Variable Major Proteins as Targets for Specific Antibodies against Borrelia miyamotoi.

Borrelia miyamotoi is a relapsing fever spirochete in Ixodes ticks that has been recently identified as a human pathogen causing hard tick-borne relapsing fever (HTBRF) across the Northern Hemisphere. No validated serologic test exists, and current serologic assays have low sensitivity in early HTBRF. To examine the humoral immune response against B. miyamotoi, we infected C3H/HeN mice with B. miyamotoi strain LB-2001 expressing variable small protein 1 (Vsp1) and demonstrated that spirochetemia was cleared after 3 d, coinciding with anti-Vsp1 IgM production. Clearance was also observed after passive transfer of immune sera to infected SCID mice. Next, we showed that anti-Vsp1 IgG eliminates Vsp1-expressing B. miyamotoi, selecting for spirochetes expressing a variable large protein (VlpC2) resistant to anti-Vsp1. The viability of Asian isolate B. miyamotoi HT31, expressing Vlp15/16 and Vlp18, was also unaffected by anti-Vsp1. Finally, in nine HTBRF patients, we demonstrated IgM reactivity to Vsp1 in two and against Vlp15/16 in four ∼1 wk after these patients tested positive for B. miyamotoi by PCR. Our data show that B. miyamotoi is able to express various variable major proteins (VMPs) to evade humoral immunity and that VMPs are antigenic in humans. We propose that serologic tests based on VMPs are of additional value in diagnosing HTBRF.

Factor Xa activation of factor V is of paramount importance in initiating the coagulation system: lessons from a tick salivary protein.

BACKGROUND: Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. METHODS AND RESULTS: Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. CONCLUSIONS: Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.

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.

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.

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.

An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands.

Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference-mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum-infected mice. A. phagocytophilum migrated normally from A. phagocytophilum-infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.

Immunization of guinea pigs with cement extract induces resistance against Ixodes scapularis ticks.

As hematophagous parasites, many tick species are important vectors of medical and veterinary disease agents. Proteins found in tick saliva and midgut have been used with some success in immunizations of animal hosts against feeding ticks, and whole saliva has been used effectively in this capacity against Ixodes scapularis, the primary vector of tickborne pathogens in the United States. Tick saliva is a complex substance containing hundreds of proteins, and the identification of specific protective antigens is ongoing. We performed a series of experiments immunizing guinea pigs with extracts prepared from midgut or attachment cement collected from adult female I. scapularis followed by challenge with nymphs of the same species. Midgut extract did not induce protective immunity, while immunization with cement extract resulted in partial protection of hosts as evidenced by premature tick detachment and 34-41% reduction in tick engorgement weights. Proteomic characterization of I. scapularis cement was performed, demonstrating that the cement extract was compositionally different from tick saliva, and vitellogenin-like lipoproteins were the most abundant proteins in cement extract (>40%). Cement was also heavily enriched with lysozymes and defensins, including those originating from both the mammalian host as well as ticks. These results demonstrate that I. scapularis cement contains immunogenic components capable of stimulating host resistance against tick feeding. Because the cement is present at the tick-host interface for an extended period of time during the feeding process, these antigens present auspicious candidates for further evaluation and potential inclusion in an anti-tick vaccine.

Tick transmission of Borrelia burgdorferi to the murine host is not influenced by environmentally acquired midgut microbiota.

