George Dimopoulos - US grants

DESCRIPTION (provided by applicant): Recent advances in Anopheles gambiae transgenic and high throughput genomic technologies have rendered it a powerful model organism for the study of host - parasite interactions and innate immunity. In addition, Anopheline mosquitoes transmit one of the most serious diseases of mankind, malaria. The lack of effective vaccines, the development of parasite resistance to drugs and mosquito resistance to insecticides contribute to the expansion of the disease and have thereby created an acute need for the development of additional novel control strategies. Control strategies based on the blocking of Plasmodium's lifecycle in the vector mosquito will require the understanding of the molecular mechanisms implicated in mosquito - parasite interactions. The mosquito's innate immune system is capable of killing large numbers of malaria parasites. Ingestion of malaria infected blood results in the activation of innate immune responses that contribute significantly to the killing of Plasmodia in the midgut. The specific elicitors of these immune responses, the immune pathways they regulate and their plasmodiocidal activity on the different stages of the parasite are as yet unknown. This application will utilize a full genome microarray for a comprehensive dissection of the systemic and midgut specific transcript responses to the different components (elicitors) that are associated with malaria infected blood and then assess the impact of these responses on Plasmodium development. These analyses will elucidate important aspects of the mosquito's infection responsive physiology and provide information on the regulation of antiplasmodial immune response. It will provide baseline assessment on the utilization of the mosquito's innate immune system for the development of malaria control strategies.

Mosquitoes utilize a variety of strategies to combat pathogens, including viruses. The long term_ goal of the PIs research is to elucidate, at the molecular level, the immune responses of the mosquito vector Aedes aegypti to infection with a range of viral pathogens, including those that could be used as agents for bioterrorism. In recent years, dengue fever has emerged as one of the most serious vector borne viral diseases. The molecular biology of dengue virus propagation in the mosquito vector is largely unknown, but the genome sequence and reverse genetic and functional genomics tools that are now available provide a means of dissecting the mosquito's anti viral defense systems in greater detail. In this proposal, we will apply these tools to characterize the Ae. aegypti mosquito's innate immune defense against the dengue virus. We have already showed that the mosquito is employing the Toll and JAK STAT immune pathways to limit dengue virus infection. We will now expand on this finding and characterize the mosquito's broader molecular immune responses to virus infection at multiple time points and tissues in Specific Aim 1. We will analyze the implication of the mosquito's immune pathways in defense against dengue virus infection in a greater detail in Specific Aim 2. In addition to the basic knowledge generated by the proposed experiments, this project should also provide a variety of tools that can be used for the development of novel antiviral control strategies.

DESCRIPTION (provided by applicant): The innate immune system is the mosquito's main line of defense against the malaria parasite Plasmodium at multiple stages of the parasite's life cycle. These immune responses are largely regulated by the TOLL and IMD pathways, which control the nuclear translocation of the NF-kappaB-like transcription factors, Rel1 and Rel2, respectively. While both pathways are implicated in anti-Plasmodium defense, we have shown that the IMD pathway is a conserved key player in regulating resistance of several Anopheles species to multiple malaria parasite species including the human pathogen P. falciparum. We have shown that the Rel2 transcription factor - mediate anti- Plasmodium action through multiple effectors and that the fitness cost of a transient induction of the Rel2 activation is minimal. As such, the IMD pathway is particularly interesting for the development of genetically modified Plasmodium resistant mosquitoes. This proposal will focus on a better understanding of the Rel2 mediated resistance to Plasmodium and thereby assess the feasibility to use this system for the development of malaria control strategies. The overall aim of this project is to develop transgenic mosquitoes that can activate Rel2 mediated anti-Plasmodium defense at an appropriate stage of infection. These mosquitoes will be used to study the regulation of this defense system and dissect the genes and mechanisms that is responsible for Plasmodium killing. PUBLIC HEALTH RELEVANCE: The Anopheles mosquito uses its innate immune system to fight against a broad spectrum of microbial pathogens including the Plasmodium parasite. We have shown that the IMD immune signaling pathway is a major player in anti-Plasmodium defense. This research proposal aims at the molecular dissection of IMD pathway mediated Plasmodium resistance in A. gambiae.

