Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-04-10
2001-01-09
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023720
Reexamination Certificate
active
06171854
ABSTRACT:
The present invention relates to a vaccine against infections caused by YF virus and its preparation by regenerating YF 17D virus from the correspondent complementary DNA (cDNA) which is present in the new plasmids pYF 5′13′ IV/G1/2 and pYFM 5.2/T3/27.
BACKGROUND OF THE INVENTION
The Flavivirus genus consists of 70 serologically cross-reactive, closely related human or veterinary pathogens causing many serious illnesses, which includes dengue fever, Japanese encephalitis (JE), tick-borne encephalitis (TBE) and yellow fever (YF). The Flaviviruses are spherical with 40-60 nm in diameter with an icosahedral capsid which contains a single positive-stranded RNA molecule.
YF virus is the prototype virus of the family of the Flaviviruses with a RNA genome of 10,862 nucleotides (nt), having a 5′ CAP structure (118 nt) and a nonpolyadenylated 3′ end (511 nt). The complete nucleotide sequence, of its RNA genome was determined by Rice, C. M. et al (1985).
The single RNA is also the viral message and its translation in the infected cell results in the synthesis of a polyprotein precursor of 3,411 amino acids which is cleaved by proteolytic processing to generate 10 virus-specific polypeptides. From the 5′ terminus, the order of the encoded proteins is: C; prM/M; E; NS1; NS2A; NS2B; NS3; NS4A; NS4B and NS5. The first 3 proteins constitute the structural proteins, i.e., they form the virus together with the packaged RNA molecule. The remainder of the genome codes for the nonstructural proteins (NS) numbered from 1 through 5, according the order of their synthesis.
The C protein, named capsid, has a molecular weight ranging from 12 to 14 kDa (12-14 kilodaitons); the membrane protein, M has a molecular weight of 8 kDa, and its precursor (prM) 18-22 kDa; the envelope protein, E, has 52-54 kDa, being all of them encoded in the first quarter of their genome.
Three of the nonstructural proteins are large and have highly conserved sequences among the flaviviruses, namely, NS1 has a molecular weight ranging from 38 to 41 kDa; NS3 has 68-70 kDa and NS100-103 kDa. No role has yet been assigned to NS1 but NS3 has been shown to be bifunctional having a protease activity needed for the processing of the polyprotein, and the other is a nucleotide triphosphatase/helicase activity which is associated with viral RNA replication. NS5, the largest and most conserved 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. NS2A has been shown to be required for proper processing of NS1 whereas NS2B has been shown to be associated with the protease activity of NS3.
Two strains of yellow fever virus (YF), isolated in 1927, gave rise to the vaccines to be used for human immunization. One, the Asibi strain, was isolated from a young african named Asibi by passage in Rhesus monkey (
Macaca mulatta
), and the other, the French Viscerotropic Virus (FVV), from a patient in Senegal.
In 1935, the Asibi strain was adapted to growth in mouse embryonic tissue. After 17 passages, the virus, named 17D, was further cultivated until passage 58 in whole chicken embryonic tissue and thereafter, until passage 114, in denervated chicken embryonic tissue only.
Theiler and Smith (Theiler, M. and Smith, H. H. (1937). “The effect of prolonged cultivation in vitro upon 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 until passages 227 and 229 and the resulting viruses, without human immune 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 passages yielded the parent 17D strain at passage level 180 (see FIG.
1
), 17D at passage 195, and the 17D-204 at passage 204. was further subcultured until passage 241 and underwent 43 additional passages in embryonated chicken eggs to yield the virus currently used for human vaccination in some countries (passage 284). The 17D-204 was further subcultured to produce Colombia 88 strain which, upon passage in embryonated chicken eggs, gave rise to different vaccine seed lots currently in use in France (I. Pasteur, at passage 235) and in the United States (Connaught, at passage 234). The 17D-213 strain was derived from 17D-204 when the primary seed lot (S1 112-6-9) from the Federal Republic of Germany (FRG 83-66) was used by the World Health Organization (WHO) to produce an avian leukosis virus-free 17D seed (S1 213/77) at passage 237.
In the late 1930's and early 1940's, mass vaccination was conducted in Brazil with the use of several substrains of 17D virus (Table I). These substrains differed in their passage history and they overlapped with regard to time of their use for inocula and/or vaccine production. The substitution of each one by the next was according to the experience gained during vaccine production, quality control and human vaccination in which the appearance of symptomatology led to the discontinuation of a given strain.
Each of these 17D-204 strains (C-204; F-204) was plaque purified in different cell lines, the virus finally amplified in SW13 cells and used for CDNA cloning and sequence analyses (Rice, C. M.; Lenches, E.; Eddy, S. R.; Shin, S. J.; Sheets, R. L. and Strauss, J. H. (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, J. P.; Girard, M. (1987). “High genetic stability of the coding region for the structural proteins of yellow fever strain 17D”. J. Gen. Virol. 68: 2245-2247).
The 17D-213 at passage 239 was tested for monkey neurovirulence (R. S. Marchevsky, personal communication, see Duarte dos Santos et al, 1995) and was the subject of sequence analysis together with 17DD (at passage 284) and comparison to previously published nucleotide sequences of other YF virus strains (Duarte dos Santos et al, 1995) (Asibi: Hahn, C. S.; Dalrymple, J. M.; Strauss, J. H. and Rice, C. M. (1987). “Comparison of the virulent Asibi strain of yellow fever virus with the 17D vaccine strain derived from it”. Proc. Natl. Acad. Sci. USA. 84: 2029-2033; 17D-204 strain, C-204: Rice. C. M.; Lenches, E. M.;
Eddy, S. R.; Shin, S. J.; Sheets, R. L. and Strauss, J. H. (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, J. P. and Girard, M. (1987). “High genetic stability of the coding region for the structural proteins of yellow fever virus strain 17D”. J. Gen. Virol. 68: 2245-2247) (see FIG.
1
).
A total of 67 nucleotide differences, corresponding to 31 amino acid changes, were originally noted between the Asibi and 17D-204 genomic sequences (see Hahn, C. S. et al, 1987). The comparison between the nucleotide sequences of 17DD and 17D-213 substrains (see Duarte dos Santos et al, 1995) and the nucleotide sequence of 17D-204 substrain (see Rice et al, 1985) showed that not all chances are common and thus not confirmed as being 17D-specific. Therefore, the 17D-substrain specific changes observed are very likely not related to attenuation but may reflect differences in behavior of these strains in monkey neurovirulence tests. In consequence, the number of changes likely to be associated with viral attenuation were reduced by 26%, i.e., to 48 nucleotide changes. From these 48 nucleotide sequence changes which are scattered along the genome, 26 are silent mutations and 22 led to amino acid substitutions. More important are the alterations noted in the E protein because it is the main target for humoral neutralizing response, i.e., it is the protein where hemag
Freire Marcos Da Silva
Galler Ricardo
Fundaco Oswaldo Cruz-Fiocruz
Pillsbury Madison & Sutro LLP
Wortman Donna C.
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