Diseases, such as malaria, dengue, leishmaniasis and tick-borne encephalitis, affect a substantial percentage of the world's population and continue to result in significant morbidity and mortality. One common aspect of these diseases is that the pathogens that cause them are transmitted by the bite of an infected arthropod (e.g. mosquito, sand fly, tick). The pathogens are delivered into the skin of the mammalian host along with arthropod saliva, which contains a wide variety of bioactive molecules. These saliva components are capable of altering hemostasis and immune responses and may contribute to the ability of the pathogen to establish an infection. The biological and immunological events that occur during pathogen transmission are poorly understood but may hold the key to novel approaches to prevent transmission and/or infection. In May 2011, 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 hosted a workshop entitled Immunological Consequences of Vector-Derived Factors which brought together experts in skin immunology, parasitology and vector biology to outline the gaps in our understanding of the process of pathogen transmission, to explore new approaches to control pathogen transmission, and to initiate and foster multidisciplinary collaborations among these investigators.
More than 14 000 species of blood-feeding arthropods have been identified that produce between 40 (sand flies) and several 100 (ticks) saliva proteins, many with unknown functions. Arthropod saliva proteins are a prime example of convergent evolution with different insect and arachnid species having independently selected non-homologous proteins with the same function (such as anti-blood clotting). The heterogeneity of salivary proteins is further increased by the evolutionary pressure that the vertebrate host exerts on arthropod-derived antigens resulting in a large number of bioactive molecules with potential for applications in pharmacology, vaccinology and basic research.
The transmission of pathogens by blood-feeding arthropods (Fig. 1 and Table 1) involves the exchange of a complex mixture of biologically active molecules between the arthropod and the vertebrate host. The arthropod injects salivary factors into the vertebrate host skin and, at the same time, the vector ingests cytokines 1, growth factors 2, complement components, antibodies and other blood-derived molecules with the blood meal. In both hosts – mammal and arthropod – these molecules can affect the survival and thus infectivity of vector-borne pathogens 3. Various bioactive molecules in the saliva of blood-feeding arthropods have evolved to facilitate feeding. Some of these arthropod-derived molecules clearly benefit vector-borne pathogens (e.g. such as yet undefined components of Tsetse fly saliva, which accelerates the onset of Trypanosoma brucei infection 4). Immunomodulatory effects of arthropod saliva could explain why the host's (skin) immune system is unable to reject the small infectious inoculum delivered during a blood meal. The events that occur at the bite-site are still poorly understood and insights into processes such as the modulation of mammalian immune functions by arthropod saliva may provide new targets to combat diseases transmitted by arthropods. These diseases include mosquito-borne malaria (more than 1 million deaths/year) 5–7 mosquito-transmitted Dengue fever (more than 20 000 deaths/year and up to 3 billion people in more than 100 countries at risk), or tick-borne encephalitis (∼12 000 reported clinical cases/year but many cases remain unreported). In May 2011, 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 hosted a workshop entitled Immunological Consequences of Vector-Derived Factors to explore new approaches to control pathogen transmission, and to initiate and foster multidisciplinary collaborations among researchers.
Table 1. Examples of important blood-feeding arthropod vector groups and diseases caused by the transmitted pathogens
Malaria, Lymphatic Filariasis, Dengue/Dengue Hemorrhagic Fever, Yellow Fever, Chickungunya Fever, West Nile Virus Encephalitis, St. Louis Encephalitis, Eastern Equine Encephalitis, Western Equine Encephalitis, Venezuelan Equine Encephalitis
Blood-feeding arthropods represent an attractive vector for pathogens. They not only provide transportation between hosts and spread the pathogen geographically, but also facilitate the pathogens' entry into the host by creating a portal. Many vector-borne pathogens enter the vertebrate host through an arthropod bite together with arthropod saliva. Most of the saliva components are still unknown and, therefore, our understanding of the critical role the arthropod's saliva plays in helping the hitchhiking pathogen establish an infection is still very limited. When studying pathogen transmission or testing vaccines against vector-borne diseases, however, concerns were raised at the workshop that natural infection differs from experimental injection of isolated pathogens by needle and syringe. Only mature (“ready-to-go”) pathogens are delivered during blood feeding while isolated (e.g. by dissecting an infected insect's salivary glands) or cultured pathogens may contain immature and non-functional organisms. Side-by-side infection studies using the natural vector of the pathogen (flies, ticks or mosquitoes) or injection of isolated pathogens by syringe revealed multiple differences in the outcome of the infection ranging from significantly different ID50 doses to differences in the pathogen load or in the lethality of the pathogen or even the complete inability to infect in the absence of vector saliva 8–12.
