MXPA98002801A - Vaccines against infections caused by virus yf, infectious cdna of yf, method to produce a recombinant yf virus from the infectious cdna of yf and plasmides to gather the infectious cdna - Google Patents

Abstract

The present invention relates to a human vaccine composition against YF infections consisting essentially of a recombinant YF virus, YFiv5.2 / DD, which is regenerated from infectious YF cDNA. New pYF plasmids 5'3'lV / G1 / 2 and pYFM 5.2 / T3 / 27 are provided, which together, have the complete sequence of said YF infectious cDNA. The method for producing recombinant YF virus and the Original, Primary and Secondary Progeny Lots are other embodiments of the present invention.

Description

Vaccines against infections caused by YF virus; YF infectious cDNA, method to produce a recombinant YF virus from the YF infectious cDNA and plasmids to bind the YF infectious cDNA The present invention relates to a vaccine against infections caused by YF virus and its preparation by regeneration of YF 17D virus of the corresponding complementary DNA (cDNA), which is present in the new plasmids pYF 5'3 'IV / G1 / 2 and PYFM 5.2 / T3 / 27.

BACKGROUND OF THE INVENTION The Flavivirus genus consists of 70 pathogens closely linked to humans or serologically cross-reactive veterinarians that cause many serious diseases, which include dengue fever, Japanese encephalitis (JE), acaricide encephalitis (TBE) and yellow fever. (YF) Flaviviruses are spherical with a diameter of 40-60 nm with an icosahedral capsid, which contains a single-stranded positive RNA molecule. The YF virus is the prototype virus of the Flavivirus family with an RNA genome of 10.862 nucleotides (nt), which has a 5 'structure CAP (18 nt) and a 3 'nopolyadenylated end (51 1 nt). The complete nucleotide sequence of its RNA genome was determined by Rice, C. et al (1985). Simple RNA is also the viral message and its translation into the infected cell results in the synthesis of a polyprotein precursor of 3.41 1 amino acids, which is cut by proteolytic processing to generate 10 virus-specific polypeptides. From the 5 'end, the order of the encoded proteins is: C; pr / M; AND; NS1; NS2A; NS2B; NS3; NS4A; NS4B and NS5. The first 3 proteins constitute the structural proteins, that is, they form the virus together with the packed RNA molecule. The rest of the genome encodes nonstructural proteins (NS) numbered from 1 to 5, according to the order of their synthesis. Protein C, called capsid, has a molecular weight that varies from 12 to 14 kDa (12-14 kilodaltons); the membrane protein, M has a molecular weight of 8 kDa, and its precursor (prM) 18-22 kDa; the envelope protein, E, is 52-54 kDa, all of them encoded in the first quarter of its genome. Three of the non-structural proteins are large and have highly conserved sequences among Flaviviruses, namely, NS1 has a molecular weight ranging from 38 to 41 kDa; NS3 has 68-70 kDa and NS5, 100-103 kDa. No paper has yet been assigned to NS1 but NS3 has been shown to be bifunctional having a protease activity necessary for polyprotein processing, and the other is a nucleotide triphosphatase / helicase activity which is associated with RNA replication viral. NS5, the most conserved and largest protein, contains several sequence motifs, which are characteristic of viral RNA polymerases. The 4 small proteins, namely NS2A, NS2B, NS4A and NS4B, are poorly conserved in their amino acid sequences, but not in their pattern of multiple hydrophobic stretches. It has been shown that NS2A is required for the appropriate processing of NS1, whereas NS2B has been shown to be associated with the protease activity of NS3. Two species of yellow fever virus (YF), isolated in 1927, motivated the vaccines to be used for human immunization. One, the Asibi species, was isolated from a young African named Asibi by passage in Rhesus monkey (Macaca mulatta), and the other, the virus French Viscerotropic (FVV), from a patient in Senegal. In 1935, the Asibi species was adapted for growth in the embryonic tissue of mice. After 17 steps, the virus, called 17D, was further cultured up to step 58 in embryonic whole chicken tissue and subsequently, up to step 14, only in chicken embryonic tissue without nerves. Theiler and Smith (Theiler, M. and Smith, H. H. (1937), "The effect of prolonged cultivation in vitro on the pathogenicity of yellow fever virus", J. Exp. Med. 65: 767-786) showed that, at this stage, there was a marked reduction in viral viscero and neurotropism when inoculated intracerebrally in monkeys. This virus was further subcultured to steps 227 and 229 and the resulting viruses, without immune human serum, were used to immunize 8 human volunteers with satisfactory results, as shown by the absence of adverse reactions and seroconversion to YF in 2 weeks. These steps produced the mother species 17D at step level 180 (see Figure 1), 17DD at step 195, and 17D-204 at step 204. 17DD was further subcultured to step 241 and experienced an additional 43 steps in eggs of embryo chicken to produce the virus currently used for human vaccination in some countries (step 284). 17D-204 was further subcultured to produce the Colombia species 88 which, over the passage in embryonic chicken eggs, motivated different batches of vaccine progeny currently in use in France (I. Pasteur, in step 235) and in the United States (Connaught, in step 234). Species 17D-213 was derived from 17D-204 when the primary progeny lot (S1 1 12-69) from the Federal Republic of Germany (FRG 83-66) was used by the World Health Organization (WHO) to produce a 17D progeny free of leukemia virus characteristic of birds (S1 213/77) in step 237. In the late 1930s and early 1940s, mass vaccination was conducted in Brazil with the use of several subspecies of 17D virus (Table I). These subspecies differed in their history of steps and overlapped with respect to the time of their use for inoculation and / or vaccine production. The substitution of each one for the next one was according to the experience gained during the production of the vaccine, the quality control and the human vaccination, in which the appearance of symptoms led to the discontinuation of a given species. Each of these species 17D-204 (C-204; F-204) was plaque purified in different cell lines, the virus was finally amplified in SW13 cells and used for cDNA cloning and sequence analysis (Rice, C. M, Lenches, E., Eddy, SR; , SJ; Sheets, RL and Strauss, JH (1985), "Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution." Science. 229: 726-733; Despres, P .; Cahour, A.; Dupuy , A., Deubel, V., Bouloy, M., Digoutte, JP, Girard, M. (1987), "High genetic stability of the coding region for the structural proteins of yellos fever strain 17D." J. Gen. Virol 68: 2245-2247). 17D-213 in step 239 was tested for monkey neurovirulence (RS Marchevsky, personal communication, see Duarte dos Santos et al, 1995) and was the subject of sequence analysis along with 17DD (in step 284) and the comparison to previously published nucleotide sequences of other YF virus species (Duarte dos Santos et al, 1995) (Asibi: Hahn, CS, Dalrymple, JM, Strauss, JH and Rice, CM (1987), "Comparison of the virulent Asibi strain of yellow fever virus with the 17D vaccine strain derived from go Proc. Nati, Acad Sci USA 84: 2029-2033; 17D-204 strain C-204: Rice, CM Lenches, Em; Eddy, SR; Shin, SJ; Sheets, RL and Strauss, JH (1985), "Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution." Science. 229: 726-733; F-204: Despres, P.; Cahour , R., Dupuy, A., Deubel, V., Bouloy, M., Digoutte, JP and Girard, M. (1987), "High genetic stability of the coding region for the structural proteins of yellow fe see 17D strain virus ", J. Gen. Virol. 68: 2245-2247) (see figure 1). A total of 67 nucleotide differences, corresponding to 31 amino acid changes, were originally noted between the genomic sequences of Asibi and 17D-204 (see Hahn, C.S. et al, 1987). The comparison between the nucleotide sequences of subspecies 17DD and 17D-213 (see Duarte dos Santos et al, 1995) and the nucleotide sequence of subspecies 17D-204 (see Rice et al, 1985) showed that not all changes They are common and in this way it is not confirmed by being 17D-specific. Therefore, the specific changes of the 17D subspecies observed are very likely not related to attenuation but may reflect differences in the behavior of these species in neurovirulence tests in monkeys. Consequently, the number of changes possibly to be associated with viral attenuation was reduced by 26%, that is, 48 nucleotide changes. Of these 48 nucleotide sequence changes, which are scattered throughout the genome, 26 are silent mutations and 22 lead to amino acid substitutions. More important are the alterations noted in the E protein because it is the main objective for the humoral neutralizing response, that is, it is the protein where the hemagglutination and neutralization epitopes are located, and mediates cell receptor recognition and cell penetration, thus directing the virus to specific cells. Importantly, protein E accumulates the highest proportion of non-conservative amino acid changes to conservatives. Altogether, eleven nucleotide substitutions were observed in the E protein gene leading to 8 amino acid changes at positions 52, 170, 173, 200, 299, 305, 331 and 380 (respectively nucleotides 1 127, 1482, 1491 , 1572, 1870, 1887, 1965 and 21 12 of the 5 'end of RNA). The alterations in amino acids 52 to 200 are located in the A domain of the E protein (domain II in the 3-D structure proposed for E protein of Favivirus-Rey, FA, Heinz, FX, Mandl, C; Kunz, C. And Harrison, SC (1995), "The envelope glycoprotein from tick-borne encephalitis virus at 2Á resolution." Nature, 375: 291-298), which is conserved among Flaviviruses and contains cross-reactive epitopes as shown by Mandl. , CW et al (Mandl, M.W.; Guirakhoo, F.; Holzmann, H .; Heinz, F.X. and Kunz, C. (1989), "Antigenic structure of the flavivirus envelope E protein at the molecular level using tick-borne encephalitis virus as a moder. J. Virol. 