Insects Go on a STING Operation to Tackle Intracellular Invaders.

Insect immunity to extracellular microbes relies largely on the TOLL and IMD pathways. In this issue of Immunity, Goto et al. (2018) report that the IKKß-Relish module of the IMD pathway hitches up the intracellular sensor STING to activate antiviral responses in Drosophila.

Natural Parasite Exposure Induces Protective Human Anti-Malarial Antibodies.

Antibodies against the NANP repeat of circumsporozoite protein (CSP), the major surface antigen of Plasmodium falciparum (Pf) sporozoites, can protect from malaria in animal models but protective humoral immunity is difficult to induce in humans. Here we cloned and characterized rare affinity-matured human NANP-reactive memory B cell antibodies elicited by natural Pf exposure that potently inhibited parasite transmission and development in vivo. We unveiled the molecular details of antibody binding to two distinct protective epitopes within the NANP repeat. NANP repeat recognition was largely mediated by germline encoded and immunoglobulin (Ig) heavy-chain complementarity determining region 3 (HCDR3) residues, whereas affinity maturation contributed predominantly to stabilizing the antigen-binding site conformation. Combined, our findings illustrate the power of exploring human anti-CSP antibody responses to develop tools for malaria control in the mammalian and the mosquito vector and provide a molecular basis for the structure-based design of next-generation CSP malaria vaccines.

High-density binding to Plasmodium falciparum circumsporozoite protein repeats by inhibitory antibody elicited in mouse with human immunoglobulin repertoire.

Antibodies targeting the human malaria parasite Plasmodium falciparum circumsporozoite protein (PfCSP) can prevent infection and disease. PfCSP contains multiple central repeating NANP motifs; some of the most potent anti-infective antibodies against malaria bind to these repeats. Multiple antibodies can bind the repeating epitopes concurrently by engaging into homotypic Fab-Fab interactions, which results in the ordering of the otherwise largely disordered central repeat into a spiral. Here, we characterize IGHV3-33/IGKV1-5-encoded monoclonal antibody (mAb) 850 elicited by immunization of transgenic mice with human immunoglobulin loci. mAb 850 binds repeating NANP motifs with picomolar affinity, potently inhibits Plasmodium falciparum (Pf) in vitro and, when passively administered in a mouse challenge model, reduces liver burden to a similar extent as some of the most potent anti-PfCSP mAbs yet described. Like other IGHV3-33/IGKV1-5-encoded anti-NANP antibodies, mAb 850 primarily utilizes its HCDR3 and germline-encoded aromatic residues to recognize its core NANP motif. Biophysical and cryo-electron microscopy analyses reveal that up to 19 copies of Fab 850 can bind the PfCSP repeat simultaneously, and extensive homotypic interactions are observed between densely-packed PfCSP-bound Fabs to indirectly improve affinity to the antigen. Together, our study expands on the molecular understanding of repeat-induced homotypic interactions in the B cell response against PfCSP for potently protective mAbs against Pf infection.

Kinetics of Plasmodium midgut invasion in Anopheles mosquitoes.

Malaria-causing Plasmodium parasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of two Anopheles species and a Plasmodium berghei fluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and Indian Anopheles species and propose that the mosquito complement-like system contributes to the species-specific dynamics of Plasmodium invasion.

Vector Immunity and Evolutionary Ecology: The Harmonious Dissonance.

Recent scientific breakthroughs have significantly expanded our understanding of arthropod vector immunity. Insights in the laboratory have demonstrated how the immune system provides resistance to infection, and in what manner innate defenses protect against a microbial assault. Less understood, however, is the effect of biotic and abiotic factors on microbial-vector interactions and the impact of the immune system on arthropod populations in nature. Furthermore, the influence of genetic plasticity on the immune response against vector-borne pathogens remains mostly elusive. Herein, we discuss evolutionary forces that shape arthropod vector immunity. We focus on resistance, pathogenicity and tolerance to infection. We posit that novel scientific paradigms should emerge when molecular immunologists and evolutionary ecologists work together.

Unbiased classification of mosquito blood cells by single-cell genomics and high-content imaging.

Mosquito blood cells are immune cells that help control infection by vector-borne pathogens. Despite their importance, little is known about mosquito blood cell biology beyond morphological and functional criteria used for their classification. Here, we combined the power of single-cell RNA sequencing, high-content imaging flow cytometry, and single-molecule RNA hybridization to analyze a subset of blood cells of the malaria mosquito Anopheles gambiae By demonstrating that blood cells express nearly half of the mosquito transcriptome, our dataset represents an unprecedented view into their transcriptional program. Analyses of differentially expressed genes identified transcriptional signatures of two cell types and provide insights into the current classification of these cells. We further demonstrate the active transfer of a cellular marker between blood cells that may confound their identification. We propose that cell-to-cell exchange may contribute to cellular diversity and functional plasticity seen across biological systems.

