Safety and immunogenicity of AGS-v PLUS, a mosquito saliva peptide vaccine against arboviral diseases: A randomized, double-blind, placebo-controlled Phase 1 trial.

BACKGROUND: Immunity to mosquito salivary proteins could provide protection against multiple mosquito-borne diseases and significantly impact public health. We evaluated the safety and immunogenicity of AGS-v PLUS, a mosquito salivary peptide vaccine, in healthy adults 18-50 years old. METHODS: We conducted a randomized, double-blind, placebo-controlled Phase 1 study of AGS-v PLUS administered subcutaneously on Days 1 and 22 at the Center for Vaccine Development and Global Health, Baltimore, MD, USA. Participants were block randomized 1:1:1:1:1 to two doses saline placebo, two doses AGS-v PLUS, AGS-v PLUS/ISA-51 and saline placebo, two doses AGS-v PLUS/ISA-51, or two doses AGS-v PLUS/Alhydrogel. Primary endpoints were safety (all participants receiving ≥1 injection) and antibody and cytokine responses (all participants with day 43 samples), analysed by intention to treat. FINDINGS: Between 26 August 2019 and 25 February 2020, 51 participants were enrolled and randomized, 11 into the single dose AGS-v PLUS/ISA-51 group and ten in other groups. Due to COVID-19, 15 participants did not return for day 43 samplings. Participants experienced no treatment-emergent or serious adverse events. All solicited symptoms in 2/10 placebo recipients and 22/41 AGS-v PLUS recipients after dose one and 1/10 placebo recipients and 22/41 AGS-v PLUS recipients after dose two were mild/moderate except for one severe fever the day after vaccination (placebo group). Only injection site pain was more common in vaccine groups (15/51 after dose 1 and 11/51 after dose 2) versus placebo. Compared to placebo, all vaccine groups had significantly greater fold change in anti-AGS-v PLUS IgG and IFN-É£ from baseline. INTERPRETATION: AGS-v PLUS had favourable safety profile and induced robust immune responses. Next steps will determine if findings translate into clinical efficacy against mosquito-borne diseases. FUNDING: UK Department of Health and Social Care.

Malaria parasite evades mosquito immunity by glutaminyl cyclase-mediated posttranslational protein modification.

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.

Adaptive Evolution as a Driving Force of the Emergence and Re-Emergence of Mosquito-Borne Viral Diseases.

Emerging and re-emerging mosquito-borne viral diseases impose a significant burden on global public health. The most common mosquito-borne viruses causing recent epidemics include flaviviruses in the family Flaviviridae, including Dengue virus (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV) and West Nile virus (WNV) and Togaviridae viruses, such as chikungunya virus (CHIKV). Several factors may have contributed to the recent re-emergence and spread of mosquito-borne viral diseases. Among these important causes are the evolution of mosquito-borne viruses and the genetic mutations that make them more adaptive and virulent, leading to widespread epidemics. RNA viruses tend to acquire genetic diversity due to error-prone RNA-dependent RNA polymerases, thus promoting high mutation rates that support adaptation to environmental changes or host immunity. In this review, we discuss recent findings on the adaptive evolution of mosquito-borne viruses and their impact on viral infectivity, pathogenicity, vector fitness, transmissibility, epidemic potential and disease emergence.

A Mosquito AgTRIO Monoclonal Antibody Reduces Early Plasmodium Infection of Mice.

Malaria begins when an infected mosquito injects saliva containing Plasmodium sporozoites into the skin of a vertebrate host. Passive immunization of mice with antiserum against the Anopheles gambiae mosquito saliva protein TRIO (AgTRIO) offers significant protection against Plasmodium infection of mice. Furthermore, passive transfer of both AgTRIO antiserum and an anti-circumsporozoite protein monoclonal antibody provides synergistic protection. In this study, we generated monoclonal antibodies against AgTRIO to delineate the regions of AgTRIO associated with protective immunity. Monoclonal antibody 13F-1 markedly reduced Plasmodium infection in mice and recognized a region (VDDLMAKFN) in the carboxyl terminus of AgTRIO. 13F-1 is an IgG2a isotype monoclonal antibody, and the Fc region is required for protection. These data will aid in the generation of future malaria vaccines that may include both pathogen and vector antigens.

Innate Immune Antagonism of Mosquito-Borne Flaviviruses in Humans and Mosquitoes.

