Estimating the effect of the wMel release programme on the incidence of dengue and chikungunya in Rio de Janeiro, Brazil: a spatiotemporal modelling study.

BACKGROUND: Introgression of genetic material from species of the insect bacteria Wolbachia into populations of Aedes aegypti mosquitoes has been shown in randomised and non-randomised trials to reduce the incidence of dengue; however, evidence for the real-world effectiveness of large-scale deployments of Wolbachia-infected mosquitoes for arboviral disease control in endemic settings is still scarce. A large Wolbachia (wMel strain) release programme was implemented in 2017 in Rio de Janeiro, Brazil. We aimed to assess the effect of this programme on the incidence of dengue and chikungunya in the city. METHODS: 67 million wMel-infected mosquitoes were released across 28 489 locations over an area of 86·8 km2 in Rio de Janeiro between Aug 29, 2017 and Dec 27, 2019. Following releases, mosquitoes were trapped and the presence of wMel was recorded. In this spatiotemporal modelling study, we assessed the effect of the release programme on the incidence of dengue and chikungunya. We used spatiotemporally explicit mathematical models applied to geocoded dengue cases (N=283 270) from 2010 to 2019 and chikungunya cases (N=57 705) from 2016 to 2019. FINDINGS: On average, 32% of mosquitoes collected from the release zones between 1 month and 29 months after the initial release tested positive for wMel. Reduced wMel introgression occurred in locations and seasonal periods in which cases of dengue and chikungunya were historically high, with a decrease to 25% of mosquitoes testing positive for wMel during months in which disease incidence was at its highest. Despite incomplete introgression, we found that the releases were associated with a 38% (95% CI 32-44) reduction in the incidence of dengue and a 10% (4-16) reduction in the incidence of chikungunya. INTERPRETATION: Stable establishment of wMel in the geographically diverse, urban setting of Rio de Janeiro seems to be more complicated than has been observed elsewhere. However, even intermediate levels of wMel seem to reduce the incidence of disease caused by two arboviruses. These findings will help to guide future release programmes. FUNDING: Bill & Melinda Gates Foundation and the European Research Council.

EVITA Dengue: a cluster-randomized controlled trial to EValuate the efficacy of Wolbachia-InfecTed Aedes aegypti mosquitoes in reducing the incidence of Arboviral infection in Brazil.

BACKGROUND: Arboviruses transmitted by Aedes aegypti including dengue, Zika, and chikungunya are a major global health problem, with over 2.5 billion at risk for dengue alone. There are no licensed antivirals for these infections, and safe and effective vaccines are not yet widely available. Thus, prevention of arbovirus transmission by vector modification is a novel approach being pursued by multiple researchers. However, the field needs high-quality evidence derived from randomized, controlled trials upon which to base the implementation and maintenance of vector control programs. Here, we report the EVITA Dengue trial design (DMID 17-0111), which assesses the efficacy in decreasing arbovirus transmission of an innovative approach developed by the World Mosquito Program for vector modification of Aedes mosquitoes by Wolbachia pipientis. METHODS: DMID 17-0111 is a cluster-randomized trial in Belo Horizonte, Brazil, with clusters defined by primary school catchment areas. Clusters (n = 58) will be randomized 1:1 to intervention (release of Wolbachia-infected Aedes aegypti mosquitoes) vs. control (no release). Standard vector control activities (i.e., insecticides and education campaigns for reduction of mosquito breeding sites) will continue as per current practice in the municipality. Participants (n = 3480, 60 per cluster) are children aged 6-11 years enrolled in the cluster-defining school and living within the cluster boundaries who will undergo annual serologic surveillance for arboviral infection. The primary objective is to compare sero-incidence of arboviral infection between arms. DISCUSSION: DMID 17-0111 aims to determine the efficacy of Wolbachia-infected mosquito releases in reducing human infections by arboviruses transmitted by Aedes aegypti and will complement the mounting evidence for this method from large-scale field releases and ongoing trials. The trial also represents a critical step towards robustness and rigor for how vector control methods are assessed, including the simultaneous measurement and correlation of entomologic and epidemiologic outcomes. Data from this trial will inform further the development of novel vector control methods. TRIAL REGISTRATION: ClinicalTrials.gov NCT04514107 . Registered on 17 August 2020 Primary sponsor: National Institute of Health, National Institute of Allergy and Infectious Diseases.

