The aim of the work described in this thesis was the evaluation of promising P. falciparum
sexual stage antigens by transient expression in N. benthamiana plants to select antigens
suitable for the generation of an improved multi domain sexual stage vaccine candidate. The
selected antigens were tackled using different strategies to reflect their diverse
characteristics. This included the design and expression of different subdomains, the
removal or reintroduction of N-linked glycosylation sites, the targeting of different subcellular
compartments and the use of heat treatment during processing. The selection of suitable
domains was based on two major empirical requirements: first, the transient expression of
stable, soluble recombinant protein at reasonable levels (>100 μg/g fresh leaf weight); and
second, the potential to use heat precipitation to remove HCPs during purification. For
Pfs48/45, the expression of a full length glyco knockout variant did not achieve detectable
levels of protein, nor did the restoration of the native glycosylation sites. For Pfs230, the N
terminal fragment Pfs230_C0 was expressed at promising levels, and heat treatment during
purification did not affect the solubility or integrity of the protein as shown by immunization
and immunofluorescence assays. PfGAP50 does not require oxidative folding, so the protein
was targeted to the plastids and this achieved a fourfold higher yield than the same protein
targeted to the ER. PfGAP50 was functional in inhibition assays but was not heat stable. To
generate a sexual stage multi target vaccine candidate, nine sexual stage fusion proteins
were designed. Only the F0 fusion protein consisting of Pfs25 and Pfs230_C0 was
expressed at high levels and heat stable during purification. Serum samples from individuals
exposed to malaria recognized the purified protein confirming its correct conformation and its
suitability as boostable component of a sexual stage vaccine candidate. Furthermore, F0
specific mouse immune sera demonstrated 100% in vitro transmission blocking activity
making F0 a promising new sexual stage vaccine candidate that will be evaluated further in
the context of a multi stage malaria vaccine cocktail produced in plants.