Mosquito-borne viruses of the Flavivirus genus (Flaviviridae family) pose an ongoing threat to global public health. For example, dengue, Japanese encephalitis, West Nile, yellow fever, and Zika viruses are transmitted by infected mosquitoes and cause severe and fatal diseases in humans. The means by which mosquito-borne flaviviruses establish persistent infection in mosquitoes and cause disease in humans are complex and depend upon a myriad of virus-host interactions, such as those of the innate immune system, which are the main focus of our review. This review also covers the different strategies utilized by mosquito-borne flaviviruses to antagonize the innate immune response in humans and mosquitoes. Given the lack of antiviral therapeutics for mosquito-borne flaviviruses, improving our understanding of these virus-immune interactions could lead to new antiviral therapies and strategies for developing refractory vectors incapable of transmitting these viruses, and can also provide insights into determinants of viral tropism that influence virus emergence into new species.

Oral susceptibility of aedine and culicine mosquitoes (Diptera: Culicidae) to Batai Orthobunyavirus.

BACKGROUND: A number of zoonotic mosquito-borne viruses have emerged in Europe in recent decades. Batai virus (BATV), a member of the genus Orthobunyavirus, is one example of a relatively newly emerged mosquito-borne virus, having been detected in mosquitoes and livestock. We conducted vector competency studies on three mosquito species at a low temperature to assess whether Aedes and Culex mosquito species are susceptible to infection with BATV. METHODS: Colonised lines of Aedes aegypti and Culex pipiens and a wild-caught species, Aedes detritus, were orally inoculated with BATV strain 53.2, originally isolated from mosquitoes trapped in Germany in 2009. Groups of blood-fed female mosquitoes were maintained at 20 °C for 7 or 14 days. Individual mosquitoes were screened for the presence of BATV in body, leg and saliva samples for evidence of infection, dissemination and transmission, respectively. BATV RNA was detected by reverse transcription-PCR, and positive results confirmed by virus isolation in Vero cells. RESULTS: Aedes detritus was highly susceptible to BATV, with an infection prevalence of ≥ 80% at both measurement time points. Disseminated infections were recorded in 30.7-41.6% of Ae. detritus, and evidence of virus transmission with BATV in saliva samples (n = 1, days post-infection: 14) was observed. Relatively lower rates of infection for Ae. aegypti and Cx. pipiens were observed, with no evidence of virus dissemination or transmission at either time point. CONCLUSIONS: This study shows that Ae. detritus may be a competent vector for BATV at 20 °C, whereas Ae. aegypti and Cx. pipiens were not competent. Critically, the extrinsic incubation period appears to be ≤ 7 days for Ae. detritus, which may increase the onward transmissibility potential of BATV in these populations.

Double peroxidase and histone acetyltransferase AgTip60 maintain innate immune memory in primed mosquitoes.

Immune priming in Anopheles gambiae is mediated by the systemic release of a hemocyte differentiation factor (HDF), a complex of lipoxin A4 bound to Evokin, a lipid carrier. HDF increases the proportion of circulating granulocytes and enhances mosquito cellular immunity. Here, we show that Evokin is present in hemocytes and fat-body cells, and messenger RNA (mRNA) expression increases significantly after immune priming. The double peroxidase (DBLOX) enzyme, present in insects but not in vertebrates, is essential for HDF synthesis. DBLOX is highly expressed in oenocytes in the fat-body tissue, and these cells increase in number in primed mosquitoes. We provide direct evidence that the histone acetyltransferase AgTip60 (AGAP001539) is also essential for a sustained increase in oenocyte numbers, HDF synthesis, and immune priming. We propose that oenocytes may function as a population of cells that are reprogrammed, and orchestrate and maintain a broad, systemic, and long-lasting state of enhanced immune surveillance in primed mosquitoes.

Insect vectors' saliva and gut microbiota as a blessing in disguise: probability versus possibility.

Drawing of host blood is a natural phenomenon during the bite of blood-probing insect vectors. Along with the blood meal, the vectors introduce salivary components and a trail of microbiota. In the case of infected vectors, the related pathogen accompanies the aforementioned biological components. In addition to Anopheles gambiae or Anopheles stephensi, the bites of other nonmalarial vectors cannot be ignored in malaria-endemic regions. Similarly, the bite incidence of Phlebotomus papatasi cannot be ignored in visceral leishmaniasis-endemic regions. Even the chances of getting bitten by uninfected vectors are higher than the infected vectors. We have discussed the probability or possibility of uninfected, infected, and/or nonvector's saliva and gut microbiota as a therapeutic option leading to the initial deterrent to pathogen establishment.

Host Factors That Control Mosquito-Borne Viral Infections in Humans and Their Vector.