DESCRIPTION (provided by applicant): Anopheles gambiae and other insects utilize a variety of strategies to combat microbial invaders. The long-term goal of the PIs research is to characterize and understand the mechanisms of innate immunity used by the mosquito against microbes. This proposal focuses on the particular role of the Down syndrome cell adhesion molecule (Dscam) as a hypervariable immune surveillance receptor. Dscam's complex genomic organization and remarkable properties suggest that it plays multiple roles in immune defense: In A. gambiae, alternative splicing of the AgDscam gene produces >31,920 alternative splice- form receptors with different adhesive characteristics and specificities. AgDscam produces microbe-specific splice-form repertoires upon immune challenge and is involved in both antibacterial and anti-Plasmodium defense. The studies outlined here will further test the hypothesis that, as previously shown for challenge with a limited number of bacteria, this phenotypic plasticity of the AgDscam gene allows it to produce receptor molecules with potent antimicrobial activity and high affinity for bacteria and the Plasmodium parasite. To test this hypothesis, the proposed studies will use RNAi silencing, immunohistochemical staining, microarray analyses, and interaction analyses to determine (in Specific Aim 1) the regulation of AgDscam splicing and production of splice form repertoire by microbe challenge and the innate immune pathways, and (in Aims 2) the specific splice-form repertoires involved in these defenses and interactions with microbes. We will finally test the anti-Plasmodium defense specificity and efficacy of AgDscam in genetically modified mosquito lines that over-express pathogen specific splice forms (in Specific Aim 3). These analyses will elucidate one of the most remarkable players of the insect innate immune system, which allows it to cope with a broad spectrum of microbes through the microbe challenge specific production of pattern recognition receptors. The study will also assess the feasibility to use AgDscam for the generation of Plasmodium resistant mosquitoes that could be used for the development of a malaria control strategy.

DESCRIPTION (provided by applicant): Mosquitoes utilize a variety of strategies to combat pathogens, including viruses. The long-term goal of the PIs research is to elucidate, at the molecular level, the immune responses of the mosquito vector Aedes aegypti to infection with a range of viral pathogens, including those that could be used as agents for bioterrorism. In recent years, dengue fever has emerged as one of the most serious vector-borne viral diseases. The molecular biology of dengue virus propagation in the mosquito vector is largely unknown, but the genome sequence, reverse genetic, transgenic and functional genomics tools that are now available provide a means of dissecting the mosquito's anti-viral defense systems in greater detail. In this proposal, we will apply these tools to characterize the implication of the Ae. aegypti JAK-STAT innate immune pathway in defense against the dengue virus. We have already showed that the mosquito is employing the JAK-STAT immune pathways to limit dengue virus infection through RNAi - based approaches. We will now use more powerful approaches, based on transgenic methodology, to expand on this finding and better characterize the JAK-STAT pathway immune responses to virus infection at multiple time points and tissues. In Specific Aim 1 we will generate and molecularly characterize the genetically modified mosquito lines that conditionally activate the JAK-STAT pathway. In Specific Aim 2 we will investigate the spatio- temporal anti-dengue activity of the recombinantly activated JAK-STAT pathway to better assess the potential use of this defense system for the development of dengue control strategies. PUBLIC HEALTH RELEVANCE: Despite the dramatic surge in dengue prevalence, surprisingly little is known about the molecular biology and genetic basis of the virus interactions with the mosquito vectors that enable transmission. This research proposal aims at the molecular characterization of the mosquito's immune defenses against virus infection that are controlled by the JAK-STAT pathway using transgenic technologies. The knowledge generated from this study can contribute to the development of control methods for dengue and other viruses such as yellow fever and West Niles viruses.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise;2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues;and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): In recent years, dengue fever has emerged as one of the most serious vector-borne viral diseases. The molecular biology of dengue virus propagation in the mosquito vector is largely unknown, but the genome sequence, reverse genetic and functional genomics tools as well as high throughput screening methodologies that are now available provide a means of dissecting the mosquito's interactions with the virus in greater detail. In this proposal, we will apply a highly innovative interdisciplinary approach to discover and develop small molecules (compounds) that can render the Aedes aegypti vector mosquito resistant to the virus and thereby incapable of transmitting disease. A previous collaboration between the groups of Drs. Garcia-Blanco and Dimopoulos identified over 300 potential dengue virus host and restriction factors through a high throughput RNAi screen in a Drosophila cell line. We will now expand on these findings and identify homologues of these factors in the Aedes mosquito in specific Aim 1. In Aim 2 we will assess the anti-dengue potential of selected factors in the Aedes cell line and mosquito to select those that are most suitable for small molecule discovery. We will then use a powerful established yeast-based platform to identify compounds that can repress dengue virus host factors, and an established insect cell line -based system to identify compounds that can activate expression of dengue virus restriction factors in Aim 3. In Aim 4 we will test a selected set of these compounds for anti-dengue activity in Aedes cell lines and adult female mosquitoes through different routes of exposure. We will finally investigate the potential influence of selected compounds on mosquito fitness parameters and assess their anti-dengue activity in field -derived mosquito strains in Aim 5. The completion of this highly innovative work will lead to a transition into translational studis on the delivery of potent anti-dengue compounds to natural dengue vector populations, as a prelude towards the development of a novel dengue control strategy.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise; 2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues; and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

