[Profiles of IgG responses against CSP, GLURP and LSA-3NR2 in urban malaria (Dakar): relations with haemoglobin levels and parasite densities]. / Profils des réponses IgG dirigées contre CSP, GLURP et LSA-3NR2 dans le paludisme urbain à Dakar : influence sur l'hémoglobinémie et les parasitémies circulantes.

Malaria remains a major health problem in sub- Saharan African countries despite substantial decreases in morbidity and mortality due to sustained control programs. Vaccines candidates were mainly tested in rural endemic setting; however increasing proportion of the population is living in urban area. Evaluation of the qualitative or quantitative immune responses to key targets of anti-Plasmodium immunity requires further investigation in urban area. In a cohort of 144 patients with mild malaria living in Dakar, we analyzed IgG responses against target antigens of P. falciparum: CSP, LSA-3NR2 and GLURP by ELISA. A mean age of 15 yrs (4-65 yrs) was found and patients were separated in 59 adults (<15yrs) and 85 children (≤15 yrs). Parasites densities (0,01-15%) did not differ between the two age groups. In contrast, haemoglobin levels appeared lower in children (4.5-16.6 g/dl) (p<0.01). For the immune results, the most recognized antigens were GLURP and CSP compared to LSA-3NR2. Levels of IgG against these antigens were significantly different between the two age groups and they were positively correlated (rho = 0.32; p<0.001). In addition, levels of IgG anti-GLURP were associated with low parasitemia (≤1%) and absence of anemia (≥11g/dl), particularly in adults (p<0.001). In a multiple regression analysis, no significant relationship was found between parasite densities and IgG responses against all the tested antigens. Our study shows the implication of IgG anti-GLURP in humoral immune response against the parasite. The present work contributes to determine IgG levels that can be used as relevant immunologic biomarkers in urban clinical malaria.

Immune responses to P. falciparum-MSP1 antigen: lack of correlation between antibody responses and the capacity of peripheral cellular immune effectors to respond to this antigen in vitro.

Protective immunity to P. falciparum blood stage infection is thought to be dependent on IgG antibodies, although the mechanisms that underlie such immunity are not clearly understood. One of the antigens thought to be involved in this protective response is MSP1. The present study has examined the levels and distribution of IgG (and IgM) antibodies to the C-terminal 19 kDa fragment of MSP1 in plasma from P. falciparum immune adult Senegalese and the capacity of the peripheral blood mononuclear cells from these patients to either proliferate or secrete IFN-gamma, IL-10 or IL-4 in vitro in response to this antigen. Specific antibodies were found in 74% of individuals' plasma; 44% of mononuclear cells proved capable of proliferating in vitro and IFN-gamma, IL-10 and IL-4 were detected in 37, 23 and 0% of culture supernatants, respectively. No significant association was found between the presence of antibodies and immune cell reactivity under the culture conditions used. This study emphasizes the complexity of the mechanisms responsible for the sustained production of potentially protective antibodies in response to proposed T-cell dependent P. falciparum blood stage antigens.

Regulation of platelet-activating factor receptor expression in human B cells and B cell lines.

We extended our previous data regarding the modulation of human platelet-activating factor receptor (hPAF-R) expression on human B cell lines as well as normal B cells. First, we showed that hPAF-R mRNA was present in B cell lines expressing membrane hPAF-R, but was absent from cell lines devoid of hPAF-R. Second, enhanced hPAF-R membrane expression induced in IM9 line by IL4 was preceeded by hPAF-R mRNA accumulation that was detectable by 8 h and which peaked at 24 h. Similar results were observed for 10 nM platelet-activating factor treatment, which increased hPAF-R mRNA content up to 120% at 48 h, whereas hPAF-R membrane expression was up-regulated by 130%. Third, our data indicate that functional hPAF-R are expressed on resting, as well as on activated, B cells and that B cell activation is required for maintaining hPAF-R membrane and mRNA expression. Thus, in normal B cells, as well as in B cell lines, transcriptional regulation and/or messenger stability control hPAF-R expression.

