Christian Roussilhon

Long-term clinical protection from falciparum malaria is strongly associated with IgG3 antibodies to merozoite surface protein 3.

Background Surrogate markers of protective immunity to malaria in humans are needed to rationalize malaria vaccine discovery and development. In an effort to identify such markers, and thereby provide a clue to the complex equation malaria vaccine development is facing, we investigated the relationship between protection acquired through exposure in the field with naturally occurring immune responses (i.e., induced by the parasite) to molecules that are considered as valuable vaccine candidates. Methods and Findings We analyzed, under comparative conditions, the antibody responses of each of six isotypes to five leading malaria vaccine candidates in relation to protection acquired by exposure to natural challenges in 217 of the 247 inhabitants of the African village of Dielmo, Senegal (96 children and 121 older adolescents and adults). The status of susceptibility or resistance to malaria was determined by active case detection performed daily by medical doctors over 6 y from a unique follow-up study of this village. Of the 30 immune responses measured, only one, antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3), was strongly associated with clinical protection against malaria in all age groups, i.e., independently of age. This immunological parameter had a higher statistical significance than the sickle cell trait, the strongest factor of protection known against Plasmodium falciparum. A single determination of antibody was significantly associated with the clinical outcome over six consecutive years in children submitted to massive natural parasite challenges by mosquitoes (over three parasite inoculations per week). Finally, the target epitopes of these antibodies were found to be fully conserved. Conclusions Since anti-MSP3 IgG3 antibodies can naturally develop along with protection against P. falciparum infection in young children, our results provide the encouraging indication that these antibodies should be possible to elicit by vaccination early in life. Since these antibodies have been found to achieve parasite killing under in vitro and in vivo conditions, and since they can be readily elicited by immunisation in naïve volunteers, our immunoepidemiological findings support the further development of MSP3-based vaccine formulations.

Association between Protection against Clinical Malaria and Antibodies to Merozoite Surface Antigens in an Area of Hyperendemicity in Myanmar: Complementarity between Responses to Merozoite Surface Protein 3 and the 220-Kilodalton Glutamate-Rich Protein

ABSTRACT We performed a longitudinal clinical and parasitological follow-up study in OoDo, a village in southeast Asia in which malaria is hyperendemic, in order to assess the association between protection against malaria attacks and antibodies to three currently evaluated vaccine candidates, merozoite surface protein 1 (MSP1), MSP3, and the 220-kDa glutamate-rich protein (GLURP) from Plasmodium falciparum. Our results showed that the levels of cytophilic immunoglobulin G3 (IgG3) antibodies against conserved regions of MSP3 and GLURP were significantly correlated with protection against clinical P. falciparum malaria. In contrast, the levels of noncytophilic IgG4 antibodies against GLURP increased with the number of malaria attacks. Furthermore, we observed a complementary effect of the MSP3- and GLURP-specific IgG3 antibodies in relation to malaria protection. In the individuals that did not respond to one of the antigens, a strong response to the other antigen was consistently detected and was associated with protection, suggesting that induction of antibodies against both MSP3 and GLURP could be important for the development of protective immunity. The complementarity of the responses to the two main targets of antibody-dependent cellular inhibition identified to date provides the first rational basis for combining these two antigens in a hybrid vaccine formulation.

Increased susceptibility to malaria during the early postpartum period.

BACKGROUND Pregnancy is associated with increased susceptibility to malaria. It is generally agreed that this increased risk ends with delivery, but the possible persistence of increased susceptibility during the puerperium had not been investigated. METHODS From June 1, 1990, to December 31, 1998, we monitored exposure to malaria, parasitemia, and morbidity among the residents of a village in Senegal in which the rate of transmission of malaria was high. In this population we analyzed 71 pregnancies in 38 women from the year before conception and through one year after delivery. RESULTS Among the 38 women, there were 58 episodes of clinical Plasmodium falciparum malaria during 61,081 person-days of observation. The incidence of malaria was 20.2 episodes per 1000 person-months during the year preceding conception and 12.0 episodes per 1000 person-months during the period from 91 to 365 days after delivery. The incidence of episodes of malaria increased significantly during the second and third trimesters of pregnancy and reached a maximum of 75.1 episodes per 1000 person-months during the first 60 days after delivery. The adjusted relative risk of an episode of malaria was 4.1 (95 percent confidence interval, 1.8 to 9.5) during the first 60 days post partum, as compared with the year preceding pregnancy. The duration of fever during the episodes of malaria was longer and the prevalence and density of asymptomatic malarial parasitemia were significantly higher during pregnancy and the early postpartum period than during the other periods. CONCLUSIONS Among women who live in areas with high rates of transmission of malaria, the susceptibility to malaria is highest during the second and third trimesters of pregnancy and the early postpartum period.

