The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane.

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

A Micronemal Protein, Scot1, Is Essential for Apicoplast Biogenesis and Liver Stage Development in Plasmodium berghei.

Plasmodium sporozoites invade hepatocytes, transform into liver stages, and replicate into thousands of merozoites that infect erythrocytes and cause malaria. Proteins secreted from micronemes play an essential role in hepatocyte invasion, and unneeded micronemes are subsequently discarded for replication. The liver-stage parasites are potent immunogens that prevent malarial infection. Late liver stage-arresting genetically attenuated parasites (GAPs) exhibit greater protective efficacy than early GAP. However, the number of late liver-stage GAPs for generating GAPs with multiple gene deletions is limited. Here, we identified Scot1 (Sporozoite Conserved Orthologous Transcript 1), which was previously shown to be upregulated in sporozoites, and by endogenous tagging with mCherry, we demonstrated that it is expressed in the sporozoite and liver stages in micronemes. Using targeted gene deletion in Plasmodium berghei, we showed that Scot1 is essential for late liver-stage development. Scot1 KO sporozoites grew normally into liver stages but failed to initiate blood-stage infection in mice due to impaired apicoplast biogenesis and merozoite formation. Bioinformatic studies suggested that Scot1 is a metal-small-molecule carrier protein. Remarkably, supplementation with metals in the culture of infected Scot1 KO cells did not rescue their phenotype. Immunization with Scot1 KO sporozoites in C57BL/6 mice confers protection against malaria via infection. These proof-of-concept studies will enable the generation of P. falciparum Scot1 mutants that could be exploited to generate GAP malaria vaccines.

The novel Plasmodium berghei protein S14 is essential for sporozoite gliding motility and infectivity.

Plasmodium sporozoites are the infective forms of the malaria parasite in the mosquito and vertebrate host. Gliding motility allows sporozoites to migrate and invade mosquito salivary glands and mammalian hosts. Motility and invasion are powered by an actin-myosin motor complex linked to the glideosome, which contains glideosome-associated proteins (GAPs), MyoA and the myosin A tail-interacting protein (MTIP). However, the role of several proteins involved in gliding motility remains unknown. We identified that the S14 gene is upregulated in sporozoite from transcriptome data of Plasmodium yoelii and further confirmed its transcription in P. berghei sporozoites using real-time PCR. C-terminal 3×HA-mCherry tagging revealed that S14 is expressed and localized on the inner membrane complex of the sporozoites. We disrupted S14 in P. berghei and demonstrated that it is essential for sporozoite gliding motility, and salivary gland and hepatocyte invasion. The gliding and invasion-deficient S14 knockout sporozoites showed normal expression and organization of inner membrane complex and surface proteins. Taken together, our data show that S14 plays a role in the function of the glideosome and is essential for malaria transmission.

A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host-pathogen interactions.

BACKGROUND: Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood. METHODS: To gain a better understanding of the protein-protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. RESULTS: A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM KD. An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. CONCLUSION: Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.

Unveiling Cryptosporidium parvum sporozoite-derived extracellular vesicles: profiling, origin, and protein composition.

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.

Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco-epidemiological settings in Ethiopia.

BACKGROUND: Malaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distribution of An. stephensi and likely household exposure in Ethiopia. METHODS: Entomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer-by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households' exposure to An. stephensi. Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plasmodium infection and mosquito blood meal source. RESULTS: Catches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75-100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a peridomestic presence of An. stephensi, ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the predominant species in 20 of the 26 sites, accounting for 42.9-100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi, and 65.4%, 46.7% and 35.8%, respectively, for An. arabiensis. None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax). CONCLUSIONS: This study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission.

Superior protection in a relapsing Plasmodium cynomolgi rhesus macaque model by a chemoprophylaxis with sporozoite immunization regimen with atovaquone-proguanil followed by primaquine.

BACKGROUND: To gain a deeper understanding of protective immunity against relapsing malaria, this study examined sporozoite-specific T cell responses induced by a chemoprophylaxis with sporozoite (CPS) immunization in a relapsing Plasmodium cynomolgi rhesus macaque model. METHODS: The animals received three CPS immunizations with P. cynomolgi sporozoites, administered by mosquito bite, while under two anti-malarial drug regimens. Group 1 (n = 6) received artesunate/chloroquine (AS/CQ) followed by a radical cure with CQ plus primaquine (PQ). Group 2 (n = 6) received atovaquone-proguanil (AP) followed by PQ. After the final immunization, the animals were challenged with intravenous injection of 104 P. cynomolgi sporozoites, the dose that induced reliable infection and relapse rate. These animals, along with control animals (n = 6), were monitored for primary infection and subsequent relapses. Immunogenicity blood draws were done after each of the three CPS session, before and after the challenge, with liver, spleen and bone marrow sampling and analysis done after the challenge. RESULTS: Group 2 animals demonstrated superior protection, with two achieving protection and two experiencing partial protection, while only one animal in group 1 had partial protection. These animals displayed high sporozoite-specific IFN-γ T cell responses in the liver, spleen, and bone marrow after the challenge with one protected animal having the highest frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. Partially protected animals also demonstrated a relatively high frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. It is important to highlight that the second animal in group 2, which experienced protection, exhibited deficient sporozoite-specific T cell responses in the liver while displaying average to high T cell responses in the spleen and bone marrow. CONCLUSIONS: This research supports the notion that local liver T cell immunity plays a crucial role in defending against liver-stage infection. Nevertheless, there is an instance where protection occurs independently of T cell responses in the liver, suggesting the involvement of the liver's innate immunity. The relapsing P. cynomolgi rhesus macaque model holds promise for informing the development of vaccines against relapsing P. vivax.

A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion.

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.

High Abundance of Haemoproteus Parasites in Culicoides (Diptera, Ceratopogonidae), with a Confirmation of Culicoides reconditus as a New Vector of These Avian Blood Parasites.

Haemoproteus parasites are the most diverse among Haemosporida. However, their natural vectors (Culicoides) are still poorly investigated and were identified for only a few parasite species and lineages. The application of an integrative approach (insect dissection, microscopic analysis, and molecular-based methods) is necessary in these studies, which have been carried out by a few research groups, mainly in Europe. The aim of this study was (i) to determine the Culicoides species that are naturally infected by Haemoproteus parasites, and which can support its complete sporogonic development, and (ii) to investigate the prevalence of Culicoides species and Haemoproteus parasite lineages in different study sites. In total, 1953 parous Culicoides females, from 11 species, were collected in four different localities in Lithuania and were dissected and analyzed using an integrative approach. The most abundant was C. pictipennis (30.3%). Parasite DNA was found in 7.9% of all investigated Culicoides, of which ~30% had sporozoites in their salivary glands, confirming their vector competence for these parasites. The Botanical Garden presented the highest number of Culicoides parous females, Culicoides species, and parasite lineages, as well as the highest positivity for sporozoites. Culicoides reconditus was confirmed as a natural vector of Haemoproteus parasites, sporozoites of six Haemoproteus lineages were reported for the first time, and 12 new interactions between Haemoproteus parasite lineages and Culicoides species were identified. Haemoproteus parasites seem to be transmitted by a high number of Culicoides species, with C. kibunensis, C. pictipennis, and C. segnis being the most important vectors.

Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes.

It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34-501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171-2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p<0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

Higher outdoor mosquito density and Plasmodium infection rates in and around malaria index case households in low transmission settings of Ethiopia: Implications for vector control.