Large-Scale Deployment and Establishment of Wolbachia Into the Aedes aegypti Population in Rio de Janeiro, Brazil.

Traditional methods of vector control have proven insufficient to reduce the alarming incidence of dengue, Zika, and chikungunya in endemic countries. The bacterium symbiont Wolbachia has emerged as an efficient pathogen-blocking and self-dispersing agent that reduces the vectorial potential of Aedes aegypti populations and potentially impairs arboviral disease transmission. In this work, we report the results of a large-scale Wolbachia intervention in Ilha do Governador, Rio de Janeiro, Brazil. wMel-infected adults were released across residential areas between August 2017 and March 2020. Over 131 weeks, including release and post-release phases, we monitored the wMel prevalence in field specimens and analyzed introgression profiles of two assigned intervention areas, RJ1 and RJ2. Our results revealed that wMel successfully invaded both areas, reaching overall infection rates of 50-70% in RJ1 and 30-60% in RJ2 by the end of the monitoring period. At the neighborhood-level, wMel introgression was heterogeneous in both RJ1 and RJ2, with some profiles sustaining a consistent increase in infection rates and others failing to elicit the same. Correlation analysis revealed a weak overall association between RJ1 and RJ2 (r = 0.2849, p = 0.0236), and an association at a higher degree when comparing different deployment strategies, vehicle or backpack-assisted, within RJ1 (r = 0.4676, p < 0.0001) or RJ2 (r = 0.6263, p < 0.0001). The frequency knockdown resistance (kdr) alleles in wMel-infected specimens from both areas were consistently high over this study. Altogether, these findings corroborate that wMel can be successfully deployed at large-scale as part of vector control intervention strategies and provide the basis for imminent disease impact studies in Southeastern Brazil.

Effectiveness of Wolbachia-infected mosquito deployments in reducing the incidence of dengue and other Aedes-borne diseases in Niterói, Brazil: A quasi-experimental study.

BACKGROUND: The introduction of the bacterium Wolbachia (wMel strain) into Aedes aegypti mosquitoes reduces their capacity to transmit dengue and other arboviruses. Evidence of a reduction in dengue case incidence following field releases of wMel-infected Ae. aegypti has been reported previously from a cluster randomised controlled trial in Indonesia, and quasi-experimental studies in Indonesia and northern Australia. METHODOLOGY/PRINCIPAL FINDINGS: Following pilot releases in 2015-2016 and a period of intensive community engagement, deployments of adult wMel-infected Ae. aegypti mosquitoes were conducted in Niterói, Brazil during 2017-2019. Deployments were phased across four release zones, with a total area of 83 km2 and a residential population of approximately 373,000. A quasi-experimental design was used to evaluate the effectiveness of wMel deployments in reducing dengue, chikungunya and Zika incidence. An untreated control zone was pre-defined, which was comparable to the intervention area in historical dengue trends. The wMel intervention effect was estimated by controlled interrupted time series analysis of monthly dengue, chikungunya and Zika case notifications to the public health surveillance system before, during and after releases, from release zones and the control zone. Three years after commencement of releases, wMel introgression into local Ae. aegypti populations was heterogeneous throughout Niterói, reaching a high prevalence (>80%) in the earliest release zone, and more moderate levels (prevalence 40-70%) elsewhere. Despite this spatial heterogeneity in entomological outcomes, the wMel intervention was associated with a 69% reduction in dengue incidence (95% confidence interval 54%, 79%), a 56% reduction in chikungunya incidence (95%CI 16%, 77%) and a 37% reduction in Zika incidence (95%CI 1%, 60%), in the aggregate release area compared with the pre-defined control area. This significant intervention effect on dengue was replicated across all four release zones, and in three of four zones for chikungunya, though not in individual release zones for Zika. CONCLUSIONS/SIGNIFICANCE: We demonstrate that wMel Wolbachia can be successfully introgressed into Ae. aegypti populations in a large and complex urban setting, and that a significant public health benefit from reduced incidence of Aedes-borne disease accrues even where the prevalence of wMel in local mosquito populations is moderate and spatially heterogeneous. These findings are consistent with the results of randomised and non-randomised field trials in Indonesia and northern Australia, and are supportive of the Wolbachia biocontrol method as a multivalent intervention against dengue, chikungunya and Zika.