BACKGROUND: Ixodes scapularis is the predominant tick vector of Borrelia burgdorferi, the agent of Lyme disease, in the USA. Molecular interactions between the tick and B. burgdorferi orchestrate the migration of spirochetes from the midgut to the salivary glands-critical steps that precede transmission to the vertebrate host. Over the last decade, research efforts have invoked a potential role for the tick microbiome in modulating tick-pathogen interactions. RESULTS: Using multiple strategies to perturb the microbiome composition of B. burgdorferi-infected nymphal ticks, we observe that changes in the microbiome composition do not significantly influence B. burgdorferi migration from the midgut, invasion of salivary glands, or transmission to the murine host. We also show that within 24 and 48 h of the onset of tick feeding, B. burgdorferi spirochetes are within the peritrophic matrix and epithelial cells of the midgut in preparation for exit from the midgut. CONCLUSIONS: This study highlights two aspects of tick-spirochete interactions: (1) environmental bacteria associated with the tick do not influence spirochete transmission to the mammalian host and (2) the spirochete may utilize an intracellular exit route during migration from the midgut to the salivary glands, a strategy that may allow the spirochete to distance itself from microbiota in the midgut lumen effectively. This may explain in part, the inability of environment-acquired midgut microbiota to significantly influence spirochete transmission. Unraveling a molecular understanding of this exit strategy will be critical to gain new insights into the biology of the spirochete and the tick. Video Abstract.

Fucosylation enhances colonization of ticks by Anaplasma phagocytophilum.

Fucosylated structures participate in a wide range of pathological processes in eukaryotes and prokaryotes. The impact of fucose on microbial pathogenesis, however, has been less appreciated in arthropods of medical relevance. Thus, we used the tick-borne bacterium Anaplasma phagocytophilum- the agent of human granulocytic anaplasmosis to understand these processes. Here we show that A. phagocytophilum uses alpha1,3-fucose to colonize ticks. We demonstrate that A. phagocytophilum modulates the expression of alpha1,3-fucosyltransferases and gene silencing significantly reduces colonization of tick cells. Acquisition but not transmission of A. phagocytophilum was affected when alpha1,3-fucosyltransferases were silenced during tick feeding. Our results uncover a novel mechanism of pathogen colonization in arthropods. Decoding mechanisms of pathogen invasion in ticks might expedite the development of new strategies to interfere with the life cycle of A. phagocytophilum.

Naturally Acquired Resistance to Ixodes scapularis Elicits Partial Immunity against Other Tick Vectors in a Laboratory Host.

In many regions where ticks negatively impact public health or economic production, multiple medically important tick species may have overlapping geographic distribution, and in North America, this includes members of Ixodes, Dermacentor, and Amblyomma genera. Acquired tick resistance is the process by which some animals develop an immune response against feeding ticks after one or more exposures. This form of immunity can restrict the ability of ticks to feed and may inhibit transmission of pathogens. Likewise, many proteins present in tick saliva are conserved among tick species, and prior studies have reported cross-protective host immunity against certain combinations of ticks. In this study, we used a guinea pig model to assess whether host resistance against Ixodes scapularis could confer protection against two other medically important tick vectors, Dermacentor variabilis and Amblyomma americanum. Tick challenges using nymphs were used to induce host resistance against a primary species, followed by additional challenge using a secondary tick species. Tick attachment to hosts and engorgement weights were reduced significantly for D. variabilis and A. americanum feeding on I. scapularis-sensitized hosts. Reciprocally, I. scapularis engorgement weights were reduced to a lesser extent, and attachment was unaffected when feeding on hosts sensitized with either D. variabilis or A. americanum. These results indicate that immunity against I. scapularis could potentially be exploited for use in an anti-tick vaccine targeting multiple tick species and their associated pathogens.

Immunomodulation by Mosquito Salivary Protein AgSAP Contributes to Early Host Infection by Plasmodium.

