B. Dubois

Human genetic factors related to susceptibility to mild malaria in Gabon.

Several human genetic factors, including red blood cell polymorphisms (ABO blood group, sickle-cell trait, G6PD deficiency) as well as point mutations in the mannose binding protein (MBP) and in the promoter regions of both the TNF-α and NOS2 genes, influence the severity of disease due to infection with Plasmodium falciparum. We assessed their impact on mild P. falciparum malaria, as part of a longitudinal investigation of clinical, parasitological and immunological parameters in a cohort of 300 Gabonese schoolchildren. We found the following frequencies: blood group O (0.54), sickle-cell trait (0.23), G6PD deficiency (0.09), MBP gene mutations (0.34), TNF-α promoter mutations (at positions −238: 0.17 and −308: 0.22) and NOS2 promoter mutation (0.18). Blood group O or hemoglobin AA were associated with protection against higher parasitemia. Girls with normal G6PD enzyme activity were protected against clinical malaria attacks. In addition, we demonstrated for the first time that the mutation at position −238 of the gene coding for the promoter region of TNF-α was positively correlated with the level of the antibody response specific for epitopes of the antigens MSA-2 and RAP-1 of P. falciparum.

MALARIA CELLULAR IMMUNE RESPONSES IN NEONATES FROM CAMEROON

T cell responses to leucoagglutinin, PPD, and seven Plasmodium falciparum blood stages antigens were investigated in 164 cord blood samples from Cameroonian neonates. In vitro T cell responses were measured by lymphocyte proliferation, and IL‐2, IFN‐γ, and IL‐4 release in the presence of crude schizont extract, purified Pf155/RESA protein, and synthetic peptides from Pf155/RESA. Following culture in presence of leucoagglutinin or PPD, proliferation and cytokine production were very low, as compared to adults from the same area. Interestingly, following stimulation of cord blood lymphocytes by malaria antigens, the percentage of responders and the mean level of positive responses were of the same order than those observed in adults for IL‐2 production, while proliferative and IL‐4 responses were only marginally decreased. Conversely, IFN‐γ production was highly reduced, as compared to adults. Our results demonstrate that prenatal immune priming to malarial antigens is common in this area and that the fetal immune system is able to respond to antigenic stimuli, as cells proliferate and generate cytokines. As cord blood lymphocytes may be induced to differentiate into effector cells producing predominantly Th1 or Th2 cytokines, malaria during pregnancy might direct the functional capacity of fetal T cells to respond to further infection.

Isotypic analysis of maternally transmitted Plasmodium falciparum-specific antibodies in Cameroon, and relationship with risk of P. falciparum infection

In malaria‐endemic areas, infants are relatively protected against malaria infection. Such protection is though to be related principally to the transplacental transfer of maternal antibodies. We measured total and Plasmodium falciparum‐specific IgG (including subclasses), IgM, and IgE antibodies in 154 paired maternal‐cord serum samples from an area of meso‐ to hyperendemic malaria in South Cameroon. Among peripheral mother blood samples, total IgG and IgM were detected in all samples, IgE in all but two. Plasmodium falciparum‐specific IgG were detected in all serum samples, IgM and IgE in < 75% of samples. The prevalence rates of anti‐P. falciparum IgG subclasses varied from 75% to 97%. With the exception of P. falciparum‐sptcifxc IgG, all antibody class and subclass levels were lower in cord blood than in peripheral mother blood. Plasmodium falciparum‐spccific IgGl and IgG3 isotypes were transferred to the offspring more often and more efficiently than IgG2 and IgG4. The detection of total and P. falciparum‐specific IgM and IgE in some cord serum samples demonstrated that fetuses can mount humoral response against malaria parasites. We also determined whether transplacentally acquired antibodies protect against malaria infection by relating the antibody levels at birth to the risk of acquiring P. falciparum infection during the first 6 months of life. Among various classes and subclasses of P. falciparum‐spccific antibodies, only IgG2 were related to a decrease in the risk of acquiring a P. falciparum peripheral blood infection from birth to 6 months of age.

