Molecular interactions between parasite and mosquito during midgut invasion as targets to block malaria transmission

Despite considerable effort, malaria remains a major public health burden. Malaria is caused by five Plasmodium species and is transmitted to humans via the female Anopheles mosquito. The development of malaria vaccines against the liver and blood stages has been challenging. Therefore, malaria elimination strategies advocate integrated measures, including transmission-blocking approaches. Designing an effective transmission-blocking strategy relies on a sophisticated understanding of the molecular mechanisms governing the interactions between the mosquito midgut molecules and the malaria parasite. Here we review recent advances in the biology of malaria transmission, focusing on molecular interactions between Plasmodium and Anopheles mosquito midgut proteins. We provide an overview of parasite and mosquito proteins that are either targets for drugs currently in clinical trials or candidates of promising transmission-blocking vaccines.

Evolutionary insights into the microneme-secreted, chitinase-containing high molecular weight protein complexes involved in Plasmodium invasion of the mosquito midgut

While general mechanisms by which Plasmodium ookinetes invade the mosquito midgut have been studied, details remain to be understood regarding the interface of the ookinete, specifically its barriers to invasion, such as the proteolytic milieu, the chitin-containing, protein cross-linked peritrophic matrix, and the midgut epithelium. Here we review knowledge of Plasmodium chitinases and the mechanisms by which they mediate the ookinete crossing the peritrophic matrix. The integration of new genomic insights into previous findings advances our understanding of Plasmodium evolution. Recently obtained Plasmodium spp. genomic data enable identification of the conserved residues in the experimentally demonstrated hetero-multimeric, high molecular weight complex comprised of a short chitinase covalently linked to binding partners, von Willebrand factor A domain-related protein (WARP) and secreted ookinete adhesive protein (SOAP). Artificial intelligence-based high-resolution structural modeling using the DeepMind AlphaFold algorithm yielded highly informative 3D structures and insights into how short chitinases, WARP, and SOAP may interact at the atomic level to form the ookinete-secreted peritrophic matrix invasion complex. Elucidating the significance of the divergence of ookinete-secreted micronemal proteins among Plasmodium species could lead to a better understanding of ookinete invasion machinery and the co-evolution of Plasmodium -mosquito interactions.

Immunopotentiation by Lymph-Node Targeting of a Malaria Transmission-Blocking Nanovaccine

A successful malaria transmission blocking vaccine (TBV) requires the induction of a high antibody titer that leads to abrogation of parasite traversal of the mosquito midgut following ingestion of an infectious bloodmeal, thereby blocking the cascade of secondary human infections. Previously, we developed an optimized construct UF6b that elicits an antigen-specific antibody response to a neutralizing epitope of Anopheline alanyl aminopeptidase N (AnAPN1), an evolutionarily conserved pan-malaria mosquito midgut-based TBV target, as well as established a size-controlled lymph node targeting biodegradable nanoparticle delivery system that leads to efficient and durable antigen-specific antibody responses using the model antigen ovalbumin. Herein, we demonstrate that co-delivery of UF6b with the adjuvant CpG oligodeoxynucleotide immunostimulatory sequence (ODN ISS) 1018 using this biodegradable nanoparticle vaccine delivery system generates an AnAPN1-specific immune response that blocks parasite transmission in a standard membrane feeding assay. Importantly, this platform allows for antigen dose-sparing, wherein lower antigen payloads elicit higher-quality antibodies, therefore less antigen-specific IgG is needed for potent transmission-reducing activity. By targeting lymph nodes directly, the resulting immunopotentiation of AnAPN1 suggests that the de facto assumption that high antibody titers are needed for a TBV to be successful needs to be re-examined. This nanovaccine formulation is stable at -20°C storage for at least 3 months, an important consideration for vaccine transport and distribution in regions with poor healthcare infrastructure. Together, these data support further development of this nanovaccine platform for malaria TBVs.

