immunology of infectious and parasitic diseases - XXXVII Congress ...

CHARACTERIZATION OF THE CONSERVATION AND ANTIGENICITY OF
NEW PROTEINS FROM L. CHAGASI WITH THE POTENTIAL FOR USE IN
THE DIAGNOSIS AND IMMUNITY AGAINST VISCERAL LEISHMANIASIS
NASCIMENTO, MARÍLIA BARBOSA. 1* ; PONTES-DE-CARVALHO, LAIN
CARLOS 2 ; OLIVEIRA, GERALDO GILENO DE SA. 2 ; DHALIA, RAFAEL 1 ;
MAGALHÃES, FRANKLIN BARBALHO. 3 AND DE MELO NETO, OSVALDO
POMPÍLIO 1 .
1 Centro de Pesquisas Aggeu Magalhães– FIOCRUZ, Recife, Brasil. 2 Centro de
Pesquisas Gonçalo Muniz– FIOCRUZ, Salvador, Brasil. 3 Associação
Caruaruense de Ensino Superior- Faculdade ASCES, Caruaru, Brasil. *E-mail:
mariliabarbosa@cpqam.fiocruz.br.
Introduction:Strategies to combat the visceral form of leishmaniasis, the most
serious form of this disease, have so far been unsuccessful. In this respect, the
identification of novel antigens for diagnosis and vaccine development against
visceral leishmaniasis becomes fundamental. Our group has been studying new
antigens from L. chagasi identified through the screening of genomic and cDNA
expression libraries using sera from infected humans and dogs. 13 different
clones were isolated encoding proteins. On ELISA pilot tests using sera from
infected individuals, and designed to study their use as diagnostic tools, these
displayed sensitivity ranging from 34% to 93%. This work focused on some of
these proteins, which have the property of being rich in repetitive motifs. The
aim was to describe aspects of their localization and conservation in different
species of trypanosomatids, as well as to better assess their antigenicity.
Methods and Results:The selected proteins, called Lci5, Lci9, Lci11, Lci12 and
Lci10, were analyzed by immunofluorescence and Western blot using rabbit
polyclonal sera. Regarding their subcellular localization, they display different
patterns with three localizing to specific regions within the cytoplasm (Lci10,
Lci12) or flagellum (Lci5), while two are uniformly dispersed throughout the
cytoplasm (Lci9, Lci11). Proteins Lci9, Lci10 and Lci11 are constitutively
expressed in growth curves of L. chagasi promastigotes and may be restricted
to Leishmania species. For Lci10, its gene sequence was then redesigned and
commercially synthesized so that, after cloning , two different protein constructs
(Lci10, complete, and Lci10-nr, non-repetitive region) could be expressed and
purified by affinity chromatography. These polypeptides were evaluated by
Indirect ELISA using sera from dogs and humans infected with L. chagasi.
When evaluated with dogs‟ sera, the results showed that Lci10-nr recognizes
96% of the sera tested and this non-repetitive region is largely responsible for
the protein antigenicity. With human samples Lci10-nr recognizes only 40% of
the sera tested, with the repetitive motifs contained in the complete protein
being responsible for increasing its antigenicity against humans‟ sera.
Conclusion:Further studies are ongoing for these proteins which show promise