Transcriptional regulation of Culex pipiens mosquitoes by Wolbachia influences cytoplasmic incompatibility.

Cytoplasmic incompatibility (CI) induced by the endosymbiont Wolbachia pipientis causes complex patterns of crossing sterility between populations of the Culex pipiens group of mosquitoes. The molecular basis of the phenotype is yet to be defined. In order to investigate what host changes may underlie CI at the molecular level, we examined the transcription of a homolog of the Drosophila melanogaster gene grauzone that encodes a zinc finger protein and acts as a regulator of female meiosis, in which mutations can cause sterility. Upregulation was observed in Wolbachia-infected C. pipiens group individuals relative to Wolbachia-cured lines and the level of upregulation differed between lines that were reproductively incompatible. Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line. Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses. The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible. Three genes in inserted or deleted regions were newly identified in the C. molestus wPip genome, one of which is a transcriptional regulator labelled wtrM. When this gene was transfected into adult Culex mosquitoes, upregulation of the grauzone homolog was observed. These data suggest that Wolbachia-mediated regulation of host gene expression is a component of the mechanism of cytoplasmic incompatibility.

Wolbachia surface protein induces innate immune responses in mosquito cells.

BACKGROUND: Wolbachia endosymbiotic bacteria are capable of inducing chronic upregulation of insect immune genes in some situations and this phenotype may influence the transmission of important insect-borne pathogens. However the molecules involved in these interactions have not been characterized. RESULTS: Here we show that recombinant Wolbachia Surface Protein (WSP) stimulates increased transcription of immune genes in mosquito cells derived from the mosquito Anopheles gambiae, which is naturally uninfected with Wolbachia; at least two of the upregulated genes, TEP1 and APL1, are known to be important in Plasmodium killing in this species. When cells from Aedes albopictus, which is naturally Wolbachia-infected, were challenged with WSP lower levels of upregulation were observed than for the An. gambiae cells. CONCLUSIONS: We have found that WSP is a strong immune elicitor in a naturally Wolbachia-uninfected mosquito species (Anopheles gambiae) while a milder elicitor in a naturally-infected species (Aedes albopictus). Since the WSP of a mosquito non-native (nematode) Wolbachia strain was used, these data suggest that there is a generalized tolerance to WSP in Ae. albopictus.

Vital functions of the malarial ookinete protein, CTRP, reside in the A domains.

The transformation of malaria ookinetes into oocysts occurs in the mosquito midgut and is a major bottleneck for parasite transmission. The secreted ookinete surface protein, circumsporozoite- and thrombospondin-related adhesive protein (TRAP)-related protein (CTRP), is essential for this transition and hence constitutes a potential target for malaria transmission blockade. CTRP is a modular multidomain protein containing six tandem von Willebrand factor A-like (A) domains and seven tandem thrombospondin type I repeat-like (TS) domains. Here we present, to our knowledge, the first structure-function analysis of CTRP using genetically modified Plasmodium berghei parasites expressing mutant versions of the ctrp gene. Our data show that the A domains of CTRP are critical for ookinete gliding motility and oocyst formation whilst, unexpectedly, its TS domains are fully redundant. These results may have important implications for the design of CTRP-based transmission blocking strategies.

Wolbachia stimulates immune gene expression and inhibits plasmodium development in Anopheles gambiae.

The over-replicating wMelPop strain of the endosymbiont Wolbachia pipientis has recently been shown to be capable of inducing immune upregulation and inhibition of pathogen transmission in Aedes aegypti mosquitoes. In order to examine whether comparable effects would be seen in the malaria vector Anopheles gambiae, transient somatic infections of wMelPop were created by intrathoracic inoculation. Upregulation of six selected immune genes was observed compared to controls, at least two of which (LRIM1 and TEP1) influence the development of malaria parasites. A stably infected An. gambiae cell line also showed increased expression of malaria-related immune genes. Highly significant reductions in Plasmodium infection intensity were observed in the wMelPop-infected cohort, and using gene knockdown, evidence for the role of TEP1 in this phenotype was obtained. Comparing the levels of upregulation in somatic and stably inherited wMelPop infections in Ae. aegypti revealed that levels of upregulation were lower in the somatic infections than in the stably transinfected line; inhibition of development of Brugia filarial nematodes was nevertheless observed in the somatic wMelPop infected females. Thus we consider that the effects observed in An. gambiae are also likely to be more pronounced if stably inherited wMelPop transinfections can be created, and that somatic infections of Wolbachia provide a useful model for examining effects on pathogen development or dissemination. The data are discussed with respect to the comparative effects on malaria vectorial capacity of life shortening and direct inhibition of Plasmodium development that can be produced by Wolbachia.