BACKGROUND: Understanding the clustering of infections for persistent malaria transmission is critical to determining how and where to target specific interventions. This study aimed to determine the density, blood meal sources and malaria transmission risk of anopheline vectors by targeting malaria index cases, their neighboring households and control villages in Arjo-Didessa, southwestern Ethiopia. METHODS: An entomological study was conducted concurrently with a reactive case detection (RCD) study from November 2019 to October 2021 in Arjo Didessa and the surrounding vicinity, southwestern Ethiopia. Anopheline mosquitoes were collected indoors and outdoors in index case households and their surrounding households (neighboring households), as well as in control households, using pyrethrum spray cache (PSC) and U.S. Centers for Disease Control and Prevention (CDC) light traps. Adult mosquitoes were morphologically identified, and speciation in the Anopheles gambiae complex was done by PCR. Mosquito Plasmodium infections and host blood meal sources were detected by circumsporozoite protein enzyme-linked immunosorbent assay (CSP-ELISA) and cytochrome b-based blood meal PCR, respectively. RESULTS: Among the 770 anopheline mosquitoes collected, An. gambiae sensu lato (A. gambiae s.l.) was the predominant species, accounting for 87.1% (n = 671/770) of the catch, followed by the Anopheles coustani complex and Anopheles pharoensis, which accounted for 12.6% (n = 97/770) and 0.26% (n = 2/770) of the catch, respectively. From the sub-samples of An. gambiae s.l.analyzed with PCR, An. arabiensis and Anopheles amharicus were identified. The overall mean density of mosquitoes was 1.26 mosquitoes per trap per night using the CDC light traps. Outdoor mosquito density was significantly higher than indoor mosquito density in the index and neighboring households (P = 0.0001). The human blood index (HBI) and bovine blood index (BBI) of An. arabiensis were 20.8% (n = 34/168) and 24.0% (n = 41/168), respectively. The overall Plasmodium sporozoite infection rate of anophelines (An. arabiensis and An. coustani complex) was 4.4% (n = 34/770). Sporozoites were detected indoors and outdoors in captured anopheline mosquitoes. Of these CSP-positive species for Pv-210, Pv-247 and Pf, 41.1% (n = 14/34) were captured outdoors. A significantly higher proportion of sporozoite-infected mosquitoes were caught in index case households (5.6%, n = 8/141) compared to control households (1.1%, n = 2/181) (P = 0.02), and in neighboring households (5.3%, n = 24/448) compared to control households (P = 0.01). CONCLUSIONS: The findings of this study indicated that malaria index cases and their neighboring households had higher outdoor mosquito densities and Plasmodium infection rates. The study also highlighted a relatively higher outdoor mosquito density, which could increase the potential risk of outdoor malaria transmission and may play a role in residual malaria transmission. Thus, it is important to strengthen the implementation of vector control interventions, such as targeted indoor residual spraying, long-lasting insecticidal nets and other supplementary vector control measures such as larval source management and community engagement approaches. Furthermore, in low transmission settings, such as the Arjo Didessa Sugarcane Plantation, providing health education to local communities, enhanced environmental management and entomological surveillance, along with case detection and management by targeting of malaria index cases and their immediate neighboring households, could be important measures to control residual malaria transmission and achieve the targeted elimination goals.

Stearoyl-CoA desaturase regulates organelle biogenesis and hepatic merozoite formation in Plasmodium berghei.

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is needed to maintain the membrane's biophysical properties and fluidity. The malaria parasite can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver-stage development. However, when Scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective pre-erythrocytic immunity and hence its utility as a GAP.

The Multiple Roles of LCCL Domain-Containing Proteins for Malaria Parasite Transmission.

Multi-protein complexes are crucial for various essential biological processes of the malaria parasite Plasmodium, such as protein synthesis, host cell invasion and adhesion. Especially during the sexual phase of the parasite, which takes place in the midgut of the mosquito vector, protein complexes are required for fertilization, sporulation and ultimately for the successful transmission of the parasite. Among the most noticeable protein complexes of the transmission stages are the ones formed by the LCCL domain-containing protein family that play critical roles in the generation of infective sporozoites. The six members of this protein family are characterized by numerous adhesive modules and domains typically found in secreted proteins. This review summarizes the findings of expression and functional studies on the LCCL domain-containing proteins of the human pathogenic P. falciparum and the rodent-infecting P. berghei and discusses the common features and differences of the homologous proteins.

Overcoming the egress block of Plasmodium sporozoites expressing fluorescently tagged circumsporozoite protein.

Plasmodium sporozoites are the highly motile and invasive forms of the malaria parasite transmitted by mosquitoes. Sporozoites form within oocysts at the midgut wall of the mosquito, egress from oocysts and enter salivary glands prior to transmission. The GPI-anchored major surface protein, the circumsporozoite protein (CSP) is important for Plasmodium sporozoite formation, egress, migration and invasion. To visualize CSP, we previously generated full-length versions of CSP internally tagged with the green fluorescent protein, GFP. However, while these allowed for imaging of sporogony in oocysts, sporozoites failed to egress. Here, we explore different strategies to overcome this block in egress and obtain salivary gland resident sporozoites that express CSP-GFP. Replacing the N-terminal and repeat region with GFP did not allow sporozoite formation. Lowering expression of CSP-GFP at the endogenous locus allowed sporozoite formation but did not overcome egress block. Crossing of CSP-GFP expressing parasites that are blocked in egress with wild-type parasites yielded a small fraction of parasites that entered salivary glands and expressed various levels of CSP-GFP. Expressing CSP-GFP constructs from a silent chromosome region from promoters that are active only post salivary gland invasion yielded normal numbers of fluorescent salivary gland sporozoites, albeit with low levels of fluorescence. We also show that lowering CSP expression by 50% allowed egress from oocysts but not salivary gland entry. In conclusion, Plasmodium berghei parasites with normal CSP expression tolerate a certain level of CSP-GFP without disruption of oocyst egress and salivary gland invasion.

CD8+ Trms against malaria liver-stage: prospects and challenges.

Attenuated sporozoites provide a valuable model for exploring protective immunity against the malarial liver stage, guiding the design of highly efficient vaccines to prevent malaria infection. Liver tissue-resident CD8+ T cells (CD8+ Trm cells) are considered the host front-line defense against malaria and are crucial to developing prime-trap/target strategies for pre-erythrocytic stage vaccine immunization. However, the spatiotemporal regulatory mechanism of the generation of liver CD8+ Trm cells and their responses to sporozoite challenge, as well as the protective antigens they recognize remain largely unknown. Here, we discuss the knowledge gap regarding liver CD8+ Trm cell formation and the potential strategies to identify predominant protective antigens expressed in the exoerythrocytic stage, which is essential for high-efficacy malaria subunit pre-erythrocytic vaccine designation.

Biting behaviour, spatio-temporal dynamics, and the insecticide resistance status of malaria vectors in different ecological zones in Ghana.

