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Is it possible to develop pan-arthropod vaccines?
Opinion
TRENDS in Parasitology
Vol.22 No.8 August 2006
Is it possible to develop pan-arthropod vaccines? J. Santiago Mejia, Jeanette V. Bishop and Richard G. Titus Department of Microbiology, Immunology and Pathology, Colorado State University, 1619 Campus Delivery, Fort Collins, CO 80523, USA
Hematophagous arthropods that transmit the etiological agents of arthropod-borne diseases have become the focus of anti-vector vaccines, targeted mainly at components of their saliva and midgut. These efforts have been directed mostly towards developing speciesspecific vaccines. An alternative is to target crossreactive epitopes that have been preserved during evolution of the arthropods. The N- and O-linked glycans that are attached to arthropod glycoproteins are one of the potential targets of this pan-arthropod vaccine approach. Here, we discuss how genetically modified Drosophila melanogaster cells can be used to synthesize and to deliver these arthropod glycans to vertebrate hosts. Arthropod-borne diseases Infections transmitted by hematophagous arthropods are responsible for some of the most devastating diseases of humans and animals worldwide, and attempts to control them markedly affect the resources of afflicted nations (see the World Health Organization, http://www.who.int/tdr/ diseases; and the World Organization for Animal Health, http://www.oie.int/eng/info/en_info.htm). Understanding the complex ecology and biology of arthropod-borne diseases (ABDs) is a major scientific challenge, but it is an essential step in the design of effective control programs. Because immunization is one of the most cost-effective interventions in public health [1], considerable effort and resources have been directed towards the development of vaccines that target the etiological agents of these diseases. So far the results have been disappointing. After decades of research, we still lack effective vaccines against some of the most important ABDs such as malaria, leishmaniasis, trypanosomiasis, filariasis and several diseases caused by tick- and mosquito-borne viruses. The vectors that transmit these diseases have now become the targets of vaccine in an effort to decrease pathogen transmission. Most attempts have focused on the induction of speciesspecific immunity, targeting proteins of the saliva and/or midgut of arthropods [2,3]. Proteins secreted into these compartments move through the Golgi system of epithelial cells, where they can be covalently modified by arthropod glycans [4]. Because arthropod glycans have not been subject to the striking diversification seen in vertebrates [5–8], they represent potential targets of Corresponding author: Mejia, J.S. ([email protected]). Available online 19 June 2006
pan-arthropod vaccines. Here we discuss this possibility and the ideal design for a vaccine required to induce safe and long-lasting pan-arthropod immunity. Arthropod hematophagy The ability to obtain and digest a blood meal from a vertebrate was a key event in the evolution of ABDs, because the reproductive efficiency of arthropods increased with this new source of nutrients. In the process, they exposed themselves to infectious agents carried by the vertebrate host – the first step leading to vector competence for the transmission of pathogens. Adaptation to hematophagy is a very complex process that has demanded several anatomical, physiological and immunological changes in hematophagous arthropods [9]. The most significant alterations occurred in (i) the mouthparts to facilitate access to blood; (ii) the salivary glands to produce molecules capable of blocking vertebrate hemostasis; and (iii) the midgut to neutralize immune injury mediated by vertebrate blood and to optimize digestion and absorption of blood components [9]. The salivary glands and midgut have attracted the attention of several groups of researchers because they are crucial determinants of vector competence and offer several targets for anti-vector vaccines [2,3]. Furthermore, they have attracted the interest of the pharmacological sector because of the extraordinary diversity of arthropod salivary molecules, which have potent effects on the vascular, neural, hemostatic and immune systems of vertebrates [10–12]. Arthropod saliva In an extraordinary example of convergent evolution, each species of hematophagous arthropod reached a particular solution to the problem of obtaining a blood meal from the host via the expression, in their saliva, of a complex and redundant mixture of inhibitors of vasoconstriction, coagulation and/or platelet aggregation [10–15]. This anti-hemostatic cocktail facilitates the flow of vertebrate blood into the arthropod midgut, but it can also have an indirect anti-inflammatory and immunomodulatory effect owing to attenuation of the defense-activation signal mediated by thrombin and other activated enzymes of the coagulation cascade [16]. This effect is further amplified by various immunomodulators that are present in arthropod saliva [17,18].