Blood feeding ensures delivery to an anatomically precise target area while isolated pathogens are often injected intravenously or intraperitoneally and this creates a highly artificial infection model. Even when injected into the skin, pathogens may not be deposited in the same cellular layer as the one the arthropod vector targets and thus a different subset of immune cells with different functions is engaged. Arthropods deliver their infectious payload together with saliva containing a wide array of biologically active, yet poorly understood molecules. Despite the small size of the inoculum and the fact that only minute amounts of saliva enter the host, the impact is significant as evidenced by the induction of antibody responses against saliva proteins. Although, in the specific case of mosquitoes, a few of the saliva proteins are indeed immunogenic, the resulting antibody response directly correlates with the number of mosquito bites, thus representing a useful biomarker for exposure to (potentially infectious) bites 13, 14. Although studies with West Nile Virus reveal that the mortality of mice correlates with the number of prior exposures to the mosquito 15, the successful induction of antibodies to certain saliva proteins, which neutralize their function, can protect the vertebrate host from the infection by a subsequent bite from the same vector species.
Battleground skin: what arthropod-borne pathogens face at the injection site
Pathogens introduced into the skin by a blood-feeding arthropod face a variety of immune defense mechanisms. Despite the importance of the skin as an entry site for many infectious agents, our current knowledge of the skin's immune system lags behind what we know about immune events in the spleen, lymph nodes or blood. A clearer understanding is needed for the contribution of individual leukocyte populations in the skin that are protective against pathogens or pathology caused by infection. Recent work with Langerin-knockout mice has significantly changed immunologists' view of Langerhans cells (LCs), the only dendritic cell population in the epidermis 16. Although the use of different knockout mice and a variety of experimental models resulted in contradictory findings, LCs now appear to function as a regulatory and suppressive cell type rather than having an immunostimulatory role. This may explain the seemingly paradoxical observation that the ablation of LCs before infection with Leishmania major reduces the parasite load in the skin and promotes healing of lesions 17. The newly proposed function of LCs is consistent with the need to prevent excessive inflammatory responses at a tissue site that is constantly exposed not only to pathogens, but also to environmental antigens and commensals. Commensals are frequently overlooked when studying immune responses to pathogens in the skin (or intestinal tract) using mice housed in animal facilities with a significantly altered composition of such organisms. The absence of commensal microorganisms, however, can drastically affect immune responses to pathogens and although the immune system of mice maintained in germ-free facilities appears to develop normally, their ability to respond is reduced in Leishmania infection of the skin. This raises concern about the validity of studies with vector-borne pathogens conducted in “clean” mice lacking commensal microorganisms.
Similar to LCs, our understanding of neutrophil function is currently undergoing significant revision. This population of professional phagocytes is not a homogenous and terminally differentiated leukocyte cell subset with only pro-inflammatory, anti-pathogen functions. Neutrophils do eliminate large numbers of bacteria and parasites before undergoing apoptosis and being removed by phagocytes, such as dermal dendritic cells. However, this process can be usurped by some infectious organisms 18. The depletion of neutrophils in a mouse model of Leishmaniasis resulted in the unexpected enhancement of T-cell priming, suggesting a regulatory role for these cells. Some components of insect saliva have already been shown to affect the function of neutrophils and further research on how this influences the ability of vector-borne pathogens to establish an infection is needed.
The other skin-resident leukocyte whose perceived function during an immune response at barrier sites needs to be revised is mast cells 19. No longer seen as mere sentinels participating in the initiation of inflammatory responses, mast cells are potent effector cells capable of directly engaging and destroying bacteria, viruses and parasites. The importance of mast cells during an arthropod's blood meal is underscored by the presence of mast cell inhibitors in the arthropod's saliva. The activation of mast cells during an infectious bite, however, may not always be beneficial to the host as shown in the case of malaria infection. Plasmodium parasites appear to have exploited the mast cell's histamine release for their own survival 20. This finding, once again, emphasizes the need to take a comprehensive view of the events occurring in the skin during the complex interaction between three different species during the transmission of pathogens by an arthropod vector.
