Medicine (RMH) - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
ItemThe role of neutrophils in immunity to malaria( 2023-07)The global malaria burden continues to remain high with most deaths occurring in young children. Understanding the protective immune mechanisms against malaria is imperative for the development of a highly efficacious vaccine since RTS,S and R21 which are recommended malaria vaccines, have modest efficacy and are limited to young children. Recent reports indicate that neutrophils may potentially play a key role in protective immunity against malaria via antibody-mediated interactions with Fc-gamma receptors (FcgR) expressed on their surface. Additionally, complement proteins may promote parasite clearance via interaction with antibodies. This thesis presents new findings on antibody interactions with complement (C1q) and FcgRs on neutrophils in immunity against severe malaria. It further presents new information on neutrophil phenotypes in response to naturally acquired malaria as well as phenotypes and transcription profiles in controlled human malaria infections (CHMI), and in vitro models. First, cytophilic IgG1 and IgG3 antibody subclasses, and antibodies that can fix C1q, or engage FcgRs to induce phagocytic clearance of merozoites were investigated. Additionally, opsonic phagocytosis of merozoites by neutrophils was quantified. These investigations were carried out on samples from young Papua New Guinean (PNG) children (n = 383) presenting with severe malaria versus uncomplicated malaria. In children presenting with severe malaria, cytophilic antibodies, antibodies that can fix C1q, or engage FcgRs against whole merozoites and neutrophil phagocytosis of merozoites opsonized by serum antibodies were lower than children with uncomplicated malaria. There was a strong association between high antibody responses to merozoites that fix complement or bind FcgRs, and reduced odds of severe disease. Second, cryopreserved neutrophils from children and adults in PNG, were phenotyped by examining receptors relevant to functions and malaria immunity. Neutrophils from children infected with P. vivax malaria (n = 37) had increased CD66b expression compared to uninfected children. Increased statistical power by combining uninfected children (n = 29) and uninfected adults (n = 35) into one group and comparing them with P. vivax and P. falciparum (n = 25) infected children further highlighted increased CD64 expression on neutrophils from malaria infected children compared to uninfected group. Neutrophils from uninfected children had distinct phenotypic differences from uninfected adults. There were also some differences identified in neutrophil phenotypes in males compared to females infected with P. falciparum. In addition, neutrophil phenotypes from uninfected adults and uninfected children varied substantially from neutrophils from malaria-naive adult Australian residents (n = 11), revealing increased CD64, CD54, and CD66b, and decreased CD62L, CD16, and CD32a expression, although CD32a expression in malaria-exposed children did not differ from malaria-naive adults. Neutrophil phenotype changes relevant to effector function and immunity to malaria model were assessed at multiple time points in a controlled human experimental P. falciparum infection trial. The study included 8 healthy Australian adult participants who were treated at day 8 of malaria infection. Neutrophil phenotypic changes occurred as early as day 4, prior to substantial blood-stage parasitemia. There were significant and substantial changes in neutrophil phenotypes following treatment to clear parasitemia and some changes persisted weeks post-treatment. To further understand neutrophil responses to malaria, phenotype changes in response to direct interaction with merozoites in an in vitro model were examined. There were no phenotype changes after neutrophils were incubated with unopsonized merozoites or with merozoites opsonized with serum from malaria-naive donors. In contrast, distinct changes in phenotypes were observed after incubation of neutrophils with merozoites opsonized by naturally-acquired antibodies from children. These changes included upregulated CD66b and downregulated CD64, CD32a, CD16, and CD62L expression. Finally, transcriptional analysis of neutrophil gene sets relevant to functions and clearance of malaria parasites were explored among samples from a controlled P. falciparum experimental infection trial in healthy Australian adults (n = 12) and using in vitro models. CHMI data revealed upregulation of gene sets including phagocytosis pathways and cytokine mediated signalling pathways. In vitro transcriptional analysis of neutrophils incubated with merozoites opsonized by purified IgG from malaria-exposed children showed upregulated gene sets including respiratory burst and cytokine activity compared to neutrophils incubated with merozoites opsonized with IgG from malaria-naive individuals. However, gene set enrichment analysis did not reveal any significant change in phagocytosis gene sets. The data generated in this thesis advances our knowledge on antibody mechanisms involved in immunity to malaria and provides new knowledge on the phenotypes and functions of neutrophils in response to malaria. Given that emerging data are increasingly indicating an important role for neutrophils in naturally-acquired and vaccine-induced immunity, this knowledge will be valuable for informing the development of more efficacious malaria vaccines.
-
ItemThe role of antibody, complement, and innate immune cells in protective immunity to malaria( 2022)Malaria continues to cause high morbidity and mortality worldwide. In Plasmodium falciparum malaria, the membrane of infected erythrocytes (IEs) is modified to express the parasite-derived protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is the main antigenic target contributing to protective immunity through antibody development. Naturally acquired antibodies are key to protection from malaria that can inhibit parasite growth and neutralise parasites, but sterile immunity is never achieved. Therefore, it is important to dissect antibody features that best correlate with protective immunity. These antibody features are driven by the antibody constant (Fc) region that could generate protective immune responses against malaria. My thesis aimed to 1) dissect the role of antibody-mediated complement activation in immunity to Plasmodium falciparum infection, 2) understand in vitro the role of antibody-dependent phagocytosis (ADP) by phagocytic cells in peripheral whole blood in parasite clearance, and 3) assess the role of ADP of IEs by neutrophils and monocytes as a clinical correlate of protection against severe malaria in children and placental malaria in pregnant women from malaria-endemic areas. For this purpose, for the first time, we developed high throughput plate-based assays to simultaneously measure ADP of P. falciparum-IEs by peripheral blood leukocytes using prediluted whole blood. We also used the developed whole blood phagocytosis assays to assess clinical correlates of protection from placental malaria in pregnant women and severe malaria in young children from malaria-endemic regions and infected with P. falciparum. Our results suggested that opsonic antibody levels directed against P. falciparum-IEs promoted antibody-opsonic phagocytosis of IEs by neutrophils and monocytes in a malaria exposure-specific manner. We also showed that opsonising antibodies that promoted ADP of IEs in whole blood mainly targeted PfEMP1 on IEs, although antibodies also target secondary antigens on the IE surface, such as the most abundant band 3 proteins. Next, we showed that antibody-complement interactions are an integral immune mechanism contributing to enhanced phagocytic clearance of P. falciparum-IEs by neutrophils and monocytes from peripheral whole blood. However, complement did not induce antibody-mediated lysis in the presence of plasma from malaria-exposed pregnant women. In malaria-infected pregnant women, opsonising antibody levels against placental binding-IEs were associated with protection against placental malaria. But the same opsonising antibodies that recognised the recombinant DBL-3 domain of VAR2CSA were not protective against placental malaria. Thus, we suggest that opsonising antibody levels that indicate protection against placental malaria are conformation-specific. Last, our results demonstrated that in young children, opsonising antibody levels against EPCR-binding P. falciparum-IEs that promoted ADP by both neutrophils and monocytes were not associated with recovery from severe malaria. Our results suggested that whole blood assays could be exploited to understand antibody and complement interactions during P. falciparum infection that could be translated to low-resource settings to identify antibody correlates of protection in future vaccine design.