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Opinion
TRENDS in Parasitology
The overall anti-hemostatic and immunomodulatory effect of arthropod saliva provides a clear evolutionary advantage, not only to the arthropod but also to microorganisms delivered along with the saliva. This phenomenon of potentiation of microbial transmission was initially documented in an experimental model of the transmission of Leishmania parasites by sandflies [19], and has been subsequently reported in other transmission models of ABDs [20–23]. Targeting salivary components provides two potential mechanisms to block the transmission of pathogens by arthropods: neutralization of the anti-hemostatic and immunomodulatory effect of arthropod saliva; and generation of a local environment leading to pathogen containment and destruction. Furthermore, given the ability of proteins to adsorb to surfaces [24], an immune response directed to salivary proteins that adsorb to pathogens can turn the microorganism into an innocent bystander of anti-salivary immunity. This pathogen-adsorption ability has been recently reported in a salivary protein (Salp15) from the hard tick Ixodes scapularis [25], making it a vaccine candidate for the control of Lyme disease. The significance of anti-salivary immunity in the epidemiology of ABDs is beginning to be explored [26]. It has been suggested that repetitive exposure to sandfly saliva explains, along with concomitant immunity, the natural resistance to Leishmania parasites that inhabitants of endemic areas develop over many years [26]. This suggestion is supported by experimental evidence indicating that exposure to the bite of non-infected arthropods protects against an infectious challenge [27,28]. Because it is not feasible to immunize individuals with arthropod saliva or dissected salivary glands, much effort has been directed towards the identification of potential vaccine candidates from the salivary gland transcriptome and proteome of selected vector species of ABDs affecting humans and other animal species [29–31]. Arthropod midgut The digestion of a blood meal in an arthropod midgut is a complex process that requires the coordinated expression of many genes involved in nutrient processing and adsorption, detoxification mechanisms and defense against the pathogens that arrive in the vertebrate blood meal [32,33]. Furthermore, several mechanisms to deactivate the innate and adaptative immune systems of the vertebrate blood must operate to protect the midgut epithelia from immune-mediated injury. The best-known example of this protection mechanism is the formation of a peritrophic membrane around the blood meal in hematophagous insects [34]. Additional mechanisms that might be significant are proteolytic inactivation caused by arthropod digestive enzymes and temperature-dependent immune attenuation caused by the decrease in blood temperature that occurs once the arthropod detaches from the host. Vaccinating against midgut components of some hematophagous arthropods has been shown to decrease vector survival, fecundity and/or ability to transmit a microorganism [35,36]. This approach has proved to be effective in controlling the cattle tick Boophilus microplus [3]. The ongoing characterization of the transcriptome and www.sciencedirect.com
Vol.22 No.8 August 2006
proteome of vectors of ABDs is likely to yield several candidates for this anti-midgut vaccine approach [31,37,38]. Selecting epitopes for a vaccine Protein epitopes It has become apparent, from tests of protein components of arthropod saliva as vaccine candidates [39,40], that antigenic polymorphism might be a problem in the design of anti-salivary vaccines. In one of the few arthropod molecules in which sequence polymorphism has been studied in detail – namely, the pituitary adenylatecyclase-activating polypeptide (PACAP) receptor agonist (maxadilan) of Lutzomyia longipalpis – the extent of polymorphism between individual flies collected from different geographical locations is very significant [41]. This polymorphism represents a mechanism that improves the fitness of the vector under the selective pressure of an anti-maxadilan immune response [42]. Genomic sequence variation is a crucial event in the adaptation process of genomes [43] and, because most salivary proteins are subject to the selective pressure of the vertebrate immune system, it can be predicted they should be highly polymorphic and encoded by rapidly evolving genes. The high frequency of gene duplications in the transcriptome of salivary glands of some species of hematophagous arthropods [29,30] supports this idea. Cross-reactive epitopes Although cross-reactive antigenicity among arthropods has not been studied systematically, it has been detected after natural or experimental exposure to arthropod saliva or midgut components and has been explained by similarities in proteins or glycans [44–46]. In addition to this ‘intra-arthropod’ antigenic cross-reactivity, some of the glycans expressed in arthropods have been found to be expressed in other microorganisms, plants and mammalian cells [47]. Carbohydrate epitopes In comparison with those of vertebrates, the N-linked and O-linked glycans of arthropods seem to have stalled at an intermediate evolutionary step, such that most of the N-linked glycans have a paucimannosidic structure