MEASURING ANTI-GAMETOCYTE IMMUNITY IN A LONGITUDINAL COHORT OF NATURALLY EXPOSED INDIVIDUALS IN KILIFI COUNTY, KENYA

Abstract

Introduction Over the last two decades, there has been a significant decrease in the global cases of malaria. However, since 2015, there has been an increase in the reported cases that has led to identification of loopholes in the current interventions deployed and aimed at elimination and/or eradication of malaria. Thus, there is increasing need for new interventions especially those targeting the transmissible stages, gametocytes, of the malaria parasite. There is on the other hand a gap in knowledge in regard to these stages as relates to development of interventions such as vaccines. Sequestration of gametocytes in the bone marrow provides an opportunity to understand the acquisition of immunity against surface antigens on gametocyte infected red blood cells (giRBCs). These surface antigens are likely to be involved in bone marrow sequestration and thus immune responses against them can inhibit (1) entry into the bone marrow; (2) retention in the bone marrow; and (3) increase clearance by immune-mediated mechanisms. Here, immune responses to gametocyte-infected erythrocytes were measured and their antibody-mediated complement function determined. Methods A total of 269 samples representing five time points (one person had four time points available) from 54 study volunteers recruited to participate in a Malaria Parasite Clearance (MALPAC) study were randomly selected. Flow cytometry assays were undertaken to determine immune responses and antibody-mediated complement fixation against genetically modified lab-adapted malaria parasite lines, NF54 and PF2004 (asexual infected red blood cells (aiRBCs) and immature giRBCs were utilised in the assays). Surface labelling of the infected red blood cells (iRBCs) was confirmed by immunofluorescence microscopy and a real-time PCR assay for determining gametocyte carriage optimized. vi Results The analysed data from MALPAC study at enrolment showed 42.6% (23/54) of the study participants tested positive for P. falciparum-specific 18s qPCR. The positivity rate fell to 22.2% (12/54) at day 7 and 16.7% (9/54) at day 28 but it rose to 37% (20/54) and 39.9% (21/54) at day 49 and day 84 respectively. Immune responses towards aiRBCs were significantly higher than giRBCs in both NF54 and PF2004 parasite strains (p=0.0019 and p<0.0001 respectively, n=269). More people were IgG positive for PF2004 aiRBCs compared to NF54 aiRBCs while prevalence in positivity for giRBCs did not vary. Responses towards +var NF54 aiRBCs (without var genes modification) were significantly higher compared to -var NF54 aiRBCs (with var genes knocked down) (p<0.0001). However, responses against -var NF54 aiRBCs was significantly higher than NF54 giRBCs (p=0.0292) (n=27). In a multivariable analysis using a mixed effect model, parasite density was found to be predictive of immune responses to aiRBCs and giRBCs. Conclusion Naturally acquired immune responses targeting the immature giRBCs are influenced by parasite density. Moreover, these antibodies have the potential to activate the classical complement pathway to enable clearance of giRBCs.