Mutantes replicativos y atenuados del virus vaccinia como candidatos vacunales frente a la leishmaniasis

• Autores: Lucas Sánchez Sampedro
• Directores de la Tesis: Mariano Esteban Rodríguez (dir. tes.), Carmen Elena Gómez Rodríguez (codir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2012
• Idioma: español
• Tribunal Calificador de la Tesis: Manuel Fresno Escudero (presid.), Rafael Blasco Lozano (secret.), Margarita del Val Latorre (voc.), Francisco Javier Moreno Nuncio (voc.), María Dolores Rodríguez Aguirre (voc.)
• Materias:
  • Ciencias de la vida
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: digitool-uam.greendata.es
• Resumen

  • Poxvirus vectors have emerged as prominent vehicles for delivering antigens of pathogens from prevalent diseases. Different strains of vaccinia virus (VACV) expressing antigens from different pathogen-causing diseases are used nowadays in preclinical and clinical trials against HIV, malaria, tuberculosis, leishmaniasis and also in cancer. The most promising vectors used in vaccination trials are the attenuated MVA and NYVAC strains. While those viruses do not produce virus progeny in human cells, which assures safety, some evidences point out that replication competent viruses with limited but amplified time of infection and expression of heterologous antigen could provide more immunogenic vaccines In this investigation we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants M65 and M101. These mutants were generated after serial passage of persistently infected Friend erythroleukemia cells (FEL). In cultured cells of different origins the mutants have growth kinetics similarly as the parental virus strain. Recombinant virus expressing Photinus pyralis luciferase gene were generated to study in vivo replication and heterologous antigen expression. In those studies, the mutant virus M65-Luc showed similar replication capacity as parental VACV Western Reserve WR-Luc virus. In the case of M101-Luc it showed more restricted phenotype, comparable to MVA-Luc in lymph nodes and spleen, although M101-Luc expression persisted longer than MVA-Luc in organs like ovaries or in cells of the peritoneal cavity. In intranasally infected mice the mutants showed different attenuation levels, and pathogenicity was strongly reduced in comparison with the parental WR strain.

    Wide genome analysis after deep sequencing revealed that both viruses shared an identical deletion in the left end of the genome comprehending some immunomodulatory but mainly host range genes. The main difference between M65 and M101 genomes is a small deletion in the right end of the genome of M101 virus that is not present in M65 genome, and covers A39R, A40R and WR208 genes.

    To further evaluate the potential of M65 and M101 as vaccine vectors against diseases, recombinant viruses that expressed the protective Leishmania LACK antigen were generated and tested in heterologous DNA prime/poxvirus boost regimen for protective efficacy against leishmaniasis. Mice immunized in DNA prime/poxvirus boost with both vectors expressing the LACK antigen of Leismania infantum resulted in protection or delay in the onset of cutaneus leishmaniasis.

    High quality CD4+ and CD8+ LACK-specific T cells were detected in adaptive immune response, early stages postchallenge and memory studies. As a remarkable difference between M65 and M101 vectors DNA-LACK/M65-LACK regimen preferently induced CD4+ T cell responses, while DNA-LACK/M101-LACK regimen preferently induced CD8+ T cell responses.

    Overall, from the replication capacity of M65 and M101 in cells of various origins and in mouse tissues, genome sequences, as well as from the immunogenic characteristics and efficacy against leishmaniasis of recombinants expressing the LACK antigen of L. infantum, we propose that M65 and M101 can be considered as novel VACV replication competent strains which can be used as vaccine vectors against pathogens.