggtaa aagtatggca aaaacaaaac gaataccgtt ttgacaacat tatggtcaac   1560 

accaaaaccg ttgcagttga gccgaaagaa gacggcgttt acgttacctt tgaaggcgcg   1620 

aacgcgccta aagagccgca acgctacgat gccgtattgg ttgccgccgg ccgcgcgccc   1680 

aacggcaaac tcatcagcgc ggaaaaagca ggcgttgccg taaccgatcg cggcttcatc   1740 

gaagtggaca aacaaatgcg taccaatgtg ccgcacatct acgccatcgg cgacatcgtc   1800 

ggtcagccga tgttggcgca caaagccgtt cacgaaggcc acgttgccgc cgaaaactgc   1860 

gccggccaca aagcctactt cgacgcacgc gtgattccgg gcgttgccta cacttccccc   1920 

gaagtggcgt gggtgggcga aaccgaactg tccgccaaag cctccggccg caaaatcacc   1980 

aaagccaact tcccgtgggc ggcttccggc cgtgcgattg ccaacggttg cgacaagccg   2040 

tttaccaagc tgatttttga tgccgaaacc ggccgcatca tcggcggcgg cattgtcggt   2100 

ccgaacggtg gcgatatgat cggcgaagtc tgccttgcca tcgaaatggg ctgcgacgcg   2160 

gcagacatcg gcaaaaccat ccacccgcac ccgggcgaat ccatcggtat ggcggcggaa   2220 

gtggcattgg gtacttgtac cgacaaaaaa aaa                                2253 

 
           
             28  
             748  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(748)  
               Aminoacidic sequence of PLL2.  
             
           
            28 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Leu Asp Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly 
     50                  55                  60 

Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Ile Val Lys Glu Ile 
 65                  70                  75                  80 

Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly 
                 85                  90                  95 

Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys 
            100                 105                 110 

Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu 
        115                 120                 125 

Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser 
    130                 135                 140 

Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe 
145                 150                 155                 160 

Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly 
                165                 170                 175 

Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 
            180                 185                 190 

Leu Gly Gly Val Asn Ser Met Asn Ser Met Asp Val Pro Ala Glu Val 
        195                 200                 205 

Ala Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser 
    210                 215                 220 

Glu Gly Gly Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala 
225                 230                 235                 240 

Pro Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala 
                245                 250                 255 

Ala Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala 
            260                 265                 270 

Glu Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala 
        275                 280                 285 

Ala Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg 
    290                 295                 300 

Tyr Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser 
305                 310                 315                 320 

Lys Ala Leu Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu 
                325                 330                 335 

Ala Ala Asn Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met 
            340                 345                 350 

Leu Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu 
        355                 360                 365 

Ala Gly Met Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly 
    370                 375                 380 

Gln Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp 
385                 390                 395                 400 

Ala Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe 
                405                 410                 415 

Lys Asn Cys Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe 
            420                 425                 430 

Ile Pro Glu Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu 
        435                 440                 445 

Lys Glu Val Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly 
    450                 455                 460 

Leu Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val 
465                 470                 475                 480 

Val Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val 
                485                 490                 495 

Lys Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val 
            500                 505                 510 

Asn Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val 
        515                 520                 525 

Thr Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala 
    530                 535                 540 

Val Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala 
545                 550                 555                 560 

Glu Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp 
                565                 570                 575 

Lys Gln Met Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile 
            580                 585                 590 

Val Gly Gln Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val 
        595                 600                 605 

Ala Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val 
    610                 615                 620 

Ile Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu 
625                 630                 635                 640 

Thr Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn 
                645                 650                 655 

Phe Pro Trp Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys 
            660                 665                 670 

Pro Phe Thr Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly 
        675                 680                 685 

Gly Gly Ile Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys 
    690                 695                 700 

Leu Ala Ile Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile 
705                 710                 715                 720 

His Pro His Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu 
                725                 730                 735 

Gly Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys 
            740                 745 

 
           
             29  
             1821  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(1821)  
               Nucleotidic sequence of the MDH in the plasmid 
      pD4  
             
           
            29 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagaaat ggacgtacct    180 

gctgaagttg caggcgtagt caaagaagtt aaagttaaag tcggcgacaa aatctctgaa    240 

ggtggtttga ttgtcgtcgt tgaagctgaa ggcacggcag ccgctcctaa agccgaagcg    300 

gctgccgccc cggcgcaaga agcccctaaa gctgccgctc ctgctccgca agccgcgcaa    360 

ttcggcggtt ctgccgatgc cgagtacgac gtggtcgtat tgggtggcgg tcccggcggt    420 

tactccgctg catttgccgc tgccgatgaa ggcttgaaag tcgccatcgt cgaacgttac    480 

aaaactttgg gcggcgtttg cctgaacgtc ggctgtatcc cttccaaagc cttgttgcac    540 

aatgccgccg ttatcgacga agtgcgccac ttggctgcca acggtatcaa ataccccgag    600 

ccggaactcg acatcgatat gcttcgcgcc tacaaagacg gcgtagtttc ccgcctcacg    660 

ggcggtttgg caggtatggc gaaaagccgt aaagtggacg ttatccaagg cgacgggcaa    720 

ttcttagatc cgcaccactt ggaagtgtcg ctgactgccg gcgacgcgta cgaacaggca    780 

gcccctaccg gcgagaaaaa aatcgttgcc ttcaaaaact gtatcattgc agcaggcagc    840 

cgcgtaacca aactgccttt cattcctgaa gatccgcgca tcatcgattc cagcggcgca    900 

ttggctctga aagaagtacc gggcaaactg ctgattatcg gcggcggcat tatcggcctc    960 

gagatgggta cggtttacag cacgctgggt tcgcgtttgg atgtggttga aatgatggac   1020 

ggcctgatgc aaggcgcaga ccgcgatttg gtaaaagtat ggcaaaaaca aaacgaatac   1080 

cgttttgaca acattatggt caacaccaaa accgttgcag ttgagccgaa agaagacggc   1140 

gtttacgtta cctttgaagg cgcgaacgcg cctaaagagc cgcaacgcta cgatgccgta   1200 

ttggttgccg ccggccgcgc gcccaacggc aaactcatca gcgcggaaaa agcaggcgtt   1260 

gccgtaaccg atcgcggctt catcgaagtg gacaaacaaa tgcgtaccaa tgtgccgcac   1320 

atctacgcca tcggcgacat cgtcggtcag ccgatgttgg cgcacaaagc cgttcacgaa   1380 

ggccacgttg ccgccgaaaa ctgcgccggc cacaaagcct acttcgacgc acgcgtgatt   1440 

ccgggcgttg cctacacttc ccccgaagtg gcgtgggtgg gcgaaaccga actgtccgcc   1500 

aaagcctccg gccgcaaaat caccaaagcc aacttcccgt gggcggcttc cggccgtgcg   1560 

attgccaacg gttgcgacaa gccgtttacc aagctgattt ttgatgccga aaccggccgc   1620 

atcatcggcg gcggcattgt cggtccgaac ggtggcgata tgatcggcga agtctgcctt   1680 

gccatcgaaa tgggctgcga cgcggcagac atcggcaaaa ccatccaccc gcacccgacc   1740 

ttgggcgaat ccatcggtat ggcggcggaa gtggcattgg gtacttgtac cgacctgcct   1800 

ccgcaaaaga aaaaaggatc c                                             1821 

 
           
             30  
             2259  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(2259)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pLL3  
             
