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Keywords:

  • Arthropod saliva;
  • immune modulation;
  • skin immunity;
  • vector-borne pathogen

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Meeting Discussion
  5. Wanted: The perfect model systems
  6. Conclusions and panel recommendations
  7. Acknowledgements
  8. Conflict of interest
  9. References

Many of the pathogens responsible for diseases that result in both economic and global health burdens are transmitted by arthropod vectors in the course of a blood meal. In the past, these vectors were viewed mainly as simple delivery vehicles but the appreciation of the role that factors in the saliva of vectors play during pathogen transmission is increasing. Vector saliva proteins alter numerous physiological events in the skin; in addition, potent immunomodulatory properties are attributed to arthropod saliva. The description of specific factors responsible for these activities and their mechanisms of action have thus far remained mostly anecdotal. The National Institute of Allergy and Infectious Diseases (NIAID) sponsored a workshop in May 2012 to explore novel approaches aimed at identifying how vector saliva components affect the function of various immune cell subsets and the subsequent impact on the transmission of vector-borne pathogens. Such knowledge could guide the development of novel drugs, vaccines and other strategies to block the transmission of vector-borne pathogens. This meeting report summarizes the discussions of the gaps/challenges which represent attractive research opportunities with significant translational potential.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Meeting Discussion
  5. Wanted: The perfect model systems
  6. Conclusions and panel recommendations
  7. Acknowledgements
  8. Conflict of interest
  9. References

Arthropod vectors transmit many pathogens that are responsible for diseases of economic and global health importance, including malaria, leishmaniasis, dengue fever, yellow fever and West Nile encephalitis; and a wide variety of tick-borne diseases caused by viruses, bacteria and parasites. Recent studies suggest that arthropod saliva not only assists the vector in obtaining a blood meal, but saliva factors also contribute to pathogen transmission, though the mechanisms have yet to be fully identified. To address challenges posed by a system that involves multiple organisms and requires input from very diverse areas of expertise, the National Institute of Allergy and Infectious Diseases (NIAID) of the US National Institutes of Health (NIH) in the Department of Health and Human Services (DHHS) hosted a series of workshops to identify scientific gaps and foster multidisciplinary collaborations to address these scientific areas. Following a workshop in May 2011 (ICVDF2011 [1]), at which researchers presented their latest insights into the biological effects of arthropod saliva molecules in vertebrate skin after a blood meal, the attendees of a meeting held in May 2012 with expertise in immunology, vector biology and infectious diseases (Table 1) were tasked with formulating novel, innovative and multidisciplinary research concepts which have the potential to significantly move the field forward. Of particular

Table 1. Names and affiliations of speakers at the “Role of Immune-Cell Subsets in the Establishment of Vector-Borne Infections” meeting, held May 2012 at the NIAID in Bethesda, USA
SpeakersAffiliationTitle of Presentation
Mathew T. Aliota/Michael A. SchmidNew York State Department of Health/School of Public Health, UC BerkeleyDendritic cells, mosquito saliva, and the immune response to dengue virus in the skin
Donald ChampagneUniversity of GeorgiaVector Saliva: Biochemical bridge between the biter and the bitten
Ian CockburnJohns Hopkins Malaria Research InstituteThe development of T and B cell responses in the context of mosquito saliva
Monica E. EmbersTulane University Health SciencesThe impact of Ixodes saliva on the acquired immune response to B. burgdorferi
Constance FinneyUniversity of CalgaryThe effect of vector microbiomes on the establishment of vector-borne infections
John E HarrisUniversity of Massachusetts Medical SchoolUsing a humanized mouse model to study the human innate and adaptive immune response at the vector-host-parasite interface
John T. HartyUniversity of IowaMessing with a serial killer
Ethan A. LernerMassachusetts General HospitalShaving with Occam's razor: Think skin
Mary-Ann McDowellUniversity of Notre DameSalivary Proteins as Immunological Triggers
Sukanya NarasimhanYale School of MedicineVAMPs – Potential role in inflammation
Fabiano OliveiraNational Institutes of Allergy and Infectious Diseases/NIHRole of sand fly saliva on the skin: Dendritic cells and their impact on leishmaniasis
Marion PepperUniversity of WashingtonCan arthropod and mammalian immune responses work in concert to inhibit parasite transmission in the mosquito?
Michael PovelonesImperial College, LondonUsing a worm to create a model mosquito
Saravanan ThangamaniUniversity of Texas Medical BranchTick-virus-host interface: Role of tick saliva induced neutrophil recruitment in the infection and dissemination of tick-borne flavivirus

interest were the questions of how different immune cells in the skin are affected by the saliva of an arthropod taking a blood meal, and how different immune cell subsets subsequently contribute to either immunity against the transmitted pathogen or the development of pathogenesis and disease. Presentations and discussions highlighted recent advances in the field, and also focused on significant gaps and challenges in our understanding of vector-borne pathogen transmission.

Meeting Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Meeting Discussion
  5. Wanted: The perfect model systems
  6. Conclusions and panel recommendations
  7. Acknowledgements
  8. Conflict of interest
  9. References

What's in a bite?

  1. Many players

    Blood is a highly attractive source of nutrients and blood-feeding evolved independently in numerous organisms [2]. Despite the differences between blood feeders and their feeding methods, blood feeders all face the same challenges during a meal, foremost blood clotting and vasoconstriction, and have thus developed effective counter-measures. A prime example is the presence of apyrases (adenosine diphoshatases) in the saliva of all blood-feeding arthropods [3]; this enzyme is implicated in the inhibition of host platelet aggregation which would otherwise slow or stop the blood flow during feeding. Despite their shared function, vector salivary proteins are structurally and evolutionarily distinct in taxonomic groups of arthropods. The independent evolution of saliva proteins combined with the strong evolutionary pressure exerted by the vertebrate host's immune response to vector saliva components complicates the search for orthologs of specific saliva components. Simple database queries (such as a BLAST search) are rather useless tools when trying to apply findings from one arthropod species to another. It may be true that general principles of (i) how blood-feeding vectors manipulate host immune responses and (ii) how blood-borne pathogens have adapted to take advantage of the vector's immunomodulatory saliva proteins apply to many different species of vectors and pathogens. However, the actual molecular pathways involved in each three-way relationship between a particular blood-feeding vector, host and microbial pathogen will have to be analyzed individually. This suggestion should not be viewed as a major obstacle but as an argument to focus more on the characterization of saliva from different blood feeders.

  2. Many targets and outcomes

    The central theme of the workshop was the question of how different immune cell subsets, particularly those in the skin, are affected by vector saliva and how an altered immune response might impact pathogen transmission, leading to either increased rejection of the pathogen or increased susceptibility of the host to infection. This information can guide the development of a new generation of vaccines which target vector saliva components, not pathogen-derived antigens (or vaccines that target both types of antigens simultaneously) in an attempt to either neutralize saliva components that promote pathogenesis by vector-borne pathogens or to mount an immune response to saliva components which result in (antigen-independent) bystander protection against the pathogen. The successful use of select sand fly saliva proteins (such as SP15, [4]) as preclinical vaccine candidates against Leishmania infection underscores the usefulness and efficacy of this alternative approach (reviewed in [5]). Similar data are starting to emerge from the use of saliva of other vectors, such as the targeting of an Ixodes sialostatin to interfere with tick feeding [6]. Insights into the functions of saliva proteins will facilitate the rational selection of vaccine candidates based on their importance for the survival of the vector-borne pathogen. One example is the Salp15 protein from Ixodes ticks which not only has immunosuppressive activity that benefits pathogens [7], but is specifically bound by Borrelia burgdorferi and protects the bacterium from antibody mediated killing [8], thus representing a very attractive vaccine target. Though very promising, the workshop participants identified a major obstacle in rapidly implementing this approach as a general strategy for blocking the transmission of blood-borne pathogens: in theory, it should be sufficient to simply determine which saliva component facilitates pathogen transmission and then select this molecule as a vaccine candidate. However, the precise role that a specific arthropod saliva molecule or saliva in general play in the transmission of a vector-borne pathogen is not easy to determine. This is underscored by seemingly contradictory results obtained in different laboratories using the same organisms (for Leishmania reviewed in [5]). A number of reasons were cited including:

    1. Redundancies in the functions of saliva proteins with multiple proteins having overlapping function [9] and/or the presence of multiple isoforms [10]. Therefore, the activity of an individual protein in salivary gland extracts may be masked when using salivary gland extract (SGE).
    2. Timing of the experiment. For example, after a first bite, sand fly saliva exacerbates the infection with Leishmania parasites. However, after multiple exposures of the same host, the presence of the same saliva promotes the rejection of parasites (reviewed in [8]).
    3. Model systems: a common challenge faced by researchers in many research areas is to find model systems that best replicate what happens in the terminal target species (usually humans). Not only do different immune cells respond differently to various arthropod saliva components, but the same immune cell type from one species can respond differently to the same saliva protein in another species. Additionally, different proteins from the same vector may serve as the main target of the immune system of different vertebrate hosts.
    4. Experimental procedures: The biological effect of saliva proteins is affected by the composition of the SGE and the procedures used to isolate saliva. The differences between preparations may include: (i) the contamination with proteins that are not normally injected during a blood meal (such as proteins found in the salivary gland tissue), or the absence in SGE of proteins normally injected during a blood meal (such as midgut proteins injected by vector species such as fleas); (ii) the vector microbiome, specifically the salivary gland microbiome which may or may not be injected during a blood meal and which likely differs between vector colonies maintained in different laboratories; (iii) the use of saliva from non-infected arthropods since the infection with a vector-borne pathogen changes the composition of vector saliva [11, 12]; and (iv) the volume of SGE or salivary proteins since it is difficult to determine the exact size of the inoculum delivered by a blood-feeding vector.
    5. Use of recombinant versus isolated salivary proteins, leading to differences in results which appear to be, in large part, due to major differences in glycosylation patterns. This has been confirmed with tick salivary proteins, which lose immunogenicity after deglycosylation, giving rise to the concept of vector associated molecular patterns (VAMPs) as a way to explain the strong immunogenicity of glycosylated saliva proteins (personal communication S. Narasimhan). This observation has major implications for the development of vaccines based on saliva proteins since many such vaccines would be recombinant proteins. VAMPs would represent a potentially new class of innate immune receptor ligands involved in the host's response against blood-feeding arthropods and thus possibly also vector-borne pathogens.
  3. The vector and host microbiome During a previous workshop on the biological effects of vector saliva [13] participants discussed how a host's microbiome—particularly the skin microbiome—can affect its susceptibility to infection with vector-borne diseases. Significant differences in the susceptibility of germ-free versus “dirty” mice to Leishmania infection underscore that ultra-clean animal facilities that prevent the establishment of a microbiome are not suited for studying vector-borne pathogen transmission in a natural setting [14]. Discussions during the current workshop were extended to how the vector's microbiome affects the susceptibility to, and transmission of, vector-borne pathogens. While the viruses, bacteria and parasites carried by a blood-feeding arthropod drastically alter its ability to transmit blood-borne pathogens [15], very little is known about this phenomenon. Insights into the underlying mechanisms could lead to new approaches to control the spread of vector-borne diseases. An example of the use of “vector probiotics” to block transmission of Dengue virus is the release of Aedes aegypti mosquitoes in Northeast Australia previously infected with a strain of Wolbachia that unexpectedly interferes with the ability of the virus to replicate in the vector [16]. Similarly, a naturally occurring Enterobacter bacterium found in the midgut of some mosquito populations renders them resistant to infection with Plasmodium falciparum (malaria) parasites [17].

Numerous factors determine the composition of an arthropod's microbiome, including its diet, environmental influences and its developmental stage. Unraveling the complexity of the vector microbiome requires a significant research commitment. While symbiotic microorganisms can directly change the susceptibility of a vector to infection by a vector-borne pathogen, it is unknown what impact the vector microbiome has on the immune response of a vertebrate host during and after a blood meal. Commensal organisms have been found in salivary glands of vectors (e.g., the Enterobacterium Sodalis glossinidius in salivary glands of tsetse flies [18]), but at this point it is unclear (i) whether certain microorganisms are not normally present in saliva but were introduced into salivary gland preparations as an experimental artifact and (ii) whether significant numbers of microorganisms from the salivary gland are injected into the vertebrate host during a blood meal and how these microorganisms alter the host's response to the co-delivered pathogens. Finding such an effect would have major implications for the interpretation of data obtained under laboratory conditions using germ-free vectors to model disease transmission.

From the thorn in the starfish to the proboscis in the dermis

Neutrophils are a major component of the innate immune system. They represent the most abundant leukocyte in blood and large numbers are present in the skin, acting as early responders to insult. However, this cell type remained understudied for a long time and was mostly described in the context of collateral tissue damage. Neutrophils are now recognized as effector cells of the innate immune system and modulators of other immune cells including macrophages, dendritic cells (DCs) and T cells. A clear understanding of how neutrophils interact with vector-borne pathogens is lacking and studies conducted so far paint a complex and somewhat confusing picture, in part due to the fact that neutrophils can be involved in both protection against vector-borne pathogens and dissemination of the pathogen. For example, after an infection with Tick-Borne Encephalitis Virus (TBEV), neutrophils are attracted to the bite site in large numbers and may be converted into viral dissemination vehicles. Similarly, neutrophils are rapidly attracted to the site of West Nile Virus (WNV) infection where they are infected and allow efficient replication of the pathogen. The significant difference in the role of neutrophils in WNV infection, i.e., supporting the pathogen at the very early stage of infection versus their involvement in clearing the virus at later stages of the infection, suggest that vector saliva factors, the effects of which would have waned at later stages of the disease, are responsible for the initial induction of neutrophils. Therefore, identifying the biological and immunological functions of arthropod saliva factors may open the door for strategies to prevent the use of immune cells as Trojan horses by vector-borne pathogens.