Arthropod-saliva and pathogens: Unholy alliance or unrealized potential?
Many arthropod-borne pathogens are injected into their host together with vector saliva. Mounting evidence indicates that these pathogens have evolved to take advantage of specific salivary proteins and sugars to facilitate the establishment of an infection. However, no clear picture of the role of arthropod saliva during infection emerges in the literature; some reports indicate that salivary factors help the pathogen 21, interfere with infection in others 22, or have no apparent effect in other models 23. Furthermore, the timing of the exposure to the saliva appears to be critical: while sand fly saliva present during infection with Leishmania exacerbates infection, pre-exposure to the same saliva components induces protective immunity 24, 25.
Only a small portion of the proteins in arthropod saliva has been studied, but they appear to influence a wide range of mammalian immune responses. Arthropod saliva contains various vasodilators and inhibitors for a broad spectrum of host functions 26 such as blood clotting (to facilitate feeding), mast cell activation (to prevent degranulation and cytokine secretion), complement activation and neutrophil activation (to control immediate responses to the process of blood feeding). Some mosquito saliva molecules interfere with adaptive immune responses by inhibiting the proliferation of B cells and T cells 27 (a phenomenon also reported for the saliva of black flies 28), and selectively suppressing Th1-type cytokine responses but not Th2-type responses 21, 27, 29. A Th2-polarization of immune responses has been reported not only for mosquito 29, but also tick saliva 30, 31. Another mechanism by which mosquito salivary proteins hamper adaptive immune responses is through the inhibition of macrophage phagocytosis and by interfering with the ability of these cells to present antigen by decreasing MHC class I and class II expression (Donald Champagne, unpublished observation). This underscores the need of the arthropod to modulate and control the vertebrate's immune response to saliva regardless of the feeding time (which only lasts seconds in the case of a mosquito, but days in the case of ticks).
Understanding the function that vector saliva has during infection with arthropod-borne pathogens is complicated by the fact that the content of the inoculum is not constant and the composition of saliva can change drastically between species and within a species in response to environmental signals:
(i)due to pressure from the mammalian host's immune system, salivary protein evolution is rapid and varies from one arthropod species to another;
(ii)co-infections (often with bacteria) of the arthropod vector affect not only the inoculum itself by adding another pathogen, but also alter the saliva composition;
(iii)the presence of infectious microorganisms in the salivary glands of blood-feeding arthropods itself alters saliva composition, such as changes in the concentration of apyrase or anti-thrombinase in infected mosquitoes 32 or Tsetse flies 33, or the selective downregulation of salivary factors in Borrelia-infected ticks 34. In contrast, the expression of the tick salivary protein Salp15 is selectively upregulated and the spirochaetes coat themselves with the protein to directly take advantage of its immunosuppressant properties;
(iv)arthropods who feed on their vertebrate host for extended periods of time (i.e. ticks) counter the host's immune response to saliva components by frequently switching expression between gene loci encoding variants of the same saliva proteins thus exposing the host to a changing pattern of saliva proteins;
(iv)a factor of great concern is the variability of saliva components between arthropods in the field and their laboratory counterparts. Because of factors that are not completely understood yet (such as genetic drift in saliva proteins due to selective pressures, the presence of diverse populations of commensal bacteria, or co-infections in blood-feeding arthropods), caution must be exercised when applying conclusions obtained from studying vector saliva proteins in laboratory arthropod strains to the conditions in the field; and
(v)the levels of at least some saliva proteins fluctuates in a circadian manner 35.
Due to this variability, studying and understanding saliva proteins may appear to be a daunting task. Workshop participants discussed systems biology approaches as a way to generate simplified computational models of the biological networks to understand how arthropod saliva proteins affect the host signaling networks. This strategy will allow investigators to generate fundamental insights into the function of arthropod saliva components as they relate to their function during infection with vector-borne pathogens and search for analogue molecules or molecules with similar function in other vector species. This approach is based on the simple assumption that the immune system of not only the mammalian host, but also that of the arthropod vector represents a common challenge to all vector-borne microorganisms. Thus, evolutionarily very distant vectors or completely unrelated pathogens (ranging from rather simple viruses to highly complex protozoan parasites) have to overcome the same kinds of evolutionarily conserved immune defense mechanisms of their hosts.