Discovery of Plasmodium modulators by genome-wide analysis of circulating hemocytes in Anopheles gambiae.

Insect hemocytes mediate important cellular immune responses including phagocytosis and encapsulation and also secrete immune factors such as opsonins, melanization factors, and antimicrobial peptides. However, the molecular composition of these important immune cells has not been elucidated in depth, because of their scarcity in the circulating hemolymph, their adhesion to multiple tissues and the lack of primary culture methods to produce sufficient material for a genome-wide analysis. In this study, we report a genome-wide molecular characterization of circulating hemocytes collected from the hemolymph of adult female Anopheles gambiae mosquitoes--the major mosquito vector of human malaria in subSaharan Africa. Their molecular profile identified 1,485 transcripts with enriched expression in these cells, and many of these genes belong to innate immune gene families. This hemocyte-specific transcriptome is compared to those of Drosophila melanogaster and two other mosquitoes, Aedes aegypti and Armigeres subalbatus. We report the identification of two genes as ubiquitous hemocyte markers and several others as hemocyte subpopulation markers. We assess, via an RNAi screen, the roles in development of Plasmodium berghei of 63 genes expressed in hemocytes and provide a molecular comparison of the transcriptome of these cells during malaria infection.

The parasite invasion marker SRPN6 reduces sporozoite numbers in salivary glands of Anopheles gambiae.

For malaria transmission to occur, Plasmodium sporozoites must infect the salivary glands of their mosquito vectors. This study reports that Anopheles gambiae SRPN6 participates in a local salivary gland epithelial response against the rodent malaria parasite, Plasmodium berghei. We showed previously that SRPN6, an immune inducible midgut invasion marker, influences ookinete development. Here we report that SRPN6 is also specifically induced in salivary glands with the onset of sporozoite invasion. The protein is located in the basal region of epithelial cells in proximity to invading sporozoites. Knockdown of SRPN6 during the late phase of sporogony by RNAi has no effect on oocyst rupture but significantly increases the number of sporozoites present in salivary glands. Despite several differences between the passage of Plasmodium through the midgut and the salivary glands, this study identifies a striking overlap in the molecular responses of these two epithelia to parasite invasion.

Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi.

During its life cycle the malarial parasite Plasmodium forms three invasive stages which have to invade different and specific cells for replication to ensue. Invasion is vital to parasite survival and consequently proteins responsible for invasion are considered to be candidate vaccine/drug targets. Plasmodium perforin-like proteins (PPLPs) have been implicated in invasion because they contain a predicted pore-forming domain. Ookinetes express three PPLPs, and one of them (PPLP3) has previously been shown to be essential for mosquito midgut invasion. In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection. Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel. The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.

An immune-responsive serpin, SRPN6, mediates mosquito defense against malaria parasites.

We have functionally analyzed the orthologous SRPN6 genes from Anopheles stephensi and Anopheles gambiae using phylogenetic, molecular, reverse genetic, and cell biological tools. The results strongly implicate SRPN6 in the innate immune response against Plasmodium. This gene belongs to a mosquito-specific gene cluster including three additional Anopheles serpins. SRPN6 expression is induced by Escherichia coli and both rodent and human malaria parasites. The gene is specifically expressed in midgut cells invaded by Plasmodium ookinetes and in circulating and attached hemocytes. Knockdown of SRPN6 expression by RNA interference in susceptible An. stephensi leads to substantially increased parasite numbers, whereas depletion in susceptible An. gambiae delays progression of parasite lysis without affecting the number of developing parasites. However, the An. gambiae SRPN6 knockdown increases the number of melanized parasites in the L3-5 refractory strain and in susceptible G3 mosquitoes depleted of CTL4. These results indicate that AsSRPN6 is involved in the parasite-killing process, whereas AgSRPN6 acts on parasite clearance by inhibiting melanization and/or promoting parasite lysis. We propose that these observed phenotypic differences are due to changed roles of the respective target serine proteases in the two mosquito species.