On the basis of sex: Impact of biological sex on the Plasmodium parasite liver stage

Abstract

The enclosed dissertation lies at the intersection of three fields of study: sex differences, malaria, and hepatology (study of the liver). Biological sex and gender are critically important factors affecting health and disease throughout the human lifespan. Malaria is a deadly disease caused by Plasmodium parasites, a pathogen with a complex lifecycle, including a brief but critical liver stage. The liver is the largest solid organ in the body, and the site of complex spatial heterogeneity and important endocrine and immunological function. Biological sex influences the liver. As a sexually dimorphic organ, the liver sustains baseline differences in function, cell composition, and gene expression profiles on the basis of sex. And the liver niche impacts the Plasmodium parasite's liver stage. As a hepatotropic pathogen, Plasmodium parasites interact within the heterogenous liver microenvironment in a series of host-pathogen interactions that permit survival in the face of innate and adaptive immune responses. However, a knowledge gap remains regarding whether biological sex impacts the liver stage of the Plasmodium parasite. Understanding this connection – between host biological sex, the hepatotropic Plasmodium parasite, and the liver – could uncover a new perspective on fundamental liver stage biology and inform the design and execution of liver-targeting vaccines. Thus, the overall goal of this dissertation is to add sex-specific hepatotropic pathogen to the definition of Plasmodium parasites. Additionally, this dissertation aims to underscore the need for sex-informed research that transcends outdated biases, ensuring that host-pathogen profiling and vaccines evolve to serve all people. This thesis demonstrates that Plasmodium parasites trigger a sex-specific host response, which holds significant implications for research and clinical interventions. Three key points will be demonstrated. First, humans exhibit sex-specific Plasmodium falciparum liver stage outcomes. Second, murine models exhibit sex-specific inflammatory and cellular responses during the rodent Plasmodium liver stage, which subsequently impacts parasite survival. Third, murine models also exhibit sex-specific protection from malaria liver stage vaccines, which is connected to the impact of androgens on CD8+ T cell effector mechanisms. Furthermore, we apply a three-part framework for understanding sex differences—encompassing the roles of sex hormones, sex chromosomes, and physiology—to offer a comprehensive, mechanistic view of how biological sex influences host responses to Plasmodium infection, with particular emphasis on the impact of sex hormones. The findings of this thesis reveal the complex impact of biological sex on liver stage immunity and Plasmodium liver stage outcomes. In humans, a pooled analysis across 13 Controlled Human Malaria Infection (CHMI) trials in malaria-naïve populations found that males are more likely to experience a delayed time to blood-stage positive parasites following challenge with Plasmodium falciparum compared to females. In mice, no difference in susceptibility of infection was identified. However, Plasmodium parasites had higher rates of survival in mice, leading to enhanced liver burden during later liver stage development. We spatially profile the liver stage and identify higher survival of infected hepatocytes in males, linking to a restricted inflammatory response and innate cell recruitment in male mice. Furthermore, the presence of androgens in males suppresses this innate immune response and alters liver stage survival. Next, this study sought to evaluate the impact of biological sex on Plasmodium liver stage vaccines. We discovered that male mice vaccinated with prime-and-trap, a whole organism-based vaccine strategy, exhibit poorer protection against Plasmodium sporozoite challenge than females. We investigated this sex difference, and identified that vaccinated male mice have fewer hepatic memory CD8+ T cells than females when scaling for liver biomass. We further investigated the impact of sex hormones, and identified that the presence of androgens did not affect memory CD8+ T cell quantity nor quality, but directly inhibited protective CD8+ T cell effector response during sporozoite challenge. The contrast in liver stage infection between mice and humans—where male mice show a higher liver parasite burden, whereas male humans experience an extended liver stage, suggesting a lower liver burden—highlights the critical role of model selection in sex difference research. Our findings reveal that baseline differences in cellular composition and tissue homeostasis must be accounted for to understand the underlying host-parasite interactions accurately. Bridging insights from mouse infection models to human observations remains essential. Overall, this research shows that sex differences in host responses significantly impact Plasmodium liver stage biology in both humans and mice. Sex hormones emerge as powerful modulators of these differences, shifting immunological baselines and influencing protection against infection in both natural-acquired and vaccine-induced immunity. This work underscores the importance of biological sex as a key variable in liver stage malaria studies and vaccine development.