Vacunación con las proteínas ácidas ribosomales de Leishmania infantum.Respuesta inmunològica y capacidad protectiva

Abstract

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 01-03-2005Despite the evidence of acquired irnrnunity and resistance to reinfection in natural Leishmania hosts, suggesting that a vaccine is feasible, to date there is no vaccine for hurnan leishrnaniasis. Recent advances in the design of vaccines are based on rnolecularly defined antigens obtained by recornbinant rnethodologies, the socalled 'second-generation vaccines". In this study, we have tested rnolecularly defined vaccines based on the acidic ribosornal proteins and the histones of Leishmania infantum. These molecules have been described as relevant irnrnunogens for the host irnrnune systern during infection with Leishmania (Requena et al., 2000). In addition, adrninistration of a rnulticornponent protein containing the antigenic detenninants of sorne of these proteins (LiP2a, LiP2b, LiPO and LiH2A), using live BCG as adjuvant, confers protection in dogs against L. infantum infection (Molano et al., 2003). Fintly. we have deterrnined that sorne of these antigens (LiPO. LiPZb, LiH2A and LiH3). expressed as recornbinant proteins in bacteria, stirnulate the in vitro proliferation of splenocytes and lyrnph node cells frorn naive rnice. This mitogenic activity is correlated with a B cell activation and cytokine synthesis (11-6 and IFNy). Secondly, probably related with the in viho effect. we present data showing that the adrninistration of the acidic ribosornal proteins, in the absence of any added adjuvant, induces a humoral response. The progressive switch frorn IgM to IgG afler the protein boosting, suggests that these are conventional secondary humoral responses requiring the participation of T cells. Since lgGl was the predorninant isotype, we conclude that adrninistration of these proteins elicited a Th2 response. Further, we analysed the adrninistration of these antigens as recornbinant proteins adjuvated with irnmunostirnulatory CpG oligodeoxynucleotides (CpG ODN). We detected an enhanced generation of lgG2a antibodies and synthesis of IFN-y following in vitro stirnulation with each antigen. Thus, we rnay conclude that that CpG ODN adjuvant is able to increase the Thl irnrnune response against these antigens. On the other hand. DNA vaccinations with either the LiPO antigen ora rnixture of four plasrnids encoding for the L. infantum histones were perfonned. It was dernonstrated that this DNA vaccines prirned for a Thl irnrnune response. which was associated with the induction of lgG2a antibodies and an antigen-specific production of IFN-y. Further, we decided to test these vaccines in the rnurine rnodel of cutaneous leishrnaniasis (CL). Effective prirnary irnrnunity against L. major in rnouse is known to require IL-12dependent production of 1FN-y frorn CD4' T cells (Thl response), which rnediates NOdependent killing by infected rnacrophages. We have dernonstrated that the adrninistration of LiPO either as protein plus CpG ODN or as a DNA vaccine. confers partial protection against cutaneous leishrnaniasis due to L. major in BALBlc rnice. These rnice exhibited a more controlled lesions but finally, progressive infection and pathology was observed. On the other hand, DNA vaccination with the rnixture of the four plasmids encoding for the L. infantum histones protects BALBlc rnice against CL due to L. major. ln addition, these rnice were protected against the progressive infection and disease, which correlates with an absence of the Th2 response against the parasite proteins. A rnodel of CL in C57BU6 rnice, that more accurately reproduces the pathology and transrnission of the hurnan disease, was used to better undentand the potency of the LiPO-vaccines. We dernonstrated that these vaccines confer protection against L. major infection. consisting of a reduction of the parasite nurnber in the infected ama and alrnost an absence of dennal pathology that correlates well with a specific Thl response. Finally. the LiPO-DNA vaccine was tested in two models of VL: BALBIc rnice and hamster infected with L. infantum. No protection was 0bse~edin these two rnodels. in spite of the fact that, at least in BALBlc rnice. a Thl response was induce