63: 564-571.) This domain II is highly cross-linked by Disulfide ligatures and low pH transition, which is related to the exposure of a hydrophobic and strictly conserved stretch of amino acids, which are supposed to be involved in the fusion of the viral envelope to the endosome membrane. amino acids 299, 305, 331 and 380 are located in domain B (domain III in the 3-D structure - see Rey, FA et al.) This domain was suggested to be involved in viral binding to a cellular receptor and consequently to be a major determinant of both host range and cellular tropism as well as virulence / attenuation.The 4 amino acid changes reported for YF are located on the distal face of domain III.This area has a loop which is a Closed return on the encephalitis virus carried by mite but contains 4 additional residues in all species carried by mosquito. Because viruses replicate in their vectors, this loop is likely to be a determinant of host range. This elongated loop contains a sequence of Arginine-Glycine-Aspartic Acid (Arg-Gly-Asp) in the 3 vaccine species YF 17D. This sequence motif is known to mediate a number of cellular interactions that include receptor ligation and is not only absent in the paternal virulent Asibi species but also in 22 other wild type YF virus species (Lepiniec, L., Dalgarno, L. Huong, VTQ, Monath, TP; Digoutte, J.P. and Deubel, V. (1994), "Geographic distribution and evolution of yellow fever viruses based on direct sequencing of genomic DNA fragments". J. Gen. Virol. 75: 417-423). This fact suggests that the mutation of Threonine (Thr) to Arginine (Arg), creating an Arg-Gly-Asp motif, is possibly to be relevant for the attenuated phenotype of the YF 17D species. Consistently, Lobigs et al (Lobigs, M .; Usha, R .; Nesterowicz, A.; Marschall, ID; Weir, RC and Dalgarno, L. (1990), "Host cell selection of Murray valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse neurovirulence. "Virology 176: 587-595) identified a sequence motif Arg-Gly-Asp (in amino acid 390), which led to the loss of virulence of encephalitis virus from Murray Valley for mice. Alterations at amino acids 170 and 173 in domain C (domain I of protein E in structure 3-D) are located very close to the position that a neutralization epitope was identified for the acaricide-borne encephalitis virus (TBE) ) (see Mandl, CW et al). A mutation in position 171 of the TBE virus E protein was shown to affect the threshold of the conformational change that activates the fusion of this protein and the 2 changes observed for the YF 17D virus may be related to the same phenomenon. It can be conceived that a lower rate of fusion can delay the degree of virus production and thus lead to a milder infection of the host. It is worthy of attention that the recent development of infectious cDNA for Japanese encephalitis virus (JE) made by Sumiyoshi, H. et al (Sumiyoshi, H., Hoke, CH and Trent, DW (1992), "Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates. "J. Virol. 66: 5425-5431) allowed the identification of a mutation (Lys by Glu) in amino acid 136 of protein E, which resulted in the loss of neurovirulence for mice (see Sumiyoshi, H., Tignor, GH and Shope, RE (1996), "Characterization of a highly attenuated Japanese encephalitis virus generated from molecularly cloned cDNA." J. Infect. Dis. 171: 1 144-1 151) . This means that domain I is an important area, which contains a critical determinant of virulence of JE virus in contrast to most data obtained from virulence analysis for other flavivurs, for which it is suggested that the III domain would be the main site for virulence / attenuation determinants. However, such analyzes of the E protein provide a framework for understanding various aspects of biology and flavivirus and suggest that it should be possible to design viruses for the development of a new live flavivirus vaccine. The issue of virulence / attenuation is of special interest for the development of vaccines but in an imaginable way the viral attenuation can result from the genetic modification in one or more viral functions. The YF virus is the ideal system to study the virulence and attenuation of flavivirus because: (i) there is a virulent species (Asibi) from which an extremely well-characterized vaccine species was derived (17D) and has been used successfully for human vaccination for over 50 years; (ii) there is an animal system which reflects human infection; (iii) the complete nucleotide sequences for both the virulent and attenuated species have been determined and (iv) the cDNA clones from which the infectious RNA can be synthesized are available. Holland, J. et al (Holland, J .; Spindler, K .; Horodyski, H .; Grabau, e; Nichol, S. and VandePol, S. (1982), "Rapid evolution of RNA genomes." Science. 215: 1577.1585) described the fact that viral RNA genomes evolve rapidly. Therefore, a given viral population, including YF vaccine virus, is possibly consisting of a population of major type sequences in which genetic variants can be detected. For YF 17D virus, this is easily seen when the virus is plated on cultured cells under a semisolid shell in which plates of different sizes are observed. The analysis of previous genomic variability using fingerprints of oligonucleotides suggested a high degree of genetic similarity between vaccines produced around the world with an estimated sequence homology of 98-100%. However, genetic changes were detected and may have occurred within 1-2 steps possibly due to the selection of subpopulations of viruses or to signal mutations. It is unknown whether the properties of the outstanding YF 17D virus vaccines are due to the existence of genetic variants in the vaccine population. In any case, the stabilization of the YF 17D genome as DNA will not only be reduced to the accumulation of mutations in the viral genome as lots of progeny are produced to replace the previous one but they will also provide a much more homogeneous population in terms of nucleotide sequence and consequently in terms of phenotypic markers including attenuation for humans, thus providing the necessary standardization of the use of YF subspecies for the production of vaccines. The ability to manipulate the flavivirus genome through infectious cloning technology has opened up new possibilities for the development of vaccines. This is because the virus can be recovered from the complementary DNA by in vitro transcription and transfection of cells cultured with RNA, and these cDNAs corresponding to the complete viral genome allow genetic modifications to be introduced at any particular site of the viral genome. The pioneering study by Racaniello and Baltimore (Racaniello, VR and Baltimore, D. (1981), "Cloned poliovirus complementary DNA is infectious in mammalian cells." Science. 214: 916-919) showed first the viability to regenerate cloned cDNA virus. . In U.S. Patent 4,719,177, Racaniello and Baltimore described, in detail, the production of viral cDNA from RNA by reverse transcribing viral RNA and inserting the resulting cDNA molecule into a recombinant DNA vector. The process was particularly concerned with the production of complementary double-stranded DNA of poliovirus (ds cDNA). They found that the transfected complete length polio virus cDNA was infectious on its own. In addition, with the development of in vitro transcription systems (see Melton, DA; Krieg, PA; Rabagliati, MR; Maniatis, T .; Zinn, K. and Green, MR (1984), "Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. "Nucí Acids. Res. 12: 7035-7056), a much higher efficiency in the synthesis of full-length viral RNA became possible, as compared to a transcript of cDNA in the cell. Additionally, the development of improved transfection methodologies such as cationic liposomes and electroporation increased the efficiency of RNA transfection of cultured cells. The construction and cloning of a stable full-length dengue cDNA copy in a species of Escherichia coli using the vector of plasmid pBR322 was described by Lai, C.J. et al (Lai, C.J.; Zhao, B .; Hori, H. and Bray, M. (1991), "Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus". Proc. Nati Acad. Sci. USA 88: 5139-5143). They verified that the RNA molecules produced by in vitro transcription of the full-length cloned DNA template were infectious, and the progeny virus recovered from the transfected cells was not distinguishable from the parent virus from which the cDNA clone was derivative. But, as mentioned in Patent Application WO 93/06214, such an infectious DNA construct and RNA transcripts generated therefrom were pathogenic, and that attenuated dengue viruses generated thus were genetically unstable and had the potential to revert to a pathogenic form with time. To solve this problem, the Applicant proposes to construct cDNA sequences that encode RNA transcripts to direct the production of chimeric dengue viruses by incorporating mutations into recombinant DNA fragments generated therefrom. A preferred mutation removes the glycosylation of the NS1 protein.