? DESCRIPTION (provided by applicant): Malaria is transmitted by Anopheles mosquitoes and has a devastating impact on human health and the socioeconomic conditions in endemic regions. The transmission of vector-borne pathogens is influenced by the vector s innate immune system which is largely regulated at the transcriptional and post-transcriptional level. The long-term goal of the PIs research is to elucidate, at the molecular level, the biological processes that take place between the Anopheles mosquito, its innate immune system and the malaria parasite, for the development of novel malaria control strategies. Our published and preliminary studies have shown that the mosquito IMD pathway - controlled innate immune response fulfills required criteria for an anti-Plasmodium effector system that could be used for the development of a malaria control strategy based on genetically modified Plasmodium-resistant mosquitoes. While micro RNAs have proven to play essential roles in regulating a variety of processes including immune response, their role in modulating mosquito resistance to human malaria parasite infection has not yet been explored. One miRNA can control the expression of a variety of genes, including those encoding immune response-regulators and effectors that block Plasmodium infection of the mosquito. We have identified several miRNAs that are predicted to target multiple transcripts encoding IMD pathway -factors and -regulated effectors. We have also shown that silencing of such miRNAs render mosquitoes more resistant to P. falciparum infection. This proposal seeks to characterize the implication of A. gambiae miRNAs in regulating anti-P. falciparum defense and asses their effectiveness for the development malaria control strategies based on genetically modified mosquitoes, using novel innovative miRNA sponge methodology.

? DESCRIPTION (provided by applicant): Malaria is caused by Plasmodium parasites and is transmitted to humans by female Anopheles mosquitoes. Current malaria control measures may not be sufficient to achieve effective control and elimination. There is an urgent need to advance our understanding of the vector's biological processes that can potentially be exploited in an effort to block malaria transmission. The mosquito's innate immunity plays a pivotal role in the interaction between the malaria parasites and the mosquito vector, and is a determining factor of vectorial capacity. In this study, we will identify microRNAs (miRNAs) used by mosquitoes to modulate their defense against Plasmodium infection and will determine whether manipulating the levels of the selected miRNAs can lead to enhanced mosquito resistance to Plasmodium infection. miRNAs are small endogenous RNA molecules that post-transcriptionally regulate gene expression. They recognize their target mRNA transcripts through partial sequence complementarity. miRNAs have been shown to act as master regulators by targeting multiple genes involved in the same biological process. Our preliminary studies clearly demonstrate that miRNAs are involved in modulating the mosquito defense response to malaria parasites. We have performed a genome-wide analysis of miRNA-mRNA interactions and discovered that the transcripts of some mosquito immune genes are differentially regulated by certain miRNAs in response to Plasmodium infection. We will investigate whether these identified miRNAs affect the survival of Plasmodium parasites, when and where the regulation takes place, and which genes are regulated (directly or indirectly) by the miRNAs during midgut invasion by Plasmodium ookinetes. The specific aims of this project are to: (1) Identify miRNA-mRNA interactions affected by P. falciparum infection of the midgut epithelium in An. gambiae, (2) Select miRNAs that play important regulatory roles in anti-Plasmodium defense, and (3) Use transgenic expression of miRNAs or miRNA sponges to enhance mosquito resistance to Plasmodium infection. Our proposed study addresses a serious gap in the understanding of miRNA function in mosquito- Plasmodium interactions, and may provide novel molecular targets for blocking malaria transmission.