Mosquito-borne viral infections are responsible for a significant degree of morbidity and mortality across the globe due to the severe diseases these infections cause, and they continue to increase each year. These viruses are dependent on the mosquito vector as the primary means of transmission to new vertebrate hosts including avian, livestock, and human populations. Due to the dynamic host environments that mosquito-borne viruses pass through as they are transmitted between vector and vertebrate hosts, there are various host factors that control the response to infection over the course of the pathogen's life cycle. In this review, we discuss these host factors that are present in either vector or vertebrate models during infection, how they vary or are conserved between hosts, and their implications in future research pertaining to disease prevention and treatment.

Genetic determinants of antiviral immunity in dipteran insects - Compiling the experimental evidence.

The genetic basis of antiviral immunity in dipteran insects is extensively studied in Drosophila melanogaster and advanced technologies for genetic manipulation allow a better characterization of immune responses also in non-model insect species. Especially, immunity in vector mosquitoes is recently in the spotlight, due to the medical impact that these insects have by transmitting viruses and other pathogens. Here, we review the current state of experimental evidence that supports antiviral functions for immune genes acting in different cellular pathways. We discuss the well-characterized RNA interference mechanism along with the less well-defined JAK-STAT, Toll, and IMD signaling pathways. Furthermore, we highlight the initial evidence for antiviral activity observed for the autophagy pathway, transcriptional pausing, as well as piRNA production from endogenous viral elements. We focus our review on studies from Drosophila and mosquito species from the lineages Aedes, Culex, and Anopheles, which contain major vector species responsible for virus transmission.

Mosquito antiviral immune pathways.

Mosquitoes are vectors of a large number of viral pathogens. In recent years, increased urbanization and climate change has expanded the range of many vector mosquitoes. The lack of effective medical interventions has made the control of mosquito-borne viral diseases very difficult. Understanding the interactions between the mosquito immune system and viruses is critical if we are to develop effective control strategies against these diseases. Mosquitoes harbor multiple conserved immune pathways that curb invading viral pathogens. Despite the conservation of these pathways, the activation and intensity of the mosquito immune response varies with the mosquito species, tissue, and the infecting virus. This article reviews major conserved antiviral immune pathways in vector mosquitoes, their interactions with invading viral pathogens, and how these interactions restrict or promote infection of these medically important viruses.

Antibody Therapy Goes to Insects: Monoclonal Antibodies Can Block Plasmodium Transmission to Mosquitoes.

Malaria eradication is a global priority but requires innovative strategies. Humoral immune responses attack different parasite stages, and antibody-based therapy may prevent malaria infection or transmission. Here, we discuss targets of monoclonal antibodies in mosquito sexual stages of Plasmodium.

Using Diverse Model Systems to Define Intestinal Epithelial Defenses to Enteric Viral Infections.

The intestine is an essential physical and immunological barrier comprised of a monolayer of diverse and specialized epithelial cells that perform functions ranging from nutrient absorption to pathogen sensing and intestinal homeostasis. The intestinal barrier prevents translocation of intestinal microbes into internal compartments. The microbiota is comprised of a complex community largely populated by diverse bacterial species that provide metabolites, nutrients, and immune stimuli that promote intestinal and organismal health. Although commensal organisms promote health, enteric pathogens, including a diverse plethora of enteric viruses, cause acute and chronic diseases. The barrier epithelium plays fundamental roles in immune defenses against enteric viral infections by integrating diverse signals, including those from the microbiota, to prevent disease. Importantly, many model systems have contributed to our understanding of this complex interface. This review will focus on the antiviral mechanisms at play within the intestinal epithelium and how these responses are shaped by the microbiota.

Research progress in RNA interference against the infection of mosquito-borne viruses.

Mosquito-borne diseases have become an important public health issue of global concern because of their high incidence and transmission rate. As a vector for mosquito-borne diseases, studying the interaction mechanism between mosquitoes and mosquito-borne viruses will help control mosquito-borne diseases. The impaired innate immunity and immune barriers evasion caused by mosquito-borne viruses in mosquitoes pose a potential risk for the persistent infection of the virus in mosquitoes and the outbreak of mosquito-borne diseases. The RNA interference (RNAi) pathway, as a powerful antiviral defense barrier in mosquitoes, can inhibit viral replication and transmission by producing a variety of small RNAs to degrade viral RNA. In this review, we summarize the related studies on the innate immune mechanism against mosquito- borne virus infection in mosquitoes about small interfering RNA (siRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA), aiming to provide a theoretical reference for the prevention and control of mosquito-borne diseases.

Characteristics of Asian Mosquito Saliva Allergens for Specific Diagnoses and Effective Therapies.