Immune response to Plasmodium falciparum liver stage antigen-1: geographical variations within Central Africa and their relationship with protection from clinical malaria

Two populations of schoolchildren from Gabon and Cameroon were tested in 1995 for their immunological reactivity to synthetic peptides (LSA-Rep, LSA-J and LSA-CTL) from Plasmodium falciparum liver stage antigen-1 (LSA-1). The prevalence and levels of both cellular (lymphocyte proliferation, tumour necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma), and interleukin-10 (IL-10)) and humoral (immunoglobulin G) responses were determined. Protection from clinical malaria, determined after a prospective 1 year study in both sites, was associated with elevated proliferative responses to LSA-Rep and LSA-CTL in the Gabonese children, as well as with higher antibody levels to both schizont extract and LSA-Rep. The prevalence of peptide-stimulated TNF-alpha secretion was higher in the Cameroonian group, but higher levels of antibodies to LSA-Rep and LSA-J were found in the Gabonese children. The immunological differences observed between children in the 2 study sites are discussed in the context of both epidemiological and individual host factors.

Immune response to Plasmodium falciparum antigens in Cameroonian primigravidae: evolution after delivery and during second pregnancy.

Mechanisms responsible for the increase in malaria susceptibility during pregnancy, and in particular during the first pregnancy, have not been elucidated. T and B cell responses to leucoagglutinin, bacille Calmette–Guérin (BCG) and to six Plasmodium falciparum antigens were longitudinally investigated in 33 pregnant women during their first pregnancy, after delivery, and during second pregnancy. Parasitological data obtained from the same women during and after the first pregnancy demonstrated the higher risk of P. falciparum infection during this pregnancy. Plasma levels of antibodies to Pf155/RESA were lower during pregnancy than after delivery. Conversely, antibodies to P. falciparum asexual blood stages were higher during pregnancy than after delivery, suggesting that during pregnancy the regulation of antibody production may be variously impaired depending upon the antigens. The most striking finding of the present study is the impairment of the IL‐2 cellular response during the first pregnancy. Conversely, proliferative responses, as well as IL‐4 and interferon‐gamma (IFN‐γ) responses, were either unaffected or moderately enhanced. No difference in humoral and cellular responses was observed between first and second pregnancy. The impairment of the IL‐2 responses involved the response to malaria peptides and proteins, as well as the response to non‐malarial antigens and to the mitogen leucoagglutinin. Thus, the alteration of malaria immunity might rather fall into the general frame of the depression of cellular immunity during pregnancy than involve a specific malaria phenomenon.

In vitro and in vivo potentiation of chloroquine against malaria parasites by an enantiomer of amlodipine.

A combination of chloroquine and amlodipine, a derivative of 1,4-dihydropyridine calcium channel blocker, was tested against Plasmodium falciparum in vitro and P. yoelii in mice. The dextrorotary enantiomer of amlodipine, practically devoid of calcium channel blocking action, increased chloroquine accumulation inside the infected mouse erythrocytes and potentiated chloroquine action against the resistant strains of P. falciparum in vitro and P. yoelii in mice. Unlike the racemate, the dextrorotary amlodipine was not toxic to the host animal, even at the highest dose of 250 mg/kg. No potentiating effect was noted in the chloroquine-susceptible strains of P. falciparum. The results of this study indicate that chloroquine potentiation of amlodipine is probably independent of calcium channels and that a combination therapy of the dextrorotary enantiomer of amlodipine and chloroquine might be a potentially useful therapeutic strategy against chloroquine-resistant falciparum malaria.

Anti‐malaria antibody‐producing B cell frequencies in adults after a Plasmodium falciparum outbreak in Madagascar

The central highlands of Madagascar offer a unique opportunity to explore the malaria immune memory, as the last murderous epidemic in the study area occurred 8 years ago. Quantification of the circulating memory B lymphocytes reacting to Plasmodium falciparum was assessed among 14 Madagascans by using a limiting dilution assay, applied to the EL4 culture system, which leads to activation, proliferation and differentia lion into antibody‐secreting cells (ASC) of most peripheral B cells. This system allowed us to observe, without any malaria‐specific restimulalion, a geometric mean frequency of one anti‐P. falciparum ASC among 2992 circulating B cells, except for one Madagascan who did not have any detectable ASC. A geometric mean frequency of one anti‐P. falciparum ASC among 1403 was obtained for six malaria hyperimmune Cameroonians, but conversely, no anti‐malaria ASC was detected in the blood of six malaria non‐immune French control subjects. Anti‐P. falciparum ASC frequencies and serum specific antibodies were strongly related. Our results indicate that anti‐malaria ASC are still present in peripheral blood of Madagascan subjects, who have not been exposed to P. falciparum for several years. These responder B cells reflect the malaria B cell memory acquired during the last epidemic.

