Vector-transmitted disease vaccines: targeting salivary proteins in transmission (SPIT)

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Opinion

Vector-transmitted disease vaccines: targeting salivary proteins in transmission (SPIT) Mary Ann McDowell Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA

More than half the population of the world is at risk for morbidity and mortality from vector-transmitted diseases, and emerging vector-transmitted infections are threatening new populations. Rising insecticide resistance and lack of efficacious vaccines highlight the need for novel control measures. One such approach is targeting the vector–host interface by incorporating vector salivary proteins in anti-pathogen vaccines. Debate remains about whether vector saliva exposure exacerbates or protects against more severe clinical manifestations, induces immunity through natural exposure or extends to all vector species and associated pathogens. Nevertheless, exploiting this unique biology holds promise as a viable strategy for the development of vaccines against vector-transmitted diseases. Vector-borne disease Blood-feeding arthropods are vectors of some of the most debilitating infections known to humans, including infamous diseases such as malaria and the plague, as well as many neglected tropical diseases like dengue, lymphatic filariasis, Chagas’ disease, and leishmaniasis. Vectorborne infections account for 17% of the global burden of infectious diseases (http://www.who.int/whr/2004/en/), causing >1 million deaths annually [1] and accounting for nearly 1 billion disability-adjusted life years [2]. Global trade, human migration, urbanization, and climate change are increasing the propensity for epidemics and expanding traditional borders for these devastating diseases. The cornerstone of vector-borne disease control has been management of vector populations through pesticides (http:// whqlibdoc.who.int/hq/2006/WHO_CDS_NTD_WHOPES_ GCDPP_2006.1_eng.pdf). Unfortunately, this approach has proven ineffective in eradicating these infections, elevating the urgency for development of innovative interventions [3]. Successful strategies will undoubtedly be combinatorial, using insecticides with novel modes of action together with nontoxic chemotherapies and effective vaccines. Vaccine discovery efforts have accelerated in recent years, however, acceptable protection in human populations has not been achieved for most vector-borne Corresponding author: McDowell, M.A. ([email protected]). Keywords: vector-transmitted disease; saliva; blood-feeding; vaccines. 1471-4922/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2015.04.011

diseases [4]. Targeting vector components, in addition to pathogen antigens (see Glossary), in multi-component vaccines is beginning to emerge as a viable vaccine platform [5]. Hematophagous disease vectors are not only vehicles for the transmission of pathogens, rather they dispense a multitude of compounds in their saliva that facilitate efficient blood feeding [6]. The influence that vector components have on arthropod-transmitted illness has only recently been appreciated [7], and evidence has emerged that immune responses to arthropod saliva can modulate the clinical manifestation or progression of vector-borne disease [8–10]. