A Viral-Vectored Multi-Stage Malaria Vaccine Regimen With Protective and Transmission-Blocking Efficacies.

Malaria parasites undergo several stages in their complex lifecycle. To achieve reductions in both the individual disease burden and malaria transmission within communities, a multi-stage malaria vaccine with high effectiveness and durability is a more efficacious strategy compared with a single-stage vaccine. Here, we generated viral-vectored vaccines based on human adenovirus type 5 (AdHu5) and adeno-associated virus serotype 1 (AAV1) expressing a fusion protein of the pre-erythrocytic stage Plasmodium falciparum circumsporozoite protein (PfCSP) and the transmission-blocking sexual stage P25 protein (Pfs25). A two-dose heterologous AdHu5-prime/AAV1-boost immunization regimen proved to be highly effective for both full protection and transmission-blocking activity against transgenic P. berghei parasites expressing the corresponding P. falciparum antigens in mice. Remarkably, the immunization regimen induced antibody responses to both PfCSP and Pfs25 for over 9 months after the boosting and also maintained high levels of transmission-reducing activity (TRA: >99%) during that period, as evaluated by a direct feeding assay. If similar efficacies on P. falciparum can be shown following vaccination of humans, we propose that this multi-stage malaria vaccine regimen will be a powerful tool for malaria control, providing greater overall protection and cost-effectiveness than single-stage vaccines.

Adeno-Associated Virus as an Effective Malaria Booster Vaccine Following Adenovirus Priming.

An ideal malaria vaccine platform should potently induce protective immune responses and block parasite transmission from mosquito to human, and it should maintain these effects for an extended period. Here, we have focused on vaccine development based on adeno-associated virus serotype 1 (AAV1), a viral vector widely studied in the field of clinical gene therapy that is able to induce long-term transgene expression without causing toxicity in vivo. Our results show the potential utility of AAV1 vectors as an extremely potent booster vaccine to induce durable immunity when combined with an adenovirus-priming vaccine in a rodent malaria model. We generated a series of recombinant AAV1s and human adenovirus type 5 (AdHu5) expressing either Plasmodium falciparum circumsporozoite protein (PfCSP) or P25 (Pfs25) protein. Heterologous two-dose immunization with an AdHu5-prime and AAV1-boost (AdHu5-AAV1) elicited robust and durable PfCSP- or Pfs25-specific functional antibodies over 280 days. Regarding protective efficacy, AdHu5-AAV1 PfCSP achieved high sterile protection (up to 80% protection rate) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. When examining transmission-blocking (TB) efficacy, we found that immunization with AdHu5-AAV1 Pfs25 maintained TB activity in vivo against transgenic P. berghei expressing Pfs25 for 287 days (99% reduction in oocyst intensity, 85% reduction in oocyst prevalence). Our data indicate that AAV1-based malaria vaccines can confer potent and durable protection as well as TB efficacy when administered following an AdHu5 priming vaccine, supporting the further evaluation of this regimen in clinical trials as a next-generation malaria vaccine platform.

Down-selecting circumsporozoite protein-based malaria vaccine: A comparison of malaria sporozoite challenge model.

Plasmodium falciparum circumsporozoite protein (PfCSP) is the main target antigen in development of pre-erythrocytic malaria vaccines. To evaluate PfCSP vaccines in animal models, challenge by intravenous sporozoite injection is preferentially used. However, in clinical trials, vaccinated human volunteers are exposed to the bites of malaria-infected mosquitoes. In this study, we down-selected Escherichia coli-produced full-length PfCSP (PfCSP-F) and its three truncated PfCSPs based on their abilities to elicit immune response and protection in mice against two challenge models. We showed that immunization with three doses of PfCSP-F elicited high anti-PfCSP antibody titres and 100% protection against the bites of infected mosquitoes. Meanwhile, three-dose truncated PfCSP induced 60%-70% protection after immunization with each truncated PfCSP. Heterologous prime-boost immunization regimen with adenovirus-PfCSP-F and R32LR greatly induced complete protection against intravenous sporozoite injection. Our results suggest that Abs to both anti-repeat and anti-nonrepeat regions induced by PfCSP-F are required to confer complete protection against challenge by the bites of infected mosquitoes, whereas anti-repeat Abs play an important role in protection against intravenous sporozoite injection. Our findings provide a potential clinical application that PfCSP-F vaccine induces potent Abs capable of neutralizing sporozoites in the dermis inoculated by infected mosquitoes and subsequently sporozoites in the blood circulation.

Anopheline antiplatelet protein from mosquito saliva regulates blood feeding behavior.

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.

Baculovirus-Induced Fast-Acting Innate Immunity Kills Liver-Stage Plasmodium.

Baculovirus (BV), an enveloped insect virus with a circular dsDNA genome, possesses unique characteristics that induce strong innate immune responses in mammalian cells. In this study, we show that BV administration in BALB/c mice not only provides complete protection against a subsequent Plasmodium berghei sporozoite infection for up to 7 d after the injection but also eliminates existing liver-stage parasites completely. The elimination of sporozoites by BV was superior to that by primaquine, and this effect occurred in a TLR9-independent manner. At 6 h after BV administration, IFN-α and IFN-γ were robustly produced in the serum, and RNA transcripts of IFN-stimulated genes were markedly upregulated in the liver compared with control mice. The in vivo passive transfer of serum after BV administration effectively eliminated liver-stage parasites, and IFN-α neutralization abolished this effect, indicating that the BV liver-stage parasite-killing mechanism is downstream of the type I IFN signaling pathway. These findings provide evidence that BV-induced, fast-acting innate immunity completely kills liver-stage parasites and, thus, may lead to new malaria drug and vaccine strategies.