Malaria is caused when Plasmodium sporozoites are injected along with saliva by an anopheline mosquito into the dermis of a vertebrate host. Arthropod saliva has pleiotropic effects that can influence local host responses, pathogen transmission, and exacerbation of the disease. A mass spectrometry screen identified mosquito salivary proteins that are associated with Plasmodium sporozoites during saliva secretions. In this study, we demonstrate that one of these salivary antigens, Anopheles gambiae sporozoite-associated protein (AgSAP), interacts directly with Plasmodium falciparum and Plasmodium berghei sporozoites. AgSAP binds to heparan sulfate and inhibits local inflammatory responses in the skin. The silencing of AgSAP in mosquitoes reduces their ability to effectively transmit sporozoites to mice. Moreover, immunization with AgSAP decreases the Plasmodium burden in mice that are bitten by Plasmodium-infected mosquitoes. These data suggest that AgSAP facilitates early Plasmodium infection in the vertebrate host and serves as a target for the prevention of malaria. IMPORTANCE Malaria is a vector-borne disease caused by Plasmodium sporozoites. When an anopheline mosquito bites its host, it releases Plasmodium sporozoites as well as saliva components. Mosquito proteins have the potential to serve as antigens to prevent or influence malaria without directly targeting the pathogen. This may help set a new paradigm for vaccine development. In this study, we have elucidated the role of a novel salivary antigen, named Anopheles gambiae sporozoite-associated protein (AgSAP). The results presented here show that AgSAP interacts with Plasmodium falciparum and Plasmodium berghei sporozoites and modulates local inflammatory responses in the skin. Furthermore, our results show that AgSAP is a novel mosquito salivary antigen that influences the early stages of Plasmodium infection in the vertebrate host. Individuals living in countries where malaria is endemic generate antibodies against AgSAP, which indicates that AgSAP can serve as a biomarker for disease prevalence and epidemiological analysis.

mRNA vaccination induces tick resistance and prevents transmission of the Lyme disease agent.

Ixodes scapularis ticks transmit many pathogens that cause human disease, including Borrelia burgdorferi. Acquired resistance to I. scapularis due to repeated tick exposure has the potential to prevent tick-borne infectious diseases, and salivary proteins have been postulated to contribute to this process. We examined the ability of lipid nanoparticle­containing nucleoside-modified mRNAs encoding 19 I. scapularis salivary proteins (19ISP) to enhance the recognition of a tick bite and diminish I. scapularis engorgement on a host and thereby prevent B. burgdorferi infection. Guinea pigs were immunized with a 19ISP mRNA vaccine and subsequently challenged with I. scapularis. Animals administered 19ISP developed erythema at the bite site shortly after ticks began to attach, and these ticks fed poorly, marked by early detachment and decreased engorgement weights. 19ISP immunization also impeded B. burgdorferi transmission in the guinea pigs. The effective induction of local redness early after I. scapularis attachment and the inability of the ticks to take a normal blood meal suggest that 19ISP may be used either alone or in conjunction with traditional pathogen-based vaccines for the prevention of Lyme disease, and potentially other tick-borne infections.

Tick immunity using mRNA, DNA and protein-based Salp14 delivery strategies.

Guinea pigs exposed to multiple infestations with Ixodes scapularis ticks develop acquired resistance to ticks, which is also known as tick immunity. The I. scapularis salivary components that contribute to tick immunity are likely multifactorial. An anticoagulant that inhibits factor Xa, named Salp14, is present in tick saliva and is associated with partial tick immunity. A tick bite naturally releases tick saliva proteins into the vertebrate host for several days, which suggests that the mode of antigen delivery may influence the genesis of tick immunity. We therefore utilized Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms. salp14 containing mRNA-LNPs vaccination elicited erythema at the tick bite site after tick challenge that occurred earlier, and that was more pronounced, compared with DNA or protein immunizations. Humoral and cellular responses associated with tick immunity were directed towards a 25 amino acid region of Salp14 at the carboxy terminus of the protein, as determined by antibody responses and skin-testing assays. This study demonstrates that the model of antigen delivery, also known as the vaccine platform, can influence the genesis of tick immunity in guinea pigs. mRNA-LNPs may be useful in helping to elicit erythema at the tick bite site, one of the most important early hallmarks of acquired tick resistance. mRNA-LNPs containing tick genes is a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases.

Fractionation of tick saliva reveals proteins associated with the development of acquired resistance to Ixodes scapularis.