The rise and fall of malaria in a west African rural community, Dielmo, Senegal, from 1990 to 2012: a 22 year longitudinal study

BACKGROUND A better understanding of the effect of malaria control interventions on vector and parasite populations, acquired immunity, and burden of the disease is needed to guide strategies to eliminate malaria from highly endemic areas. We monitored and analysed the changes in malaria epidemiology in a village community in Senegal, west Africa, over 22 years. METHODS Between 1990 and 2012, we did a prospective longitudinal study of the inhabitants of Dielmo, Senegal, to identify all episodes of fever and investigate the relation between malaria host, vector, and parasite. Our study included daily medical surveillance with systematic parasite detection in individuals with fever. We measured parasite prevalence four times a year with cross-sectional surveys. We monitored malaria transmission monthly with night collection of mosquitoes. Malaria treatment changed over the years, from quinine (1990-94), to chloroquine (1995-2003), amodiaquine plus sulfadoxine-pyrimethamine (2003-06), and finally artesunate plus amodiaquine (2006-12). Insecticide-treated nets (ITNs) were introduced in 2008. FINDINGS We monitored 776 villagers aged 0-101 years for 2 378 150 person-days of follow-up. Entomological inoculation rate ranged from 142·5 infected bites per person per year in 1990 to 482·6 in 2000, and 7·6 in 2012. Parasite prevalence in children declined from 87% in 1990 to 0·3 % in 2012. In adults, it declined from 58% to 0·3%. We recorded 23 546 fever episodes during the study, including 8243 clinical attacks caused by Plasmodium falciparum, 290 by Plasmodium malariae, and 219 by Plasmodium ovale. Three deaths were directly attributable to malaria, and two to severe adverse events of antimalarial drugs. The incidence of malaria attacks ranged from 1·50 attacks per person-year in 1990 to 2·63 in 2000, and to only 0·046 in 2012. The greatest changes were associated with the replacement of chloroquine and the introduction of ITNs. INTERPRETATION Malaria control policies combining prompt treatment of clinical attacks and deployment of ITNs can nearly eliminate parasite carriage and greatly reduce the burden of malaria in populations exposed to intense perennial malaria transmission. The choice of drugs seems crucial. Rapid decline of clinical immunity allows rapid detection and treatment of novel infections and thus has a key role in sustaining effectiveness of combining artemisinin-based combination therapy and ITNs despite increasing pyrethroid resistance. FUNDING Pasteur Institutes of Dakar and Paris, Institut de Recherche pour le Développement, and French Ministry of Cooperation.

Reduced Immune Activation and T Cell Apoptosis in Human Immunodeficiency Virus Type 2 Compared with Type 1: Correlation of T Cell Apoptosis with β2 Microglobulin Concentration and Disease Evolution

This study analyzes the degree of immune activation and characterizes apoptosis in lymphocytes from healthy West African donors or patients infected with human immunodeficiency virus (HIV)-1 or -2. The lower decline of CD4 T cells in HIV-2- compared with HIV-1-infected donors is associated with lower levels of immune activation, evaluated by HLA-DR expression on lymphocytes and sera concentrations of IgG and beta2 microglobulin (beta2m). Ex vivo apoptosis was found in both infections in all lymphocyte subsets, including CD4 and CD8 T cells, as well as B cells, but was lower in HIV-2 than in HIV-1 infection. Interestingly, high correlations were found in HIV-2- and HIV-1-infected donors between the level of CD4 T cell apoptosis and beta2m concentration and progression of the disease. These observations support the hypothesis that long-term activation of the immune system, weaker in HIV-2 infection, significantly contributes to T cell deletion and disease evolution.

Identification of a Conserved Region of Plasmodium falciparum MSP3 Targeted by Biologically Active Antibodies to Improve Vaccine Design

Merozoite surface protein 3 (MSP3) is a target of antibody-dependent cellular inhibition (ADCI), a protective mechanism against Plasmodium falciparum malaria. From the C-terminal half of the molecule, 6 overlapping peptides were chosen to characterize human immune responses. Each peptide defined at least 1 non-cross-reactive B cell epitope. Distinct patterns of antibody responses, by level and IgG subclass distribution, were observed in inhabitants of a malaria-endemic area. Antibodies affinity purified toward each peptide differed in their functional capacity to mediate parasite killing in ADCI assays: 3 of 6 overlapping peptides had a major inhibitory effect on parasite growth. This result was confirmed by the passive transfer of anti-MSP3 antibodies in vivo in a P. falciparum mouse model. T helper cell epitopes were identified in each peptide. Antigenicity and functional assays identified a 70-amino acid conserved domain of MSP3 as a target of biologically active antibodies to be included in future vaccine constructs based on MSP3.