           
            30 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagaaat ggacgtacct    180 

gctgaagttg caggcgtagt caaagaagtt aaagttaaag tcggcgacaa aatctctgaa    240 

ggtggtttga ttgtcgtcgt tgaagctgaa ggcacggcag ccgctcctaa agccgaagcg    300 

gctgccgccc cggcgcaaga agcccctaaa gctgccgctc ctgctccgca agccgcgcaa    360 

ttcggcggtt ctgccgatgc cgagtacgac gtggtcgtat tgggtggcgg tcccggcggt    420 

tactccgctg catttgccgc tgccgatgaa ggcttgaaag tcgccatcgt cgaacgttac    480 

aaaactttgg gcggcgtttg cctgaacgtc ggctgtatcc cttccaaagc cttgttgcac    540 

aatgccgccg ttatcgacga agtgcgccac ttggctgcca acggtatcaa ataccccgag    600 

ccggaactcg acatcgatat gcttcgcgcc tacaaagacg gcgtagtttc ccgcctcacg    660 

ggcggtttgg caggtatggc gaaaagccgt aaagtggacg ttatccaagg cgacgggcaa    720 

ttcttagatc cgcaccactt ggaagtgtcg ctgactgccg gcgacgcgta cgaacaggca    780 

gcccctaccg gcgagaaaaa aatcgttgcc ttcaaaaact gtatcattgc agcaggcagc    840 

cgcgtaacca aactgccttt cattcctgaa gatccgcgca tcatcgattc cagcggcgca    900 

ttggctctga aagaagtacc gggcaaactg ctgattatcg gcggcggcat tatcggcctc    960 

gagatgggta cggtttacag cacgctgggt tcgcgtttgg atgtggttga aatgatggac   1020 

ggcctgatgc aaggcgcaga ccgcgatttg gtaaaagtat ggcaaaaaca aaacgaatac   1080 

cgttttgaca acattatggt caacaccaaa accgttgcag ttgagccgaa agaagacggc   1140 

gtttacgtta cctttgaagg cgcgaacgcg cctaaagagc cgcaacgcta cgatgccgta   1200 

ttggttgccg ccggccgcgc gcccaacggc aaactcatca gcgcggaaaa agcaggcgtt   1260 

gccgtaaccg atcgcggctt catcgaagtg gacaaacaaa tgcgtaccaa tgtgccgcac   1320 

atctacgcca tcggcgacat cgtcggtcag ccgatgttgg cgcacaaagc cgttcacgaa   1380 

ggccacgttg ccgccgaaaa ctgcgccggc cacaaagcct acttcgacgc acgcgtgatt   1440 

ccgggcgttg cctacacttc ccccgaagtg gcgtgggtgg gcgaaaccga actgtccgcc   1500 

aaagcctccg gccgcaaaat caccaaagcc aacttcccgt gggcggcttc cggccgtgcg   1560 

attgccaacg gttgcgacaa gccgtttacc aagctgattt ttgatgccga aaccggccgc   1620 

atcatcggcg gcggcattgt cggtccgaac ggtggcgata tgatcggcga agtctgcctt   1680 

gccatcgaaa tgggctgcga cgcggcagac atcggcaaaa ccatccaccc gcacccgacc   1740 

ttgggcgaat ccatcggtat ggcggcggaa gtggcattgg gtacttgtac cgacctgcct   1800 

ccgcaaaaga aaaaaggatc cgacaggctg agaatggaca aactacagct caaaggaatg   1860 

tcatactcta tgtgtacagg aaagtttaaa attgtgaagg aaatagcaga aacacaacat   1920 

ggaacaatag ttatcagagt acaatatgaa ggggacggct ctccatgtaa gatccctttt   1980 

gagataatgg atttggaaaa aagacacgtc ttaggtcgcc tgattacagt taacccgatc   2040 

gtaacagaaa aagatagccc agtcaacata gaagcagaac ctccattcgg agacagctac   2100 

atcatcatag gagtagagcc gggacaattg aaactcaact ggtttaagaa aggaagttcc   2160 

atcggccaaa tgtttgagac aacaatgaga ggagcgaaga gaatggccat tttaggtgac   2220 

acagcctggg attttgggtc tctgggtggt taaggatcc                          2259 

 
           
             31  
             745  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(745)  
               Aminoacidic sequence of PLL3  
             
           
            31 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Met Asn Ser Met Asp Val Pro Ala Glu Val Ala Gly Val 
     50                  55                  60 

Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 
 65                  70                  75                  80 

Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 
                 85                  90                  95 

Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 
            100                 105                 110 

Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 
        115                 120                 125 

Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala 
    130                 135                 140 

Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr 
145                 150                 155                 160 

Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu 
                165                 170                 175 

Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn 
            180                 185                 190 

Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala 
        195                 200                 205 

Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met 
    210                 215                 220 

Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu 
225                 230                 235                 240 

Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu 
                245                 250                 255 

Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys 
            260                 265                 270 

Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu 
        275                 280                 285 

Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val 
    290                 295                 300 

Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 
305                 310                 315                 320 

Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 
                325                 330                 335 

Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 
            340                 345                 350 

Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 
        355                 360                 365 

Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu 
    370                 375                 380 

Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val 
385                 390                 395                 400 

Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala 
                405                 410                 415 

Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met 
            420                 425                 430 

Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln 
        435                 440                 445 

Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu 
    450                 455                 460 

Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly 
465                 470                 475                 480 

Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu 
                485                 490                 495 

Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp 
            500                 505                 510 

Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr 
        515                 520                 525 

Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile 
    530                 535                 540 

Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 
545                 550                 555                 560 

Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 
                565                 570                 575 

Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys 
            580                 585                 590 

Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Arg Leu Arg Met Asp 
        595                 600                 605 

Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met Cys Thr Gly Lys Phe 
    610                 615                 620 

Lys Ile Val Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile Val Ile 
625                 630                 635                 640 

Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu 
                645                 650                 655 

Ile Met Asp Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile Thr Val 
            660                 665                 670 

Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu Ala Glu 
        675                 680                 685 

Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln 
    690                 695                 700 

Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser Ile Gly Gln Met Phe 
705                 710                 715                 720 

Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala Ile Leu Gly Asp Thr 
                725                 730                 735 

Ala Trp Asp Phe Gly Ser Leu Gly Gly 
            740                 745 

 
           
             32  
             429  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(429)  
               Nucleotidic sequence coding for the aminoacids 
      286 to 426 of the DEN-1 envelope protein  
             
           
            32 

agactaaaaa tggataaact gactttaaaa ggggtatcat atgtaatgtg cacagggtca     60 

ttcaagttag agaaggaagt ggctgagacc cagcatggaa ctgttctagt gcaggttaaa    120 

tacgaaggaa cagatgcacc atgcaagatc cccttctcgt cccaagatga gaaaggagta    180 

acccagaatg ggagattgat aacagccaac cccatagtca ttgacaaaga aaaaccagtc    240 

aacattgaag cggagccacc ttttggtgag agctatattg tggtaggagc aggtgaaaaa    300 

gctttgaaac taagctggtt caagaaggga agcagtatag ggaaaatgtt tgaagcaact    360 

gcccgtggag cacgaaggat ggccatcctg ggagacaccg catgggactt cggttctata    420 

ggagggtaa                                                            429 

 
           
             33  
             615  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(615)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pLH1  
             
           
            33 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagac    180 

aggctcaaaa tggataaact gactttaaaa ggggtatcat atgtaatgtg cacagggtca    240 

ttcaagttag agaaggaagt ggctgagacc cagcatggaa ctgttctagt gcaggttaaa    300 

tacgaaggaa cagatgcacc atgcaagatc cccttctcgt cccaagatga gaaaggagta    360 

acccagaatg ggagattgat aacagccaac cccatagtca ttgacaaaga aaaaccagtc    420 

aacattgaag cggagccacc ttttggtgag agctatattg tggtaggagc aggtgaaaaa    480 

gctttgaaac taagctggtt caagaaggga agcagtatag ggaaaatgtt tgaagcaact    540 

gcccgtggag cacgaaggat ggccatcctg ggagacaccg catgggactt cggttctatt    600 

ggcgggtaag gatcc                                                     615 

 
           
             34  
             174  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(174)  
               Aminoacidic sequence of the PLH1  
             
           
            34 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Leu Asp Phe Lys Leu Glu Lys Glu Val Ala Glu Thr Gln 
     50                  55                  60 

His Gly Thr Val Leu Val Gln Val Lys Tyr Gln Gly Thr Asp Ala Pro 
 65                  70                  75                  80 

Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly Val Thr Gln Asn 
                 85                  90                  95 

Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp Lys Glu Lys Pro Val 
            100                 105                 110 

Asn Ile Glu Thr Glu Pro Pro Phe Gly Glu Ser Tyr Ile Val Val Gly 
        115                 120                 125 

Ala Gly Glu Lys Ala Leu Lys Gln Cys Trp Phe Lys Lys Gly Ser Ser 
    130                 135                 140 

Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg Arg Met Ala 
145                 150                 155                 160 

Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Ile Gly Gly 
                165                 170 

 
           
             35  
             2253  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(2253)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pLH2  
             
