Sandra do Lago Moraes de Ávila

Immune responses to multiple antigen peptides containing T and B epitopes from Plasmodium falciparum circumsporozoite protein of Brazilian individuals naturally exposed to malaria

We have evaluated the immune responses of individuals living in a malaria endemic area of Brazil to the (T1B)4, a multiple antigen peptide (MAP) from Plasmodium falciparum circumsporozoite (CS) protein and the related monoepitope MAPs, B4 and (T1)4, and the linear peptides, T1B and B. The highest antibody frequencies were against MAPs containing the B cell epitope sequence (T1B)4 (42·2%) and B4 (28·8%), while the highest lymphoproliferative response frequencies were against the MAPs containing the T cell epitope sequence (T1)4 (47%) and (T1B)4 (36·4%). We analysed individual responses considering lymphoproliferative response to (T1)4 MAP and IgG antibody titre to (T1B)4 as patterns of ideal cellular and humoral responses, respectively. The frequency of responders, cellular and/or humoral was 66·6%, significantly higher than non responders (P = 0·003). We also determined the HLA class II haplotype of each individual but no association between these and immune response patterns to the MAPs was observed. The results showed that individuals primed against P. falciparum in their natural habitat, present a very diverse array of responses against the same peptide antigens, varying from no response in one‐third of the individuals to cognate B and T cell responses. Our study underlines the importance of previous studies of vaccine candidates to guarantee that the immunization will be capable of reverting inefficient or absent responses to malaria epitopes.

Malaria serology: performance of six Plasmodium falciparum antigen extracts and of three ways of determining serum titers in IgG and IgM-ELISA

The study evaluated six Plasmodium falciparum antigen extracts to be used in the IgG and IgM enzyme-linked immunosorbent assays (ELISA), for malaria diagnosis and epidemiological studies. Results obtained with eighteen positive and nine negative control sera indicated that there were statistically significant differences among these antigen extracts (Multifactor ANOVA, p < 0.0001). Urea, sodium deoxycholate and Zwittergent antigen extracts performed better than did the three others, their features being very similar for the detection of IgG antibodies. Urea, alkaline and sodium deoxycholate antigen extracts proved to be better than the others for the detection of IgM antibodies. A straight line relationship was found between the optical densities (or their respective log10) and the log10 of antibody dilutions, with a very constant slope. Thus serum titers could be determined by direct titration and by two different equations, needing only one serum dilution. For IgM antibody detections, log10 expression gave results that better correlated with direct titration (95% Bonferroni). For IgG antibody detections, the titer differences were not significant. The reproducibility of antibody titers and antigen batches was also evaluated, giving satisfactory results.

Evaluation of different methods for Plasmodia detection, in well defined population groups in an endemic area of Brazil

Mais de 500.000 casos novos de malaria foram notificados no Brasil, em 1992. P.falciparum and P.vivax sao as especies responsaveis por 99,3% dos casos. O diagnostico precoce e indispensavel para inicio do tratamento adequado. Neste trabalho, apresentamos os resultados do metodo tradicional para deteccao de plasmodios, gota espessa, e os resultados de metodos alternativos estudados: imunofluorescencia indireta com anticorpo policlonal anti-P.falciparum e QBC-metodo, em grupos populacionais bem definidos. A analise dos resultados foi feita em relacao a presenca ou ausensia de sintomas clinicos de malaria. Tambem, diferentes classes de imunoglobulinas anti-P.falciparum foram quantificadas para auxiliar na analise global dos resultados, principalmente nos resultados discrepantes. Nos concluimos que os metodos alternativos sao mais sensiveis do que a gota espessa e que a associacao das informacoes epidemiologicas, clinicas e laboratoriais e necessaria para definir a presenca de malaria.

STANDARDIZATION OF PROCEDURES OF Plasmodium falciparum ANTIGEN PREPARATION FOR SEROLOGIC TESTS

The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20 degrees C up to 10 days or at -70 degrees C for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.