PROJECT SUMMARY The malaria parasite Plasmodium has to accomplish its sporogonic development in the mosquito vector in order for malaria transmission to occur. The long-term goal of the PI's research is to elucidate the interactions between the mosquito vector and malaria parasite to identify transmission-blocking targets suitable for the development of disease control strategies. During its journey through the mosquito, the parasite engages in intimate interactions with the mosquito midgut, hemolymph, and salivary gland and relies on numerous mosquito-derived host factors (agonists). Plasmodium can be impaired in the vector by agonist deletion through gene editing (GM mosquito population replacement), depletion through RNA interference (RNAi-based control through GM mosquito population replacement or external application of dsRNAs or analogues), or blocking/interference/inhibition through antibodies (transmission blocking vaccines) or small molecules (transmission blocking drugs or mosquito exposure). Hence, an obvious advantage of studying Plasmodium agonists for malaria control is that they can be targeted through multiple means. The recently developed CRISPR/CAS9-based gene editing in Anopheles mosquitoes provide new and promising opportunities for the study of agonist function through gene deletion, and for malaria control through gene drive. Here we will focus on 10 pro-Plasmodium factors (agonists) to assess their potential for malaria control and further our knowledge of their involvement in Plasmodium transmission. In Aim 1, we will validate 4 putative Plasmodium agonists (that have previously only been tested with non-human parasites) for P. falciparum transmission-blocking potential in A. gambiae using RNAi-mediated gene silencing. In Aim 2, we will develop CRISPR/CAS9- mediated gene KO mosquitoes for selected P. falciparum agonists. In Aim 3, we will assess these KO A. gambiae lines for resistance to P. falciparum infection and their fitness impact in terms of mosquito longevity and fecundity.

The long-term goal of the principal investigator?s research is to elucidate the interactions between the mosquito vector and Zika virus (ZIKV) and Dengue virus (DENV), to identify transmission-blocking targets suitable for the development of disease control strategies. During their journey through the vector, the viruses engage in intimate interactions with the mosquito midgut and other tissues, relying on numerous mosquito-derived agonists (host factors). ZIKV and DENV can be impaired in the vector by agonist deletion through gene editing, depletion through RNA interference, or blocking / interference / inhibition through antibodies. Hence, an obvious advantage of studying virus agonists for disease control is that they can be targeted through multiple means. The recently developed CRISPR/CAS9-based gene editing in Aedes mosquitoes provide new and promising opportunities for the study of agonist function through gene deletion, and could be used to develop novel ZIKV and DENV control strategies. Here we will focus on 10 virus agonists to assess their potential for disease control and further our knowledge of their involvement in ZIKV and DENV infection. In Aim 1, we will validate 6 putative DENV agonists for ZIKV blocking potential in Ae. aegypti using RNAi-mediated gene silencing. In Aim 2, we will develop CRISPR/CAS9-mediated conditional bloodmeal-inducible midgut-specific gene KO mosquitoes for selected ZIKV and DENV agonists. In Aim 3, we will assess these KO Ae. aegypti lines for resistance to virus infection and their fitness impact in terms of mosquito longevity and fecundity.