and the O-linked glycans are limited to the core glycan structures known as Tn and T antigens (GalNAc-Ser/Thr and Galb1-3GalNAc-Ser/Thr, respectively) [5–7]. The structure and immunogenicity of N-linked glycans of arthropods have been studied extensively in lepidopteran insect cells because the biotechnology industry relies on them for the production of various proteins that require glycosylation for appropriate folding and optimal pharmacological properties [5]. The high immunogenicity of these glycans and their limited evolutionary diversification place them as key candidates for a vaccine capable of inducing immunity to salivary and midgut glycoproteins of all species of hematophagous arthropod. This approach has been suggested as a way to develop vaccines to control ABDs [48]. Much less information is available on arthropod O-linked glycans [6,7], and their potential as targets for
Opinion
TRENDS in Parasitology
anti-vector immunity has not been explored. Interestingly, the only O-linked glycans so far described in arthropods, the Tn and T antigens, have been extensively studied as pan-carcinoma antigens [49]. The expression of these antigens in both arthropods and mammalian tumor cells represents an interesting link between anti-arthropod and anti-cancer immunity, and offers an opportunity to apply advances derived from attempts to generate anti-cancer immunity by targeting O-linked glycans [50] to the design of anti-vector vaccines. Vaccine design The presence in arthropod saliva of several molecules that mediate anti-hemostatic and immunomodulatory effects represents functional redundancy akin to that of snake venom [51]. To neutralize the biological activity of snake venom, it is necessary to use antisera prepared against whole venom [52]. A similar approach might be required to neutralize the biological activity of arthropod saliva. Because it is impractical to immunize individuals or animals with whole saliva derived from hematophagous arthropods, however, other alternatives must be considered. Cell lines derived from the salivary gland of arthropods would be the ideal immunogen, providing broad immunity with relevant epitopes in the context of xenoreactivity. Arthropod cell lines derived from salivary glands are not available, but cell lines derived from embryos or ovaries could be used as platforms for the synthesis of salivary and/or midgut glycoproteins of hematophagous arthropods. In fact, Drosophila melanogaster cells have been modified to improve their capacity to present antigens to the vertebrate immune system [53]. This approach could be useful in the design of anti-vector vaccines because the modified Drosophila cell lines express the required N- and O-linked glycans. Furthermore, to optimize immunogenicity and to minimize adverse side-effects, the cells could be transformed to express glycoproteins with the appropriate structure and clustering of glycans. Safety concerns In healthy populations of vertebrates, the cores of the Nand O-linked glycans are extensively modified and not accessible to antibodies directed to arthropod-derived glycans. Because alteration of N- and O-linked glycans in vertebrates is linked to several autoimmune diseases [54], however, a chief concern is that immunization with arthropod-derived glycans might amplify the ongoing immunopathology in individuals with autoimmune disease. Another concern – namely, the induction of allergic response to arthropod glycans – can be minimized by removing the a1–3 fucosylated N-linked glycans that have been identified as the main epitopes involved in allergic responses to insect glycoproteins [55]. These concerns must be addressed in experimental models before any attempt is made to use glycan-based panarthropod vaccines. Concluding remarks Despite big gaps in our understanding of arthropod hematophagy, great strides have been made in this field www.sciencedirect.com
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through functional genomics analysis of the salivary glands and midgut of hematophagous arthropods. Nevertheless, much remains to be learned about the extent of functional redundancy in these compartments and how it can be affected by antibodies directed to crossreactive epitopes. Because N- and O-linked glycans are among the most promising candidates for pan-arthropod vaccines, there is an urgent need to characterize these structures in arthropods, especially those arthropods that are vectors of ABDs. Selected glycans with the appropriate structure and clustering can be expressed in insect cells that have been modified to improve their capacity to present antigens to the immune system of vertebrates. One of the advantages of an approach based on modified insect cells is that the immunity induced can be boosted by natural exposure to the saliva of any hematophagous arthropod, regardless of species. A vaccine with such characteristics would be a considerable weapon in the arsenal to control ABDs worldwide.
Acknowledgements We are grateful to Stephen Wikel, Jesus Valenzuela, Stephen Higgs, Barbara Drolet, Keith Nelson and Tereza Magalhaes for critically reviewing the manuscript; and thank Carolina Barillas-Mury, Diana Sierra, Tess Brodie and Claudia Bernal for helpful suggestions. This work was supported by grants from the National Institutes of Health (RO1 534411 and RCE 534724).
