Komi Gbedande

L'objectif général de ce travail était d'étudier des mécanismes immunologiques de la mise en place d'une mémoire spécifique et protectrice au candidat vaccin contre le paludisme associé à la grossesse (PAG). Deux projets complémentaires (STOPPAM et PLACMALVAC) ont été mis en place par des consortiums européens et africains en vue du développement d'un vaccin contre le PAG. Le projet STOPPAM est un suivi de cohortes de 1037 femmes enceintes et de 222 nouveau-nés avec comme objectif de caractériser les parasites de P. falciparum pour identifier un candidat vaccin contre le PAG et aussi d'étudier les conséquences du PAG sur la santé de la mère et du nouveau-né. L'objectif du projet PLACMALVAC est de réaliser un essai clinique de phase I chez des femmes volontaires saines et en parallèle préparer un essai clinique de phase II par une étude de faisabilité avec un suivi de cohorte de 50 femmes enceintes primigestes. Dans une première partie, nous avons étudié les conséquences du PAG sur les profils de réponses immunologiques des nouveau-nés de 0 à 12 mois. Deux études ont été ainsi réalisées chez les enfants issus des mères présentant différentes histoires palustres au cours de la grossesse et donc des contacts in utero avec des antigènes palustres à différents moments de la grossesse. Nous avons étudiés l'évolution de 0 à 12 mois : des réponses cytokiniques à des ligands de TLR ainsi que les fréquences des lymphocytes CD4, CD8, Treg, NK et NKT. Nos résultats montrent que ce sont les infections à l'accouchement qui pourraient modifier et altérer l'activation du système immunitaire inné et adaptative. Cette altération influencerait la mise en place d'une immunité protectrice chez les nouveau-nés pendant leurs premières années de vie. La deuxième partie de notre travail s'est intéressée à l'évaluation des réponses immunologiques des cellules T et B mémoires spécifiques du candidat vaccin VAR2CSA. Deux études ont été réalisées pour évaluer: i) les réponses des cellules T mémoires chez 2 sous groupes de femmes enceintes du projet STOPPAM prélevées au début de la grossesse et à l'accouchement. Nous avons caractérisé des réponses des cellules T mémoires spécifique du VAR2CSA-DBL5 via la fréquence des cellules T sécrétrices de l'IFN-g et la mesure de 5 cytokines dans les surnageants de culture après stimulation des cellules mononucléées. Nos résultats montrent que l'infection palustre au cours de la grossesse est associée à une plus forte production d'IFN-g à l'accouchement en réponse à la stimulation in vitro avec le VAR2CSA-DBL5 et la PHA. ii) les réponses des cellules T et B mémoires chez les primigestes du projet PLACMALVAC afin d'étudier la construction naturelle de la mémoire spécifique au VAR2CSA (ID1-DBL2-ID2a) durant la première grossesse. Les cellules mononucléées ont été isolées et congelées à partir des prélèvements du sang veineux collectés à 4 différents moments au cours du suivi (premier et cinquième mois de grossesse, à l'accouchement et 6 mois après accouchement). Nous avons quantifié les cellules B mémoires spécifiques de l'antigène vaccinal VAR2CSA et aussi mesuré les cytokines dans les surnageants de culture après stimulation des cellules. L'infection palustre au cours de la grossesse est associée à une augmentation de la fréquence des cellules B mémoires. Ces résultats suggèrent, l'acquisition via des infections naturelles au cours de la première grossesse, d'une mémoire anti-VAR2CSA protectrice pour la prévention du PAG. ? L'ensemble des travaux réalisés au cours de cette thèse a permis de mieux comprendre les mécanismes immunologiques impliqués dans la plus grande susceptibilité des nouveau-nés aux infections liées au PAG pendant l'enfance et aussi l'acquisition naturelle d'une mémoire immunologique contre le PAG spécifique du candidat vaccin actuel VAR2CSA (ID1-DBL2-ID2a) qui est en cours d'essai clinique de phase I au Bénin et en Allemagne.

