Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion

Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and species-specific transmembrane adhesins, which interact with an intracellular motor complex shared across parasite stages and species. How gliding is regulated by extracellular factors and intracellular signalling mechanisms is largely unknown, but current evidence suggests an important role for cytosolic calcium as a second messenger. Studying a Plasmodium berghei gene deletion mutant, we here provide evidence that a calcium-dependent protein kinase, CDPK3, has an important function in regulating motility of the ookinete in the mosquito midgut. We show that a cdpk3(–) parasite clone produces morphologically normal ookinetes, which fail to engage the midgut epithelium, due to a marked reduction in their ability to glide productively, resulting in marked reduction in malaria transmission to the mosquito. The mutant was successfully complemented with an episomally maintained cdpk3 gene, restoring mosquito transmission to wild-type level. cdpk3(–) ookinetes maintain their full genetic differentiation potential when microinjected into the mosquito haemocoel and cdpk3(–) sporozoites produced in this way are motile and infectious, suggesting an ookinete-limited essential function for CDPK3

Safety and efficacy of novel malaria vaccine regimens of RTS,S/AS01B alone, or with concomitant ChAd63-MVA-vectored vaccines expressing ME-TRAP

We assessed a combination multi-stage malaria vaccine schedule in which RTS,S/AS01B was given concomitantly with viral vectors expressing multiple-epitope thrombospondin-related adhesion protein (ME-TRAP) in a 0-month, 1-month, and 2-month schedule. RTS,S/AS01B was given as either three full doses or with a fractional (1/5th) third dose. Efficacy was assessed by controlled human malaria infection (CHMI). Safety and immunogenicity of the vaccine regimen was also assessed. Forty-one malaria-naive adults received RTS,S/AS01B at 0, 4 and 8 weeks, either alone (Groups 1 and 2) or with ChAd63 ME-TRAP at week 0, and modified vaccinia Ankara (MVA) ME-TRAP at weeks 4 and 8 (Groups 3 and 4). Groups 2 and 4 received a fractional (1/5th) dose of RTS,S/AS01B at week 8. CHMI was delivered by mosquito bite 11 weeks after first vaccination. Vaccine efficacy was 6/8 (75%), 8/9 (88.9%), 6/10 (60%), and 5/9 (55.6%) of subjects in Groups 1, 2, 3, and 4, respectively. Immunological analysis indicated significant reductions in anti-circumsporozoite protein antibodies and TRAP-specific T cells at CHMI in the combination vaccine groups. This reduced immunogenicity was only observed after concomitant administration of the third dose of RTS,S/AS01B with the second dose of MVA ME-TRAP. The second dose of the MVA vector with a four-week interval caused significantly higher anti-vector immunity than the first and may have been the cause of immunological interference. Co-administration of ChAd63/MVA ME-TRAP with RTS,S/AS01B led to reduced immunogenicity and efficacy, indicating the need for evaluation of alternative schedules or immunization sites in attempts to generate optimal efficacy

Morphogenesis of Plasmodium zoites is uncoupled from tensile strength.

A shared feature of the motile stages (zoites) of malaria parasites is a cortical cytoskeletal structure termed subpellicular network (SPN), thought to define and maintain cell shape. Plasmodium alveolins comprise structural components of the SPN, and alveolin gene knockout causes morphological abnormalities that coincide with markedly reduced tensile strength of the affected zoites, indicating the alveolins are prime cell shape determinants. Here, we characterize a novel SPN protein of Plasmodium berghei ookinetes and sporozoites named G2 (glycine at position 2), which is structurally unrelated to alveolins. G2 knockout abolishes parasite transmission and causes zoite malformations and motility defects similar to those observed in alveolin null mutants. Unlike alveolins, however, G2 contributes little to tensile strength, arguing against a cause-effect relationship between tensile strength and cell shape. We also show that G2 null mutant sporozoites display an abnormal arrangement of their subpellicular microtubules. These results provide important new understanding of the factors that determine zoite morphogenesis, as well as the potential roles of the cortical cytoskeleton in gliding motility