Chanel Milène Naar

In our effort to design an effective ultralow-dose attenuated whole sporozoite (SPZ) vaccine, we explored the use of chemical adjuvants to increase the immunogenicity of sporozoites. Critical to the potency of attenuated sporozoites is their ability to migrate to and infect the liver. We thus used an in vivo mouse challenge model to compare the effects of external and internal chemical modifications to sporozoites. We loaded sporozoites with CL307 using two strategies: a supramolecular surface modification adjuvant system (SPZ-SAS(CL307)) and a mitochondrial membrane potential system (SPZ-MMP(CL307)). SPZ-SAS(CL307) contained 200 times more CL307 as compared to SPZ-MMP(CL307). However, SPZ-SAS(CL307) could not establish a liver infection after intravenous injection (25 000 sporozoites), suggesting loss of function of the sporozoites. On the contrary, SPZ-MMP(CL307) could establish an equivalent liver infection to the control already at a dose of 5000 sporozoites. When the immunized mice were challenged, SPZ-MMP(CL307) equaled the protective efficacy of control sporozoites. Our findings indicate that the MMP system can be used to chemically augment live cells, such as sporozoites, with adjuvants without negatively affecting their infectivity and protective efficacy in vivo. For malaria, further testing of different types of adjuvants at different concentrations is needed to ultimately develop vaccines with increased potency. However, in essence, this platform provides a proof-of-concept for a Trojan horse adjuvanting or drug loading concept for any whole-cell vaccine or therapy, within the infectious diseases space or beyond in oncological applications.

Argonautes are ancient proteins with well-characterised functions in cell-autonomous gene regulation and genome defence, but less clear roles in non-cell-autonomous processes. Extracellular Argonautes have been reported across plants, animals and protozoa, yet their biochemical and functional properties remain elusive. Here, we demonstrate that an extracellular Argonaute (exWAGO) released by the rodent-infective nematode Heligmosomoides bakeri is detectable inside mouse cells during the natural infection. We show that exWAGO is released from H. bakeri in both vesicular and non-vesicular forms that have different resistances to proteolysis, different accessibilities to antibodies and associate with different subsets of secondary siRNAs. Using recombinant exWAGO protein, we demonstrate that non-vesicular exWAGO is internalised by mouse cells in vitro and that immunisation of mice with exWAGO confers partial protection against subsequent H. bakeri infection and generates antibodies that block exWAGO uptake into cells. Finally, we show that properties of exWAGO are conserved across Clade V nematodes that infect humans and livestock. Together, this work expands the context in which Argonautes function and illuminates an RNA-binding protein as a vaccine target for parasitic nematodes.

Argonautes are ancient proteins with well-characterised functions in cell-autonomous gene regulation and genome defense but less clear roles in non-cell-autonomous processes. Extracellular Argonautes have been reported across plants, animals and protozoa yet their biochemical and functional properties remain elusive. Here we demonstrate that an extracellular Argonaute (exWAGO) released by the rodent-infective parasitic nematode Heligmosomoides bakeri is detectable inside mouse cells during the natural infection. We further show that exWAGO is released from H.bakeri in both vesicular and non-vesicular forms that have different resistances to proteolysis, different accessibilities to antibodies and associate with different subsets of secondary siRNAs. Using recombinant exWAGO protein we demonstrate that non-vesicular exWAGO is directly internalised by mouse cells in vitro and that immunisation of mice with exWAGO confers partial protection against subsequent H. bakeri infection and generates antibodies that block exWAGO uptake into cells. Finally, we show that properties of exWAGO are conserved across Clade V nematodes that infect humans and livestock. Together this work expands the context in which Argonautes function and illuminates an RNA-binding protein as a vaccine target for parasitic nematodes.

Malaria vaccines consisting of metabolically active Plasmodium falciparum (Pf) sporozoites can offer improved protection compared with currently deployed subunit vaccines. In a previous study, we demonstrated the superior protective efficacy of a three-dose regimen of late-arresting genetically attenuated parasites administered by mosquito bite (GA2-MB) compared with early-arresting counterparts (GA1-MB) against a homologous controlled human malaria infection. Encouraged by these results, we explored the potency of a single GA2-MB immunization in a placebo-controlled randomized trial. Primary outcomes were safety and tolerability, time-to-parasitemia and protective efficacy. Humoral and cellular immunological results were considered secondary outcomes. Here we report the safe administration of GA2-MB with no breakthrough malaria and sterile protection in nine of ten participants at 6 weeks after a single immunization with 50 GA2-infected mosquitoes, compared with none of five mock-immunized participants, against a homologous controlled human malaria infection. Immunization increased circulating Pf-specific polyfunctional effector memory CD4⁺ T cells coexpressing tumor necrosis factor and interleukin-2. This unprecedented 90% protective efficacy after a single low-dose immunization holds great promise for the potency of GA2 immunization. Future studies should demonstrate whether GA2 is similarly efficacious in pre-exposed populations and whether the favorable safety profile reported here holds up in larger groups. ClinicalTrials.gov registration: NCT05468606.

Despite promising results in malaria-naïve individuals, whole sporozoite (SPZ) vaccine efficacy in malaria-endemic settings has been suboptimal. Vaccine hypo-responsiveness due to previous malaria exposure has been posited as responsible, indicating the need for SPZ vaccines of increased immunogenicity. To this end, we here demonstrate a proof-of-concept for altering SPZ immunogenicity, where supramolecular chemistry enables chemical augmentation of the parasite surface with a TLR7 agonist-based adjuvant (SPZ-SAS(CL307)). In vitro, SPZ-SAS(CL307) remained well recognized by immune cells and induced a 35-fold increase in the production of pro-inflammatory IL-6 (p < 0.001). More promisingly, immunization of mice with SPZ-SAS(CL307) yielded improved SPZ-specific IFN-γ production in liver-derived NK cells (percentage IFN-γ⁺ cells 11.1 ± 1.8 vs. 9.4 ± 1.5%, p < 0.05), CD4⁺ T cells (4.7 ± 4.3 vs. 1.8 ± 0.7%, p < 0.05) and CD8⁺ T cells (3.6 ± 1.4 vs. 2.5 ± 0.9%, p < 0.05). These findings demonstrate the potential of using chemical augmentation strategies to enhance the immunogenicity of SPZ-based malaria vaccines.