The construction of the full-length YF 17D cDNA template that can be transcribed in vitro to produce infectious YF virus RNA was described by Rice et al (Rice, CM; Grakoui, A.; Galler, R. and Chambers, T (1989), "Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation." The New Biologist 1: 285-296). Due to the instability of full-length YF cDNA clones and their toxic effects on Escherichia coli, they developed a strategy in which full-length templates for transcription were constructed by in vitro ligation of appropriate restriction fragments. Furthermore, they found that the YF virus recovered from cDNA was indistinguishable from the parent virus by several criteria. The YF infectious cDNA is derived from subspecies 17D-204. Although the YF virus generated from the known YF infectious cDNA is preferably attenuated, it can not be used for human vaccination due to its residual neurovirulence, as determined by Marchevsky, R.S. et al (Marchevsky, RS, Mariano, J. Ferreira, VS, Almeida, E., Cerqueira, MJ, Carvalho, R., Pissurno, JW Travassos da Rosa, A. PA .; Simdes, MC; Santos, CND; Ferreira , ll Muyiaert, IR; Mann, GF; Rice, CM and Galler, R. (1995), "Phenotypic analysis of yellow fever virus derived from complementary DNA." Am. J. Trop. Med. Hyg. 52 (1): 75-80). In brief, to obtain a YF vaccine virus using recombinant DNA techniques, it is necessary, cumulatively: (1) to genetically modify the existing YF infectious cDNA; (2) ensure that the construct of infectious DNA and RNA transcripts generated from it motivate a virus that is not pathogenic, and, furthermore, that it does not have the potential to revert to a pathogenic form; (3) the YF virus generated from the cloned cDNA, in addition to being attenuated, must retain its immunological properties. Accordingly, an improved YF virus vaccine without neurovirulence and immunogenic generated from an infectious cDNA of cloned YF should be developed for human immunization.

SUMMARY OF THE INVENTION An objective of the present invention is to provide an efficient and safe YF virus vaccine obtained from a cloned cDNA having the phenotypic characteristics of the 17DD species, mainly its attenuation and immunogenicity. In one embodiment, the present invention relates to a new version of YF infectious cDNA clone that is similar to 17DD, which is the most genetically stable subspecies of the YF 17D species. In another embodiment of the present invention, new YF plasmids are provided, which have the complete sequence of the YF infectious cDNA. Another embodiment of the present invention is a recombinant YF virus, which is regenerated from an infectious YF cDNA.

In another embodiment, the present invention relates to a process for the production of a virus of a YF vaccine by transfecting host cells and recovering similar viruses to 17DD. The cDNA template of the present invention resulted from nine mutations, which have been introduced into the infectious cDNA called YFiv5.2 (see Rice et al., 1989). New plasmids, called pYF5'a'IV / G1 / 2 and pYF5.2 / T3 / 27, and a method to obtain them, are provided to achieve the mentioned mutations.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the history of the passage of the original YF Asibi species and derivation of the YF 17D vaccine species. Figure 2, the use of YF 17DD virus for human immunization during the establishment of the 17D species in Brazil. Figure 3 shows the analysis of the N-linked glycosylation of viral E protein in denaturing polyacrylamide gel. Figure 4 shows the structure of plasmid pYF5'3'IV / G1 / 2 carrying the final 5 'and 3' end sequences of the infectious cDNA of the present invention. Figure 5 shows the structure of plasmid pYFM5.2 / T3 / 27 which carries the middle region of the infectious YF cDNA genome of the present invention. Figure 6 sets forth the complete nucleotide sequence of the YF infectious cDNA of the present invention.

Figure 7 illustrates the methodology for the regeneration of the YF virus from the cloned cDNA of the present invention. Figure 8 shows the comparative plate size analysis between YF 17D viruses.