A New Role of the Mosquito Complement-like Cascade in Male Fertility in Anopheles gambiae.

Thioester-containing protein 1 (TEP1) is a key immune factor that determines mosquito resistance to a wide range of pathogens, including malaria parasites. Here we report a new allele-specific function of TEP1 in male fertility. We demonstrate that during spermatogenesis TEP1 binds to and removes damaged cells through the same complement-like cascade that kills malaria parasites in the mosquito midgut. Further, higher fertility rates are mediated by an allele that renders the mosquito susceptible to Plasmodium. By elucidating the molecular and genetic mechanisms underlying TEP1 function in spermatogenesis, our study suggests that pleiotropic antagonism between reproduction and immunity may shape resistance of mosquito populations to malaria parasites.

Comparative Proteomics and Functional Analysis Reveal a Role of Plasmodium falciparum Osmiophilic Bodies in Malaria Parasite Transmission.

An essential step in the transmission of the malaria parasite to the Anopheles vector is the transformation of the mature gametocytes into gametes in the mosquito gut, where they egress from the erythrocytes and mate to produce a zygote, which matures into a motile ookinete. Osmiophilic bodies are electron dense secretory organelles of the female gametocytes which discharge their contents during gamete formation, suggestive of a role in gamete egress. Only one protein with no functional annotation, Pfg377, is described to specifically reside in osmiophilic bodies in Plasmodium falciparum Importantly, Pfg377 defective gametocytes lack osmiophilic bodies and fail to infect mosquitoes, as confirmed here with newly produced pfg377 disrupted parasites. The unique feature of Pfg377 defective gametocytes of lacking osmiophilic bodies was here exploited to perform comparative, label free, global and affinity proteomics analyses of mutant and wild type gametocytes to identify components of these organelles. Subcellular localization studies with fluorescent reporter gene fusions and specific antibodies revealed an osmiophilic body localization for four out of five candidate gene products analyzed: the proteases PfSUB2 (subtilisin 2) and PfDPAP2 (Dipeptidyl aminopeptidase 2), the ortholog of the osmiophilic body component of the rodent malaria gametocytes PbGEST and a previously nonannotated 13 kDa protein. These results establish that osmiophilic bodies and their components are dispensable or marginally contribute (PfDPAP2) to gamete egress. Instead, this work reveals a previously unsuspected role of these organelles in P. falciparum development in the mosquito vector.

Intracellular immune responses of dipteran insects.

Vector-borne diseases, transmitted by bloodsucking arthropods, pose worldwide socio-medical and economical problems. Some of the major human infectious diseases, such as malaria, Dengue fever, and yellow fever, are transmitted by mosquitoes. While the majority of pathogens enjoy extracellular life styles in insects, viruses and some endosymbionts are strictly intracellular. Here, we summarize our knowledge on defense reactions against intracellular microorganisms in dipteran insects and discuss the potential of insects as models to study human pathogens.

AP-1/Fos-TGase2 axis mediates wounding-induced Plasmodium falciparum killing in Anopheles gambiae.

Anopheline mosquitoes are the only vectors of human malaria worldwide. It is now widely accepted that mosquito immune responses play a crucial role in restricting Plasmodium development within the vector; therefore, further dissection of the molecular mechanisms underlying these processes should inform new vector control strategies urgently needed to roll back the disease. Here, using genome-wide transcriptional profiling, bioinformatics, and functional gene analysis, we identify a new axis of mosquito resistance to monoclonal Plasmodium falciparum infections that includes the AP-1 transcription factor Fos and the transglutaminase 2 (TGase2), a cross-linking enzyme with known roles in wound responses. We demonstrate that Fos regulates induction of TGase2 expression after wounding but does not affect expression of the components of the well characterized complement-like system. Silencing of Fos or of TGase2 aborts the wounding-induced mosquito killing of P. falciparum. These results reveal multiple signaling pathways that are required for efficient Plasmodium killing in Anopheles gambiae.

Midgut microbiota of the malaria mosquito vector Anopheles gambiae and interactions with Plasmodium falciparum infection.

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.

The major yolk protein vitellogenin interferes with the anti-plasmodium response in the malaria mosquito Anopheles gambiae.