Studies of the Parasite-Midgut Interaction Reveal Plasmodium Proteins Important for Malaria Transmission to Mosquitoes

Malaria transmission relies on parasite-mosquito midgut interaction. The interactive proteins are hypothesized to be ideal targets to block malaria transmission to mosquitoes. We chose 76 genes that contain signal peptide-coding regions and are upregulated and highly abundant at sexual stages. Forty-six of these candidate genes (60%) were cloned and expressed using the baculovirus expression system in insect cells. Six of them, e.g., PF3D7_0303900, PF3D7_0406200 (Pfs16), PF3D7_1204400 (Pfs37), PF3D7_1214800, PF3D7_1239400, and PF3D7_1472800 were discovered to interact with blood-fed mosquito midgut lysate. Previous works showed that among these interactive proteins, knockout the orthologs of Pfs37 or Pfs16 in P. berghei reduced oocysts in mosquitoes. Here we further found that anti-Pfs16 polyclonal antibody significantly inhibited P. falciparum transmission to Anopheles gambiae. Investigating these candidate proteins will improve our understanding of malaria transmission and discover new targets to break malaria transmission.

Differential transcriptomic response of Anopheles arabiensis to Plasmodium vivax and Plasmodium falciparum infection

Plasmodium vivax malaria is now recognized as the second most dangerous parasitic threat to human health with the regular decrease of Plasmodium falciparum worldwide over recent decades. A very limited numbers of studies address the interaction of P. vivax with its Anopheles mosquito vectors. Those studies were conducted in P. vivax endemic countries with P.vivax local major vectors for which limited genomic and genetic tools are available. Despite the presence of P. vivax in several African countries and increasing reports on its occurrence in many others, there is virtually no data on the molecular responses of Anopheles arabiensis, a major African mosquito vector, to P. vivax, which limits the development of further mosquito-targeted interventions aimed at reducing P. vivax transmission. Taking advantage of the situation of Madagascar where P. falciparum, P. vivax and An. arabiensis are present, we explore the molecular responses of An. arabiensis towards these two human malaria parasites. RNA sequencing on RNAs isolated from mosquito midguts dissected at the early stage of infection (24 hours) was performed using mosquitoes fed on the blood of P. vivax and P. falciparum gametocyte carriers in a field station. From a de novo assembly of An. arabiensis midgut total RNA transcriptome, the comparative analysis revealed that a greater number of genes were differentially expressed in the mosquito midgut in response to P. vivax (209) than to P. falciparum (81). Among these, 15 common genes were identified to be significantly expressed in mosquito midgut 24 hours after ingesting P. vivax and P. falciparum gametocytes, including immune responsive genes and genes involved in amino-acid detoxification pathways. Importantly, working with both wild mosquitoes and field circulating parasites, our analysis revealed a strong mosquito genotype by parasite genotype interaction. Our study also identified 51 putative long non-coding RNAs differentially expressed in An. arabiensis mosquito infected midgut. Among these, several mapped to the published An. arabiensis genome at genes coding immune responsive genes such as gambicin 1, leucine-rich repeat containing genes, either on sense or antisense strands. This study constitutes the first comparison of An. arabiensis molecular interaction with P. vivax and P. falciparum, investigating both coding and long non-coding RNAs for the identification of potential transcripts, that could lead to the development of novel approaches to simultaneously block the transmission of vivax and falciparum malaria.

Aedes aegypti SGS1 is critical for Plasmodium gallinaceum infection of both the mosquito midgut and salivary glands

Background The invasion of the mosquito salivary glands by Plasmodium sporozoites is a critical step that defines the success of malaria transmission and a detailed understanding of the molecules responsible for salivary gland invasion could be leveraged towards control of vector-borne pathogens. Antibodies directed against the mosquito salivary gland protein SGS1 have been shown to reduce Plasmodium gallinaceum sporozoite invasion of Aedes aegypti salivary glands, but the specific role of this protein in sporozoite invasion and in other stages of the Plasmodium life cycle remains unknown. Methods RNA interference and CRISPR/Cas9 were used to evaluate the role of A. aegypti SGS1 in the P. gallinaceum life cycle. Results Knockdown and knockout of SGS1 disrupted sporozoite invasion of the salivary gland. Interestingly, mosquitoes lacking SGS1 also displayed fewer oocysts. Proteomic analyses confirmed the abolishment of SGS1 in the salivary gland of SGS1 knockout mosquitoes and revealed that the C-terminus of the protein is absent in the salivary gland of control mosquitoes. In silico analyses indicated that SGS1 contains two potential internal cleavage sites and thus might generate three proteins. Conclusion SGS1 facilitates, but is not essential for, invasion of A. aegypti salivary glands by P. gallinaceum and has a dual role as a facilitator of parasite development in the mosquito midgut. SGS1 could, therefore, be part of a strategy to decrease malaria transmission by the mosquito vector, for example in a transgenic mosquito that blocks its interaction with the parasite.