Early high avidity specific IgG production in experimental hamster visceral leishmaniasis.

Visceral leishmaniasis (VL) by Leishmania (Leishmania) infantum is epidemic in Brazil. Hypergammaglobulinemia appears early in patients with VL and is ineffective. Usually, high-affinity IgG B cells are selected during most infections, a critical step for an effective humoral response. The avidity of IgG antibodies in VL is unexplored due to the absence of temporal parameters in most patients, associated to low clinical significance. Experimental infection models overcome this fact, allowing the monitoring of the disease temporal evolution. In this study, the avidity of IgG antibodies was evaluated in experimental models, in infection in hamsters, and in immunization in rabbits. Specific IgG antibodies were detected by ELISA, using chaotropic solution to determine avidity, as reported for viral infections. The levels of IgG antibodies correlated with the progression of experimental infection in hamsters or antigenic stimulation in immunized rabbits. However, IgG avidity was high early in infected animals, even in early periods (> 80%), while in immunized rabbits, they had early antibodies of low avidity with progressive maturation, similar as other infections. These data suggest that the affinity maturation of the avidity of anti-Leishmania IgG antibodies promoted at an early stage, influencing the appropriate interaction between antigens and affecting the disease progression. This fact could be associated to monovalent immune complexes, as reported in human and experimental VL. This scenario may be related to an independent process of immune cell activation by the parasite but absent in antigen preparation used as immunogens.

High levels of serum glycans monovalent IgG immune complexes detected by dissociative ELISA in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is epidemic in Brazil with an increasing incidence of human cases and canine reservoirs, with host hypergammaglobulinemia. Conventional enzyme-linked immunosorbent assay (cELISA) based on several parasitic antigens is the main method for diagnosis and indication of treatment. Dissociative ELISA (dELISA) uses acidic treatment to free immunoglobulin G (IgG) from immune complexes, and its use revealed a significant positive fraction of suspected cases with negative serology. Looking for small molecules or haptens that block IgG antibodies, we purified by molecular exclusion chromatography, 1000-3000 MW molecules from promastigote soluble extract, mostly oligosaccharides comprising 6-13 sugar residues using MALDI-TOF analysis. Glycan-BSA complex (GBC) was constructed by conjugating promastigote glycans to BSA molecules, allowing their use in the solid support in cELISA or dELISA. Sera from experimentally infected hamsters showed higher levels of blocked monomeric IgG during infection, mostly against GBC, which was also present in lower concentrations in the promastigote soluble extract dELISA. Those data show that most of the specific monomeric IgG in serum are blocked by haptens composed by glycans produced by the parasite, better detected in the high dilution of sera in the dELISA assays. dELISA is a useful technique for detecting blocked monomeric antibodies that could have difficult clearance from blood, which could result in hypergammaglobulinemia.