Vaccines to persistent parasite infections have been challenging, and current iterations lack long-term protection. Cytomegalovirus ( CMV) chronic vaccine vectors drive protection against SIV, tuberculosis and liver-stage malaria correlated with antigen-specific CD8 T cells with a Tem phenotype. This phenotype is likely driven by a combination of antigen-specific and innate adjuvanting effects of the vector, though these mechanisms are less well understood. Sterilizing immunity from live Plasmodium chabaudi vaccination lasts less than 200 days. While P. chabaudi -specific antibody levels remain stable after vaccination, the decay of parasite-specific T cells correlates with loss of challenge protection. Therefore, we enlisted murine CMV as a booster strategy to prolong T cell responses against malaria. To study induced T cell responses, we included P. chabaudi MSP-1 epitope B5 (MCMV-B5). We found that MCMV vector alone significantly protected against a challenge P. chabaudi infection 40-60 days later, and that MCMV-B5 was able to make B5-specific Teff, in addition to previously-reported Tem, that survive to the challenge timepoint. Used as a booster, MCMV-B5 prolonged protection from heterologous infection beyond day 200, and increased B5 TCR Tg T cell numbers, including both a highly-differentiated Tem phenotype and Teff, both previously reported to protect. B5 epitope expression was responsible for maintenance of Th1 and Tfh B5 T cells. In addition, the MCMV vector had adjuvant properties, contributing non-specifically through prolonged stimulation of IFN-γ. In vivo neutralization of IFN-γ, but not IL-12 and IL-18, late in the course of MCMV, led to loss of the adjuvant effect. Mechanistically, sustained IFN-γ from MCMV increased CD8α ⁺ dendritic cell numbers, and led to increased IL-12 production upon Plasmodium challenge. In addition, neutralization of IFN-γ before challenge reduced the polyclonal Teff response to challenge. Our findings suggest that, as protective epitopes are defined, an MCMV vectored booster can prolong protection through the innate effects of IFN-γ. Authors’ summary: Malaria represents a challenging target for vaccination. This is in part because of the requirement for CD4 T cell immunity in addition to the standard B cell responses current vaccines induce. However, human malaria vaccine approaches thus far have limited longevity of protection due to decay of T cell responses. This includes the most advanced malaria vaccine, a virus-like particle expressing one recombinant liver-stage antigen (RTS,S), and liver-stage parasites attenuated by radiation (PfSPZ), as well as live vaccination with drug-treatment. Our work seeks to prolong this protection using MCMV, a promising vaccine vector known to promote CD8 T cell responses. We observed that boosting the live malaria vaccine with MCMV including a Plasmodium antigen led to longer protection from P. chabaudi parasitemia, and can be used to promote maintenance of antigen-specific CD4 T cells. In investigating the mechanisms of the MCMV booster, we found that the cytokine IFN-γ is required for prolonged protection and enhances priming of the innate immune system for prolonged protection from malaria. Our research informs both the quest for a longer-lived malaria vaccine and that to understand mechanisms of protection from persistent infection.

CD4 T cells are required, along with antibodies, for complete protection from blood-stage infection with Plasmodium spp., which cause malaria. Without continuous exposure, as on emigration of people from endemic areas, protection from malaria decays.

