An atlas of human vector-borne microbe interactions reveals pathogenicity mechanisms.

Vector-borne diseases are a leading cause of death worldwide and pose a substantial unmet medical need. Pathogens binding to host extracellular proteins (the "exoproteome") represents a crucial interface in the etiology of vector-borne disease. Here, we used bacterial selection to elucidate host-microbe interactions in high throughput (BASEHIT)-a technique enabling interrogation of microbial interactions with 3,324 human exoproteins-to profile the interactomes of 82 human-pathogen samples, including 30 strains of arthropod-borne pathogens and 8 strains of related non-vector-borne pathogens. The resulting atlas revealed 1,303 putative interactions, including hundreds of pairings with potential roles in pathogenesis, including cell invasion, tissue colonization, immune evasion, and host sensing. Subsequent functional investigations uncovered that Lyme disease spirochetes recognize epidermal growth factor as an environmental cue of transcriptional regulation and that conserved interactions between intracellular pathogens and thioredoxins facilitate cell invasion. In summary, this interactome atlas provides molecular-level insights into microbial pathogenesis and reveals potential host-directed targets for next-generation therapeutics.

Two mosquito salivary antigens demonstrate promise as biomarkers of recent exposure to P. falciparum infected mosquito bites.

Background: Measuring malaria transmission intensity using the traditional entomological inoculation rate is difficult. Antibody responses to mosquito salivary proteins such as SG6 have previously been used as biomarkers of exposure to Anopheles mosquito bites. Here, we investigate four mosquito salivary proteins as potential biomarkers of human exposure to mosquitoes infected with P. falciparum: mosGILT, SAMSP1, AgSAP, and AgTRIO. Methods: We tested population-level human immune responses in longitudinal and cross-sectional plasma samples from individuals with known P. falciparum infection from low and moderate transmission areas in Senegal using a multiplexed magnetic bead-based assay. Results: AgSAP and AgTRIO were the best indicators of recent exposure to infected mosquitoes. Antibody responses to AgSAP, in a moderate endemic area, and to AgTRIO in both low and moderate endemic areas, were significantly higher than responses in a healthy non-endemic control cohort (p-values = 0.0245, 0.0064, and <0.0001 respectively). No antibody responses significantly differed between the low and moderate transmission area, or between equivalent groups during and outside the malaria transmission seasons. For AgSAP and AgTRIO, reactivity peaked 2-4 weeks after clinical P. falciparum infection and declined 3 months after infection. Discussion: Reactivity to both AgSAP and AgTRIO peaked after infection and did not differ seasonally nor between areas of low and moderate transmission, suggesting reactivity is likely reflective of exposure to infectious mosquitos or recent biting rather than general mosquito exposure. Kinetics suggest reactivity is relatively short-lived. AgSAP and AgTRIO are promising candidates to incorporate into multiplexed assays for serosurveillance of population-level changes in P. falciparum-infected mosquito exposure.

mRNA vaccination of rabbits alters the fecundity, but not the attachment, of adult Ixodes scapularis.

19ISP is a nucleoside-modified mRNA-lipid nanoparticle vaccine that targets 19 Ixodes scapularis proteins. We demonstrate that adult I. scapularis have impaired fecundity when allowed to engorge on 19ISP-immunized rabbits. 19ISP, therefore, has the potential to interrupt the tick reproductive cycle, without triggering some of the other effects associated with acquired tick resistance. This may lead to the development of new strategies to reduce I. scapularis populations in endemic areas.

mosGILT controls innate immunity and germ cell development in Anopheles gambiae.

Gene-edited mosquitoes lacking a gamma-interferon-inducible lysosomal thiol reductase-like protein, namely (mosGILTnull) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILTnull Anopheles gambiae was therefore compared to wild type (WT) mosquitoes by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILTnull A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg, an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILTnull mosquitoes. These results provide a crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.

Signaling between mammalian adiponectin and a mosquito adiponectin receptor reduces Plasmodium transmission.

IMPORTANCE: When a female mosquito takes a blood meal from a mammalian host, components of the blood meal can affect mosquito fitness and indirectly influence pathogen infectivity. We identified a pathway involving an Anopheles gambiae adiponectin receptor, which, triggered by adiponectin from an incoming blood meal, decreases Plasmodium infection in the mosquito. Activation of this pathway negatively regulates lipophorin expression, an important lipid transporter that both enhances egg development and Plasmodium infection. This is an unrecognized cross-phyla interaction between a mosquito and its vertebrate host. These processes are critical to understanding the complex life cycle of mosquitoes and Plasmodium following a blood meal and may be applicable to other hematophagous arthropods and vector-borne infectious agents.