DETAILED DESCRIPTION OF THE INVENTION The phenotypic test of the virus recovered from the cDNA of the subspecies 17D-204 described by Rice et al., 1989, showed that the virus is suitable for localizing the virulence determinants with respect to the type phenotype. natural. However, the slightly higher clinical record observed in neurovirulence tests suggests caution in its use for human vaccination (Marchevsky et al, 1995). It is also to be considered that the subspecies YF 204, used for the preparation of the cDNA library and the infectious cDNA (Rice et al., 1985, 1989), is closely related in terms of lineage and number of steps for other subspecies YF 204 , which cause the majority of cases of postvaccination encephalitis in the vaccinated (Schoub, BD, Dommann, CJ; Johnson, S.; Downie, C. and Patel, PL (1990), "Encephalitis in a 1 S-year following boy following 17D YF vaccine1 '." J. Infection 21: 105-106; Merlo, C, Steffen, R.; Landis, T. , Tsai, T. and Karabatsos, N. (1993), "Possible association of encephalitis and a 17D YF vaccination in a 29-year old traveler." Vaccine. 1 1: 691). In contrast, no case of post-vaccine encephalitis was registered with subspecies 17DD, even in the first days of vaccination (Fox, JP, Lennette, E., Manso, C, and Souza Aguiar, J. R. (1942 ), "Encephalitis in man following vaccination with yellow fever 17D virus, Am. J. Hyg. 36: 17-142; Fox, JP, and Penna, HA (1943)," Behavior of 17D yellow fever virus in Rhesus monkeys. To substrain, dose and neural or extraneural inoculation. "A, J. Hyg. 38: 52-172.) As is evident from Table 1 and Figure 2, the subspecies 17DD was by far the most commonly used subspecies for both inoculum for vaccine production with special emphasis for the subspecies EPIow which is still used to date The complete nucleotide sequence of the genome of the subspecies 17DD EPIow has been recently derived (Duarte dos Santos et al, 1995) and its comparison with the sequences available for other similar subspecies 17D-204 (Rice et al, 1985; Despres et al. 1987) and 17D-213 (Duarte dos Santos et al. 1995) provides an estimate of the degree of genetic variability among these species. The average number of fixed amino acid or nucleotide sequence changes per virus passage is significantly lower for DD, suggesting that the 17DD species is genetically more stable than the others (Galler, R., et al, Vaccine, in press) and this it may be of importance with respect to the production of YF vaccines.

Table 1: Estimated quantities of inocula and vaccines produced with different subspecies during the period of 1937-1942 in Brazil Vaccine means that the inoculum for the subsequent vaccine production was also a batch of vaccines. The amount of inoculum in these cases is not available. + Currently in use for vaccine production in FIOCRUZ = NA not available Therefore, in the present invention, mutations have been introduced into the YF infectious clone cDNA, mainly YFiv5.2, to make it similar to DD. These mutations are located at the following nucleotide / gene / amino acid position: • 1140 / E / 36 (T (ti ina) => Val (Valine)? C (Cytosine) = Ala (Alanine)), • 1436, 1437 / E / 155 (G (Guanine), A (Adenine) = > Asp (Aspartic Acid), A (Adenine), G (Guanine) = > Ser (Serine)), • 1946 / E / 335 (T (thymine) = - > Ser (Serine) -? C (Cytosine) = > Pro (Proline)), • 2219, 220 / E / 409 (A (Adenine) .C (Cytosine) => Thr (Threonine)? G (Guanine), T (Thymine) = Val (Valine)), • 8808 / NS5 / 391 (A (Adenine) = > - Asn (Asparagine)? G (Guanine) = * Ser (Serine)), • 9605 / NS5 / 657 (G (Guanine) => Asp (Aspartic Acid)? A (Adenine) = >Asn (Asparagine)).

In addition to these mutations, one coding mutation and four silent mutations occurred fortuitously in the following nucleotide / gene 2356 / E (T (Thymine)? C (Cytosine)), 2602 / NS1 (T (Thymine) C (Cytosine)), 2677 / NS1 (C (Cytosine) - T (Thymine)), 2681 / NS1 (G (Guanine) = > - Ala (Alanine)? A (Adenine) = > - Thr (Threonine)), and 10722 (G (Guanine)? A (Adenine)). Finally, the mutation that occurred in nucleotide / gene 8656 / NS5 (A (adenine)? C (cytosine)) was necessary to create a BstEII site that allows the appropriate ligation and regeneration of the whole genome and, consequently, the recovery of the virus.

In protein E, the creation of an N-linked glycosylation site at amino acid E / 155 (nt>, which is located in domain I (as defined in Rey, FA, et al), could influence the fusogenic activity of protein E as observed for a type 2 dengue virus that had that site eliminated by mutation, this is so, because the E proteins, in the absence of the sugar portion, have a higher pH threshold and therefore both would merge to the endosomal membrane more easily and thereby allow the viral cycle to proceed.In this regard, YF virus vaccine 17D-204 consists of a mixed population of viruses with and without that glycosylation site, in contrast to the 17DD and 17D-213 viruses (see Post, PR, Santos, CND, Carvalho, R., Cruz, ACR, Rice, CM and Galler, R. (1992, "Heterogeneity in envelope protein sequence and N-linked glycosylation among Yellow Fever virus vaccine strains. "Virology 188: 160-167.) For the construction of the YF infectious DNA, a population devoid of this site was selected. It should be noted that a 17D-204 virus that caused a fatal human case of post-vaccine encephalitis also did not have an N-linked glycosylation site due to the mutation (see Jennings, AD; Gibson, CA; Miller, BR; Mathews, JH; Mitchell, CJ; Roehrig, JT; Wood, DJ; Taffs, F .; Sil, BK; Whitby, SN; Monath, TP; Minor, PD; Sanders, PG, and Barrett, ADT (1994), "Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis. "J. Infecí., Dis. 169: 512-518). Figure 3 shows an immunoprecipitation gel of viral proteins labeled with [35S] methionine from two species of Yellow Fever vaccine and infectious clones of Yellow Fever Virus. VERO cells were infected at an MOI of 1. At 48 hours post-infection, the cell monolayers were pulse labeled with [35S] methionine for 1 hour. Extracts of detergent cells were immunoprecipitated with mouse hyperimmune antiserum specific for yellow fever (obtained from ATCC). All the immunoprecipitates were collected using protein A sepharose. The samples were analyzed by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis and by fluorography and exposure at -70 ° C. The numbers on the trails correspond to: (1> species of vaccine 17DD; (2) infectious clone A5 / 2-T3; (3) infectious clone A5 / T3; (4) vaccine species 17D-213; (5) infectious clone G1 / 5.2; (6) infectious clone A5-T3 / 27; (7) infectious clone G1 / 2-T3 N / S; (8) infectious clone G1 / 2-T3 / 27. molecular weight are shown on the left and viral proteins of yellow fever on the right.The potential role of the other changes in the protein sequence E for the complete attenuation of the virus recovered from cDNA is less clear. With respect to the 2 alterations in the NS5 protein, there are no structural analyzes available to date and therefore, it is difficult to predict the effect of any particular amino acid change in its conformation / function. However, the mutation at amino acid 657 from the amino terminus of NS5 is only 8 amino acids away from the putative catalytic site of the RNA replicase, the Gly-Asp-Asp motif (Glycine-Aspartic Acid-Aspartic Acid), which is conserved through the viral RNA polymerases of the plant to the viruses of animals. It would not be surprising if this mutation has somewhat altered the activity of the enzyme leading to better kinetics of RNA replication in the infected cell and consequently higher viral production. Mutations at nt 1140 sites, 1436, 1437, 1946, 2219, 2220, 8808 and 9605 have been introduced into YFiv5.2 of YF infectious cDNA. The YF infectious cDNA in its known version exists in the form of two plasmids carrying the final 5 * and 3 'end sequences (pYF5'3'IV) and the middle region of the genome (pYFM5.2) as described by Rice. , CM, et al (Rice et al, 1989). The construction of these YF plasmids required the ligation of several cDNA fragments present in different plasmids of the cDNA library. The virus that motivated this cDNA library has been double-purified by plaque in CEF cultures and the titer amplified by consecutive steps in Vero, BHK and SW13 cells, once each (see Figure 1). The degree of genetic variability in the viral population used for RNA extraction is not known. However, the complete nucleotide sequence analysis of the final infectious cDNA plasmids provided the identification of nucleotide changes not present in any other 17D virus for which genomic sequences are available. Due to stability problems, it was impossible to include the complete YF genome in a single plasmid and therefore a two plasmid system and in vitro ligation of purified restriction fragments to regenerate the entire genome was established (see Rice, CM, et. al, 1989). Plasmid pYF5'3'IV contains the 5 'terminal sequence of YF (nt 1 -2271) adjacent to the SP6 phage polymerase promoter and the 3' terminal sequence (nt 8276-10862) adjacent to the Xhol site used for the production of running transcripts (see figure 2 in Rice, CM, et al, 1989). Plasmid pYFM5.2 contains cDNA YF 17D from nt 1372 to 8704. Plasmid pYF5'3'IV / G1 / 2 is prepared from pYF5'3'IV by creating changes, in the 5 'terminal sequence of YF, in nucleotides 1 140 and 1436/1437, and in the 3 'terminal sequence, in nucleotides 8656 and 9605 (see Table 2). The rest of the plasmid consists of pBR322 with a deletion of the AatlI to Eco0109 sites, which resulted in the destruction of both sites. This plasmid contains a unique Aatll site corresponding to nt 8406 of the YD 17D cDNA. To achieve the changes, two separate turns of cloning / mutagenesis steps were necessary to create the relevant mutations in the E and NS5 proteins. The E mutations were introduced by cloning a Xbal / Pstl fragment into pAlter ™ (Promega, Inc.) and a restriction fragment exchanging with Apal / Notl. The NS5 changes were introduced by cloning / mutagenesis of an EcoRI / Sstl fragment into pAlter and triturating it in the original plasmid using the same enzymes. The structure of plasmid pYF5'3'IV / G1 / 2 is shown in Figure 4.