           
            35 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagac    180 

aggctcaaaa tggataaact gactttaaaa ggggtatcat atgtaatgtg cacagggtca    240 

ttcaagttag agaaggaagt ggctgagacc cagcatggaa ctgttctagt gcaggttaaa    300 

tacgaaggaa cagatgcacc atgcaagatc cccttctcgt cccaagatga gaaaggagta    360 

acccagaatg ggagattgat aacagccaac cccatagtca ttgacaaaga aaaaccagtc    420 

aacattgaag cggagccacc ttttggtgag agctatattg tggtaggagc aggtgaaaaa    480 

gctttgaaac taagctggtt caagaaggga agcagtatag ggaaaatgtt tgaagcaact    540 

gcccgtggag cacgaaggat ggccatcctg ggagacaccg catgggactt cggatctata    600 

ggaggggtga attcgatgaa ttcgatggac gtacctgctg aagttgcagg cgtagtcaaa    660 

gaagttaaag ttaaagtcgg cgacaaaatc tctgaaggtg gtttgattgt cgtcgttgaa    720 

gctgaaggca cggcagccgc tcctaaagcc gaagcggctg ccgccccggc gcaagaagcc    780 

cctaaagctg ccgctcctgc tccgcaagcc gcgcaattcg gcggttctgc cgatgccgag    840 

tacgacgtgg tcgtattggg tggcggtccc ggcggttact ccgctgcatt tgccgctgcc    900 

gatgaaggct tgaaagtcgc catcgtcgaa cgttacaaaa ctttgggcgg cgtttgcctg    960 

aacgtcggct gtatcccttc caaagccttg ttgcacaatg ccgccgttat cgacgaagtg   1020 

cgccacttgg ctgccaacgg tatcaaatac cccgagccgg aactcgacat cgatatgctt   1080 

cgcgcctaca aagacggcgt agtttcccgc ctcacgggcg gtttggcagg tatggcgaaa   1140 

agccgtaaag tggacgttat ccaaggcgac gggcaattct tagatccgca ccacttggaa   1200 

gtgtcgctga ctgccggcga cgcgtacgaa caggcagccc ctaccggcga gaaaaaaatc   1260 

gttgccttca aaaactgtat cattgcagca ggcagccgcg taaccaaact gcctttcatt   1320 

cctgaagatc cgcgcatcat cgattccagc ggcgcattgg ctctgaaaga agtaccgggc   1380 

aaactgctga ttatcggcgg cggcattatc ggcctcgaga tgggtacggt ttacagcacg   1440 

ctgggttcgc gtttggatgt ggttgaaatg atggacggcc tgatgcaagg cgcagaccgc   1500 

gatttggtaa aagtatggca aaaacaaaac gaataccgtt ttgacaacat tatggtcaac   1560 

accaaaaccg ttgcagttga gccgaaagaa gacggcgttt acgttacctt tgaaggcgcg   1620 

aacgcgccta aagagccgca acgctacgat gccgtattgg ttgccgccgg ccgcgcgccc   1680 

aacggcaaac tcatcagcgc ggaaaaagca ggcgttgccg taaccgatcg cggcttcatc   1740 

gaagtggaca aacaaatgcg taccaatgtg ccgcacatct acgccatcgg cgacatcgtc   1800 

ggtcagccga tgttggcgca caaagccgtt cacgaaggcc acgttgccgc cgaaaactgc   1860 

gccggccaca aagcctactt cgacgcacgc gtgattccgg gcgttgccta cacttccccc   1920 

gaagtggcgt gggtgggcga aaccgaactg tccgccaaag cctccggccg caaaatcacc   1980 

aaagccaact tcccgtgggc ggcttccggc cgtgcgattg ccaacggttg cgacaagccg   2040 

tttaccaagc tgatttttga tgccgaaacc ggccgcatca tcggcggcgg cattgtcggt   2100 

ccgaacggtg gcgatatgat cggcgaagtc tgccttgcca tcgaaatggg ctgcgacgcg   2160 

gcagacatcg gcaaaaccat ccacccgcac ccgggcgaat ccatcggtat ggcggcggaa   2220 

gtggcattgg gtacttgtac cgacaaaaaa aaa                                2253 

 
           
             36  
             727  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(727)  
               Aminoacidic sequence of the PLH2  
             
           
            36 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Leu Asp Phe Lys Leu Glu Lys Glu Val Ala Glu Thr Gln 
     50                  55                  60 

His Gly Thr Val Leu Val Gln Val Lys Tyr Gln Gly Thr Asp Ala Pro 
 65                  70                  75                  80 

Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly Val Thr Gln Asn 
                 85                  90                  95 

Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp Lys Glu Lys Pro Val 
            100                 105                 110 

Asn Ile Glu Thr Glu Pro Pro Phe Gly Glu Ser Tyr Ile Val Val Gly 
        115                 120                 125 

Ala Gly Glu Lys Ala Leu Lys Gln Cys Trp Phe Lys Lys Gly Ser Ser 
    130                 135                 140 

Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg Arg Met Ala 
145                 150                 155                 160 

Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Ile Gly Gly Val Asn 
                165                 170                 175 

Ser Met Asn Ser Met Asp Val Pro Ala Glu Val Ala Gly Val Val Lys 
            180                 185                 190 

Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly Leu Ile 
        195                 200                 205 

Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala Glu Ala 
    210                 215                 220 

Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro Ala Pro 
225                 230                 235                 240 

Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp Val Val 
                245                 250                 255 

Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala Ala Ala 
            260                 265                 270 

Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr Leu Gly 
        275                 280                 285 

Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu Leu His 
    290                 295                 300 

Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn Gly Ile 
305                 310                 315                 320 

Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala Tyr Lys 
                325                 330                 335 

Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met Ala Lys 
            340                 345                 350 

Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu Asp Pro 
        355                 360                 365 

His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu Gln Ala 
    370                 375                 380 

Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys Ile Ile 
385                 390                 395                 400 

Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu Asp Pro 
                405                 410                 415 

Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val Pro Gly 
            420                 425                 430 

Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met Gly Thr 
        435                 440                 445 

Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met Met Asp 
    450                 455                 460 

Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp Gln Lys 
465                 470                 475                 480 

Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys Thr Val 
                485                 490                 495 

Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu Gly Ala 
            500                 505                 510 

Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val Ala Ala 
        515                 520                 525 

Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala Gly Val 
    530                 535                 540 

Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met Arg Thr 
545                 550                 555                 560 

Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln Pro Met 
                565                 570                 575 

Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu Asn Cys 
            580                 585                 590 

Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly Val Ala 
        595                 600                 605 

Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu Ser Ala 
    610                 615                 620 

Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp Ala Ala 
625                 630                 635                 640 

Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr Lys Leu 
                645                 650                 655 

Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile Val Gly 
            660                 665                 670 

Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile Glu Met 
        675                 680                 685 

Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His Pro Gly 
    690                 695                 700 

Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys Thr Asp 
705                 710                 715                 720 

Leu Pro Pro Gln Lys Lys Lys 
                725 

 
           
             37  
             2250  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(2250)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pLH3  
             
           
            37 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagaaat ggacgtacct    180 

gctgaagttg caggcgtagt caaagaagtt aaagttaaag tcggcgacaa aatctctgaa    240 

ggtggtttga ttgtcgtcgt tgaagctgaa ggcacggcag ccgctcctaa agccgaagcg    300 

gctgccgccc cggcgcaaga agcccctaaa gctgccgctc ctgctccgca agccgcgcaa    360 

ttcggcggtt ctgccgatgc cgagtacgac gtggtcgtat tgggtggcgg tcccggcggt    420 

tactccgctg catttgccgc tgccgatgaa ggcttgaaag tcgccatcgt cgaacgttac    480 

aaaactttgg gcggcgtttg cctgaacgtc ggctgtatcc cttccaaagc cttgttgcac    540 

aatgccgccg ttatcgacga agtgcgccac ttggctgcca acggtatcaa ataccccgag    600 

ccggaactcg acatcgatat gcttcgcgcc tacaaagacg gcgtagtttc ccgcctcacg    660 

ggcggtttgg caggtatggc gaaaagccgt aaagtggacg ttatccaagg cgacgggcaa    720 

ttcttagatc cgcaccactt ggaagtgtcg ctgactgccg gcgacgcgta cgaacaggca    780 

gcccctaccg gcgagaaaaa aatcgttgcc ttcaaaaact gtatcattgc agcaggcagc    840 

cgcgtaacca aactgccttt cattcctgaa gatccgcgca tcatcgattc cagcggcgca    900 

ttggctctga aagaagtacc gggcaaactg ctgattatcg gcggcggcat tatcggcctc    960 

gagatgggta cggtttacag cacgctgggt tcgcgtttgg atgtggttga aatgatggac   1020 

ggcctgatgc aaggcgcaga ccgcgatttg gtaaaagtat ggcaaaaaca aaacgaatac   1080 

cgttttgaca acattatggt caacaccaaa accgttgcag ttgagccgaa agaagacggc   1140 

gtttacgtta cctttgaagg cgcgaacgcg cctaaagagc cgcaacgcta cgatgccgta   1200 

ttggttgccg ccggccgcgc gcccaacggc aaactcatca gcgcggaaaa agcaggcgtt   1260 

gccgtaaccg atcgcggctt catcgaagtg gacaaacaaa tgcgtaccaa tgtgccgcac   1320 

atctacgcca tcggcgacat cgtcggtcag ccgatgttgg cgcacaaagc cgttcacgaa   1380 

ggccacgttg ccgccgaaaa ctgcgccggc cacaaagcct acttcgacgc acgcgtgatt   1440 

ccgggcgttg cctacacttc ccccgaagtg gcgtgggtgg gcgaaaccga actgtccgcc   1500 

aaagcctccg gccgcaaaat caccaaagcc aacttcccgt gggcggcttc cggccgtgcg   1560 

attgccaacg gttgcgacaa gccgtttacc aagctgattt ttgatgccga aaccggccgc   1620 

atcatcggcg gcggcattgt cggtccgaac ggtggcgata tgatcggcga agtctgcctt   1680 

gccatcgaaa tgggctgcga cgcggcagac atcggcaaaa ccatccaccc gcacccgacc   1740 

ttgggcgaat ccatcggtat ggcggcggaa gtggcattgg gtacttgtac cgacctgcct   1800 

ccgcaaaaga aaaaaggatc cagactaaaa atggataaac tgactttaaa aggggtatca   1860 

tatgtaatgt gcacagggtc attcaagtta gagaaggaag tggctgagac ccagcatgga   1920 

actgttctag tgcaggttaa atacgaagga acagatgcac catgcaagat ccccttctcg   1980 

tcccaagatg agaaaggagt aacccagaat gggagattga taacagccaa ccccatagtc   2040 

attgacaaag aaaaaccagt caacattgaa gcggagccac cttttggtga gagctatatt   2100 

gtggtaggag caggtgaaaa agctttgaaa ctaagctggt tcaagaaggg aagcagtata   2160 

gggaaaatgt ttgaagcaac tgcccgtgga gcacgaagga tggccatcct gggagacacc   2220 

gcatgggact tcggttctat aggtgggtaa                                    2250 

 
           
             38  
             724  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(724)  
               Aminoacidic sequence of the PLH3  
             
           
            38 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Met Asn Ser Met Asp Val Pro Ala Glu Val Ala Gly Val 
     50                  55                  60 

Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 
 65                  70                  75                  80 

Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 
                 85                  90                  95 

Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 
            100                 105                 110 

Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 
        115                 120                 125 

Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala 
    130                 135                 140 

Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr 
145                 150                 155                 160 

Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu 
                165                 170                 175 

Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn 
            180                 185                 190 

Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala 
        195                 200                 205 

Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met 
    210                 215                 220 

Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu 
225                 230                 235                 240 

Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu 
                245                 250                 255 

Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys 
            260                 265                 270 

Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu 
        275                 280                 285 

Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val 
    290                 295                 300 

Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 
305                 310                 315                 320 

Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 
                325                 330                 335 

Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 
            340                 345                 350 

Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 
        355                 360                 365 

Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu 
    370                 375                 380 

Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val 
385                 390                 395                 400 

Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala 
                405                 410                 415 

Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met 
            420                 425                 430 

Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln 
        435                 440                 445 

Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu 
    450                 455                 460 

Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly 
465                 470                 475                 480 

Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu 
                485                 490                 495 

Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp 
            500                 505                 510 

Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr 
        515                 520                 525 

Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile 
    530                 535                 540 

Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 
545                 550                 555                 560 

Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 
                565                 570                 575 

Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys 
            580                 585                 590 

Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Phe Lys Leu Glu Lys 
        595                 600                 605 

Glu Val Ala Glu Thr Gln His Gly Thr Val Leu Val Gln Val Lys Tyr 
    610                 615                 620 

Gln Gly Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu 
625                 630                 635                 640 

Lys Gly Val Thr Gln Asn Arg Leu Ile Thr Ala Asn Pro Ile Val Thr 
                645                 650                 655 

Asp Lys Glu Lys Pro Val Asn Ile Glu Thr Glu Pro Pro Phe Gly Glu 
            660                 665                 670 

Ser Tyr Ile Val Val Gly Ala Gly Glu Lys Ala Leu Lys Gln Cys Trp 
        675                 680                 685 

Phe Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg 
    690                 695                 700 

Gly Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly 
705                 710                 715                 720 

Ser Ile Gly Gly 

 
           
             39  
             426  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(426)  
               Nucleotidic sequence coding for the aminoacids 
      286 to 426 of the DEN-3 envelope protein  
             
           
            39 

agactcaaga tggacaaatt gaaactcaag gggatgagct atgcaatgtg cttgaatacc     60 

tttgtgttga agaaagaagt ctccgaaacg cagcatggga caatactcat taaggttgag    120 

tacaaagggg aagatgcacc ctgcaagatt cctttctcca cggaggatgg acaagggaaa    180 

gctcacaatg gcagactgat cacagccaat ccagtggtga ccaagaagga ggagcctgtc    240 

aacattgagg ctgaacctcc ttttggggaa agtaatatag taattggaat tggagacaaa    300 

gccctgaaaa tcaactggta caggaaggga agctcgattg ggaagatgtt cgaggccact    360 

gccagaggtg caaggcgcat ggccatcttg ggagacacag cctgggactt tggatcagtg    420 

ggtggt                                                               426 

 
           
             40  
             615  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(615)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pAZ1  
             
           
            40 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagat    180 

agactcaaga tggacaaatt gaaactcaag gggatgagct atgcaatgtg cttgaatacc    240 

tttgtgttga agaaagaagt ctccgaaacg cagcatggga caatactcat taaggttgag    300 

tacaaagggg aagatgcacc ctgcaagatt cctttctcca cggaggatgg acaagggaaa    360 

gctcacaatg gcagactgat cacagccaat ccagtggtga ccaagaagga ggagcctgtc    420 

aacattgagg ctgaacctcc ttttggggaa agtaatatag taattggaat tggagacaaa    480 

gccctgaaaa tcaactggta caggaaggga agctcgattg ggaagatgtt cgaggccact    540 

gccagaggtg caaggcgcat ggccatcttg ggagacacag cctgggactt tggttcagtg    600 

ggtggttaag gatcc                                                     615 

 
           
             41  
             194  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(194)  
               Aminoacidic sequence of the PAZ1  
             
           
            41 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Leu Asp Arg Leu Lys Met Asp Lys Leu Lys Leu Lys Gly 
     50                  55                  60 

Met Ser Tyr Ala Met Cys Leu Asn Thr Phe Val Leu Lys Lys Glu Val 
 65                  70                  75                  80 

Ser Glu Thr His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu 
                 85                  90                  95 

Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys 
            100                 105                 110 

Ala His Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys 
        115                 120                 125 

Glu Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn 
    130                 135                 140 

Ile Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Arg 
145                 150                 155                 160 

Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala 
                165                 170                 175 

Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val 
            180                 185                 190 

Gly Gly 

 
           
             42  
             2253  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(2253)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pAZ2  
             
           
            42 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagat    180 

agactcaaga tggacaaatt gaaactcaag gggatgagct atgcaatgtg cttgaatacc    240 

tttgtgttga agaaagaagt ctccgaaacg cagcatggga caatactcat taaggttgag    300 

tacaaagggg aagatgcacc ctgcaagatt cctttctcca cggaggatgg acaagggaaa    360 

gctcacaatg gcagactgat cacagccaat ccagtggtga ccaagaagga ggagcctgtc    420 

aacattgagg ctgaacctcc ttttggggaa agtaatatag taattggaat tggagacaaa    480 

gccctgaaaa tcaactggta caggaaggga agctcgattg ggaagatgtt cgaggccact    540 

gccagaggtg caaggcgcat ggccatcttg ggagacacag cctgggactt tggatctgtg    600 

ggtggtgtga attcgatgaa ttcgatggac gtacctgctg aagttgcagg cgtagtcaaa    660 

gaagttaaag ttaaagtcgg cgacaaaatc tctgaaggtg gtttgattgt cgtcgttgaa    720 

gctgaaggca cggcagccgc tcctaaagcc gaagcggctg ccgccccggc gcaagaagcc    780 

cctaaagctg ccgctcctgc tccgcaagcc gcgcaattcg gcggttctgc cgatgccgag    840 

tacgacgtgg tcgtattggg tggcggtccc ggcggttact ccgctgcatt tgccgctgcc    900 

gatgaaggct tgaaagtcgc catcgtcgaa cgttacaaaa ctttgggcgg cgtttgcctg    960 

aacgtcggct gtatcccttc caaagccttg ttgcacaatg ccgccgttat cgacgaagtg   1020 

cgccacttgg ctgccaacgg tatcaaatac cccgagccgg aactcgacat cgatatgctt   1080 

cgcgcctaca aagacggcgt agtttcccgc ctcacgggcg gtttggcagg tatggcgaaa   1140 

agccgtaaag tggacgttat ccaaggcgac gggcaattct tagatccgca ccacttggaa   1200 

gtgtcgctga ctgccggcga cgcgtacgaa caggcagccc ctaccggcga gaaaaaaatc   1260 

gttgccttca aaaactgtat cattgcagca ggcagccgcg taaccaaact gcctttcatt   1320 

cctgaagatc cgcgcatcat cgattccagc ggcgcattgg ctctgaaaga agtaccgggc   1380 

aaactgctga ttatcggcgg cggcattatc ggcctcgaga tgggtacggt ttacagcacg   1440 

ctgggttcgc gtttggatgt ggttgaaatg atggacggcc tgatgcaagg cgcagaccgc   1500 

gatttggtaa aagtatggca aaaacaaaac gaataccgtt ttgacaacat tatggtcaac   1560 

accaaaaccg ttgcagttga gccgaaagaa gacggcgttt acgttacctt tgaaggcgcg   1620 

aacgcgccta aagagccgca acgctacgat gccgtattgg ttgccgccgg ccgcgcgccc   1680 

aacggcaaac tcatcagcgc ggaaaaagca ggcgttgccg taaccgatcg cggcttcatc   1740 

gaagtggaca aacaaatgcg taccaatgtg ccgcacatct acgccatcgg cgacatcgtc   1800 

ggtcagccga tgttggcgca caaagccgtt cacgaaggcc acgttgccgc cgaaaactgc   1860 

gccggccaca aagcctactt cgacgcacgc gtgattccgg gcgttgccta cacttccccc   1920 

gaagtggcgt gggtgggcga aaccgaactg tccgccaaag cctccggccg caaaatcacc   1980 

aaagccaact tcccgtgggc ggcttccggc cgtgcgattg ccaacggttg cgacaagccg   2040 

tttaccaagc tgatttttga tgccgaaacc ggccgcatca tcggcggcgg cattgtcggt   2100 

ccgaacggtg gcgatatgat cggcgaagtc tgccttgcca tcgaaatggg ctgcgacgcg   2160 

gcagacatcg gcaaaaccat ccacccgcac ccgggcgaat ccatcggtat ggcggcggaa   2220 

gtggcattgg gtacttgtac cgacaaaaaa aaa                                2253 

 
           
             43  
             747  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(747)  
               Aminoacidic sequence of the PAZ2  
             
           
            43 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Leu Asp Arg Leu Lys Met Asp Lys Leu Lys Leu Lys Gly 
     50                  55                  60 

Met Ser Tyr Ala Met Cys Leu Asn Thr Phe Val Leu Lys Lys Glu Val 
 65                  70                  75                  80 

Ser Glu Thr His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu 
                 85                  90                  95 

Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys 
            100                 105                 110 

Ala His Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys 
        115                 120                 125 

Glu Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn 
    130                 135                 140 