Metabolomic changes associated with acquired resistance to Ixodes scapularis.

Guinea pigs repeatedly exposed to Ixodes scapularis develop acquired resistance to the ticks (ATR). The molecular mechanisms of ATR have not been fully elucidated, and partially involves immune responses to proteins in tick saliva. In this study, we examined the metabolome of sera of guinea pigs during the development of ATR. Induction of components of the tyrosine metabolic pathway, including hydroxyphenyllactic acid (HPLA), were associated with ATR. We therefore administered HPLA to mice, an animal that does not develop ATR, and exposed the animals to I. scapularis. We also administered nitisinone, a known inhibitor of tyrosine degradation, to another group of mice. The mortality of I. scapularis that fed on mice given HPLA or nitisinone was 26 % and 72 % respectively, compared with 2 % mortality among ticks that fed on control animals. These data indicate that tick bites alter the guinea pig metabolome, and that the tyrosine metabolism pathway can potentially be targeted for I. scapularis control.

Adiponectin in the mammalian host influences ticks' acquisition of the Lyme disease pathogen Borrelia.

Arthropod-borne pathogens cause some of the most important human and animal infectious diseases. Many vectors acquire or transmit pathogens through the process of blood feeding. Here, we report adiponectin, the most abundant adipocyte-derived hormone circulating in human blood, directly or indirectly inhibits acquisition of the Lyme disease agent, Borrelia burgdorferi, by Ixodes scapularis ticks. Rather than altering tick feeding or spirochete viability, adiponectin or its associated factors induces host histamine release when the tick feeds, which leads to vascular leakage, infiltration of neutrophils and macrophages, and inflammation at the bite site. Consistent with this, adiponectin-deficient mice have diminished pro-inflammatory responses, including interleukin (IL)-12 and IL-1ß, following a tick bite, compared with wild-type animals. All these factors mediated by adiponectin or associated factors influence B. burgdorferi survival at the tick bite site. These results suggest a host adipocyte-derived hormone modulates pathogen acquisition by a blood-feeding arthropod.

A ticking time bomb hidden in plain sight.

The deer tick transmits nearly half of the known tick-borne pathogens in the United States, and its expanding geographic range increases the risk of human infection. To decrease the abundance of and infection risk from deer ticks, approaches that include vaccines for human use and for animal hosts are desired.

Bulk and single-nucleus RNA sequencing highlight immune pathways induced in individuals during an Ixodes scapularis tick bite.

Ticks are hematophagous arthropods that use a complex mixture of salivary proteins to evade host defenses while taking a blood meal. Little is known about the immunological and physiological consequences of tick feeding on humans. Here, we performed the first bulk and single-nucleus RNA sequencing (snRNA-seq) of skin and blood of four persons presenting with naturally acquired, attached Ixodes scapularis ticks. Pathways and individual genes associated with innate and adaptive immunity were identified based on bulk RNA sequencing, including interleukin-17 signaling and platelet activation pathways at the site of tick attachment or in peripheral blood. snRNA-seq further revealed that the Hippo signaling, cell adhesion, and axon guidance pathways were involved in the response to an I. scapularis bite in humans. Features of the host response in these individuals also overlapped with that of laboratory guinea pigs exposed to I. scapularis and which acquired resistance to ticks. These findings offer novel insights for the development of new biomarkers for I. scapularis exposure and anti-tick vaccines for human use.

Development of an mRNA-lipid nanoparticle vaccine against Lyme disease.

Lyme disease is the most common vector-borne infectious disease in the United States, in part because a vaccine against it is not currently available for humans. We propose utilizing the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform to generate a Lyme disease vaccine like the successful clinical vaccines against SARS-CoV-2. Of the antigens expressed by Borrelia burgdorferi, the causative agent of Lyme disease, outer surface protein A (OspA) is the most promising candidate for vaccine development. We have designed and synthesized an OspA-encoding mRNA-LNP vaccine and compared its immunogenicity and protective efficacy to an alum-adjuvanted OspA protein subunit vaccine. OspA mRNA-LNP induced superior humoral and cell-mediated immune responses in mice after a single immunization. These potent immune responses resulted in protection against bacterial infection. Our study demonstrates that highly efficient mRNA vaccines can be developed against bacterial targets.

Anopheles gambiae mosGILT regulates innate immune genes and zpg expression.

Gene-edited mosquitoes lacking a g amma-interferon-inducible lysosomal thiol reductase-like protein, namely ( mosGILT null ) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILT null A. gambiae was therefore compared to wild type (WT) by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILT null A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg , an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILT null mosquitoes. These results provide the crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.

Metabolomic changes associated with acquired resistance to Ixodes scapularis.

