With the increasing prevalence of artemisinin-resistant malaria parasites, a highly efficacious and durable vaccine for malaria is urgently required. We have developed an experimental virus-vectored vaccine platform based on an envelope-modified baculovirus dual-expression system (emBDES). Here, we show a conceptually new vaccine platform based on an adenovirus-prime/emBDES-boost heterologous immunization regimen expressing the Plasmodium falciparum circumsporozoite protein (PfCSP). A human adenovirus 5-prime/emBDES-boost heterologous immunization regimen consistently achieved higher sterile protection against transgenic P. berghei sporozoites expressing PfCSP after a mosquito-bite challenge than reverse-ordered or homologous immunization. This high protective efficacy was also achieved with a chimpanzee adenovirus 63-prime/emBDES-boost heterologous immunization regimen against an intravenous sporozoite challenge. Thus, we show that the adenovirus-prime/emBDES-boost heterologous immunization regimen confers sterile protection against sporozoite challenge by two individual routes, providing a promising new malaria vaccine platform for future clinical use.

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control. Malaria, transmitted by anopheline mosquitoes, represents an important global public health challenge. The salivary glands of mosquitoes are an attractive target tissue for malaria and vector control. We produced a transgenic strain inducing cell death in the salivary glands with a cell death effector molecule in the Asian malaria vector mosquito, Anopheles stephensi. This transgenic strain contained extremely low amounts of saliva. An analysis of this strain revealed that saliva plays an important role in probing as well as malaria infection in the midgut in a rodent malaria model. The dysfunction in the salivary glands enabled the inhibition of malaria transmission to mosquito midguts. Therefore, salivary components are also important in malaria control.

Previous studies have shown that the baculovirus-vectored vaccine based on the “baculovirus dual expression system (BDES)” is an effective vaccine delivery platform for malaria. However, a point of weakness remaining for use of this vaccine platform in vivo concerns viral inactivation by serum complement. In an effort to achieve complement resistance, the gene encoding the human decay-accelerating factor (hDAF) was incorporated into the BDES malaria vaccine expressing the Plasmodium falciparum circumsporozoite protein (PfCSP). The newly-developed BDES vaccine, designated BDES-sPfCSP2-Spider, effectively displayed hDAF and PfCSP on the surface of the viral envelope, resulting in complement resistance both in vitro and in vivo. Importantly, upon intramuscular inoculation into mice, the BDES-sPfCSP2-Spider vaccine had a higher protective efficacy (60%) than that of the control vaccine BDES-sPfCSP2-Spier (30%) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. DAF-shielded BDES-vaccines offer great potential for development as a new malaria vaccine platform against the sporozoite challenge.

Background: Blood-sucking phlebotomine sand flies are vectors of the protozoan parasites Leishmania spp. Although the intestinal microbiota is involved in a wide range of biological and physiological processes and has the potential to alter vector competence, little is known about the factors that modify the gut microbiota composition of sand flies. As a key step toward addressing this issue, we investigated the impact of host species on the gut bacterial composition in Phlebotomus and Lutzomyia sand flies reared under the same conditions. Methods: Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing were used to characterize the overall bacterial composition of three laboratory-reared sandflies: Phlebotomus papatasi, Ph. duboscqi, and Lutzomyia longipalpis. Results: Our results showed that the larvae of the three sand fly species harbored almost the same microbes but had different relative abundances. Adult Ph. papatasi and Ph. duboscqi revealed similar microbiome compositions, which were distinct from that of adult Lu. longipalpis. Furthermore, we showed that Ph. papatasi and Ph. duboscqi are hosts for different bacterial genera. The experiment was repeated twice to improve accuracy and increase reliability of the data, and the same results were obtained even when a distinct composition of the microbiome among the same species was identified probably because of the use of different larvae food batch. Conclusions: The present study provides key insights into the role of host species in the gut microbial content of different sand fly species reared under the same conditions, which may influence their susceptibility to Leishmania infection. Graphical

The saliva of hematophagous arthropods is enriched with a complex mixture of antihemostatic molecules, the biological functions of which are largely unknown. Anopheline antiplatelet protein (AAPP) from malaria vector mosquito exhibits strong antiplatelet activity when bound directly to host collagen by its C-terminus and through its N-terminus with Ca²⁺-binding activity. To investigate the biological functions of AAPP in blood feeding behavior and malaria transmission, we generated transgenic Anopheles stephensi mosquito lines expressing anti-AAPP antibody single-chain fragment (scFv) in their salivary glands. The AAPP-specific collagen-binding activity was completely abolished by AAPP-scFv complex formation in the saliva. Probing and prediuresis time, feeding success, blood meal size, and fecundity, which are all fitness characteristics, were significantly reduced in the transgenic mosquitoes. However, oocysts number in these mosquitoes were not significantly reduced following blood meal intake from Plasmodium berghei-infected mice. These results show that although AAPP plays an important role in mosquito blood feeding, its neutralizing activity did not affect sporogonic development in our laboratory model, but its high fitness cost would pose a survival risk for parasite-infected mosquitoes in nature.