SUMMARY Arboviruses remain an immense public health threat, causing yearly large epidemics. The overarching aim of this research is to better understand the role of the Aedes aegypti RNA interference (RNAi) pathway in antiviral defense and innate immunity, and to generate knowledge and tools for the development of new methods to control arbovirus transmission. Population replacement of wild-type with genetically modified mosquitoes incapable of pathogen transmission is emerging as a promising complement to other disease control methods. Because Ae. aegypti transmits multiple arbovirus pathogens that are frequently sympatric, it is important for a transgenic mosquito to be resistant to multiple pathogens. The siRNA pathway-mediated antiviral defense system is known to act against a broad range of viruses. However, despite the RNAi pathway's emergence as the major pan-antiviral defense system it remains understudied in mosquitoes. Our research plan is designed to test the overarching hypothesis that the A. aegypti proteins, Dicer-2 (Dcr-2), R2D2 and Argonaute-2 (Ago-2), are key components mediating an RNA silencing response that is broadly protective against arbovirus infections. We will use of siRNA-deficient loss-of-function mutants and transgenic mosquitoes over-expressing Dcr-2, Ago-2 and R2D2 that will be generated in Aim 1. With these genetic tools we will clarify the temporal and spatial specificity of the antiviral response in Aim 2, and investigate possible inter-tissue signaling and regulation of vertical transmission in Aim 3. In Aim 4 we will address interactions between the RNAi pathway and other innate immunity defense systems. This project utilizes the complementary expertise of Drs Dimopoulos and Myles with the arbovirus infection systems, mosquito transgenesis, mosquito innate immunity and RNAi/small RNA biology. Our proposed project will also generate powerful tools for studying other aspects of the RNAi pathway in Ae. aegypti biology.

SUMMARY Mosquito associated bacteria have a major impact on larval development, life span, vector competence and mating preferences. Multiple studies have deciphered the taxonomic diversity of bacteria associated with various mosquito species. It has been shown that altered microbiota affects multiple mosquito life traits. Despite these studies, the functional role of individual bacterial taxa and their interactions with each other in the mosquito gut remains unknown. To study the role of individual bacteria from a complex community, targeted modulation of the mosquito microbiota is desired. Bacteriophages provide a perfect tool for selective removal of targeted bacteria from microbiota. This proposal aims to utilize bacteriophages to selectively modulate the mosquito microbiota and study its effect of various mosquito life-history traits. Based on literature and our own studies, we have identified five bacterial taxa that are frequently associated with various mosquito species. In aim 1, we will isolate bacteriophages from various environmental sources capable of infecting selected mosquito gut bacteria. In our preliminary experiments we have already successfully isolated multiple bacteriophages capable of selectively infecting various mosquito gut bacteria. In aim 2, we will use isolated bacteriophages to selectively modulate the microbiota of adult Aedes and Anopheles mosquitoes. The effect of this altered microbiota on mosquito life span, fecundity and vector competence will be studied. In aim 3, we will use isolated bacteriophages to selectively modulate the microbiota in larval breeding water. We will evaluate the effect of altered microbiota on larval development, and vector competence, lifespan and fecundity of adult mosquitoes. Our preliminary data with gnotobiotic larvae has shown that addition of bacteriophages to larval breeding water can affect the larval development. This project will be the first ever use of bacteriophages in the mosquito microbiota research and has the potential to be developed into a novel technique to control mosquito borne diseases.

ABSTRACT This proposal will elucidate how two specific proteins in mosquito saliva influence the early stage of Plasmodium infection in mice. Our group previously developed a general screen to identify proteins in Anopheles gambiae saliva that associate with sporozoites during movement out the mosquito and deposition into the skin of a mammalian host (Schleicher Graham TR, Yang J, Freudzon M, Rembisz A, Craft S, Hamilton M, M, Mlambo G, Tripathi AK, Li Y, Cresswell P, Sinnis P, Dimopoulos G, Fikrig E. Nat Commun. 2018 25;9(1):2908). Several proteins were identified, including sporozoite-associated mosquito saliva protein (SAMSP) 1 and SAMSP2. Our preliminary data shows that SAMSP1 plays a role in facilitating Plasmodium infection in mice and that SAMSP2 has immunomodulatory activity. This proposal will now examine the importance of SAMSP1 and SAMPS2 in influencing the initial stage of Plasmodium infection in mice. Gene silencing and antibody blocking studies will assess the role of these proteins in the early stages of murine infection. In vivo two-photon microscopy of the dermis, and in vitro assays to assess movement and immune modulation, will delineate the effects of SAMSP1 and SAMSP2 on sporozoite activity. Understanding the importance of SAMSP1 and SAMSP2 in Plasmodium infection will hopefully lead to new strategies to combat malaria.