Guinea pigs repeatedly exposed to Ixodes scapularis develop acquired resistance to the ticks (ATR). The molecular mechanisms of ATR have not been fully elucidated, and partially involve immune responses to proteins in tick saliva. In this study, we examined the metabolome of sera of guinea pigs during the development of ATR. Induction of components of the tyrosine metabolic pathway, including hydroxyphenyllactic acid (HPLA), were associated with ATR. We therefore administered HPLA to mice, an animal that does not develop ATR, and exposed the animals to I. scapularis . We also administered nitisinone, a known inhibitor of tyrosine degradation, to another group of mice. The mortality of I. scapularis that fed on mice given HPLA or nitisinone was 26% and 72% respectively, compared with 2% mortality among ticks that fed on control animals. These data indicate that metabolic changes that occur after tick bites contribute to ATR.

Specific mRNA lipid nanoparticles and acquired resistance to ticks.

Acquired resistance to ticks can develop when animals are repeatedly exposed to ticks. Recently, acquired resistance to Ixodes scapularis was induced in guinea pigs immunized with an mRNA-lipid nanoparticle vaccine (19ISP) encoding 19 I. scapularis proteins. Here, we evaluated specific mRNAs present in 19ISP to identify critical components associated with resistance to ticks. A lipid nanoparticle containing 12 mRNAs which included all the targets within 19ISP that elicited strong humoral responses in guinea pigs, was sufficient to induce robust resistance to ticks. Lipid nanoparticles containing fewer mRNAs or a single mRNA were not able to generate strong resistance to ticks. All lipid nanoparticles containing salp14 mRNA, however, were associated with increased redness at the tick bite site - which is the first manifestation of acquired resistance to ticks. This study demonstrates that more than one I. scapularis target within 19ISP is required for resistance to ticks, and that additional targets may also play a role in this process.

A mosquito AgTRIO mRNA vaccine contributes to immunity against malaria.

Malaria begins when an infected mosquito injects saliva containing Plasmodium sporozoites into the skin of a vertebrate host. To prevent malaria, vaccination is the most effective strategy and there is an urgent need for new strategies to enhance current pathogen-based vaccines. Active or passive immunization against a mosquito saliva protein, AgTRIO, contributes to protection against Plasmodium infection of mice. In this study, we generated an AgTRIO mRNA-lipid nanoparticle (LNP) and assessed its potential usefulness as a vaccine against malaria. Immunization of mice with an AgTRIO mRNA-LNP generated a robust humoral response, including AgTRIO IgG2a isotype antibodies that have been associated with protection. AgTRIO mRNA-LNP immunized mice exposed to Plasmodium berghei-infected mosquitoes had markedly reduced initial Plasmodium hepatic infection levels and increased survival compared to control mice. In addition, as the humoral response to AgTRIO waned over 6 months, additional mosquito bites boosted the AgTRIO IgG titers, including IgG1 and IgG2a isotypes, which offers a unique advantage compared to pathogen-based vaccines. These data will aid in the generation of future malaria vaccines that may include both pathogen and vector antigens.

Vaccinia Virus Strain MVA Expressing a Prefusion-Stabilized SARS-CoV-2 Spike Glycoprotein Induces Robust Protection and Prevents Brain Infection in Mouse and Hamster Models.

The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we reported on the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino-acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino-acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and was correctly processed and transported to the cell surface, where it efficiently produced cell-cell fusion. Version Spf, however, was not proteolytically processed, and despite being transported to the plasma membrane, it failed to induce cell-cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin-converting enzyme 2 (K18-hACE2) in mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the level of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine.

Frankenbacteriosis targeting interactions between pathogen and symbiont to control infection in the tick vector.

Tick microbiota can be targeted for the control of tick-borne diseases such as human granulocytic anaplasmosis (HGA) caused by model pathogen, Anaplasma phagocytophilum. Frankenbacteriosis is inspired by Frankenstein and defined here as paratransgenesis of tick symbiotic/commensal bacteria to mimic and compete with tick-borne pathogens. Interactions between A. phagocytophilum and symbiotic Sphingomonas identified by metaproteomics analysis in Ixodes scapularis midgut showed competition between both bacteria. Consequently, Sphingomonas was selected for frankenbacteriosis for the control of A. phagocytophilum infection and transmission. The results showed that Franken Sphingomonas producing A. phagocytophilum major surface protein 4 (MSP4) mimic pathogen and reduce infection in ticks by competition and interaction with cell receptor components of infection. Franken Sphingomonas-MSP4 transovarial and trans-stadial transmission suggests that tick larvae with genetically modified Franken Sphingomonas-MSP4 could be produced in the laboratory and released in the field to compete and replace the wildtype populations with associated reduction in pathogen infection/transmission and HGA disease risks.