Table 2: Genetic modification of plasmid YF. Plasmid Nucleotide changes YF5'3'IV G1 / 2 1140 1436/1437 8656.9605 YFM5.2 T3 / 27 1946 (Plasmid extended to 719 2219/2220 nucleotides for the T3 series with 8656 site Sali only) 8808 a. All changes were confirmed by nucleotide sequence determination in the plasmid DNA and recovered virus cDNA.

The plasmid pYFM5.2 / T3 / 27 is prepared from pYFM5.2 by introducing the changes in nucleotides 1946, 2219/2220, 8656 and 8808. To achieve the nucleotide changes, an AatlI fragment was introduced into pYFM5.2. / Sall spanning the nucleotides of YF 8406-9423 making this plasmid 719 nucleotides larger than its parent plasmid pYFM5.2. Since Salí is also present in the same nucleotide position in the YF sequences contained in the other YF plasmid (pYF5'3'IV) it became possible to use a combination of Apal or Nsil and Sali to produce the relevant restriction enzyme fragments . The intermediate plasmid pYFM5.2 / T3 was used to derive plasmid T3 / 27 which contains the changes in nucleotides 1946 (T? - C), 2219 (A? G) and 2220 (C? T) as compared to YFiv 5.2 parent (see Table 3). The structure of plasmid pYFM5.2 / T3 / 27 is shown in Figure 5.

Table 3: Comparison of sequences of clones of YF infectious plasmids. NT / gene YFiv5.2a DDb YFiv5.2 / DDc NT = > AA 1 140 / E T C C T = Val? C = > To 1436, 1437 / E G, A A, G A, G G, A = > Asp-? A, G = > Be 1946 / E T C C T = > S? C = P 2219.2220 / E A, C G, T G.T A, C = Thr? G, T = > VAL 2356 / E T T C - 2602 / NS1 T T C - 2677 / NS1 C C T - 2681 / NS1 G G A G = > Ala? A = - Thr 8656 / NS5 A A C - 8808 / NS5 A G G A = Asn? G = > Be 9605 / NS5 G A A A = > Asp? G = > Asn 10454 G A A - 10722 G G A - RICE ET AL (1989) b Duarte dos Santos et al (1995) c Ferreira, I I and Galler, R. (unpublished).

The EstEII site in nucleotide 8656 of YF was created in both YF plasmids and in its digestion with Apal or Nsil and BstEII provides the appropriate restriction enzyme fragments for ligation and regeneration of the complete genome of the virus. This characteristic constitutes another genetic marker for this new version of the YF infectious cDNA. The complete nucleotide sequence of YF infectious cDNA (YFiv5.2 / DD) is shown in Figure 6. The deposit of plasmids pYF5'3'IV / G1 / 2 and pYFM5.2 / T3 / 27 has been made in The American Type Culture Collection and are identified by Accession No. ATCC 97771 and 97772, respectively. Figure 7 shows the methodology for the YF virus regeneration from the cloned complementary DNA. Plasmids G1 / 2 and T3 / 27 shown in Figures 4 and 5 are digested with Apal and Sali to produce restriction fragments, which are purified, ligated and digested with XhoI. The resulting DNA corresponds to the full-length YF cDNA template that can be used for in vitro transcription with SP6 polymerase to produce infectious RNA transcripts on transfection of cultured vertebrate cells. The black area corresponding to the sequences of the vector contains the beta-lactamase gene and the origin of replication. The position of the SP6 promoter is shown and is adjacent to the first 5 'nucleotide of the YF genome. Nucleotides 1-1603 (below the Apal site) and 9424 (Sali) for 10862 (Xhol) come from plasmid G1 / 2 while nucleotides from 1604 (Apal) to 9423 (Sali) come from plasmid T3 / 27. In addition to pBR322, other vectors, which provide stabilization of the YF virus genome can be used to prepare the plasmids of the present invention. Specific examples include plasmids such as pBR 325, pBR 327, pBR 328, pUC 7, pUC 8, pUC 9, pUC 19, phages such as? phage, M13 phage and the like. The templates were prepared from pYF5'3'IV / G1 / 2 and pYFM5.2 / T3 / 27 using Apal / Sall and Nsil / Sall to produce the restriction fragments for in vitro ligation. After digestion with XhoI to linearize the ligated DNA, the template was used for in vitro transcription. The virus has been recovered after transfection of RNA from cultured animal cells. The regenerated virus of the plasmids pYF5 '$' IV / G1 / 2 and pYFM5.2 / T3 / 27 will be referred to hereafter as YFiv5.2 / DD. Similar to YF 17DD and 17D-213, the new virus produces large plates in Vero cells in contrast to the 17D virus recovered from the original cDNA (YFiv5.2; see figure 8). In ten consecutive steps of this virus in CEF cells, this large plaque phenotype showed to be stable. In addition, there was no alteration in their neurovirulence for mice as compared to other well-known YF 17D vaccine controls (Table 4).