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TRENDS in Parasitology
Vol.22 No.8 August 2006
Is it possible to develop pan-arthropod vaccines? J. Santiago Mejia, Jeanette V. Bishop and Richard G. Titus Department of Microbiology, Immunology and Pathology, Colorado State University, 1619 Campus Delivery, Fort Collins, CO 80523, USA
Hematophagous arthropods that transmit the etiological agents of arthropod-borne diseases have become the focus of anti-vector vaccines, targeted mainly at components of their saliva and midgut. These efforts have been directed mostly towards developing speciesspecific vaccines. An alternative is to target crossreactive epitopes that have been preserved during evolution of the arthropods. The N- and O-linked glycans that are attached to arthropod glycoproteins are one of the potential targets of this pan-arthropod vaccine approach. Here, we discuss how genetically modified Drosophila melanogaster cells can be used to synthesize and to deliver these arthropod glycans to vertebrate hosts. Arthropod-borne diseases Infections transmitted by hematophagous arthropods are responsible for some of the most devastating diseases of humans and animals worldwide, and attempts to control them markedly affect the resources of afflicted nations (see the World Health Organization, http://www.who.int/tdr/ diseases; and the World Organization for Animal Health, http://www.oie.int/eng/info/en_info.htm). Understanding the complex ecology and biology of arthropod-borne diseases (ABDs) is a major scientific challenge, but it is an essential step in the design of effective control programs. Because immunization is one of the most cost-effective interventions in public health [1], considerable effort and resources have been directed towards the development of vaccines that target the etiological agents of these diseases. So far the results have been disappointing. After decades of research, we still lack effective vaccines against some of the most important ABDs such as malaria, leishmaniasis, trypanosomiasis, filariasis and several diseases caused by tick- and mosquito-borne viruses. The vectors that transmit these diseases have now become the targets of vaccine in an effort to decrease pathogen transmission. Most attempts have focused on the induction of speciesspecific immunity, targeting proteins of the saliva and/or midgut of arthropods [2,3]. Proteins secreted into these compartments move through the Golgi system of epithelial cells, where they can be covalently modified by arthropod glycans [4]. Because arthropod glycans have not been subject to the striking diversification seen in vertebrates [5–8], they represent potential targets of Corresponding author: Mejia, J.S. ([email protected]). Available online 19 June 2006
pan-arthropod vaccines. Here we discuss this possibility and the ideal design for a vaccine required to induce safe and long-lasting pan-arthropod immunity. Arthropod hematophagy The ability to obtain and digest a blood meal from a vertebrate was a key event in the evolution of ABDs, because the reproductive efficiency of arthropods increased with this new source of nutrients. In the process, they exposed themselves to infectious agents carried by the vertebrate host – the first step leading to vector competence for the transmission of pathogens. Adaptation to hematophagy is a very complex process that has demanded several anatomical, physiological and immunological changes in hematophagous arthropods [9]. The most significant alterations occurred in (i) the mouthparts to facilitate access to blood; (ii) the salivary glands to produce molecules capable of blocking vertebrate hemostasis; and (iii) the midgut to neutralize immune injury mediated by vertebrate blood and to optimize digestion and absorption of blood components [9]. The salivary glands and midgut have attracted the attention of several groups of researchers because they are crucial determinants of vector competence and offer several targets for anti-vector vaccines [2,3]. Furthermore, they have attracted the interest of the pharmacological sector because of the extraordinary diversity of arthropod salivary molecules, which have potent effects on the vascular, neural, hemostatic and immune systems of vertebrates [10–12]. Arthropod saliva In an extraordinary example of convergent evolution, each species of hematophagous arthropod reached a particular solution to the problem of obtaining a blood meal from the host via the expression, in their saliva, of a complex and redundant mixture of inhibitors of vasoconstriction, coagulation and/or platelet aggregation [10–15]. This anti-hemostatic cocktail facilitates the flow of vertebrate blood into the arthropod midgut, but it can also have an indirect anti-inflammatory and immunomodulatory effect owing to attenuation of the defense-activation signal mediated by thrombin and other activated enzymes of the coagulation cascade [16]. This effect is further amplified by various immunomodulators that are present in arthropod saliva [17,18].