Ile Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Arg 
145                 150                 155                 160 

Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala 
                165                 170                 175 

Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val 
            180                 185                 190 

Gly Gly Val Asn Ser Met Asn Ser Met Asp Val Pro Ala Glu Val Ala 
        195                 200                 205 

Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu 
    210                 215                 220 

Gly Gly Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro 
225                 230                 235                 240 

Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala 
                245                 250                 255 

Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu 
            260                 265                 270 

Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala 
        275                 280                 285 

Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr 
    290                 295                 300 

Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys 
305                 310                 315                 320 

Ala Leu Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala 
                325                 330                 335 

Ala Asn Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu 
            340                 345                 350 

Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala 
        355                 360                 365 

Gly Met Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln 
    370                 375                 380 

Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala 
385                 390                 395                 400 

Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys 
                405                 410                 415 

Asn Cys Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile 
            420                 425                 430 

Pro Glu Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys 
        435                 440                 445 

Glu Val Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu 
    450                 455                 460 

Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val 
465                 470                 475                 480 

Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys 
                485                 490                 495 

Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn 
            500                 505                 510 

Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr 
        515                 520                 525 

Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val 
    530                 535                 540 

Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu 
545                 550                 555                 560 

Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys 
                565                 570                 575 

Gln Met Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val 
            580                 585                 590 

Gly Gln Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala 
        595                 600                 605 

Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile 
    610                 615                 620 

Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr 
625                 630                 635                 640 

Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe 
                645                 650                 655 

Pro Trp Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro 
            660                 665                 670 

Phe Thr Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly 
        675                 680                 685 

Gly Ile Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu 
    690                 695                 700 

Ala Ile Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His 
705                 710                 715                 720 

Pro His Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly 
                725                 730                 735 

Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys 
            740                 745 

 
           
             44  
             2256  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(2256)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pAZ3  
             
           
            44 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagaaat ggacgtacct    180 

gctgaagttg caggcgtagt caaagaagtt aaagttaaag tcggcgacaa aatctctgaa    240 

ggtggtttga ttgtcgtcgt tgaagctgaa ggcacggcag ccgctcctaa agccgaagcg    300 

gctgccgccc cggcgcaaga agcccctaaa gctgccgctc ctgctccgca agccgcgcaa    360 

ttcggcggtt ctgccgatgc cgagtacgac gtggtcgtat tgggtggcgg tcccggcggt    420 

tactccgctg catttgccgc tgccgatgaa ggcttgaaag tcgccatcgt cgaacgttac    480 

aaaactttgg gcggcgtttg cctgaacgtc ggctgtatcc cttccaaagc cttgttgcac    540 

aatgccgccg ttatcgacga agtgcgccac ttggctgcca acggtatcaa ataccccgag    600 

ccggaactcg acatcgatat gcttcgcgcc tacaaagacg gcgtagtttc ccgcctcacg    660 

ggcggtttgg caggtatggc gaaaagccgt aaagtggacg ttatccaagg cgacgggcaa    720 

ttcttagatc cgcaccactt ggaagtgtcg ctgactgccg gcgacgcgta cgaacaggca    780 

gcccctaccg gcgagaaaaa aatcgttgcc ttcaaaaact gtatcattgc agcaggcagc    840 

cgcgtaacca aactgccttt cattcctgaa gatccgcgca tcatcgattc cagcggcgca    900 

ttggctctga aagaagtacc gggcaaactg ctgattatcg gcggcggcat tatcggcctc    960 

gagatgggta cggtttacag cacgctgggt tcgcgtttgg atgtggttga aatgatggac   1020 

ggcctgatgc aaggcgcaga ccgcgatttg gtaaaagtat ggcaaaaaca aaacgaatac   1080 

cgttttgaca acattatggt caacaccaaa accgttgcag ttgagccgaa agaagacggc   1140 

gtttacgtta cctttgaagg cgcgaacgcg cctaaagagc cgcaacgcta cgatgccgta   1200 

ttggttgccg ccggccgcgc gcccaacggc aaactcatca gcgcggaaaa agcaggcgtt   1260 

gccgtaaccg atcgcggctt catcgaagtg gacaaacaaa tgcgtaccaa tgtgccgcac   1320 

atctacgcca tcggcgacat cgtcggtcag ccgatgttgg cgcacaaagc cgttcacgaa   1380 

ggccacgttg ccgccgaaaa ctgcgccggc cacaaagcct acttcgacgc acgcgtgatt   1440 

ccgggcgttg cctacacttc ccccgaagtg gcgtgggtgg gcgaaaccga actgtccgcc   1500 

aaagcctccg gccgcaaaat caccaaagcc aacttcccgt gggcggcttc cggccgtgcg   1560 

attgccaacg gttgcgacaa gccgtttacc aagctgattt ttgatgccga aaccggccgc   1620 

atcatcggcg gcggcattgt cggtccgaac ggtggcgata tgatcggcga agtctgcctt   1680 

gccatcgaaa tgggctgcga cgcggcagac atcggcaaaa ccatccaccc gcacccgacc   1740 

ttgggcgaat ccatcggtat ggcggcggaa gtggcattgg gtacttgtac cgacctgcct   1800 

ccgcaaaaga aaaaaggatc cagactcaag atggacaaat tgaaactcaa ggggatgagc   1860 

tatgcaatgt gcttgaatac ctttgtgttg aagaaagaag tctccgaaac gcagcatggg   1920 

acaatactca ttaaggttga gtacaaaggg gaagatgcac cctgcaagat tcctttctcc   1980 

acggaggatg gacaagggaa agctcacaat ggcagactga tcacagccaa tccagtggtg   2040 

accaagaagg aggagcctgt caacattgag gctgaacctc cttttgggga aagtaatata   2100 

gtaattggaa ttggagacaa agccctgaaa atcaactggt acaggaaggg aagctcgatt   2160 

gggaagatgt tcgaggccac tgccagaggt gcaaggcgca tggccatctt gggagacaca   2220 

gcctgggact ttggttcagt gggtggttaa ggatcc                             2256 

 
           
             45  
             744  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(744)  
               Aminoacidic sequence of the PAZ3  
             
           
            45 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Met Asn Ser Met Asp Val Pro Ala Glu Val Ala Gly Val 
     50                  55                  60 

Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 
 65                  70                  75                  80 

Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 
                 85                  90                  95 

Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 
            100                 105                 110 

Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 
        115                 120                 125 

Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala 
    130                 135                 140 

Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr 
145                 150                 155                 160 

Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu 
                165                 170                 175 

Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn 
            180                 185                 190 

Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala 
        195                 200                 205 

Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met 
    210                 215                 220 

Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu 
225                 230                 235                 240 

Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu 
                245                 250                 255 

Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys 
            260                 265                 270 

Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu 
        275                 280                 285 

Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val 
    290                 295                 300 

Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 
305                 310                 315                 320 

Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 
                325                 330                 335 

Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 
            340                 345                 350 

Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 
        355                 360                 365 

Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu 
    370                 375                 380 

Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val 
385                 390                 395                 400 

Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala 
                405                 410                 415 

Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met 
            420                 425                 430 

Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln 
        435                 440                 445 

Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu 
    450                 455                 460 

Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly 
465                 470                 475                 480 

Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu 
                485                 490                 495 

Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp 
            500                 505                 510 

Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr 
        515                 520                 525 

Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile 
    530                 535                 540 

Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 
545                 550                 555                 560 

Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 
                565                 570                 575 

Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys 
            580                 585                 590 

Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Arg Leu Lys Met Asp 
        595                 600                 605 

Lys Leu Lys Leu Lys Gly Met Ser Tyr Ala Met Cys Leu Asn Thr Phe 
    610                 615                 620 

Val Leu Lys Lys Glu Val Ser Glu Thr His Gly Thr Ile Leu Ile Lys 
625                 630                 635                 640 

Val Glu Tyr Lys Gly Glu Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr 
                645                 650                 655 

Glu Asp Gly Gln Gly Lys Ala His Asn Gly Arg Leu Ile Thr Ala Asn 
            660                 665                 670 

Pro Val Val Thr Lys Lys Glu Glu Pro Val Asn Ile Glu Ala Glu Pro 
        675                 680                 685 

Pro Phe Gly Glu Ser Asn Ile Val Ile Gly Ile Gly Asp Lys Ala Leu 
    690                 695                 700 

Lys Ile Asn Trp Tyr Arg Lys Gly Ser Ser Ile Gly Lys Met Phe Glu 
705                 710                 715                 720 

Ala Thr Ala Arg Gly Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala 
                725                 730                 735 

Trp Asp Phe Gly Ser Val Gly Gly 
            740 

 
           
             46  
             426  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(426)  
               Nucleotidic sequence coding for the aminoacids 
      286 to 426 of the DEN-4 envelope protein  
             
           
            46 

aaagtccgta tggagaaatt gagaatcaag ggaatgtcat acacgatgtg ttcaggaaag     60 

ttttcaattg acaaagagat ggcagaaaca cagcatggga caacagtggt gaaagtcaag    120 

tatgaaggtg ctggagctcc gtgtaaagtc cccatagaga taagagatgt aaacaaggaa    180 

aaagtggttg ggcgtatcat ctcatccacc cctttggctg agaataccaa cagtgtaacc    240 

aacatagaat tagaaccccc ctttggggac agctacatag tgataggtgt tggaaacagc    300 

gcattaacac tccattggtt caggaaaggg agttccattg gcaagatgtt tgagtccaca    360 

tacagaggtg caaaacgaat ggccattcta ggtgaaacag cttgggattt tggttccgtt    420 

ggtgga                                                               426 

 
           