Neutrophils, attracted and activated by injury and infection (as shown in Figure 1 in the case of a tick bite), employ several strategies to neutralize pathogens including the recently discovered neutrophil extracellular traps (NETs) [19]. NETs are networks of extracellular fibers, primarily composed of DNA from neutrophils, which bind pathogens. These NETs may interfere with blood-feeding as suggested by the presence of endonucleases in the saliva of blood-feeding arthropods. Endonucleases in arthropod saliva, however, also appear to benefit vector-borne pathogens by interfering with neutrophils recruited to the bite-site (personal communication L.F. Oliveira). This finding further underscores the importance of studying infection by vector-borne pathogens in the presence of saliva.

image

Figure 1. Histopathology of the site of a tick (nymphal Ixodes scapularis) bite 6 hours post infestation is shown [27]. The tick's calcified hypostome (maxilla) is clearly shown embedded in the skin and surrounded by a cellular, neutrophil-dominated infiltrate. This cellular infiltrate was triggered by the cutaneous tissue injury and functionally affected by various biologically active compounds in the arthropod's saliva (Image courtesy of S. Thangamani, original magnification 100×).

Download figure to PowerPoint

Skin dendritic cell subsets complicate the story

The complexity of the dermal DC system has only recently been recognized and new animal models have led to a concept in which these different DC populations exhibit very distinct functions [20]. Langerhans cells (LCs), the only DC population found in the epidermis, were first described 150 years ago and have until recently been considered the main antigen presenting cell of the skin. In a still emerging model, LCs may play a regulatory (i.e., immunosuppressive) role in some situations [21], while directing the type of inflammatory response in others [22]. Discussions at the workshop revealed major gaps in our understanding of how different DC populations in the skin respond to immune-modulatory components of vector saliva and how this affects pathogen transmission. Significant effects of saliva on DC function have been described already, including the induction of inflammatory responses by Borrelia, but suppression of those responses as soon as tick saliva is added [23]. However, such insights were gained in vitro, often using cell lines and therefore cannot be translated to the complex situation that exists in the skin. Other in vitro studies have indicated that the exposure of DCs to mosquito saliva can prevent subsequent infection of these cells with WNV, suggesting activation of DCs by saliva components. In contrast, others have shown that select salivary proteins can block the antigen-presenting function of DCs, resulting in a strong reduction of the ability of DCs to prime T-cell responses, as already discussed during the 2011 workshop on the immune consequences of vector-derived factors [1] (summarized in [13]). In conclusion, it will be imperative to conduct studies in vivo with a focus on the contribution of individual skin DC populations to the overall immune response in the presence or absence of certain salivary components.

Wanted: The perfect model systems

  1. Top of page
  2. Abstract
  3. Introduction
  4. Meeting Discussion
  5. Wanted: The perfect model systems
  6. Conclusions and panel recommendations
  7. Acknowledgements
  8. Conflict of interest
  9. References

A major roadblock for the study of vector-borne disease transmission in vivo is the availability of appropriate animal models. This shortcoming affects three distinct organisms involved in the transmission of vector-borne diseases: the vector, the pathogen and the vertebrate host. In terms of the vector, laboratory-raised vectors are quite dissimilar to their counterparts in the wild, particularly when looking at the composition of their saliva or their immune status, both of which affect pathogen transmission. Using salivary extracts instead of the vector to deliver immunologically active salivary proteins introduces an additional set of variables and complexities as discussed above. In terms of the pathogen, strains of patnogens used in laboratory studies may not be representative of the pathogen found in the wild, having been subject to very different evolutionary pressure. Furthermore, the strains or species used to infect laboratory animals are aften different from those that infect humans. Malaria is a prime example, with rodent malaria strains such as P. yoelii differing drastically from P. falciparum, the major cause of human malaria, for which it serves as a “model”. In terms of the vertebrate host, the choice of an appropriate vertebrate model in which human immune responses to an infection can be replicated still remains the most significant challenge. Some vector-borne human pathogens do not infect conventional laboratory animals, and the response to the pathogens that do infect laboratory models is often different in different host species. This may be due to species differences resulting in differential expression of receptors on immune cells or even the number or anatomical location of subsets of immune cells. Therefore, short of using human subjects, there is an urgent need for better humanized mouse models in which key tissues and cells involved in the response being studied (immune cells, skin, etc.) have been replaced with their human counterparts. This research area is still in its infancy but growing fast [24]. Workshop participants recommended the increased utilization as well as refinement of such new humanized models by researchers working on the transmission of vector-borne pathogens.