CD4 T cells are required, along with antibodies, for complete protection from blood-stage infection with Plasmodium spp ., which cause malaria. Without continuous exposure, as on emigration of people from endemic areas, protection from malaria decays. As in other persistent infections, low-level P. chabaudi protects the host from re-infection at two months post-infection, a phenomenon termed premunition. Premunition is correlated with T cell responses, rather than antibody levels. We previously showed that while both effector T cells (Teff) and memory T cells (Tmem) are present after infection, Teff protect better than Tmem. Here we studied T cell kinetics post-infection by labelling dividing Ifng ⁺ T cells with BrdU in infected Ifng- reporter mice. A large drop in specific T cell numbers and Ifng ⁺ cells upon clearance of parasite suggest a mechanism for decay of protection. Although protection decays, CD4 Tmem persist, including a highly-differentiated CD27 ^- Effector Memory (Tem) subset that maintains some Ifng expression. In addition, pre-treatment of chronically-infected animals with neutralizing antibody to IFN-γ, or clodronate liposomes before re-infection decrease premonition supporting a role for Th1-type immunity to re-infection. A pulse/chase experiment comparing chronically infected to treated animals showed that recently divided Ifng ⁺ T cells, particularly IFN-γ ⁺ TNF ⁺ IL-2 ^- T cells, are promoted by persistent infection. These data suggest that low-level persistent infection reduces CD4 ⁺ Tmem survival and multi-functional Teff but promotes IFN-γ ⁺ TNF ⁺ IL-2 ^- Late Effector Memory and Terminally Differentiated Effector T cells and prolongs immunity.

Malaria, blood-borne filarial worms and intestinal parasites are all endemic in Gabon. This geographical co-distribution leads to polyparasitism and, consequently, the possibility of immune-mediated interactions among different parasite species. Intestinal protozoa and helminths could modulate antimalarial immunity, for example, thereby potentially increasing or reducing susceptibility to malaria. The aim of the study was to compare the cytokine levels and cytokine ratios according to parasitic profiles of the population to determine the potential role of co-endemic parasites in the malaria susceptibility of populations. Blood and stool samples were collected during cross-sectional surveys in five provinces of Gabon. Parasitological diagnosis was performed to detect plasmodial parasites, Loa loa, Mansonella perstans, intestinal helminths (STHs) and protozoan parasites. Nested PCR was used to detect submicroscopic plasmodial infection in individuals with negative blood smears. A cytometric bead array was used to quantify interleukin (IL)-6, IL-10 and tumour necrosis factor (TNF)-α in the plasma of subjects with different parasitological profiles. Median IL-6 and IL-10 levels and the median IL-10/TNF-α ratio were all significantly higher among individuals with Plasmodium (P.) falciparum infection than among other participants (pP. falciparum-intestinal protozoa co-infection (p = 0.04), respectively. IL-6 (r = -0.37; PPPP. falciparum/intestinal protozoa co-infections were associated with an enhanced IL-10 response. Immunity against malaria could differ according to age and carriage of other parasites. Helminths and intestinal protozoa can play a role in the high susceptibility to malaria currently observed in some areas of Gabon, but further investigations are necessary.

Introduction: Neonatal sepsis outreaches all causes of neonatal mortality worldwide and remains a major societal burden in low and middle income countries. In addition to limited resources, endemic morbidities, such as malaria and prematurity, predispose neonates and infants to invasive infection by altering neonatal immune response to pathogens. Nevertheless, thoughtful epidemiological, diagnostic and immunological evaluation of neonatal sepsis and the impact of gestational malaria have never been performed. Methods and analysis: A prospective longitudinal multicentre follow-up of 580 infants from birth to 3 months of age in urban and suburban Benin will be performed. At delivery, and every other week, all children will be examined and clinically evaluated for occurrence of sepsis. At delivery, cord blood systematic analysis of selected plasma and transcriptomic biomarkers (procalcitonin, interleukin (IL)-6, IL-10, IP10, CD74 and CX3CR1 ) associated with sepsis pathophysiology will be evaluated in all live births as well as during the follow-up, and when sepsis will be suspected. In addition, whole blood response to selected innate stimuli and extensive peripheral blood mononuclear cells phenotypic characterisation will be performed. Reference intervals specific to sub-Saharan neonates will be determined from this cohort and biomarkers performances for neonatal sepsis diagnosis and prognosis tested. Ethics and dissemination: Ethical approval has been obtained from the Comité d’Ethique de la Recherche – Institut des Sciences Biomédicales Appliquées (CER-ISBA 85 - 5 April 2016, extended on 3 February 2017). Results will be disseminated through international presentations at scientific meetings and publications in peer-reviewed journals. Trial registration number: ClinicalTrials.gov registration number: NCT03780712 .