NIAID hosted the workshop entitled Immunological Consequences of Vector-Derived Factors based on the recognition that research into vector-borne diseases is mostly conducted by scientists focusing on one of the three organisms involved in the transmission of vector-borne pathogens (vertebrate, pathogen or arthropod vector), while largely disregarding the contribution of the other organism(s) such as the potent biological (and specifically immunological) effects of arthropod saliva. Speakers invited to this workshop (Table 2) included individuals with expertise in vector biology, vector-borne disease and human immunology who had an opportunity to interact and exchange information about the complex process of pathogen transmission. Successful vaccines and strategies to prevent or control vector-borne diseases, however, require a comprehensive understanding of the complex events that occur during the transmission of arthropod-delivered pathogens. A textbook example of this challenge is Leishmania vaccines: while numerous experimental (subunit) vaccines protect animals against infection by isolated parasites injected through a needle, none work under natural challenge conditions, where the parasite is injected (together with arthropod saliva) by a sand fly 36. Leishmania is also the first infectious disease model in which immune responses against select sand fly saliva proteins induced by DNA vaccines have been shown to protect against infection with naturally transmitted Leishmania37. This represents a novel and fundamentally different approach to combating vector-borne diseases. The next generation of vaccines against vector-borne diseases may need to comprise components of the pathogen as well as vector saliva factors to induce robust protection, which can bypass the antigenic polymorphism of pathogen-derived vaccine targets. The surprisingly strong immunogenicity of the small amount of saliva proteins injected by both infected and non-infected blood-feeding arthropods offers another innovative but not widely used application, i.e. the correlation of humoral responses to saliva proteins with the frequency of insect bites 35, 38. This allows for the rapid and very straightforward determination of the success of vector-control and -eradication strategies.
Table 2. Names and affiliations of speakers from the ICVDF meeting, May 2011
National Institutes of Health, USA
Inst. Trop. Med. Antwerp & Vrije Univ., Belgium
University of Georgia, USA
National Institutes of Health, USA
University of California San Diego, USA
Kansas State University, USA
Julián F. Hillyer
Vanderbilt University, USA
University of Minnesota, USA
University of California Davis, USA
Mary Ann McDowell
Notre Dame University, USA
Mt. Sinai School of Medicine, USA
Texas A&M, USA
IRD – UMR MiVEGEC, France
National Institutes of Health, USA
National Institutes of Health, USA
IRD – UMR MiVEGEC, France
Quinnipiac University, USA
National Institutes of Health, USA
University of Pittsburgh, USA
While the relevance of understanding the biological and immunological activity of vector saliva components during infection with pathogens is obvious, a second promising – but understudied – area of research is the development of arthropod saliva components into novel drugs (“drugs from bugs”). These molecules have powerful biological activities, which have been refined through a lengthy evolutionary process to work at extremely low concentrations. Considering that more than 14 000 species of blood-feeding arthropods exist, each producing up to several hundred different saliva proteins, the sialome represents an enormous, but mostly untapped resource of potential drugs. Even less research than on saliva proteins has been carried out on the lipid and carbohydrate content of arthropod saliva, which may hold yet more surprises.
Research on the transmission of vector-borne diseases has progressed disappointingly slowly with effective vaccines against major vector-borne diseases, except Yellow Fever, still not available. Workshop participants concluded that progress in basic research of these diseases as well as vaccine development will require significantly intensified interactions between multiple disciplines, including entomology, immunology – in particular (human) skin immunobiology – viral/bacterial/parasite biology and systems biology. For basic immunologists, such collaborations with experts in the area of vector-borne diseases provide an opportunity for the discovery of novel pathogen escape mechanisms, which may provide unprecedented insights into immune mechanisms. From a practical standpoint, this may be achieved by encouraging the cross-training, e.g. of postdoctoral fellows, through exchange programs between laboratories specialized in the different research areas to prepare them for a career as independent researchers in a niche area with tremendous growth potential and relevance for human health.