Caracterização fenotípica e molecular dos vírus quiméricos febre amarela / dengue candidatos a uma vacina tetravalente recombinante contra dengue

Abstract

Dengue virus infection is a public health problem worldwide, with recurrent epidemics in tropical regions of Asia, Africa and the Americas. The development of vaccines against dengue is of paramount importance, since to date there are no licensed vaccines. Our ultimate intention is to be able to develop a vaccine against dengue based on recombinant yellow fever 17D vaccine virus. Recombinant viruses were constructed by exchanging structural genes prM / E of yellow fever virus by the respective genes of dengue virus serotypes 1, 2, 3, and 4. This study describes the genotypic and phenotypic characteristics of such Yellow Fever / Dengue recombinant viruses built previously. This characterization was focused on plaque size phenotype, growth in Vero cell culture in serum free medium, replication in Vero cells grown in Cell Factory (medium scale), genetic stability, clonal selection and protection against a lethal challenge in a murine model. The constructions of the recombinant viruses YFV/DENV prM/E produced viruses that replicate efficiently in stationary T-175cm ² flasks and Cell Fatory with replication and plaque morphology similar to parental wild dengue virus, suggesting that the exchange of prM/E of yellow fever virus for the same region of dengue can affect this phenotypic trait After clonal purification steps viral clones had their genome sequenced and compared with the genome of the P2 virus in the working seed lot. Some changes were observed between the amino acid sequences of passages P2 and CP2 in all clones except for clones from YFV/DENV4. Clones CP2 were further passed through eight passages in Vero cell to determine whether the virus was genetically stable. After the passages we noted that many changes were of adaptive nature to Vero cell. The clones were characterized with respect to plaque morphology as revealed by imunofocus. Clones YFV/DENV1 (PE), YFV/DENV2 (PE), YFV/DENV4 were shown to be homogeneous and YFV/DENV3 H87 was rather heterogeneous, even after cloning. Aiming to evaluate the neurotropism (attenuation) of viral clones, two experiments were conducted with three week-old mice with viral input of 10³ PFU/mouse in the first experiment and the second with undiluted viral stock. Suckling mice received a viral input of 10³ PFU/mouse. The results showed no difference between the two tests with three week-old mice, in which none of the chimeric viruses were neurovirulent. However, in suckling mice a significant degree of neurovirulence was noted. We studied the immunogenicity of the YFV/DENV2 (PE) viruses in a murine model and immunized animals challenged with an otherwise lethal dose of neurotropic DENV2 virus. Clone YFV/DENV2C provided 93% protection whereas YFV/DENV2A and YFV/DENV2B reached 81% and 75%, respectively. The results are of great importance for the development of a tetravalent vaccine.