Malaria during pregnancy is a major cause of maternal morbidity as well as fetal and neonatal mortality. Previous studies including our own suggested that placental and peripheral cytokines and chemokines levels measured at delivery can be used as biomarkers for pregnancy outcomes. However, the timing of malaria infection during pregnancy matters and these studies lack in addressing the effect of different cytokines in peripheral blood plasma samples taken at early- mid-pregnancy and at delivery. Here we aimed to investigate whether peripheral plasma cytokine levels were associated with pregnancy outcomes in a cohort of 400 Beninese pregnant women. Using a high sensitivity cytometry-based method, we quantified the levels of IL-4, IL-5, IL-10, IL-12p70 and IFN-γ in peripheral plasma samples taken at two time-points during pregnancy and at delivery in various groups of pregnant women identified with P. falciparum infection, with anaemia, with preterm births, or those giving birth to babies small for gestational age. We found that, consistently at all time-points, elevated levels of IL-10 were strongly and significantly associated with P. falciparum infection, whilst the levels of IFN-γ at inclusion and delivery were weakly but also significantly associated. Low levels of IL-5 at delivery were associated with a greater risk of both pre-term births and small for gestational age babies. The immunosuppressive effects of IL-10 likely affect the overall cytokine equilibrium during pregnancy in women harbouring P. falciparum infections. Our findings highlight the peripheral signature of pregnancy outcomes and strengthen the idea of using cytokines as diagnostic or prognostic markers.

Plasmodium falciparum-infected erythrocytes bind to specific endothelial cell receptors via members of the PfEMP1 family exported onto the erythrocyte surface. These interactions are mediated by different types of cysteine-rich interdomain region (CIDR) domains found in the N-terminal region of all PfEMP1. CIDRα1 domains bind endothelial protein C receptor (EPCR), CIDRα2–6 domains bind CD36, whereas the receptor specificity of CIDRβ/γ/δ domains is unknown. In this study, we investigated the level of immunoglobulin (Ig)G targeting the different types of PfEMP1 CIDR during the first year of life. We used plasma collected longitudinally from children of pregnant women who had been followed closely through pregnancy. Antibodies to CIDRα1 domains were more frequent in cord blood compared with antibodies to CIDRα2–6 domains. Higher IgG levels to EPCR-binding CIDRα1 variants positively correlated with the timing of first infections. Antibodies to all PfEMP1 types declined at similar rates to the point of disappearance over the first 6 months of life. At 12 months, children had acquired antibody to all types of CIDR domains, mostly in children with documented P falciparum infections. These observations agree with the notion that the timing and phenotype of first P falciparum infections in life are influenced by the immune status of the mother.

Plasmodium falciparum infection and malaria remain a risk for millions of children and pregnant women. Here, we seek to integrate knowledge of mouse and human T helper cell (Th) responses to blood‐stage Plasmodium infection to understand their contribution to protection and pathology. Although there is no complete Th subset differentiation, the adaptive response occurs in two phases in non‐lethal rodent Plasmodium infection, coordinated by Th cells. In short, cellular immune responses limit the peak of parasitemia during the first phase; in the second phase, humoral immunity from T cell–dependent germinal centers is critical for complete clearance of rapidly changing parasite. A strong IFN‐γ response kills parasite, but an excess of TNF compared with regulatory cytokines (IL‐10, TGF‐β) can cause immunopathology. This common pathway for pathology is associated with anemia, cerebral malaria, and placental malaria. These two phases can be used to both understand how the host responds to rapidly growing parasite and how it attempts to control immunopathology and variation. This dual nature of T cell immunity to Plasmodium is discussed, with particular reference to the protective nature of the continuous generation of effector T cells, and the unique contribution of effector memory T cells.