[Antibodies specific to Plasmodium falciparum antigens in immune individuals: III. Seasonal course of the response to a major antigen of the asexual blood forms in two sites of different malaria exposure]. / Anticorps spécifiques d'antigènes de Plasmodium falciparum chez les sujets immuns: III.-Evolution saisonnière des réponses vis à vis d'un antigène majeur des formes sanguines asexuées dans deux sites d'expositions palustres différentes.

Specific IgG1 and IgG3 antibody responses to a major Plasmodium falciparum blood stage antigen i.e. MSP1 have been measured in plasma obtained from immune individuals living in areas with different endemicities and sampled at different periods corresponding to different levels of parasite transmission. The study shows a significant imbalance between IgG1 and IgG3 antibody responses in Dielmo vs. Ndiop, and a differential regulation of IgG1 and IgG3 responses both in subclasses and in titers (low: 1/200, and high: 1/2,000) depending upon intensity of parasite exposure.

[Specific antibodies against Plasmodium falciparum antigens in immune subjects: II. Screening of responses against the merozoite major surface antigen (MSP!)]. / Anticorps spécifiques d'antigènes de Plasmodium falciparum chez les sujets immuns: II. Criblage des réponses vis à vis d'un antigène majeur de la surface des mérozoïtes (MSP1).

Specific immune responses to asexual blood stages of P. falciparum antigens (a lysate of parasitized red blood cells and a characterized vaccine candidate i.e. MSP1 p19) were analyzed in plasma samples from immune adult individuals living in three different areas of Senegal, where malaria transmission is different. Most individuals in the three sites had specific IgG and IgM to total P. falciparum antigens, whereas approximately 50% had either IgG or IgM specific to MSP1 p19. Further, no anti-MSP1 p19 IgG2 and IgG4 antibody was noticed in any individual whereas the distribution of anti-MSP1 p19 IgG1 and IgG3 was different upon the epidemiological context. In addition, no relationship was found between antibody responses and in vitro T cell responses against P. falciparum antigens upon those experimental conditions. These data stress on the relatively elevated distribution of specific antibodies to MSP1 p19 in P. falciparum hyperendemic areas and suggest a differential regulation of isotypes depending on individual parasite exposure.

Experimental IgG antibody production in vitro by peripheral blood and tonsil surface gamma+ B lymphocytes from Plasmodium falciparum-immune West Africans.

Antigen reactive B cells in tonsil specimens from teenagers from a region moderately exposed to P. falciparum were capable of being differentiated in vitro and producing specific immunoglobulin (Ig)G in up to 33% of individual experiments. Mononuclear cells or purified (s)gamma+ CD19+ B cells from peripheral blood or tonsil specimens from P falciparum-immune Senegalese subjects produced antigen-specific IgG upon appropriate stimulation in vitro. One fraction of this IgG was produced de novo by differentiated B cells and another fraction was likely bound on the surface of circulating or resident CD19+ sgamma+ B cells which were found in significantly greater numbers in individuals from rural Senegal as compared to nonimmune European controls. This study further documents the baseline levels of in vitro driven anti-P. falciparum IgG antibody production by mononuclear cells from blood and tonsils in immune populations exposed to P. falciparum differentially. Furthermore, this study demonstrates the relevance and potential utility of tonsils as a source of B lymphocytes to characterize further specific antibody responses to P. falciparum antigens in immune populations.

Regulation of paf-acether receptor expression in human B cells.