Table 4: Neurovirulence and neuroinvasion of mice "of YF virus 17D Virus Intracerebral Intranasal Inoculation Route Inoculation Deaths Inoculum Time Deaths Time of (PFU / mlb) c survival (PFU / ml) c average average survival 17DDd 6.5x10S 7/8 10.43 ± 1.13 6.5x106 1/10 NAh 17D-2136 2x106 8/8 8.50 ± 1.07 2.1x107 2/10 16.5 DAYS 17D-204 / A5-T3f 4.75x10S 8/8 8.50 ± 0.93 4.75x106 0/9 NAh 17D-204 / G1 / 2-T3 / 279 1.3x106 8/8 8.50 ± 0.93 1.3x107 0/14 N.Ah a Swiss innate mice, 3 weeks of age (11g) b.pfu = plaque forming unit 00 c. 20 μl each ic and imd Current subspecies used for production of YF vaccines, the sequence on which the genetic modifications introduced in the first infectious cDNA of YF are based.A species of early experimental tissue culture vaccine, also is the subspecies WHO.F. This subspecies corresponds to the original virus derived from the first version of YF infectious cDNA. of the progeny of the genetically modified infectious cDNA clone virus, h. N.A. = not available.

The culture of animal cells used herein can be any culture as long as the 17D species of YF virus can replicate. Specific examples include, Hela (derived from human cervical carcinoma), CV-1 (derived from monkey kidney), BSC- (monkey kidney derivative), RK 13 (rabbit kidney derivative), L929 (derived from of mouse connective tissue), CE cell (chicken embryo), CEF (chicken embryo fibroblast), SW-13 (derived from human adrenocortical carcinoma), BHK-21 (baby hamster kidney), Vero (monkey kidney) African green), LLC-MK2 (derived from Rhesus monkey kidney (Macaca mulata)), etc. Accordingly, according to one of the embodiments of the present invention, a protocol was established by driving nucleic acid to production of YF vaccine virus under Good Manufacturing Practices (GMP) by transfecting certified cells for the production of human vaccines and recovering viruses similar to 17DD. This virus was then used to produce batches of primary and secondary progeny in primary cultures of chicken embryo fibroblasts under GMP. The virus resulting from the progeny batches was tested for neurovirulence in monkeys. This work should set the precedent for the production of new live attenuated flaviviruses from cloned cDNA considering that infectious clones are now available for several of them with special emphasis on Japanese encephalitis and dengue. The work done to produce the original primary and secondary progeny batches is detailed in Example 5.

The production of YF vaccine based on the progeny lot system has arisen from the need to have reliable virus with respect to human vaccination. Thus, the concept of the progeny lot system was the first advance in the development of the YF vaccine. In the period of 1937-1942, scientists who were working with the establishment of YF vaccine production used a number of viral 17D subspecies (17D Rio, 17Dlow, 17D2RÍO, 17D3R? O, 17DD, 17Ddhigh, 17Ddlow, EP, EPIow , Ephigh, NY102, NY104, NY310 / 318). As the production and vaccination campaigns continued and the complications with the vaccines were noticed, the possibility of phenotypic selection of viruses was understood through serial steps in tissue culture. When verifying early records of vaccine production in FIOCRUZ, we have noted that for all subspecies more inoculum was prepared than what is actually used for vaccine production, and therefore, the vials of the specific steps were usually available (Post, PR, and Galler, R., unpublished). This procedure of operation together with the use of multiple species at a time in a way allowed them to have an uninterrupted production. With the observation of post-vaccine complications in humans and also the failure of some viruses in the quality control tests (neurovirulence for monkeys) as described by Fox et al (1942) and Fox and Penna (1943), it became imperative reduce the variables during the production of the vaccine. One of the possibilities was to reduce the viral steps used for production. This was achieved by establishing the progeny lot system, in which the virus is maintained at levels of defined steps while the particular step is tested in quality. In the records we have noted that the primary fact that led to this development was the observation that DD subspecies high passages (called DDhigh) led to the loss of immunogenicity in vaccinated humans with poor coverage of the local population against wild type YF (Fox & Penna, 1943). This observation led the scientists back to the initial steps of the subspecies DD, more precisely to 229/230, which was then used to prepare 8 consecutive inocula (called DD1 to 8). Each of these DO subspecies were cultivated for no more than 3f_t steps. Each step was actually used for vaccine production but to different degrees. The procedure produced reliable viruses with respect to human vaccination. At this stage, subspecies NY104 was the one that was used for the majority of vaccine production and therefore this change in operation was implemented and NY104 was the first subspecies to be fully employed in the progeny lot system. It is illustrative for such a practice that only 3 batches of progeny (E688, E694 and E716) were used to produce almost 2 million doses of vaccine during a 10-month time interval. Unfortunately, some of these batches of vaccines became extremely neurotropic (Fox et al, 1942, Fox &Penna, 1943) and the use of this subspecies was discontinued. The next species in the line was EPIow, which was used in sc243 to produce inocula in eggs with an embryo instead of embryonic chicken tissue without a nervous system. There were 150 serial steps of EPIow in embryo eggs, but only 7 of these steps were used for vaccine production. The virus produced in this way performed well in tests of neurovirulence in mono and in human trials. Therefore, EPIow in the passage of chicken embryos 35 was used to prepare the EPF374 vaccine, which subsequently motivated the primary and secondary progeny batches under current use, which are only 2 steps ahead of the original progeny over a 50 year time period. In a preferred embodiment of the present invention, to achieve the spread of YF 17D vaccine virus in cells certified for the production of human vaccines, primary cultures of chicken embryo fibroblasts (CEF) were used in all production steps. There are several reasons for using CEF cells: these cells have been used successfully for the production of measles vaccines for years with extensive experience in their preparation and quality controls; A number of Standard Operating Practices (SOPs) is available. Moreover, the production of YF vaccine in CEF cultures led to 3 consecutive batches of vaccines that passed all tests including neurovirulence for monkeys. The stabilization of the YF 17D genome according to the DNA will not only reduce the accumulation of mutations in the viral genome as the lots of progeny are produced to replace the previous one, but it will also provide a much more homogeneous population in terms of nucleotide sequence and consequently in term of phenotypic markers including attenuation for humans. As mentioned above, in a preferred embodiment, all batches were prepared in primary cultures of chicken embryo fibroblasts (CEF), using eggs derived from SPF (Specific Pathogen Free) group. The cell cultures were established in a suitable medium and used subsequently to post-sowing. The viruses were recovered after incubation by centrifugation and removal of cellular debris. To the supernatant containing the viruses was added a stabilizer, which is, for those skilled in the art, known to enhance the stability of viral immunogenic compositions. All viruses were stored at -70 ° C. The following examples are illustrative of the invention and represent preferred embodiments. Those skilled in the art may know, or be able to find themselves using no more routine experimentation, to employ other appropriate materials and techniques, such as the aforementioned vectors, cultured cells and transfection methods.