www.sciencedirect.com 1471-4922/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2006.06.001
368
Opinion
TRENDS in Parasitology
The overall anti-hemostatic and immunomodulatory effect of arthropod saliva provides a clear evolutionary advantage, not only to the arthropod but also to microorganisms delivered along with the saliva. This phenomenon of potentiation of microbial transmission was initially documented in an experimental model of the transmission of Leishmania parasites by sandflies [19], and has been subsequently reported in other transmission models of ABDs [20–23]. Targeting salivary components provides two potential mechanisms to block the transmission of pathogens by arthropods: neutralization of the anti-hemostatic and immunomodulatory effect of arthropod saliva; and generation of a local environment leading to pathogen containment and destruction. Furthermore, given the ability of proteins to adsorb to surfaces [24], an immune response directed to salivary proteins that adsorb to pathogens can turn the microorganism into an innocent bystander of anti-salivary immunity. This pathogen-adsorption ability has been recently reported in a salivary protein (Salp15) from the hard tick Ixodes scapularis [25], making it a vaccine candidate for the control of Lyme disease. The significance of anti-salivary immunity in the epidemiology of ABDs is beginning to be explored [26]. It has been suggested that repetitive exposure to sandfly saliva explains, along with concomitant immunity, the natural resistance to Leishmania parasites that inhabitants of endemic areas develop over many years [26]. This suggestion is supported by experimental evidence indicating that exposure to the bite of non-infected arthropods protects against an infectious challenge [27,28]. Because it is not feasible to immunize individuals with arthropod saliva or dissected salivary glands, much effort has been directed towards the identification of potential vaccine candidates from the salivary gland transcriptome and proteome of selected vector species of ABDs affecting humans and other animal species [29–31]. Arthropod midgut The digestion of a blood meal in an arthropod midgut is a complex process that requires the coordinated expression of many genes involved in nutrient processing and adsorption, detoxification mechanisms and defense against the pathogens that arrive in the vertebrate blood meal [32,33]. Furthermore, several mechanisms to deactivate the innate and adaptative immune systems of the vertebrate blood must operate to protect the midgut epithelia from immune-mediated injury. The best-known example of this protection mechanism is the formation of a peritrophic membrane around the blood meal in hematophagous insects [34]. Additional mechanisms that might be significant are proteolytic inactivation caused by arthropod digestive enzymes and temperature-dependent immune attenuation caused by the decrease in blood temperature that occurs once the arthropod detaches from the host. Vaccinating against midgut components of some hematophagous arthropods has been shown to decrease vector survival, fecundity and/or ability to transmit a microorganism [35,36]. This approach has proved to be effective in controlling the cattle tick Boophilus microplus [3]. The ongoing characterization of the transcriptome and www.sciencedirect.com
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proteome of vectors of ABDs is likely to yield several candidates for this anti-midgut vaccine approach [31,37,38]. Selecting epitopes for a vaccine Protein epitopes It has become apparent, from tests of protein components of arthropod saliva as vaccine candidates [39,40], that antigenic polymorphism might be a problem in the design of anti-salivary vaccines. In one of the few arthropod molecules in which sequence polymorphism has been studied in detail – namely, the pituitary adenylatecyclase-activating polypeptide (PACAP) receptor agonist (maxadilan) of Lutzomyia longipalpis – the extent of polymorphism between individual flies collected from different geographical locations is very significant [41]. This polymorphism represents a mechanism that improves the fitness of the vector under the selective pressure of an anti-maxadilan immune response [42]. Genomic sequence variation is a crucial event in the adaptation process of genomes [43] and, because most salivary proteins are subject to the selective pressure of the vertebrate immune system, it can be predicted they should be highly polymorphic and encoded by rapidly evolving genes. The high frequency of gene duplications in the transcriptome of salivary glands of some species of hematophagous arthropods [29,30] supports this idea. Cross-reactive epitopes Although cross-reactive antigenicity among arthropods has not been studied systematically, it has been detected after natural or experimental exposure to arthropod saliva or midgut components and has been explained by similarities in proteins or glycans [44–46]. In addition to this ‘intra-arthropod’ antigenic cross-reactivity, some of the glycans expressed in arthropods have been found to be expressed in other microorganisms, plants and mammalian cells [47]. Carbohydrate epitopes In comparison with those of vertebrates, the N-linked and O-linked glycans of arthropods seem to have stalled at an intermediate evolutionary step, such that most of the N-linked glycans have a paucimannosidic structure