Paf-acether (paf) is a phospholipid cytokine alloted with potent inflammatory and immunoregulatory properties. Recent reports indicated that in human B cell lines, paf modulated both early and late activation events. In our study, we showed that four of six human B cell lines specifically bound [3H]paf irrespective of the stage of differentiation, the presence of EBV genome or cell surface phenotype. Binding was saturated and fit a one receptor model with a dissociation constant ranging from 1 to 6 nM and a number of sites per cell ranging from approximately equal to 4000 in Rjc13 to approximately equal to 30,000 in Raji or IM9. In addition, our data indicate that 1) maximal expression occurred during the log phase growth; 2) paf itself (10-100 nM) or rIL-4 (100 U/ml) up-regulated by two- to threefold the number of paf binding sites without affecting the affinity. Finally, we found that activated normal B lymphocytes exhibited a higher capacity than resting B cells to incorporate and metabolize [3H]paf at 37 degrees C. Resting B lymphocytes lacked specific binding capacity for paf, yet specific paf receptors were induced upon stimulation via Staphylococcus aureus Cowan I or phorbol 12,13 dibutyrate plus ionomycin. These results suggest that B cell activation is a critical event for paf receptor expression and modulation.

Plasmodium falciparum- and merozoite surface protein 1-specific antibody isotype balance in immune Senegalese adults.

This study shows markedly different isotype distributions of antibodies to asexual blood stages of Plasmodium falciparum and to merozoite surface protein 1 in clinically immune Senegalese adults depending on the study site. The relationships between immunoglobulin M (IgM) and IgG and between IgG3 and IgG1 antibodies differed in settings where transmission is perennial compared to settings where it is seasonal. This suggests a role for antibody class and/or subclass production and utilization in the regulation of protective immunity to such antigens.

Secretion of parasite-specific immunoglobulin G by purified blood B lymphocytes from immune individuals after in vitro stimulation with recombinant Plasmodium falciparum merozoite surface protein-119 antigen.

The C-terminal 19 000 MW fragment of merozoite surface protein-1 (MSP119) is one of the most promising candidate antigens for a malaria vaccine. Baculovirus recombinant Plasmodium falciparum MSP119 has been used to define conditions for the in vitro production of specific antibodies by purified human blood B cells in a culture system where T-cell signals were provided by the engagement of CD40 molecules and exogenous cytokines. MSP119 preferentially induced surface immunoglobulin G (IgG) -positive (sgamma+) B lymphocytes from P. falciparum-immune donors to differentiate and produce antigen-specific IgG. In contrast, naïve B cells or cells from non-immune donors could not be induced to secrete parasite-specific IgG in vitro. Although IgG secretion was obtained in the absence of exogenous cytokines, it was dependent on B-cell-derived interleukin-10 (IL-10) and/or B-cell factor(s) under the control of IL-10, since IgG levels were significantly decreased in the presence of neutralizing anti-IL-10 antibodies. These results demonstrate at the cellular level that a single malaria vaccine candidate polypeptide can direct parasite-specific antibody production mediated by the secretion of potentiating factors.

Different Plasmodium falciparum recombinant MSP1(19) antigens differ in their capacities to stimulate in vitro peripheral blood T lymphocytes in individuals from various endemic areas.

This study reports on T-cell proliferative responses to the 19-kDa C-terminal domain of the Plasmodium falciparum merozoite surface protein (MSP1(19)). Three different recombinant proteins were used: an Escherichia coli product expressing the first EGF-like domain and Saccharomyces cerevisiae and baculovirus/insect-cell-produced proteins containing both EGF-like domains, the latter protein being produced with or without N-glycosylation. Cell donors were P. falciparum-immune adults with no recent history of clinical malaria and recruited from three Senegalese settings with different epidemiological parasite transmission. Each mononuclear-blood-cell preparation was stimulated with a range of concentrations of the three proteins. Most subjects' mononuclear cells were reactive to at least one protein, but significant differences in lymphoproliferation were seen between the settings and within individual cultures depending on the protein source and concentration. Importantly, lymphoproliferation indices correlated inversely with the intensity of P. falciparum malaria transmission. When purified T lymphocytes were cultured in the presence of MSP1(19) plus autologous monocytes, B lymphocytes or a proposed CD1+ dendritic-cell population as costimulatory cells, significant differences were observed depending on the individual's previous exposure to parasites. This study shows that the stimulation of lymphocyte proliferation in vitro with MSP1(19) depends on several factors, including epidemiological conditions and protein preparations.