When taking a blood meal on a person infected with malaria, female Anopheles gambiae mosquitoes, the major vector of human malaria, acquire nutrients that will activate egg development (oogenesis) in their ovaries. Simultaneously, they infect themselves with the malaria parasite. On traversing the mosquito midgut epithelium, invading Plasmodium ookinetes are met with a potent innate immune response predominantly controlled by mosquito blood cells. Whether the concomitant processes of mosquito reproduction and immunity affect each other remains controversial. Here, we show that proteins that deliver nutrients to maturing mosquito oocytes interfere with the antiparasitic response. Lipophorin (Lp) and vitellogenin (Vg), two nutrient transport proteins, reduce the parasite-killing efficiency of the antiparasitic factor TEP1. In the absence of either nutrient transport protein, TEP1 binding to the ookinete surface becomes more efficient. We also show that Lp is required for the normal expression of Vg, and for later Plasmodium development at the oocyst stage. Furthermore, our results uncover an inhibitory role of the Cactus/REL1/REL2 signaling cassette in the expression of Vg, but not of Lp. We reveal molecular links that connect reproduction and immunity at several levels and provide a molecular basis for a long-suspected trade-off between these two processes.

A heterodimeric complex of the LRR proteins LRIM1 and APL1C regulates complement-like immunity in Anopheles gambiae.

The leucine-rich repeat (LRR) proteins LRIM1 and APL1C control the function of the complement-like protein TEP1 in Anopheles mosquitoes. The molecular structure of LRIM1 and APL1C and the basis of their interaction with TEP1 represent a new type of innate immune complex. The LRIM1/APL1C complex specifically binds and solubilizes a cleaved form of TEP1 without an intact thioester bond. The LRIM1 and APL1C LRR domains have a large radius of curvature, glycosylated concave face, and a novel C-terminal capping motif. The LRIM1/APL1C complex is a heterodimer with a single intermolecular disulfide bond. The structure of the LRIM1/APL1C heterodimer reveals an interface between the two LRR domains and an extensive C-terminal coiled-coil domain. We propose that a cleaved form of TEP1 may act as a convertase for activation of other TEP1 molecules and that the LRIM1/APL1C heterodimer regulates formation of this TEP1 convertase.

Evolutionary modelling indicates that mosquito metabolism shapes the life-history strategies of Plasmodium parasites.

Within-host survival and between-host transmission are key life-history traits of single-celled malaria parasites. Understanding the evolutionary forces that shape these traits is crucial to predict malaria epidemiology, drug resistance, and virulence. However, very little is known about how Plasmodium parasites adapt to their mosquito vectors. Here, we examine the evolution of the time Plasmodium parasites require to develop within the vector (extrinsic incubation period) with an individual-based model of malaria transmission that includes mosquito metabolism. Specifically, we model the metabolic cascade of resource allocation induced by blood-feeding, as well as the influence of multiple blood meals on parasite development. Our model predicts that successful vector-to-human transmission events are rare, and are caused by long-lived mosquitoes. Importantly, our results show that the life-history strategies of malaria parasites depend on the mosquito's metabolic status. In our model, additional resources provided by multiple blood meals lead to selection for parasites with slow or intermediate developmental time. These results challenge the current assumption that evolution favors fast developing parasites to maximize their chances to complete their within-mosquito life cycle. We propose that the long sporogonic cycle observed for Plasmodium is not a constraint but rather an adaptation to increase transmission potential.

Molecular and functional properties of human Plasmodium falciparum CSP C-terminus antibodies.

Human monoclonal antibodies (mAbs) against the central repeat and junction domain of Plasmodium falciparum circumsporozoite protein (PfCSP) have been studied extensively to guide malaria vaccine design compared to antibodies against the PfCSP C terminus. Here, we describe the molecular characteristics and protective potential of 73 germline and mutated human mAbs against the highly immunogenic PfCSP C-terminal domain. Two mAbs recognized linear epitopes in the C-terminal linker with sequence similarity to repeat and junction motifs, whereas all others targeted conformational epitopes in the α-thrombospondin repeat (α-TSR) domain. Specificity for the polymorphic Th2R/Th3R but not the conserved RII+/CS.T3 region in the α-TSR was associated with IGHV3-21/IGVL3-21 or IGLV3-1 gene usage. Although the C terminus specific mAbs showed signs of more efficient affinity maturation and class-switching compared to anti-repeat mAbs, live sporozoite binding and inhibitory activity was limited to a single C-linker reactive mAb with cross-reactivity to the central repeat and junction. The data provide novel insights in the human anti-C-linker and anti-α-TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs.

Glycosylated nanoparticle-based PfCSP vaccine confers long-lasting antibody responses and sterile protection in mouse malaria model.