EXAMPLE 1 Preparation of plasmids of DNAs: a) Derivation of plasmid pYF5'3'IV As described in Rice et al, 1989, plasmid pYF5'3'IV contains the 5 'terminal YF sequence (nt 1-2271) adjacent to the promoter SP6, and the 3 'terminal sequence (nt 8276-10862) adjacent to the Xhol site, which is used to linearize the template and thereby allow the production of running transcripts, all introduced in the pBR322 sequence. The original plasmids from which the 5 'terminal YF sequence was derived are: pYF5'ext # 20 (nt 1-536), p28m (nt 537-1964), and p10 (nt 1965-2271). P35", (nt 8276-8732), p34", (nt 9658-10223), pYF3'ext. # 17 (nt 10224-10708) and pYF3'1 # 12 (nt 10709-10862) were the original plasmids to derive the terminal 3 'YF sequence (Rice et al, 1989). Plasmid pYF5'3'IV contains a unique Aatll site corresponding to nt 8406 of the YF 17D cDNA. b) Derivation of plasmid pYF5'3'IV / G1 / 2 pYF5'3'IV / G1 / 2 was prepared from pYF5'3'IV by performing two separate rounds of cloning / mutagenesis to create the changes in the nucleotides 1 140, 1436/1437 in protein E, and in nucleotides 8656, 8808 and 9605 in protein NS5. The above genetic changes were made by cloning a Xbal / Pstl fragment into pAlter (Promega Corp.) and replacing the original sequence with the mutant by restriction fragment exchange using Apal / Notl. Changes of NS5 were introduced by cloning / mutagenesis of an EcoRI / Sstl fragment into pAlter and troquering it into the original plasmid using the same enzymes. c) Preparation of plasmid pYFM5.2 The plasmid pYFM5.2 used was also described in Rice et al, 1989. The original plasmids from which the nucleotide sequence 1372-8704 of cDNA of YF 17D was derived are: p9" (nt 1372-1603), p10", (nt 1604-3828), p3", (nt 3824-6901), p9"(nt 6902-7888) and p35," Xho "# 19 (nt 7889-8704). p35m Xho_ # 19 was constructed from p35 in which a silent change from C to T in nt 8212 was introduced to destroy the Xhol site in the YF cDNA in order to allow the use of Xhol to linearize DNA templates and consequently the production of running transcripts. d) Derivation of plasmid pYFM5.2 / T3 / 27 pYFM5.2 / T3 / 27 was prepared from pYFM5.2 by introducing an Aatll / Sal fragment, which covers nts of YF 8406-9423 and creating a BstEII site in nt 8656 of YF. A second mutation was introduced at position 8808 on the NS5 protein. Since the digestion of plasmid DNAs with Aatll is difficult because it is an expensive and very finicky enzyme, a BstEII site was created in nt 8656 of YF in both YF plasmids following a digestion with Apal or Nsil to produce the fragments of restriction enzyme appropriate for the ligation and regeneration of the complete genome, allowing the recovery of the virus. This can also be achieved when the restriction fragments are produced by digestion with Apal or Nsil and Sali. The structure of each plasmid is shown in Figures 3 and 4 and the complete sequence of the YF coding sequences for each plasmid is given in Figure 5. A comparison between the sequences of the original YF infectious plasmid clone, the species YF 17DD and the infectious cDNA clone of YF, YFiv5.2 / DD, are shown in Table 3. e) Preparation of large amounts of plasmid DNA To prepare plasmid DNAs from bacteria, glycerol supports of E. coli harboring each of the two YF plasmids should be available. The 50% glycerol medium - Luria Balance is used in the preparation of the supports, which are stored at -70 ° C.

Frozen aliquots of the pDNA are also available. Bacteria are grown in 5 ml of LB containing ampicillin (15 μg / ml) overnight at 37 ° C. This is used to inoculate large 1: 100 volumes of LB (usually 100-200 ml). At OD6oo of 0.8, chloramphenicol is added at 250 μg / ml for amplification of the plasmid DNA overnight. The plasmid is extracted using the alkaline lysis method. The final DNA precipitate is resuspended in TE (Tris-EDTA buffer) and cesium chloride is added until a refractive index of 1.3890 is reached. Plasmid DNA is pooled by ultracentrifugation for 24 hours. Clustered DNA is recovered by drilling the tube, extracting with butanol and extensive dialysis.

EXAMPLE 2 Preparation of DNA template: The template to be used for the YF 17D virus regeneration is prepared by digesting the plasmid DNA with Nsil and Salí (Promega Inc.) in the same buffer conditions, as recommended by the manufacturer. Ten μg of each plasmid are digested with both enzymes (the amount required is calculated in terms of the number of "pmol hits" present in each pDNA in order to achieve complete digestion in 2 hours). Digestion is verified by removing an aliquot (200 ng) and running it in 0.8% agarose / TAE gels. When the digestion is complete, the restriction enzymes are inactivated by heating. The DNA fragments are ligated to a concentration of 20 μg / ml for each fragment and T4 DNA ligase for 5 U / ml. The ligation is allowed to proceed overnight at 15 ° C. The ligation mixture is heated at 65 ° C for 20 minutes to inactivate the T4DNA ligase and an aliquot is taken (200 ng). Additional digestion of the DNA resulting from the ligation is carried out through the use of Xhol, and is carried out with buffer conditions adjusted according to the manufacturer's specifications (Promega) in order to linearize the template. The resulting product was subsequently extracted with phenol-chloroform and precipitated with ethanol. The precipitate is washed with 80% ethanol and resuspended in sterile RNase-free Tris-EDTA buffer. A template aliquot is taken for agarose gel analysis along with commercial markers for band size and quantification. The template is stored at -20 ° C until its use for in vitro transcription.

EXAMPLE 3 Transcription of RNA from the cDNA template of the present invention: RNA transcripts were prepared by using the DNA template of the present invention, in a manner similar to that described in Konarska et al., 1984 and in Rice. et al, 1987 (Konarska, MM; Padgett, RA; Sharp, PA (1984) "Recognition of cap structure in in vitro splicing of mRNA precursors." Cell 38: 731-736; Rice, CM; Levis R., Strauss, JH; Huang, HV (1987) "Production of infectious RNA transcripts from Sindbis virus cDNA clones: Mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to genérate defined mutants. J Virol 61: 3809-3819). The reaction was allowed to proceed at 39 ° C for 1 hour.The mixture was extracted with phenol-chloroform twice and the nucleic acid was recovered by ethanol precipitation.The precipitate was resuspended in sterile RNase-free water and he took an aliquot to determine No specific infectivity.

EXAMPLE 4 RNA transfection: CEF transfection is performed using LipofectAMI NE® (Life Technologies catalog # 18324-012. It is a 3: 1 (w / w) liposome formulation of 2,3-dioleyloxy-N- [2 (sperminecaboxyamido) ethyl] -NN trifluoroacetate, di methyl-2,3-bis (9-octadecenyloxy) - 1-polypathionic propanaminium lipid) and neutral dioleoylphosphatidyl ethanolamine (DOPE) in membrane filtered water) at a concentration of 20 μg / ml in RNase-free PBS. PBS - saline solution buffered with phosphate - is prepared and sterilized by autoclaving at 121 ° C for 20 minutes. The LipofectAMI NE is pipetted into a 5 ml tube of polystyrene containing PBS. The primary CEF cells are seeded and used 24 hours after sowing. The transcription mixture is added to the cell culture monolayer in the form of drops. The cells are incubated for 20 minutes at room temperature. Subsequently, the mixture is removed by aspiration, washed with PBS and incubated for 72 hours in medium 199. The supernatant of the culture constitutes the viral support after the addition of the stabilizer. Viral support is tested for sterility, toxicity, potency and for the presence of foreign agents. Viral support is the original progeny lot. The specific infectivity of the transcripts is deduced from experiments in which serial dilutions of the RNA are transfected in Vero cells, and these are superimposed with a semi-solid medium. Staining with violet crystal for the count of plates and knowing the amount of RNA, as determined by the measurement of OD (optical density) at 260 nm, will allow the determination of specific infectivity of the transcripts (PFU / μg of total RNA) . This determination is important to establish the multiplicity of infection cases that lead to the original support. It assures an acceptable number of cases equivalent to infection with live virus in order to reduce the probability of accumulation of mutations in the viral genome given the high number of replication cycles due to low RNA entry. There are currently two methods for efficient transfection of cells cultured with RNA. One is mediated by lipids and the other is electroporation. In the lipid mediated method, LipofectAMINE or Lipofectin are normally used. A series of experiments using viral RNA extracted from Vero cells infected with YFiv5.2 / DD allowed comparison of the efficiency of lipofectin and LipofectAMINE as well as the amount of RNA, which provides the highest transfection yields (measured by titers of specific infectivity). ) as it relates to the concentration of lipids used (concentrations ranging from 10 to 40 μg / ml). LipofectAMINE is the reagent used in the present invention because it had the highest performance. The amount of RNA to be used can not be very high. There is a limit on the amount of RNA that can be mixed with a corresponding amount of lipid to achieve the best transfection efficiency, and, consequently, the highest specific infectivity. In the present invention, a proportion of the reagents ranging from 0.5-2 μg of total RNA: 10-40 μg of LipofectAMINE per 1 ml of PBS is used.