Human TcRγδ+ lymphocyte response on primary exposure to Plasmodium falciparum

In 29 patients experiencing their first P. falciparum malarial attack. blood levels of TcRγδ+ lymphocytes were studied from the onset of infection to up to 6‐9 months later. Blood TcRγδ+ lymphocytes, revealed using the TcRδ1 monoclonal antibody (MoAb) were increased both in absolute and relative numbers. Alterations lasted for up to 3‐4 months following the attack. A TiγA/ BB3 reactive Vγ/9 subset was preferentially amplified, in vitro, TcRγδ+ lymphocytes from both malaria‐sensitized and unprimed donors responded to P. falciparum schizont extract (PhSE). PFSE‐stimulated polyclonal T cell lines consisted principally in TcRγδ+ cells with a TiγAd+/BB‐3+ phenotype. Several TcRγδ+ T cell clones obtained from patients recovering from acute malarial attack were maintained in the presence of PFSE and autologous irradiated PBL. They belong to the Vγ9 subset. In long‐term cultures. TcRγδ+ clones progressively lost their capacity to react to PFSE antigen while they were able to proliferate and to exert cytotoxic activity in response to autologous TcRγδ+, PFSE‐specific T lymphocyte clones. This suggests that regulatory interactions occur between activated TcRγδ+ and TcRγβ+ cells generated by P. falciparum. Sequential variations in blood TcRγδ+ and TcRγβ+ lymphocyte levels after primary exposure to P. falciparum suggest that such regulatory interactions may occur in vivo.

Acute Plasmodium falciparum infection is associated with increased percentages of apoptotic cells

The impact of acute malaria infection on the level of spontaneous apoptosis, i.e., the percentage of apoptotic cells detectable in lymphocytes cultured without any exogenous stimulus for 3 days in vitro, was evaluated. Quantitation of apoptosis was performed by staining of lymphocyte nuclei with propidium iodide and analysis of the fluorescence by cytometry. The mean apoptosis of 23 HIV-negative patients (15 Africans and 8 Europeans) determined during a confirmed Plasmodium falciparum attack was 27.2% (95% confidence interval (CI) = 23.5-30.7%) i.e., 2.2 times the mean level found in 49 controls (12.4%, CI = 11.1-13.6). These controls included age- and sex-matched Africans (n = 37) and Europeans (n = 12) differing only by their previous level of exposure to P. falciparum. Naive (European) as well as previously exposed (African) subjects showed dramatically elevated levels of spontaneous apoptosis during the malaria attack (mean = 22.5%, CI = 20.7-24.4 for Europeans; mean = 29.7%, CI = 24.6-34.7 for Africans). Such unusually raised levels were observed for at least 1.5 months and were probably detectable for longer periods as suggested by the fact that the mean level of spontaneous apoptosis in healthy Africans was basically higher (13.8%, CI = 12.5-15) than the one found in healthy Europeans (8.2%, CI = 6.3-10.1) (P = 0.0001). Selective immunomagnetic cell isolations carried out immediately before apoptosis quantitation showed that this process affected not only the alpha beta T cells (CD4+ T cells as well as CD8+ T cells) but also the gamma delta T cells and the B-lymphocyte subset.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis modulation in mononuclear cells recovered from individuals exposed to Plasmodium falciparum infection.

In endemic areas, asymptomatic infection by the malaria parasite Plasmodium falciparum was found associated with elevated percentages of human hosts mononuclear cell spontaneous in‐vitro apoptosis. In Dielmo, a village where malaria is holoendemic, apoptosis was age‐and parasite‐dependent. In‐vitro exposure of peripheral blood mononuclear cells (PBMC) to the parasite extract induced a marked increase in the mononuclear cell membrane expression of functional CD95 antigen: a 3‐h exposure of the mononuclear cells to anti‐CD95 antibodies led to a detectable increase in the mean percentage of apoptotic nuclei found in the cultures carried out in the presence of P. falciparum extracts compared to control cultures. IL‐2, IL‐4, IL‐6 and IL‐10 promoted the viability of PBMC in cultures while IL‐1α or IFN‐γ had no obvious impact and TNFα gave conflicting results. IL‐2 was the most efficient cytokine at rescuing PBMC from cell death and this effect was associated with a strong increase in T cell activation. In contrast, IL‐4 and IL‐10 had no such effect on T cell activation, hence they acted as survival factors and not through their mitogenic activity. Taken together, these different observations suggested that the levels of in‐vitro apoptosis observed were not only associated with parasite infection, but also potentially modulated by the human host through different pathways.

Plasmodium falciparum Merozoite Surface Protein 6 Displays Multiple Targets for Naturally Occurring Antibodies That Mediate Monocyte-Dependent Parasite Killing

ABSTRACT Plasmodium falciparum MSP6 is a merozoite surface antigen that shows organization and sequence homologies similar to those of MSP3. Within its C-terminus conserved region, it presents some epitopes that are cross-reactive with MSP3 and others that are not, both being targets of naturally occurring antibodies that block the P. falciparum erythrocytic cycle in cooperation with monocytes.