BACKGROUND: A significant decrease in malaria morbidity and mortality has been attained using long-lasting insecticide-treated nets and indoor residual spraying. Selective pressure from these control methods influences changes in vector bionomics and behavioural pattern. There is a need to understand how insecticide resistance drives behavioural changes within vector species. This study aimed to determine the spatio-temporal dynamics and biting behaviour of malaria vectors in different ecological zones in Ghana in an era of high insecticide use for public health vector control. METHODS: Adult mosquitoes were collected during the dry and rainy seasons in 2017 and 2018 from five study sites in Ghana in different ecological zones. Indoor- and outdoor-biting mosquitoes were collected per hour from 18:00 to 06:00 h employing the human landing catch (HLC) technique. Morphological and molecular species identifications of vectors were done using identification keys and PCR respectively. Genotyping of insecticide-resistant markers was done using the TaqMan SNP genotyping probe-based assays. Detection of Plasmodium falciparum sporozoites was determined using PCR. RESULTS: A total of 50,322 mosquitoes belonging to four different genera were collected from all the study sites during the sampling seasons in 2017 and 2018. Among the Anophelines were Anopheles gambiae s.l. 93.2%, (31,055/33,334), An. funestus 2.1%, (690/33,334), An. pharoensis 4.6%, (1545/33,334), and An. rufipes 0.1% (44/33,334). Overall, 76.4%, (25,468/33,334) of Anopheles mosquitoes were collected in the rainy season and 23.6%, (7866/33,334) in the dry season. There was a significant difference (Z = 2.410; P = 0.0160) between indoor-biting (51.1%; 15,866/31,055) and outdoor-biting An. gambiae s.l. (48.9%; 15,189/31,055). The frequency of the Vgsc-1014F mutation was slightly higher in indoor-biting mosquitoes (54.9%) than outdoors (45.1%). Overall, 44 pools of samples were positive for P. falciparum CSP giving an overall sporozoite rate of 0.1%. CONCLUSION: Anopheles gambiae s.l. were more abundant indoors across all ecological zones of Ghana. The frequency of G119S was higher indoors than outdoors from all the study sites, but with higher sporozoite rates in outdoor mosquitoes in Dodowa and Kpalsogu. There is, therefore, an urgent need for a supplementary malaria control intervention to control outdoor-biting mosquitoes.

House Screening Reduces Exposure to Indoor Host-Seeking and Biting Malaria Vectors: Evidence from Rural South-East Zambia.

This study evaluated the impact of combining house screens with long-lasting insecticidal nets (LLINs) on mosquito host-seeking, resting, and biting behavior. Intervention houses received house screens and LLINs, while control houses received only LLINs. Centre for Disease Control light traps, pyrethrum spray collections and human landing catches were used to assess the densities of indoor and outdoor host-seeking, indoor resting, and biting behavior of malaria vectors in 15 sentinel houses per study arm per sampling method. The protective efficacy of screens and LLINs was estimated through entomological inoculation rates (EIRs). There were 68% fewer indoor host-seeking Anopheles funestus (RR = 0.32, 95% CI 0.20-0.51, p < 0.05) and 63% fewer An. arabiensis (RR = 0.37, 95% CI 0.22-0.61, p < 0.05) in screened houses than unscreened houses. There was a significantly higher indoor biting rate for unscreened houses (6.75 bites/person/h [b/p/h]) than for screened houses (0 b/p/h) (χ2 = 6.67, df = 1, p < 0.05). The estimated indoor EIR in unscreened houses was 2.91 infectious bites/person/six months, higher than that in screened houses (1.88 infectious bites/person/six months). Closing eaves and screening doors and windows has the potential to reduce indoor densities of malaria vectors and malaria transmission.

High human blood meal index of mosquitoes in Arba Minch town, southwest Ethiopia: an implication for urban mosquito-borne disease transmission.

Unplanned human population shifts in urban areas are expected to increase the prevalence of vector-borne diseases. This study aimed to investigate mosquito species composition, blood meal sources, and malaria vectors in an urban area. Indoor-resting adult mosquitoes were collected using Prokopack and host-seeking mosquitoes using Centers for Disease Control and Prevention light traps in Arba Minch town. Larval collection from artificial containers was done in those houses selected for adult mosquito collection. Anopheles adults collected and emerged from larvae were identified morphologically using a taxonomic key. ELISA was used to identify blood meal sources in freshly fed Anopheles and Culex mosquitoes, and CSP of Anopheles mosquitoes. A total of 16,756 female mosquitoes were collected. Of these, 93% (15,571) were Culex, 6% (1016) were Anopheles, and 1% (169) were Aedes mosquitoes. Out of the 130 adult mosquitoes that were raised from larvae collected from the containers, 20% were An. rhodesiensis, while the remaining 80% were Aedes mosquitoes. Out of 823 mosquitoes tested for blood meal origins, 86.3% (710/823) tested positive for human blood, 2.2% (18/823) tested positive for bovine blood, and 11.5% (95/823) were negative for human and bovine antibodies. Anopheles gambiae complex had a human blood meal index (HBI) of 50% (90/180; CI 42.3-57.5%) and a bovine blood meal index (BBI) of only 0.5% (95% CI 0.01-3.1%). Culex HBI was 96.7% (620/641), and its BBI index was 2.4% (15/641). While it was low (0.8%) in Culex, the proportion of An. gambiae complex with unidentified blood meal sources was 49.5% (95 CI% 41.9-56.9%). Among the 1016 Anopheles mosquitoes tested, a single An. gambiae complex (0.1%; 1/1016) was positive for P. vivax CSP. The high HBI indicates frequent contact between humans and vectors. To reduce human exposure, personal protection tools should be implemented.