EXAMPLE 5 Preparation of progeny batches All batches were prepared in primary CEF cultures using eggs derived from SPF (Specific Pathogen Free) groups.

The viruses were recovered by combining the medium present in each flask T in centrifuge bottles and rotating at low speed to remove the cellular waste. The supernatant was aspirated in flasks of 11 containing stabilizer at a 1: 1 ratio and slowly frozen by rotating on a dry ice bath of ethanol after the removal of all the quality control aliquots. All viruses were stored at -70 ° C.

Preparation of the original progeny lot The original progeny lots consisted of 3 separate transcription / transfection experiments performed on different days with different batches of primary CEF cultures. Fibroblasts from primary chicken embryos were seeded. A total of 3 disposable 175 cm2 T flasks containing RNA transcribed in vitro were transfected into CEF cells using LipofectAMine ™. Each flask T provided a total of 80 ml of culture supernatant. In three separate transfections performed on different days and therefore with different batches of CEF cell titres for the original progeny batches were: T1, 104-66 (4.66 log.o pfu / ml); T2, 104 87 (4.87 log. Or pfu / ml); T3, 105 46 (5.46 log10 pfu / ml). Each batch provided a total volume of 480 ml of original virus. Eighty my were used for quality control, subtracting 400 mi for the preparation of primary progeny batches.

Preparation of primary progeny batch Two batches of primary progeny were prepared and named LP1 and LP2. LP1 is derived from the original progeny lot T3 while LP2 is derived from T2. Each was tested for sterility, potency and foreign agents with satisfactory results. The volumes obtained and titles are: Progeny lot Volume (mi) Title (Iog10 pfu / ml) LP1 1,200 6.22 LP2 1,600 6.20 pfu = plaque forming unit Secondary progeny batches Three batches of secondary progeny were prepared and named LS1, LS2 and LS3. LS1 and LS2 are derived from the primary progeny lot LP1 while LS3 is derived from LP2. Each was tested for sterility, potency and foreign agents with satisfactory results. The volumes obtained and titles are: Progeny lot Volume (mi) Title (log10 pfu / ml) LS1 5.200 6.20 LS2 5.600 6.05 LS3 5.200 6.73 Each of these lots of progeny should be sufficient for YF vaccine production using the current manufacturing methodology (embryo eggs) or the cellular system (CEF cells) for almost 50 years at a rate of at least 50 million doses / year. The foregoing provides a description of the preferred embodiments, however, it should be noted that numerous structural changes and modifications can be made without departing from the spirit and scope of the present invention.

Claims (22)

1. A vaccine composition for humans against yellow fever infection comprising a recombinant YF virus, which is regenerated from infectious YF cDNA having the base sequence set forth in Figure 6 and equivalents thereto containing different codons for the Same amino acid sequences or equivalent sequences.

2. A vaccine composition according to claim 1, wherein the recombinant YF virus is a virus recovered from a culture of transfected cells from chicken embryo fibroblasts.

3. A vaccine composition according to claims 1 and 2, wherein the recombinant YF virus is present in said composition in an amount sufficient to induce immunity against said infection, and wherein said composition further comprises a pharmaceutically acceptable carrier.

4. A vaccine composition according to claim 3, which further comprises a pharmaceutically acceptable stabilizer.

5. An infectious YF cDNA having the base sequence set forth in Figure 6 and equivalents therefor containing different codons for the same amino acid sequences or equivalent sequences.

6. A DNA construct consisting essentially of a vector and a DNA segment carrying the extreme 5 'and 3' end sequences of the YF infectious cDNA of claim 5.

7. The DNA construct according to claim 6, wherein said vector is selected from the group consisting of pBR322, pBR325, BR327, pBR328, pUC7, pUC8, pUC9, pUC19,? phage, M13 phage or others, in which the YF genome is genetically stable.

8. The DNA construct according to claim 6, which has the structure of the plasmid pYF5'3'IV / G1 / 2.

9. The DNA construct consisting essentially of a vector and a segment of DNA that carries the middle region of the YF infectious cDNA genome of claim 5.

The DNA construct according to claim 9, which has the structure of the plasmid pYFM5.2 / T3 / 27. eleven .

The DNA construct according to claims 8, 9 and 10, wherein said vector is selected from the group consisting of pBR322, pBR325, BR327, pBR328, pUC7, pUC8, pUC9, pUC19,? phage, M13 phage or others in which the YF genome is genetically stable, either in parts or carrying the complete sequence set forth in claim 5.

12. A recombinant YF virus, which is regenerated from YF infectious cDNA having the base sequence set forth in Figure 6 and equivalents therefor which contain different codons for the same amino acid sequences or equivalent sequences.

13. The recombinant YF virus according to claim 12, which is a virus recovered from a transfected cell culture of chicken embryo fibroblasts.

14. The recombinant YF virus according to claims 12 and 13, which is YFiv5.2 / DD.

15. A method for producing recombinant YF virus comprising the steps of: a. transfecting cells with infectious YF cDNA of claim 5; b. culturing said cells under conditions sufficient for the cellular production of recombinant YF virus; and c. collect said recombinant YF virus, which is a YF virus similar to 17DD.

The method according to claim 15, wherein said YF infectious cDNA is the cDNA as set forth in Figure 6.

The method according to claims 15 and 16, wherein said cells are selected from the group consisting of Hela (derived from human cervical carcinoma), CV-1 (derived from monkey kidney), BSC-1 (derived from monkey kidney), RK 13 (derived from rabbit kidney), L929 (derived from mouse connective tissue), CE cell (chicken embryo), CEF (chicken embryo fibroblast), SW-13 (derived from human adrenocortical carcinoma), BHK-21 (baby hamster kidney), Vero (green monkey kidney) African), LLC-MK2 (derived from Rhesus monkey kidney (Macaca mulata)) or others, where the YF virus can replicate.

18. The method according to claim 15, wherein said cells are CEF.

19. The method according to claim 15, which is performed under Good Manufacturing Practices (GMP).

20. An original progeny lot comprising YFiv5.2 / DD, which is produced according to claim 19.

21. A batch of primary progeny comprising YFiv5.2 / DD, which is produced according to claim 19.

22. A batch of secondary progeny comprising YFiv5.2 / DD, which is produced according to the claim 19