             47  
             615  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(615)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pID1  
             
           
            47 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagattt ggatctagac    180 

aaagtgcgta tggagaaatt gagaatcaag ggaatgtcat acacgatgtg ttcaggaaag    240 

ttttcaattg acaaagagat ggcagaaaca cagcatggga caacagtggt gaaagtcaag    300 

tatgaaggtg ctggagctcc gtgtaaagtc cccatagaga taagagatgt aaacaaggaa    360 

aaagtggttg ggcgtatcat ctcatccacc cctttggctg agaataccaa cagtgtaacc    420 

aacatagaat tagaaccccc ctttggggac agctacatag tgataggtgt tggaaacagc    480 

gcattaacac tccattggtt caggaaaggg agttccattg gcaagatgtt tgagtccaca    540 

tacagaggtg caaaacgaat ggccattcta ggtgaaacag cttgggattt tggttccgtt    600 

ggtggataag gatcc                                                     615 

 
           
             48  
             192  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(192)  
               Aminoacidic sequence of the PID1  
             
           
            48 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly Met Ser 
     50                  55                  60 

Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met Ala Glu 
 65                  70                  75                  80 

Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly Ala Gly 
                 85                  90                  95 

Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys Glu Lys 
            100                 105                 110 

Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn Thr Asn 
        115                 120                 125 

Ser Val Thr Asn Ile Glu Leu Glu Arg Pro Leu Asp Ser Tyr Ile Val 
    130                 135                 140 

Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg Lys Gly 
145                 150                 155                 160 

Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly Ala Lys Arg 
                165                 170                 175 

Met Ala Ile Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser Val Gly Gly 
            180                 185                 190 

 
           
             49  
             2241  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(2241)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pID2  
             
           
            49 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg aacagaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagacaa agtccgtatg    180 

gagaaattga gaatcaaggg aatgtcatac acgatgtgtt caggaaagtt ttcaattgac    240 

aaagagatgg cagaaacaca gcatgggaca acagtggtga aagtcaagta tgaaggtgct    300 

ggagctccgt gtaaagtccc catagagata agagatgtaa acaaggaaaa agtggttggg    360 

cgtatcatct catccacccc tttggctgag aataccaaca gtgtaaccaa catagaatta    420 

gaacccccct ttggggacag ctacatagtg ataggtgttg gaaacagcgc attaacactc    480 

cattggttca ggaaagggag ttccattggc aagatgtttg agtccacata cagaggtgca    540 

aaacgaatgg ccattctagg tgaaacagct tgggattttg gtagcgttgg tggactgaat    600 

tcgatgaatt cgatggacgt acctgctgaa gttgcaggcg tagtcaaaga agttaaagtt    660 

aaagtcggcg acaaaatctc tgaaggtggt ttgattgtcg tcgttgaagc tgaaggcacg    720 

gcagccgctc ctaaagccga agcggctgcc gccccggcgc aagaagcccc taaagctgcc    780 

gctcctgctc cgcaagccgc gcaattcggc ggttctgccg atgccgagta cgacgtggtc    840 

gtattgggtg gcggtcccgg cggttactcc gctgcatttg ccgctgccga tgaaggcttg    900 

aaagtcgcca tcgtcgaacg ttacaaaact ttgggcggcg tttgcctgaa cgtcggctgt    960 

atcccttcca aagccttgtt gcacaatgcc gccgttatcg acgaagtgcg ccacttggct   1020 

gccaacggta tcaaataccc cgagccggaa ctcgacatcg atatgcttcg cgcctacaaa   1080 

gacggcgtag tttcccgcct cacgggcggt ttggcaggta tggcgaaaag ccgtaaagtg   1140 

gacgttatcc aaggcgacgg gcaattctta gatccgcacc acttggaagt gtcgctgact   1200 

gccggcgacg cgtacgaaca ggcagcccct accggcgaga aaaaaatcgt tgccttcaaa   1260 

aactgtatca ttgcagcagg cagccgcgta accaaactgc ctttcattcc tgaagatccg   1320 

cgcatcatcg attccagcgg cgcattggct ctgaaagaag taccgggcaa actgctgatt   1380 

atcggcggcg gcattatcgg cctcgagatg ggtacggttt acagcacgct gggttcgcgt   1440 

ttggatgtgg ttgaaatgat ggacggcctg atgcaaggcg cagaccgcga tttggtaaaa   1500 

gtatggcaaa aacaaaacga ataccgtttt gacaacatta tggtcaacac caaaaccgtt   1560 

gcagttgagc cgaaagaaga cggcgtttac gttacctttg aaggcgcgaa cgcgcctaaa   1620 

gagccgcaac gctacgatgc cgtattggtt gccgccggcc gcgcgcccaa cggcaaactc   1680 

atcagcgcgg aaaaagcagg cgttgccgta accgatcgcg gcttcatcga agtggacaaa   1740 

caaatgcgta ccaatgtgcc gcacatctac gccatcggcg acatcgtcgg tcagccgatg   1800 

ttggcgcaca aagccgttca cgaaggccac gttgccgccg aaaactgcgc cggccacaaa   1860 

gcctacttcg acgcacgcgt gattccgggc gttgcctaca cttcccccga agtggcgtgg   1920 

gtgggcgaaa ccgaactgtc cgccaaagcc tccggccgca aaatcaccaa agccaacttc   1980 

ccgtgggcgg cttccggccg tgcgattgcc aacggttgcg acaagccgtt taccaagctg   2040 

atttttgatg ccgaaaccgg ccgcatcatc ggcggcggca ttgtcggtcc gaacggtggc   2100 

gatatgatcg gcgaagtctg ccttgccatc gaaatgggct gcgacgcggc agacatcggc   2160 

aaaaccatcc acccgcaccc gggcgaatcc atcggtatgg cggcggaagt ggcattgggt   2220 

acttgtaccg acaaaaaaaa a                                             2241 

 
           
             50  
             747  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(747)  
               Aminoacidic sequence of the PID2  
             
           
            50 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Leu Asp Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly 
     50                  55                  60 

Met Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met 
 65                  70                  75                  80 

Ala Glu Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly 
                 85                  90                  95 

Ala Gly Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys 
            100                 105                 110 

Glu Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn 
        115                 120                 125 

Thr Asn Ser Val Thr Asn Ile Glu Leu Glu Arg Pro Leu Asp Ser Tyr 
    130                 135                 140 

Ile Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg 
145                 150                 155                 160 

Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly Ala 
                165                 170                 175 

Lys Arg Met Ala Ile Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser Val 
            180                 185                 190 

Gly Gly Leu Asn Ser Met Asn Ser Met Asp Val Pro Ala Glu Val Ala 
        195                 200                 205 

Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu 
    210                 215                 220 

Gly Gly Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro 
225                 230                 235                 240 

Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala 
                245                 250                 255 

Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu 
            260                 265                 270 

Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala 
        275                 280                 285 

Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr 
    290                 295                 300 

Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys 
305                 310                 315                 320 

Ala Leu Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala 
                325                 330                 335 

Ala Asn Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu 
            340                 345                 350 

Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala 
        355                 360                 365 

Gly Met Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln 
    370                 375                 380 

Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala 
385                 390                 395                 400 

Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys 
                405                 410                 415 

Asn Cys Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile 
            420                 425                 430 

Pro Glu Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys 
        435                 440                 445 

Glu Val Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu 
    450                 455                 460 

Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val 
465                 470                 475                 480 

Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys 
                485                 490                 495 

Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn 
            500                 505                 510 

Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr 
        515                 520                 525 

Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val 
    530                 535                 540 

Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu 
545                 550                 555                 560 

Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys 
                565                 570                 575 

Gln Met Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val 
            580                 585                 590 

Gly Gln Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala 
        595                 600                 605 

Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile 
    610                 615                 620 

Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr 
625                 630                 635                 640 

Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe 
                645                 650                 655 

Pro Trp Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro 
            660                 665                 670 

Phe Thr Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly 
        675                 680                 685 

Gly Ile Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu 
    690                 695                 700 

Ala Ile Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His 
705                 710                 715                 720 

Pro His Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly 
                725                 730                 735 

Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys 
            740                 745 

 
           
             51  
             2256  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(2256)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pID3  
             