Selected P. falciparum specific immune responses are maintained in AIDS adults in Burkina Faso

In tropical areas where Plasmodium falciparum malaria is endemic, co‐infection with HIV‐1 does not lead to a worsening of malaria, raising questions about the immunological interactions between both infections. Alterations of immune response to malaria during HIV‐1 infection was investigated in the town of Bobo Dioulasso, Burkina Faso. Sixty‐six adults were enrolled, including 37 HIV‐1 positive subjects with <250 CD4+ cells/μl and clinical AIDS, and 29 HIV‐1 negative healthy subjects. In vitro lymphocyte proliferation and cytokine (IFN‐γ, IL‐2 and IL‐4) production were assessed in isolated mononuclear cells (PBMC) in presence of PHA, PPD or three malarial antigens: the baculovirus‐expressed protein from P. falciparum Merozoite Surface Protein‐1, a P. falciparum in vitro culture and a crude schizont extract. Compared with healthy subjects, AIDS patients presented with decreased levels of cell proliferation and of IFN‐γ and IL‐2 production, in response to all antigens except the schizont extract. Similar levels of IL‐4 production were obtained in both groups. Mitogenic stimulation of whole blood cultures was also performed, and revealed similar trends in cytokine production as in PBMC cultures. These results show that some components of the specific human immune responses to falciparum parasites may not be modified during AIDS, in spite of the strong cellular alterations induced by HIV, namely the decrease of the CD4+ lymphocyte subset.

Quantification of antibody‐secreting lymphocytes that react with Pf155/RESA from Plasmodium falciparum: an ELISPOT assay for field studies

We have adapted the enzyme‐linked immunospot assay (ELISPOT) to enumerate the cells from Plasmodium falciparum‐primed donors that produce IgG in vitro in response to malaria antigens. In vitro activation of cell cultures with two synthetic peptides (EENVEHDA)4, and (LGRSGGDIIKMQTL) corresponding to immunodominant T cell epitopes of the ring‐infected erythrocyte surface antigen (Pf 155/RESA) gave specific antibody‐secreting cells (ASC) in five and six of the 15 P. falciparum‐primed donors from Cameroon. Antibodies produced after a stimulation by synthetic peptides reacted also with total parasite proteins. However, crude P. falciparum antigen did not trigger a higher number of cells than did synthetic peptides. The absence of significant relation between the presence of sera antibodies and in vitro ASC against the same peptide suggests that the kinetics of circulating primed lymphocytes and antibodies are different. We evaluated 0–04–0–29% of peripheral blood B cells to be the frequency of memory cells specific to a single Pfl 55/RESA epitope in these donors. This study suggests that the ELISPOT assay should permit the analysis of B cell responses to malarial antigens at the single‐cell level and its applicability to epidemiological field studies. This assay should be well suited to the identification of T helper epitopes capable of inducing the production of antibodies by human B cells, and will constitute an important tool for the selection of immunogens to be included in a subunit vaccine.

Temporal variations in immune responses to conserved regions of Plasmodium falciparum merozoite surface proteins related to the severity of a prior malaria episode in Gabonese children

We measured cellular and humoral responses to conserved regions of Plasmodium falciparum merozoite surface proteins 1 and 2 (MSP-1 and MSP-2) at different times during and after acute infection in matched groups of Gabonese children who presented with either mild or severe malaria. We used an MSP-1(19) recombinant protein and peptides corresponding to conserved epitopes in MSP-1 and MSP-2 N- and C-terminal regions. The prevalences of peptide-induced cell-mediated responses were maximal in both groups when they were healthy, but were consistently higher in the mild malaria group, whereas peptide-specific antibody responses were consistently highest in the severe malaria group. Recombinant MSP-1(19) protein-specific antibody levels in the 2 groups were similar both prior to and 1 month post-treatment but declined later when the children were healthy and parasite-free, to a significantly lower level in those admitted with severe malaria, reflecting the profile of the predominant MSP-1(19)-specific immunoglobulin G1 isotype. This finding implies a defect in the ability of children with a history of severe malaria to maintain an antibody response putatively associated with immunity to P. falciparum malaria.