Tick-borne diseases pose a global medical problem. As transmission of tick-borne pathogens to their hosts occurs during tick feeding, development of vaccines thwarting this process could potentially prevent transmission of multiple tick-borne pathogens. The idea of tick vaccines is based on the phenomenon of acquired tick immunity, rejection of ticks feeding on hosts which were repeatedly infested by ticks. Recently, we demonstrated that saliva of the blacklegged tick Ixodes scapularis, which is the main vector of tick-borne pathogens in northeast USA, is sufficient for induction of tick immunity in the guinea pig model and that immunity directed against tick glycoproteins is important in this phenomenon. Nevertheless, immunity elicited against individual tick salivary antigens, which have been identified and tested so far, provided only modest tick rejection. We therefore now tested fractions of tick saliva produced by liquid chromatography for their ability to induce tick immunity in the guinea pig model. Immunization with all individual fractions elicited antibodies that reacted with tick saliva, however only some fractions displayed the ability to induce robust protective tick immunity. Mass spectrometry analysis led to identification of 24 proteins present only in saliva fractions which were able to induce tick immunity, suggesting suitable candidates for development of a tick vaccine.

Inhibition of neutrophil function by two tick salivary proteins.

The saliva of hematophagous arthropods contains potent anti-inflammatory and antihemostatic activities that promote acquisition of the blood meal and enhance infection with pathogens. We have shown that polymorphonuclear leukocytes (PMN) treated with the saliva of the tick Ixodes scapularis have reduced expression of beta(2) integrins, impaired PMN adherence, and reduced killing of Borrelia burgdorferi, the causative agent of Lyme disease. Here we describe two Ixodes proteins that are induced upon tick feeding and expressed predominantly in the salivary glands. Using saliva harvested from ticks with reduced levels of ISL 929 and ISL 1373 through targeted RNA interference knockdown, as well as purified recombinant proteins, we show the effects of these proteins on downregulation of PMN integrins and inhibition of the production of O(2)(-) by PMN in vitro. Mice immunized with ISL 929/1373 had increased numbers of PMN at the site of tick attachment and a lower spirochete burden in the skin and joints 21 days after infection compared to control-immunized animals. Our results suggest that ISL 929 and ISL 1373 contribute to the inhibition of PMN functions shown previously with tick saliva and support important roles for these inhibitory proteins in the modulation of PMN function in vivo.

Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks.

Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.

Host-specific expression of Ixodes scapularis salivary genes.

Ixodes scapularis vectors several pathogens including Borrelia burgdorferi, the agent of Lyme disease. Nymphal and larval stages, and the pathogens transmitted by I. scapularis are maintained in a zoonotic cycle involving rodent reservoir hosts, predominantly Peromyscus leucopus. Humans are not reservoir hosts, however, accidental encounters of infected ticks with humans, results in pathogen transmission to the human host. Laboratory models of non-reservoir hosts such as guinea pigs develop a strong immune response to tick salivary proteins and reject ticks upon repeated tick infestations. Anecdotal and scientific evidence suggests that humans that get frequent tick bites might also develop resistance to ticks. Mus musculus, the laboratory model of natural host, does not develop resistance to I. scapularis upon repeated tick infestations. Addressing this dichotomy in vector-host interaction, we present data that suggest that the salivary transcriptome and proteome composition is different in mouse and guinea pig-fed I. scapularis, and that these differences might contribute to differences in host immune responses. These findings reveal a new insight into vector-host interactions and offer a functional paradigm to better understand the phenomenon of acquired tick-resistance.

Infection-derived lipids elicit an immune deficiency circuit in arthropods.

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.

Disruption of the salivary protein 14 in Ixodes scapularis nymphs and impact on pathogen acquisition.

We previously examined the physiological role of the anticoagulant salivary protein 14 (salp14) in adult Ixodes scapularis and showed that Salp14 played a role in tick feeding and engorgement. We now analyze whether the disruption of the salp14 family expression by RNA interference affects tick weight in naïve nymph I. scapularis. Salp14 expression after dsRNA injection was significantly reduced, as shown by mRNA and protein analysis. However, nymph engorgement weight was not altered in salp9pac (salp14 paralog) dsRNA-injected ticks. We also determined Borrelia burgdorferi and Anaplasma phagocytophilum acquisition in I. scapularis nymphs that had reduced Salp14 expression. B. burgdorferi and A. phagocytophilum acquisition was not affected 72 hours after feeding. Our results suggest that different mechanisms govern nymph and adult feeding in I. scapularis.