Croquemort elicits activation of the immune deficiency pathway in ticks.

The immune deficiency (IMD) pathway directs host defense in arthropods upon bacterial infection. In Pancrustacea, peptidoglycan recognition proteins sense microbial moieties and initiate nuclear factor-κB-driven immune responses. Proteins that elicit the IMD pathway in non-insect arthropods remain elusive. Here, we show that an Ixodes scapularis homolog of croquemort (Crq), a CD36-like protein, promotes activation of the tick IMD pathway. Crq exhibits plasma membrane localization and binds the lipid agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. Crq regulates the IMD and jun N-terminal kinase signaling cascades and limits the acquisition of the Lyme disease spirochete B. burgdorferi. Additionally, nymphs silenced for crq display impaired feeding and delayed molting to adulthood due to a deficiency in ecdysteroid synthesis. Collectively, we establish a distinct mechanism for arthropod immunity outside of insects and crustaceans.

Mosquito Salivary Proteins and Arbovirus Infection: From Viral Enhancers to Potential Targets for Vaccines.

Arthropod-borne viruses present important public health challenges worldwide. Viruses such as DENV, ZIKV, and WNV are of current concern due to an increasing incidence and an expanding geographic range, generating explosive outbreaks even in non-endemic areas. The clinical signs associated with infection from these arboviruses are often inapparent, mild, or nonspecific, but occasionally develop into serious complications marked by rapid onset, tremors, paralysis, hemorrhagic fever, neurological alterations, or death. They are predominately transmitted to humans through mosquito bite, during which saliva is inoculated into the skin to facilitate blood feeding. A new approach to prevent arboviral diseases has been proposed by the observation that arthropod saliva facilitates transmission of pathogens. Viruses released within mosquito saliva may more easily initiate host invasion by taking advantage of the host's innate and adaptive immune responses to saliva. This provides a rationale for creating vaccines against mosquito salivary proteins, especially because of the lack of licensed vaccines against most of these viruses. This review aims to provide an overview of the effects on the host immune response by the mosquito salivary proteins and how these phenomena alter the infection outcome for different arboviruses, recent attempts to generate mosquito salivary-based vaccines against flavivirus including DENV, ZIKV, and WNV, and the potential benefits and pitfalls that this strategy involves.

Malaria: influence of Anopheles mosquito saliva on Plasmodium infection.

Malaria is caused by Plasmodium protozoa that are transmitted by anopheline mosquitoes. Plasmodium sporozoites are released with saliva when an infected female mosquito takes a blood meal on a vertebrate host. Sporozoites deposited into the skin must enter a blood vessel to start their journey towards the liver. After migration out of the mosquito, sporozoites are associated with, or in proximity to, many components of vector saliva in the skin. Recent work has elucidated how Anopheles saliva, and components of saliva, can influence host-pathogen interactions during the early stage of Plasmodium infection in the skin. Here, we discuss how components of Anopheles saliva can modulate local host responses and affect Plasmodium infectivity. We hypothesize that therapeutic strategies targeting mosquito salivary proteins can play a role in controlling malaria and other vector-borne diseases.

Repeated Tick Infestations Impair Borrelia burgdorferi Transmission in a Non-Human Primate Model of Tick Feeding.

The blacklegged tick, Ixodes scapularis, is the predominant vector of Borrelia burgdorferi, the agent of Lyme disease in the USA. Natural hosts of I. scapularis such as Peromyscus leucopus are repeatedly infested by these ticks without acquiring tick resistance. However, upon repeated tick infestations, non-natural hosts such as guinea pigs, mount a robust immune response against critical tick salivary antigens and acquire tick resistance able to thwart tick feeding and Borrelia burgdorferi transmission. The salivary targets of acquired tick resistance could serve as vaccine targets to prevent tick feeding and the tick transmission of human pathogens. Currently, there is no animal model able to demonstrate both tick resistance and diverse clinical manifestations of Lyme disease. Non-human primates serve as robust models of human Lyme disease. By evaluating the responses to repeated tick infestation, this animal model could accelerate our ability to define the tick salivary targets of acquired resistance that may serve as vaccines to prevent the tick transmission of human pathogens. Towards this goal, we assessed the development of acquired tick resistance in non-human primates upon repeated tick infestations. We report that following repeated tick infestations, non-human primates do not develop the hallmarks of acquired tick resistance observed in guinea pigs. However, repeated tick infestations elicit immune responses able to impair the tick transmission of B. burgdorferi. A mechanistic understanding of the protective immune responses will provide insights into B. burgdorferi-tick-host interactions and additionally contribute to anti-tick vaccine discovery.