An allergic reaction to mosquitoes can result in severe or abnormal local or systemic reactions such as anaphylaxis, angioedema, and general urticarial or wheezing. The aim of this review is to provide information on mosquito saliva allergens that can support the production of highly specific recombinant saliva allergens. In particular, candidate allergens of mosquitoes that are well suited to the ecology of mosquitoes that occur mainly in East Asia will be identified and introduced. By doing so, the diagnosis and treatment of patients with severe sensitivity to mosquito allergy will be improved by predicting the characteristics of East Asian mosquito allergy, presenting the future direction of production of recombinant allergens, and understanding the difference between East and West.

Developmental and comparative perspectives on mosquito immunity.

Diseases spread by mosquitoes have killed more people than those spread by any other group of arthropod vectors and remain an important factor in determining global health and economic stability. The mosquito innate immune system can act to either modulate infection with human pathogens or fight off entomopathogens and increase the fitness and longevity of infected mosquitoes. While work remains towards understanding the larval immune system and the development of the mosquito immune system, it has recently become clearer that environmental factors heavily shape the developing mosquito immune system and continue to influence the adult immune system as well. The adult immune system has been well-studied and is known to involve multiple tissues and diverse molecular mechanisms. This review summarizes and synthesizes what is currently understood about the development of the mosquito immune system and includes comparisons of immune components unique to mosquitoes among the blood-feeding arthropods as well as important distinguishing factors between the anopheline and culicine mosquitoes. An explanation is included for how mosquito immunity factors into vector competence and vectorial capacity is presented along with a model for the interrelationships between nutrition, microbiome, pathogen interactions and behavior as they relate to mosquito development, immune status, adult female fitness and ultimately, vectorial capacity. Novel discoveries in the fields of mosquito ecoimmunology, neuroimmunology, and intracellular antiviral responses are highlighted.

Mosquito-fungus interactions and antifungal immunity.

The mosquito immune system has evolved in the presence of continuous encounters with fungi that range from food to foes. Herein, we review the field of mosquito-fungal interactions, providing an overview of current knowledge and topics of interest. Mosquitoes encounter fungi in their aquatic and terrestrial habitats. Mosquito larvae are exposed to fungi on plant detritus, within the water column, and at the water surface. Adult mosquitoes are exposed to fungi during indoor and outdoor resting, blood and sugar feeding, mating, and oviposition. Fungi enter the mosquito body through different routes, including ingestion and through active or passive breaches in the cuticle. Oral uptake of fungi can be beneficial to mosquitoes, as yeasts hold nutritional value and support larval development. However, ingestion of or surface contact with fungal entomopathogens leads to colonization of the mosquito with often lethal consequences to the host. The mosquito immune system recognizes fungi and mounts cellular and humoral immune responses in the hemocoel, and possibly epithelial immune responses in the gut. These responses are regulated transcriptionally through multiple signal transduction pathways. Proteolytic protease cascades provide additional regulation of antifungal immunity. Together, these immune responses provide an efficient barrier to fungal infections, which need to be overcome by entomopathogens. Therefore, fungi constitute an excellent tool to examine the molecular underpinnings of mosquito immunity and to identify novel antifungal peptides. In addition, recent advances in mycobiome analyses can now be used to examine the contribution of fungi to various mosquito traits, including vector competence.

Mosquito antiviral defense mechanisms: a delicate balance between innate immunity and persistent viral infection.

Mosquito-borne diseases are associated with major global health burdens. Aedes spp. and Culex spp. are primarily responsible for the transmission of the most medically important mosquito-borne viruses, including dengue virus, West Nile virus and Zika virus. Despite the burden of these pathogens on human populations, the interactions between viruses and their mosquito hosts remain enigmatic. Viruses enter the midgut of a mosquito following the mosquito's ingestion of a viremic blood meal. During infection, virus recognition by the mosquito host triggers their antiviral defense mechanism. Of these host defenses, activation of the RNAi pathway is the main antiviral mechanism, leading to the degradation of viral RNA, thereby inhibiting viral replication and promoting viral clearance. However, whilst antiviral host defense mechanisms limit viral replication, the mosquito immune system is unable to effectively clear the virus. As such, these viruses can establish persistent infection with little or no fitness cost to the mosquito vector, ensuring life-long transmission to humans. Understanding of the mosquito innate immune response enables the discovery of novel antivectorial strategies to block human transmission. This review provides an updated and concise summary of recent studies on mosquito antiviral immune responses, which is a key determinant for successful virus transmission. In addition, we will also discuss the factors that may contribute to persistent infection in mosquito hosts. Finally, we will discuss current mosquito transmission-blocking strategies that utilize genetically modified mosquitoes and Wolbachia-infected mosquitoes for resistance to pathogens.