           
            51 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagaaat ggacgtacct    180 

gctgaagttg caggcgtagt caaagaagtt aaagttaaag tcggcgacaa aatctctgaa    240 

ggtggtttga ttgtcgtcgt tgaagctgaa ggcacggcag ccgctcctaa agccgaagcg    300 

gctgccgccc cggcgcaaga agcccctaaa gctgccgctc ctgctccgca agccgcgcaa    360 

ttcggcggtt ctgccgatgc cgagtacgac gtggtcgtat tgggtggcgg tcccggcggt    420 

tactccgctg catttgccgc tgccgatgaa ggcttgaaag tcgccatcgt cgaacgttac    480 

aaaactttgg gcggcgtttg cctgaacgtc ggctgtatcc cttccaaagc cttgttgcac    540 

aatgccgccg ttatcgacga agtgcgccac ttggctgcca acggtatcaa ataccccgag    600 

ccggaactcg acatcgatat gcttcgcgcc tacaaagacg gcgtagtttc ccgcctcacg    660 

ggcggtttgg caggtatggc gaaaagccgt aaagtggacg ttatccaagg cgacgggcaa    720 

ttcttagatc cgcaccactt ggaagtgtcg ctgactgccg gcgacgcgta cgaacaggca    780 

gcccctaccg gcgagaaaaa aatcgttgcc ttcaaaaact gtatcattgc agcaggcagc    840 

cgcgtaacca aactgccttt cattcctgaa gatccgcgca tcatcgattc cagcggcgca    900 

ttggctctga aagaagtacc gggcaaactg ctgattatcg gcggcggcat tatcggcctc    960 

gagatgggta cggtttacag cacgctgggt tcgcgtttgg atgtggttga aatgatggac   1020 

ggcctgatgc aaggcgcaga ccgcgatttg gtaaaagtat ggcaaaaaca aaacgaatac   1080 

cgttttgaca acattatggt caacaccaaa accgttgcag ttgagccgaa agaagacggc   1140 

gtttacgtta cctttgaagg cgcgaacgcg cctaaagagc cgcaacgcta cgatgccgta   1200 

ttggttgccg ccggccgcgc gcccaacggc aaactcatca gcgcggaaaa agcaggcgtt   1260 

gccgtaaccg atcgcggctt catcgaagtg gacaaacaaa tgcgtaccaa tgtgccgcac   1320 

atctacgcca tcggcgacat cgtcggtcag ccgatgttgg cgcacaaagc cgttcacgaa   1380 

ggccacgttg ccgccgaaaa ctgcgccggc cacaaagcct acttcgacgc acgcgtgatt   1440 

ccgggcgttg cctacacttc ccccgaagtg gcgtgggtgg gcgaaaccga actgtccgcc   1500 

aaagcctccg gccgcaaaat caccaaagcc aacttcccgt gggcggcttc cggccgtgcg   1560 

attgccaacg gttgcgacaa gccgtttacc aagctgattt ttgatgccga aaccggccgc   1620 

atcatcggcg gcggcattgt cggtccgaac ggtggcgata tgatcggcga agtctgcctt   1680 

gccatcgaaa tgggctgcga cgcggcagac atcggcaaaa ccatccaccc gcacccgacc   1740 

ttgggcgaat ccatcggtat ggcggcggaa gtggcattgg gtacttgtac cgacctgcct   1800 

ccgcaaaaga aaaaaggatc caaagtgcgt atggagaaat tgagaatcaa gggaatgtca   1860 

tacacgatgt gttcaggaaa gttttcaatt gacaaagaga tggcagaaac acagcatggg   1920 

acaacagtgg tgaaagtcaa gtatgaaggt gctggagctc cgtgtaaagt ccccatagag   1980 

ataagagatg taaacaagga aaaagtggtt gggcgtatca tctcatccac ccctttggct   2040 

gagaatacca acagtgtaac caacatagaa ttagaacccc cctttgggga cagctacata   2100 

gtgataggtg ttggaaacag cgcattaaca ctccattggt tcaggaaagg gagttccatt   2160 

ggcaagatgt ttgagtccac atacagaggt gcaaaacgaa tggccattct aggtgaaaca   2220 

gcttgggatt ttggttcggt tggtggctaa ggatcc                             2256 

 
           
             52  
             744  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(744)  
               Aminoacidic sequence of the PID3  
             
           
            52 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Met Asn Ser Met Asp Val Pro Ala Glu Val Ala Gly Val 
     50                  55                  60 

Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu Gly Gly 
 65                  70                  75                  80 

Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro Lys Ala 
                 85                  90                  95 

Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala Ala Pro 
            100                 105                 110 

Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu Tyr Asp 
        115                 120                 125 

Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala Phe Ala 
    130                 135                 140 

Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr Lys Thr 
145                 150                 155                 160 

Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu 
                165                 170                 175 

Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala Ala Asn 
            180                 185                 190 

Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu Arg Ala 
        195                 200                 205 

Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala Gly Met 
    210                 215                 220 

Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln Phe Leu 
225                 230                 235                 240 

Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala Tyr Glu 
                245                 250                 255 

Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys Asn Cys 
            260                 265                 270 

Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile Pro Glu 
        275                 280                 285 

Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys Glu Val 
    290                 295                 300 

Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met 
305                 310                 315                 320 

Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val Glu Met 
                325                 330                 335 

Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys Val Trp 
            340                 345                 350 

Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn Thr Lys 
        355                 360                 365 

Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr Phe Glu 
    370                 375                 380 

Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val Leu Val 
385                 390                 395                 400 

Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu Lys Ala 
                405                 410                 415 

Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys Gln Met 
            420                 425                 430 

Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val Gly Gln 
        435                 440                 445 

Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala Ala Glu 
    450                 455                 460 

Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile Pro Gly 
465                 470                 475                 480 

Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr Glu Leu 
                485                 490                 495 

Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe Pro Trp 
            500                 505                 510 

Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro Phe Thr 
        515                 520                 525 

Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly Gly Ile 
    530                 535                 540 

Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu Ala Ile 
545                 550                 555                 560 

Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His Pro His 
                565                 570                 575 

Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly Thr Cys 
            580                 585                 590 

Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Lys Val Arg Met Glu 
        595                 600                 605 

Lys Leu Arg Ile Lys Gly Met Ser Tyr Thr Met Cys Ser Gly Lys Phe 
    610                 615                 620 

Ser Ile Asp Lys Glu Met Ala Glu Thr Gln His Gly Thr Thr Val Val 
625                 630                 635                 640 

Lys Val Lys Tyr Glu Gly Ala Gly Ala Pro Cys Lys Val Pro Ile Glu 
                645                 650                 655 

Ile Arg Asp Val Asn Lys Glu Lys Val Val Gly Arg Ile Ile Ser Ser 
            660                 665                 670 

Thr Pro Leu Ala Glu Asn Thr Asn Ser Val Thr Asn Ile Glu Leu Glu 
        675                 680                 685 

Arg Pro Leu Asp Ser Tyr Ile Val Ile Gly Val Gly Asn Ser Ala Leu 
    690                 695                 700 

Thr Leu His Trp Phe Arg Lys Gly Ser Ser Ile Gly Lys Met Phe Glu 
705                 710                 715                 720 

Ser Thr Tyr Arg Gly Ala Lys Arg Met Ala Ile Leu Gly Glu Thr Ala 
                725                 730                 735 

Trp Asp Phe Gly Ser Val Gly Gly 
            740 

 
           
             53  
             2694  
             DNA  
             Escherichia coli  
             
               gene  
               (1)..(2694)  
               Nucleotidic sequence coding for the quimeric 
      protein in the plasmid pD4D2  
             
