The Expression Of Recombinant Plasmodium Falciparum Sexual-Stage Proteins And Assessment Of Naturally Acquired Transmission-Blocking Immunity

Abstract

Persons residing in regions endemic to malaria have been shown to develop antibodies against gametocytes, which could subsequently prevent the progression of the parasite within the mosquito vector. The acquired immune ability to affect gametocyte infectivity within the mosquito vector and in-turn block transmission of parasites to the vector is termed as transmission-blocking immunity. Various gametocyte-specific antigens of Plasmodium falciparum have been characterised and evaluated as candidates for a possible transmission-blocking vaccine. This study aimed to express gametocyte-specific surface antigens with the cell -free wheat germ system and mammalian HEK293 systems of expression and assess the ability of antibodies from sera of malaria-exposed persons to recognize the recombinant proteins. The most highly expressed surface localized proteins of mature (Stage V) gametocytes according to published proteome data (Lasonder et al., 2016), were amplified and expressed for immunoprofiling by ELISA. Seven gametocyte-specific surface antigens were expressed using the WGCFS to express the proteins MDV-1, PSOP25_3D7, PSOP25_10668 and CPP4, while the mammalian cells HEK293 expression system was used to express the proteins CVMPPP, PEB-P and PSOP1. The plasma samples used in this analysis were those that were collected during two previous studies, that is, the Longitudinal assessment of malaria transmissibility (LAMB, N = 284) and the cross-sectional study, Assessment of the infectious reservoir of malaria (AFIRM = 413). xii The observations presented in this thesis show the ability to not only produce recombinant gametocyte-specific proteins using various expression systems, but to produce proteins that are recognized by the sera of persons exposed to malaria. The findings show that host responses to gametocyte proteins are influenced by the participant‘s age, as well as the density and presence of parasites. Antibody responses among individuals older than 15- years were consistently higher for all antigens when compared the younger individuals. Gametocyte-positive individuals had higher responses (p<0.05) as opposed to the gametocyte-negative individuals, for all the antigens. The results also reveal significant cross-correlations between gametocyte density (p<0.001) and the responses to all the recombinant gametocyte antigens evaluated. Furthermore, for the AFIRM study, a model was fitted to predict the odds of one presenting with gametocytes. The following predictors were included in the logistic model: age category, gametocyte status, season, parasite density (as measured by qPCR18S, NASBA18s and NASBAPfs25), responses to AMA-1, sickle gene and α-thalassemia. The model shows that individuals in whom parasites were detected by qPCR18s, had 1% (p<0.05) higher odds of being gametocyte positive compared to those in whom no parasites could be detected. On the other hand, participants who were under 5 years of age had 99% (p<0.05) odds of being gametocyte negative. Individuals who responded to the antigens MDV1 and AMA1(a marker for exposure to parasites), had higher odds of being gametocyte positive, in that for every one-unit increase in the mean response to the two recombinant proteins, an increase of 160% and 93% in the odds of gametocyte positivity was seen and this was statistically significant (p < 0.05). Participants who were sampled during the dry season had 2% (p < 0.05) higher odds of being gametocyte positive compared to those sampled within a malaria season, while adjusted for parasite density as measured by qPCR18s. xiii Antibody responses among individuals older than 15-year were consistently higher for all antigens in comparison to responses for the other two age groups. A similar trend was observed with AMA1, which serves as a marker for exposure to asexual parasites. When assessing for the role of season in influencing responses, there was no difference in responses among those samples during peak malaria season and those sampled after the malaria season had passed. This can be attributed to the maintenance of the parasite during the dry season by undetectable sub-microscopic or low-density gametocytes. An increase in responses to AMA-1, as revealed by the model, resulted in an increase in responses to recombinant proteins when adjusted for all other predictors. The longitudinal LAMB study enabled evaluation of responses over six different time points. A multivariable logistic regression model to predict parasitaemia, showed that oneunit increase in the mean response to AMA1, increased the odds of parasitaemia by 56% (p<0.05). An increase in age increased odds of being negative for parasites by 96% (p<0.05). This study is unique as it offers a look at responses based on both a cross-sectional as well as longitudinal study. In future, it would be important to further add on to the observations by measuring transmission-blocking activity using of mosquito feeding assay. Moreover, understanding the mechanism underlying the recognition of gametocyte-specific antigens, and further finding a highly immunogenic target(s) will go a long way in advancing the development of a vaccine to block transmission.