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Joice, Regina Carol

Due to an increase in malaria control programs in the last decade, the world has witnessed dramatic reductions in the number of infections and deaths caused by the malaria parasite. With malaria eradication on the global health agenda, a shift toward transmission-focused research has led to a renewed focus on a previously neglected stage of malaria: the sexual stage (gametocyte). Malaria's sexual stages are the only stages in the human host that are transmitted to the mosquito vector, and are therefore of critical importance for blocking transmission of this devastating disease. The process through which developing gametocytes sequester outside of the bloodstream during their 8-10 day maturation is not well understood and stands to be exploited as a potential target for therapeutic intervention. In Chapter 1, we discuss the current state of knowledge on the development of these stages in the human host. In Chapter 2, we investigate anatomical enrichment sites for developing gametocytes in the human host using autopsy tissue from cases of fatal malaria. Immunohistochemistry (IHC) and quantitative reverse transcriptase PCR-based assessments identified the bone marrow as a preferential enrichment site of developing gametocytes. Co-localization with host proteins revealed the enrichment of gametocytes inside the extravascular space of the bone marrow, often observed in contact with erythroblastic island structures. In vitro experiments with erythrocyte precursor cells, as well as in vivo co-localization studies demonstrated that gametocytes can develop within the cells of the hematopoietic system of the bone marrow. In Chapter 3, we present an assay and analysis tool for inferring the presence of young and mature asexual and sexual stages in the peripheral blood of infected patients based on gene expression data. We apply this assay to malaria patient cohorts and in vitro drug perturbation time course experiments, and demonstrate its use in identifying young and mature gametocyte carriers, as well as characterizing the effect of a given perturbation on parasite development. This body of work aims to contribute to the overall knowledge base for malaria’s elusive gametocytes as well as to establish tools for performing future assessments on these transmissible stages.

In the current era of malaria eradication, reducing transmission is critical. Assessment of transmissibility requires tools that can accurately identify the various developmental stages of the malaria parasite, particularly those required for transmission (sexual stages). Here, we present a method for estimating relative amounts of Plasmodium falciparum asexual and sexual stages from gene expression measurements. These are modeled using constrained linear regression to characterize stage-specific expression profiles within mixed-stage populations. The resulting profiles were analyzed functionally by gene set enrichment analysis (GSEA), confirming differentially active pathways such as increased mitochondrial activity and lipid metabolism during sexual development. We validated model predictions both from microarrays and from quantitative RT-PCR (qRT-PCR) measurements, based on the expression of a small set of key transcriptional markers. This sufficient marker set was identified by backward selection from the whole genome as available from expression arrays, targeting one sentinel marker per stage. The model as learned can be applied to any new microarray or qRT-PCR transcriptional measurement. We illustrate its use in vitro in inferring changes in stage distribution following stress and drug treatment and in vivo in identifying immature and mature sexual stage carriers within patient cohorts. We believe this approach will be a valuable resource for staging lab and field samples alike and will have wide applicability in epidemiological studies of malaria transmission.

The growing prevalence of antimicrobial resistance in major pathogens is outpacing discovery of new antimicrobial classes. Vaccines mitigate the effect of antimicrobial resistance by reducing the need for treatment, but vaccines for many drug-resistant pathogens remain undiscovered or have limited efficacy, in part because some vaccines selectively favor pathogen strains that escape vaccine-induced immunity. A strain with even a modest advantage in vaccinated hosts can have high fitness in a population with high vaccine coverage, which can offset a strong selection pressure such as antimicrobial use that occurs in a small fraction of hosts. We propose a strategy to target vaccines against drug-resistant pathogens, by using resistance-conferring proteins as antigens in multicomponent vaccines. Resistance determinants may be weakly immunogenic, offering only modest specific protection against resistant strains. Therefore, we assess here how varying the specific efficacy of the vaccine against resistant strains would affect the proportion of drug-resistant vs. –sensitive strains population-wide for three pathogens – Streptococcus pneumoniae, Staphylococcus aureus, and influenza virus – in which drug resistance is a problem. Notably, if such vaccines confer even slightly higher protection (additional efficacy between 1% and 8%) against resistant variants than sensitive ones, they may be an effective tool in controlling the rise of resistant strains, given current levels of use for many antimicrobial agents. We show that the population-wide impact of such vaccines depends on the additional effect on resistant strains and on the overall effect (against all strains). Resistance-conferring accessory gene products or resistant alleles of essential genes could be valuable as components of vaccines even if their specific protective effect is weak.