           
            53 

atgggccacc accaccacca ccacgccatg gtagataaaa gaatggcttt agttgaattg     60 

aaagtgcccg acattggcgg acacgaaaat gtagatatta tcgcggttga agtaaacgtg    120 

ggcgacacta ttgctgtgga cgataccctg attactttgg atctagacaa agtccgtatg    180 

gagaaattga gaatcaaggg aatgtcatac acgatgtgtt caggaaagtt ttcaattgac    240 

aaagagatgg cagaaacaca gcatgggaca acagtggtga aagtcaagta tgaaggtgct    300 

ggagctccgt gtaaagtccc catagagata agagatgtaa acaaggaaaa agtggttggg    360 

cgtatcatct catccacccc tttggctgag aataccaaca gtgtaaccaa catagaatta    420 

gaacccccct ttggggacag ctacatagtg ataggtgttg gaaacagcgc attaacactc    480 

cattggttca ggaaagggag ttccattggc aagatgtttg agtccacata cagaggtgca    540 

aaacgaatgg ccattctagg tgaaacagct tgggattttg gttccgttgg tggtcttcta    600 

gaaatggacg tacctgctga agttgcaggc gtagtcaaag aagttaaagt taaagtcggc    660 

gacaaaatct ctgaaggtgg tttgattgtc gtcgttgaag ctgaaggcac ggcagccgct    720 

cctaaagccg aagcggctgc cgccccggcg caagaagccc ctaaagctgc cgctcctgct    780 

ccgcaagccg cgcaattcgg cggttctgcc gatgccgagt acgacgtggt cgtattgggt    840 

ggcggtcccg gcggttactc cgctgcattt gccgctgccg atgaaggctt gaaagtcgcc    900 

atcgtcgaac gttacaaaac tttgggcggc gtttgcctga acgtcggctg tatcccttcc    960 

aaagccttgt tgcacaatgc cgccgttatc gacgaagtgc gccacttggc tgccaacggt   1020 

atcaaatacc ccgagccgga actcgacatc gatatgcttc gcgcctacaa agacggcgta   1080 

gtttcccgcc tcacgggcgg tttggcaggt atggcgaaaa gccgtaaagt ggacgttatc   1140 

caaggcgacg ggcaattctt agatccgcac cacttggaag tgtcgctgac tgccggcgac   1200 

gcgtacgaac aggcagcccc taccggcgag aaaaaaatcg ttgccttcaa aaactgtatc   1260 

attgcagcag gcagccgcgt aaccaaactg cctttcattc ctgaagatcc gcgcatcatc   1320 

gattccagcg gcgcattggc tctgaaagaa gtaccgggca aactgctgat tatcggcggc   1380 

ggcattatcg gcctcgagat gggtacggtt tacagcacgc tgggttcgcg tttggatgtg   1440 

gttgaaatga tggacggcct gatgcaaggc gcagaccgcg atttggtaaa agtatggcaa   1500 

aaacaaaacg aataccgttt tgacaacatt atggtcaaca ccaaaaccgt tgcagttgag   1560 

ccgaaagaag acggcgttta cgttaccttt gaaggcgcga acgcgcctaa agagccgcaa   1620 

cgctacgatg ccgtattggt tgccgccggc cgcgcgccca acggcaaact catcagcgcg   1680 

gaaaaagcag gcgttgccgt aaccgatcgc ggcttcatcg aagtggacaa acaaatgcgt   1740 

accaatgtgc cgcacatcta cgccatcggc gacatcgtcg gtcagccgat gttggcgcac   1800 

aaagccgttc acgaaggcca cgttgccgcc gaaaactgcg ccggccacaa agcctacttc   1860 

gacgcacgcg tgattccggg cgttgcctac acttcccccg aagtggcgtg ggtgggcgaa   1920 

accgaactgt ccgccaaagc ctccggccgc aaaatcacca aagccaactt cccgtgggcg   1980 

gcttccggcc gtgcgattgc caacggttgc gacaagccgt ttaccaagct gatttttgat   2040 

gccgaaaccg gccgcatcat cggcggcggc attgtcggtc cgaacggtgg cgatatgatc   2100 

ggcgaagtct gccttgccat cgaaatgggc tgcgacgcgg cagacatcgg caaaaccatc   2160 

cacccgcacc cgaccttggg cgaatccatc ggtatggcgg cggaagtggc attgggtact   2220 

tgtaccgacc tgcctccgca aaagaaaaaa ggatccgaca ggctgagaat ggacaaacta   2280 

cagctcaaag gaatgtcata ctctatgtgt acaggaaagt ttaaaattgt gaaggaaata   2340 

gcagaaacac aacatggaac aatagttatc agagtacaat atgaagggga cggctctcca   2400 

tgtaagatcc cttttgagat aatggatttg gaaaaaagac acgtcttagg tcgcctgatt   2460 

acagttaacc cgatcgtaac agaaaaagat agcccagtca acatagaagc agaacctcca   2520 

ttcggagaca gctacatcat cataggagta gagccgggac aattgaaact caactggttt   2580 

aagaaaggaa gttccatcgg ccaaatgttt gagacaacaa tgagaggagc gaagagaatg   2640 

gccattttag gtgacacagc ctgggatttt gggtctctgg gtggttaagg atcc         2694 

 
           
             54  
             891  
             PRT  
             Escherichia coli  
             
               CHAIN  
               (1)..(891)  
               Aminoacidic sequence of the PD4D2  
             
           
            54 

His His His His His His Met Val Asp Lys Arg Met Ala Leu Val Glu 
  1               5                  10                  15 

Leu Lys Val Pro Asp Ile Gly Gly His Glu Asn Val Asp Ile Ile Ala 
             20                  25                  30 

Val Glu Val Asn Val Gly Asp Thr Ile Ala Val Asp Asp Thr Leu Ile 
         35                  40                  45 

Thr Leu Asp Leu Asp Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly 
     50                  55                  60 

Met Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met 
 65                  70                  75                  80 

Ala Glu Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly 
                 85                  90                  95 

Ala Gly Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys 
            100                 105                 110 

Glu Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn 
        115                 120                 125 

Thr Asn Ser Val Thr Asn Ile Glu Leu Glu Arg Pro Leu Asp Ser Tyr 
    130                 135                 140 

Ile Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg 
145                 150                 155                 160 

Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly Ala 
                165                 170                 175 

Lys Arg Met Ala Ile Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser Val 
            180                 185                 190 

Gly Gly Leu Leu Glu Met Asn Ser Met Asp Val Pro Ala Glu Val Ala 
        195                 200                 205 

Gly Val Val Lys Glu Val Lys Val Lys Val Gly Asp Lys Ile Ser Glu 
    210                 215                 220 

Gly Gly Leu Ile Val Val Val Glu Ala Glu Gly Thr Ala Ala Ala Pro 
225                 230                 235                 240 

Lys Ala Glu Ala Ala Ala Ala Pro Ala Gln Glu Ala Pro Lys Ala Ala 
                245                 250                 255 

Ala Pro Ala Pro Gln Ala Ala Gln Phe Gly Gly Ser Ala Asp Ala Glu 
            260                 265                 270 

Tyr Asp Val Val Val Leu Gly Gly Gly Pro Gly Gly Tyr Ser Ala Ala 
        275                 280                 285 

Phe Ala Ala Ala Asp Glu Gly Leu Lys Val Ala Ile Val Glu Arg Tyr 
    290                 295                 300 

Lys Thr Leu Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro Ser Lys 
305                 310                 315                 320 

Ala Leu Leu His Asn Ala Ala Val Ile Asp Glu Val Arg His Leu Ala 
                325                 330                 335 

Ala Asn Gly Ile Lys Tyr Pro Glu Pro Glu Leu Asp Ile Asp Met Leu 
            340                 345                 350 

Arg Ala Tyr Lys Asp Gly Val Val Ser Arg Leu Thr Gly Gly Leu Ala 
        355                 360                 365 

Gly Met Ala Lys Ser Arg Lys Val Asp Val Ile Gln Gly Asp Gly Gln 
    370                 375                 380 

Phe Leu Asp Pro His His Leu Glu Val Ser Leu Thr Ala Gly Asp Ala 
385                 390                 395                 400 

Tyr Glu Gln Ala Ala Pro Thr Gly Glu Lys Lys Ile Val Ala Phe Lys 
                405                 410                 415 

Asn Cys Ile Ile Ala Ala Gly Ser Arg Val Thr Lys Leu Pro Phe Ile 
            420                 425                 430 

Pro Glu Asp Pro Arg Ile Ile Asp Ser Ser Gly Ala Leu Ala Leu Lys 
        435                 440                 445 

Glu Val Pro Gly Lys Leu Leu Ile Ile Gly Gly Gly Ile Ile Gly Leu 
    450                 455                 460 

Glu Met Gly Thr Val Tyr Ser Thr Leu Gly Ser Arg Leu Asp Val Val 
465                 470                 475                 480 

Glu Met Met Asp Gly Leu Met Gln Gly Ala Asp Arg Asp Leu Val Lys 
                485                 490                 495 

Val Trp Gln Lys Gln Asn Glu Tyr Arg Phe Asp Asn Ile Met Val Asn 
            500                 505                 510 

Thr Lys Thr Val Ala Val Glu Pro Lys Glu Asp Gly Val Tyr Val Thr 
        515                 520                 525 

Phe Glu Gly Ala Asn Ala Pro Lys Glu Pro Gln Arg Tyr Asp Ala Val 
    530                 535                 540 

Leu Val Ala Ala Gly Arg Ala Pro Asn Gly Lys Leu Ile Ser Ala Glu 
545                 550                 555                 560 

Lys Ala Gly Val Ala Val Thr Asp Arg Gly Phe Ile Glu Val Asp Lys 
                565                 570                 575 

Gln Met Arg Thr Asn Val Pro His Ile Tyr Ala Ile Gly Asp Ile Val 
            580                 585                 590 

Gly Gln Pro Met Leu Ala His Lys Ala Val His Glu Gly His Val Ala 
        595                 600                 605 

Ala Glu Asn Cys Ala Gly His Lys Ala Tyr Phe Asp Ala Arg Val Ile 
    610                 615                 620 

Pro Gly Val Ala Tyr Thr Ser Pro Glu Val Ala Trp Val Gly Glu Thr 
625                 630                 635                 640 

Glu Leu Ser Ala Lys Ala Ser Gly Arg Lys Ile Thr Lys Ala Asn Phe 
                645                 650                 655 

Pro Trp Ala Ala Ser Gly Arg Ala Ile Ala Asn Gly Cys Asp Lys Pro 
            660                 665                 670 

Phe Thr Lys Leu Ile Phe Asp Ala Glu Thr Gly Arg Ile Ile Gly Gly 
        675                 680                 685 

Gly Ile Val Gly Pro Asn Gly Gly Asp Met Ile Gly Glu Val Cys Leu 
    690                 695                 700 

Ala Ile Glu Met Gly Cys Asp Ala Ala Asp Ile Gly Lys Thr Ile His 
705                 710                 715                 720 

Pro His Pro Gly Glu Ser Ile Gly Met Ala Ala Glu Val Ala Leu Gly 
                725                 730                 735 

Thr Cys Thr Asp Leu Pro Pro Gln Lys Lys Lys Gly Ser Arg Leu Arg 
            740                 745                 750 

Met Asp Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met Cys Thr Gly 
        755                 760                 765 

Lys Phe Lys Ile Val Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile 
    770                 775                 780 

Val Ile Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro 
785                 790                 795                 800 

Phe Glu Ile Met Asp Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile 
                805                 810                 815 

Thr Val Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu 
            820                 825                 830 

Ala Glu Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro 
        835                 840                 845 

Gly Gln Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser Ile Gly Gln 
    850                 855                 860 

Met Phe Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala Ile Leu Gly 
865                 870                 875                 880 

Asp Thr Ala Trp Asp Phe Gly Ser Leu Gly Gly 
                885                 890