Anopheles arabiensis continues to be the primary vector of Plasmodium falciparum after decades of malaria control in southwestern Ethiopia.

BACKGROUND: Investigating the species distribution and their role in malaria transmission is important as it varies from place to place and is highly needed to design interventions appropriate to the site. The current study aimed to investigate the Anopheles mosquito species distribution and their infection rate in southwestern Ethiopia. METHODS: The study was conducted in 14 malaria-endemic kebeles (the smallest administrative unit), which were situated in eight different malaria-endemic districts and four zones in southwestern Ethiopia. Ten per cent of households in each village were visited to collect adult mosquitoes using Centers for Disease Control and Prevention (CDC) light traps. The larval and pupal collection was done from breeding sites within the villages, and reared to adults. Female mosquitoes were morphologically identified. The head and thorax of adult Anopheles mosquitoes were tested for circumsporozoite proteins (CSPs) using ELISA. At the same time, legs, wings, and abdomen were used to identify sibling species using PCR targeting the rDNA intergenic spacers region for species typing of the Anopheles funestus group and the internal transcribed spacer 2 region genes for Anopheles gambiae complex. RESULTS: A total of 1445 Anopheles mosquitoes comprising eight species were collected. Of 813 An. gambiae complex tested by PCR, 785 (97%) were Anopheles arabiensis, and the remaining 28 (3%) were not amplified. There were 133 An. funestus group captured and tested to identify the species, of which 117 (88%) were positive for Anopheles parensis, and 15 (11%) were not amplified. A single specimen (1%) showed a band with a different base pair length from the known An. funestus group species. Sequencing revealed this was Anopheles sergentii. Among 1399 Anopheles tested for CSPs by ELISA, 5 (0.4%) An. arabiensis were positive for Plasmodium falciparum and a single (0.07%) was positive for Plasmodium vivax. CONCLUSIONS: Anopheles arabiensis continues to play the principal role in malaria transmission despite implementing indoor-based interventions for decades. Sequencing results suggest that An. sergentii was amplified by the An. funestus group primer, producing PCR amplicon size of different length. Therefore, relying solely on amplifying a specific gene of interest in grouping species could be misleading, as different species may share the same gene.

Malaria sporozoites evade host complement attack.

Complement is the first line of the host innate immune response against bacterial and viral infections; however, its role in the development of the malaria liver stage remains undefined. We found that sporozoite infection by either a mosquito bite or intravenous injection activated systemic complement, but neither depletion of C3 nor knockout of C3 had a significant effect on malaria liver stage development. Incubation of mouse serum with trypsin-treated sporozoites, but not naive sporozoites, led to the deposition of a membrane attack complex (MAC) on the surface of sporozoites and greatly reduced the number of exo-erythrocytic forms (EEF). Further studies have shown that the recruitment of complement H factor (CFH) may be associated with the prevention of MAC deposition on the surface of naïve sporozoites. Our data strongly suggest that sporozoites can escape complement attacks and provide us with a novel strategy to prevent malaria infection.