Conclusions and panel recommendations

  1. Top of page
  2. Abstract
  3. Introduction
  4. Meeting Discussion
  5. Wanted: The perfect model systems
  6. Conclusions and panel recommendations
  7. Acknowledgements
  8. Conflict of interest
  9. References

Deciphering the complex interactions that occur between three distinct organisms — vertebrate host, arthropod vector and vector-borne pathogen — during the disease transmission process is a significant challenge which is further complicated by the addition of the effect the vector microbiome has on pathogen transmission [25]. Workshop participants made several recommendations to advance the field:

  1. Improved model systems:

    The lack of strong model systems was a recurring theme as it affects all three organisms involved in the disease transmission process as discussed above. The field would particularly benefit from better mouse models which involve human immune cells and human tissues (such as the skin) to minimize artifacts. There is also a lot of room for the improvement of vector models, some of which suffer from artifacts resulting from long-term maintenance in laboratories (such as alterations in — or lack of — their microbiome [26]). Furthermore, the lack of gene knockout models for arthropod saliva molecules slows research into the function and relevance of individual saliva components which in turn slows their exploration as vaccine candidates.

  2. Experimental design:

    1. Work conducted with arthropod saliva thus far indicates a major role of arthropod salivary factors in the transmission of vector-borne pathogens. However, some of the results are confusing and difficult to interpret because of: the use of artificial in vitro systems (e.g., cell lines instead of primary immune cell subsets); the use of salivary gland extracts, which differ from the salivary inoculum of the blood-feeding arthropod; and the use of recombinant salivary proteins which e.g., lack the appropriate glycosylation pattern.
    2. Most of the information regarding the impact that arthropod saliva factors have on the function of immune cells comes from in vitro studies, employing cell lines. However, such studies do not take into account the enormous diversity of some cell types, such as DCs and the differential impact of different cell subsets on an immune response, or interactions between immune cells in situ which cannot be replicated in vitro. Considering the differences in the functions and responses of different DC subpopulations it is essential to immediately replicate and confirm in primary cells the results obtained with cell lines to establish the biological relevance of those findings.

      Pathogen transmission by a vector is a complex, two-way street with the delivery of pathogenic organisms, various immmunomodulatory saliva proteins and possibly arthropod commensals into the skin of a vertebrate vector, and the simultaneous uptake of vertebrate immune cells and blood factors (cytokines, antibodies, hormones, etc.) into the arthropod's midgut during the blood meal. Both inocula affect the recipients’ immune response but the impact of mammalian blood-derived factors on the arthropods immune system and subsequently the survival of pathogens in the vector are still very poorly understood and will be the focus of a follow-up workshop.

  3. Multidisciplinary teams:

    Research on vector-borne pathogens and their transmission is primarily conducted by experts in one of the research areas, i.e., vector biologists, parasitologists, virologists or bacteriologists. To move the field forward significantly, it will be essential for those researchers to come together as multi-disciplinary research teams with an increased involvement of immunologists. Furthermore, the field needs to move away from the narrowly focused studies of the different organisms involved in the transmission of vector-borne pathogens — vector, mammalian host and pathogen — in isolation, to a more holistic approach which is admittedly more complicated, but may provide a more integrated understanding of the disease process.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Meeting Discussion
  5. Wanted: The perfect model systems
  6. Conclusions and panel recommendations
  7. Acknowledgements
  8. Conflict of interest
  9. References

The conference organizers would like to thank the following researchers who provided subject matter expertise to the presenters for the development of multi-disciplinary research proposals which were presented at the workshop: Drs. John Andersen (NIAID/NIH), Elke Bergmann-Leitner (Walter Reed Army Institute of Research), Richard Cummings (Emory University), Marcelo Jacobs-Lorena (Johns Hopkins School of Public Health), Shaden Kamhawi (NIAID/NIH), Ethan Lerner (Massachusetts General Hospital), Polly Matzinger (NIAID/NIH), Esther von Stehbut (University of Mainz, Germany), Mark Udey (NCI/NIH), Jesus Valenzuela (NIAID/NIH), and Fidel Zavala (Johns Hopkins University).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Meeting Discussion
  5. Wanted: The perfect model systems
  6. Conclusions and panel recommendations
  7. Acknowledgements
  8. Conflict of interest
  9. References