The development of an effective and durable vaccine remains a central goal in the fight against malaria. Circumsporozoite protein (CSP) is the major surface protein of sporozoites and the target of the only licensed Plasmodium falciparum (Pf) malaria vaccine, RTS,S/AS01. However, vaccine efficacy is low and short-lived, highlighting the need for a second-generation vaccine with superior efficacy and durability. Here, we report a Helicobacter pylori apoferritin-based nanoparticle immunogen that elicits strong B cell responses against PfCSP epitopes that are targeted by the most potent human monoclonal antibodies. Glycan engineering of the scaffold and fusion of an exogenous T cell epitope enhanced the anti-PfCSP B cell response eliciting strong, long-lived and protective humoral immunity in mice. Our study highlights the power of rational vaccine design to generate a highly efficacious second-generation anti-infective malaria vaccine candidate and provides the basis for its further development.

High-throughput sorting of mosquito larvae for laboratory studies and for future vector control interventions.

BACKGROUND: Mosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever. Genetic control strategies require the release of large number of male mosquitoes into field populations, whether they are based on the use of sterile males (sterile insect technique, SIT) or on introducing genetic traits conferring refractoriness to disease transmission (population replacement). However, the current absence of high-throughput techniques for sorting different mosquito populations impairs the application of these control measures. METHODS: A method was developed to generate large mosquito populations of the desired sex and genotype. This method combines flow cytometry and the use of Anopheles gambiae transgenic lines that differentially express fluorescent markers in males and females. RESULTS: Fluorescence-assisted sorting allowed single-step isolation of homozygous transgenic mosquitoes from a mixed population. This method was also used to select wild-type males only with high efficiency and accuracy, a highly desirable tool for genetic control strategies where the release of transgenic individuals may be problematic. Importantly, sorted males showed normal mating ability compared to their unsorted brothers. CONCLUSIONS: The developed method will greatly facilitate both laboratory studies of mosquito vectorial capacity requiring high-throughput approaches and future field interventions in the fight against infectious disease vectors.

Molecular and cellular components of the mating machinery in Anopheles gambiae females.

Anopheles gambiae mosquitoes are the principal vectors of malaria. A major determinant of the capacity of these mosquitoes as disease vectors is their high reproductive rate. Reproduction depends on a single insemination, which profoundly changes the behavior and physiology of females. To identify factors and mechanisms relevant to the fertility of A. gambiae, we performed a comprehensive analysis of the molecular and cellular machinery associated with copulation in females. Initial whole-body microarray experiments comparing virgins with females at 2 h, 6 h, and 24 h after mating detected large transcriptional changes. Analysis of tissue localization identified a subset of genes whose expression was strikingly regulated by mating in the lower reproductive tract and, surprisingly, the gut. In the atrium of virgin females, where the male seminal fluid is received, our studies revealed a "mating machinery" consisting of molecular and structural components that are turned off or collapse after copulation, suggesting that this tissue loses its competence for further insemination. In the sperm storage organ, we detected a number of mating-responsive genes likely to have a role in the maintenance and function of stored sperm. These results identify genes and mechanisms regulating the reproductive biology of A. gambiae females, highlighting considerable differences with Drosophila melanogaster. Our data inform vector control strategies and reveal promising targets for the manipulation of fertility in field populations of these important disease vectors.

Critical Steps of Plasmodium falciparum Ookinete Maturation.

The egress and fertilization of Plasmodium gametes and development of a motile ookinete are the first crucial steps that mediate the successful transmission of the malaria parasites from humans to the Anopheles vector. However, limited information exists about the cell biology and regulation of this process. Technical impediments in the establishment of in vitro conditions for ookinete maturation in Plasmodium falciparum and other human malaria parasites further constrain a detailed characterization of ookinete maturation. Here, using fluorescence microscopy and immunolabeling, we compared P. falciparum ookinete maturation in Anopheles coluzzii mosquitoes in vivo and in cell culture in vitro. Our results identified two critical steps in ookinete maturation that are regulated by distinct mosquito factors, thereby highlighting the role of the mosquito environment in the transmission efficiency of malaria parasites.

Evolution of protective human antibodies against Plasmodium falciparum circumsporozoite protein repeat motifs.

The circumsporozoite protein of the human malaria parasite Plasmodium falciparum (PfCSP) is the main target of antibodies that prevent the infection and disease, as shown in animal models. However, the limited efficacy of the PfCSP-based vaccine RTS,S calls for a better understanding of the mechanisms driving the development of the most potent human PfCSP antibodies and identification of their target epitopes. By characterizing 200 human monoclonal PfCSP antibodies induced by sporozoite immunization, we establish that the most potent antibodies bind around a conserved (N/D)PNANPN(V/A) core. High antibody affinity to the core correlates with protection from parasitemia in mice and evolves around the recognition of NANP motifs. The data suggest that the rational design of a next-generation PfCSP vaccine that elicits high-affinity antibody responses against the core epitope will promote the induction of protective humoral immune responses.