and the O-linked glycans are limited to the core glycan structures known as Tn and T antigens (GalNAc-Ser/Thr and Galb1-3GalNAc-Ser/Thr, respectively) [5–7]. The structure and immunogenicity of N-linked glycans of arthropods have been studied extensively in lepidopteran insect cells because the biotechnology industry relies on them for the production of various proteins that require glycosylation for appropriate folding and optimal pharmacological properties [5]. The high immunogenicity of these glycans and their limited evolutionary diversification place them as key candidates for a vaccine capable of inducing immunity to salivary and midgut glycoproteins of all species of hematophagous arthropod. This approach has been suggested as a way to develop vaccines to control ABDs [48]. Much less information is available on arthropod O-linked glycans [6,7], and their potential as targets for
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anti-vector immunity has not been explored. Interestingly, the only O-linked glycans so far described in arthropods, the Tn and T antigens, have been extensively studied as pan-carcinoma antigens [49]. The expression of these antigens in both arthropods and mammalian tumor cells represents an interesting link between anti-arthropod and anti-cancer immunity, and offers an opportunity to apply advances derived from attempts to generate anti-cancer immunity by targeting O-linked glycans [50] to the design of anti-vector vaccines. Vaccine design The presence in arthropod saliva of several molecules that mediate anti-hemostatic and immunomodulatory effects represents functional redundancy akin to that of snake venom [51]. To neutralize the biological activity of snake venom, it is necessary to use antisera prepared against whole venom [52]. A similar approach might be required to neutralize the biological activity of arthropod saliva. Because it is impractical to immunize individuals or animals with whole saliva derived from hematophagous arthropods, however, other alternatives must be considered. Cell lines derived from the salivary gland of arthropods would be the ideal immunogen, providing broad immunity with relevant epitopes in the context of xenoreactivity. Arthropod cell lines derived from salivary glands are not available, but cell lines derived from embryos or ovaries could be used as platforms for the synthesis of salivary and/or midgut glycoproteins of hematophagous arthropods. In fact, Drosophila melanogaster cells have been modified to improve their capacity to present antigens to the vertebrate immune system [53]. This approach could be useful in the design of anti-vector vaccines because the modified Drosophila cell lines express the required N- and O-linked glycans. Furthermore, to optimize immunogenicity and to minimize adverse side-effects, the cells could be transformed to express glycoproteins with the appropriate structure and clustering of glycans. Safety concerns In healthy populations of vertebrates, the cores of the Nand O-linked glycans are extensively modified and not accessible to antibodies directed to arthropod-derived glycans. Because alteration of N- and O-linked glycans in vertebrates is linked to several autoimmune diseases [54], however, a chief concern is that immunization with arthropod-derived glycans might amplify the ongoing immunopathology in individuals with autoimmune disease. Another concern – namely, the induction of allergic response to arthropod glycans – can be minimized by removing the a1–3 fucosylated N-linked glycans that have been identified as the main epitopes involved in allergic responses to insect glycoproteins [55]. These concerns must be addressed in experimental models before any attempt is made to use glycan-based panarthropod vaccines. Concluding remarks Despite big gaps in our understanding of arthropod hematophagy, great strides have been made in this field www.sciencedirect.com
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through functional genomics analysis of the salivary glands and midgut of hematophagous arthropods. Nevertheless, much remains to be learned about the extent of functional redundancy in these compartments and how it can be affected by antibodies directed to crossreactive epitopes. Because N- and O-linked glycans are among the most promising candidates for pan-arthropod vaccines, there is an urgent need to characterize these structures in arthropods, especially those arthropods that are vectors of ABDs. Selected glycans with the appropriate structure and clustering can be expressed in insect cells that have been modified to improve their capacity to present antigens to the immune system of vertebrates. One of the advantages of an approach based on modified insect cells is that the immunity induced can be boosted by natural exposure to the saliva of any hematophagous arthropod, regardless of species. A vaccine with such characteristics would be a considerable weapon in the arsenal to control ABDs worldwide.
Acknowledgements We are grateful to Stephen Wikel, Jesus Valenzuela, Stephen Higgs, Barbara Drolet, Keith Nelson and Tereza Magalhaes for critically reviewing the manuscript; and thank Carolina Barillas-Mury, Diana Sierra, Tess Brodie and Claudia Bernal for helpful suggestions. This work was supported by grants from the National Institutes of Health (RO1 534411 and RCE 534724).
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