Cross-protective immunity to Leishmania amazonensis is mediated by CD4+and CD8+epitopes of Leishmania donovani nucleoside hydrolase terminal domains

The nucleoside hydrolase (NH) of Leishmania donovani (NH36) is a phylogenetic marker of high homology among Leishmania parasites. in mice and dog vaccination, NH36 induces a CD4+ T cell-driven protective response against Leishmania chagasi infection directed against its C-terminal domain (F3). the C-terminal and N-terminal domain vaccines also decreased the footpad lesion caused by Leishmania amazonensis. We studied the basis of the crossed immune response using recombinant generated peptides covering the whole NH36 sequence and saponin for mice prophylaxis against L. amazonensis. the F1 (amino acids 1-103) and F3 peptide (amino acids 199-314) vaccines enhanced the IgG and IgG2a anti-NH36 antibodies to similar levels. the F3 vaccine induced the strongest DTH response, the highest proportions of NH36-specific CD4+ and CD8+ T cells after challenge and the highest expression of IFN-gamma and TNF-alpha. the F1 vaccine, on the other hand, induced a weaker but significant DTH response and a mild enhancement of IFN-gamma and TNF-alpha levels. the in vivo depletion with anti-CD4 or CD8 monoclonal antibodies disclosed that cross-protection against L. amazonensis infection was mediated by a CD4+ T cell response directed against the C-terminal domain (75% of reduction of the size of footpad lesion) followed by a CD8+T cell response against the N-terminal domain of NH36 (57% of reduction of footpad lesions). Both vaccines were capable of inducing long-term cross-immunity. the amino acid sequence of NH36 showed 93% identity to the sequence of the NH A34480 of L amazonensis, which also showed the presence of completely conserved predicted epitopes for CD4+ and CD8+ T cells in F1 domain, and of CD4+ epitopes differing by a single amino acid, in F1 and F3 domains. the identification of the C-terminal and N-terminal domains as the targets of the immune response to NH36 in the model of L. amazonensis infection represents a basis for the rationale development of a bivalent vaccine against leishmaniasis.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Univ Fed Rio de Janeiro, Inst Microbiol Paulo Goes, Dept Microbiol Geral, Lab Biol & Bioquim Leishmania, BR-21941902 Rio de Janeiro, RJ, BrazilUniv Fed Rio de Janeiro, Inst Microbiol Paulo Goes, Lab Imunol, BR-21941902 Rio de Janeiro, RJ, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Biol Geral, Belo Horizonte, MG, BrazilUniv Fed Rio de Janeiro, Fac Med, Programa Pos Grad Clin Med, BR-21941902 Rio de Janeiro, RJ, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilCNPq: 301215-2007-3CNPq: 302039/2010-4CNPq: 559756/2010-0CNPq: 404400/2012-4FAPERJ: 102733/2008FAPERJ: 102957/2011FAPERJ: E-26/102415/2010FAPERJ: E-26/110535/2010Web of Scienc

Leishmania donovani Nucleoside Hydrolase terminal domains in cross-protective immunotherapy against Leishmania amazonensis murine infection

Nucleoside hydrolases of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. Leishmania donovani Nucleoside hydrolase (NH36) induced a main CD4+ T cell driven protective response against Leishmania chagasi infection in mice which is directed against its C-terminal domain. In this study, we used the three recombinant domains of NH36: N-terminal domain (F1, amino acids 1-103), central domain (F2 aminoacids 104-198) and C-terminal domain (F3 amino acids 199-314) in combination with saponin and assayed their immunotherapeutic effect on Balb/c mice previously infected with L. amazonensis. We identified that the F1 and F3 peptides determined strong cross-immunotherapeutic effects, reducing the size of footpad lesions to 48% and 64%, and the parasite load in footpads to 82.6% and 81%, respectively. The F3 peptide induced the strongest anti-NH36 antibody response and intradermal response (IDR) against L. amazonenis and a high secretion of IFN-γ and TNF-α with reduced levels of IL-10. The F1 vaccine, induced similar increases of IgG2b antibodies and IFN-γ and TNF-α levels, but no IDR and no reduction of IL-10. The multiparameter flow cytometry analysis was used to assess the immune response after immunotherapy and disclosed that the degree of the immunotherapeutic effect is predicted by the frequencies of the CD4+ and CD8+ T cells producing IL-2 or TNF-α or both. Total frequencies and frequencies of double-cytokine CD4 T cell producers were enhanced by F1 and F3 vaccines. Collectively, our multifunctional analysis disclosed that immunotherapeutic protection improved as the CD4 responses progressed from 1+ to 2+, in the case of the F1 and F3 vaccines, and as the CD8 responses changed qualitatively from 1+ to 3+, mainly in the case of the F1 vaccine, providing new correlates of immunotherapeutic protection against cutaneous leishmaniasis in mice based on T-helper TH1 and CD8+ mediated immune responses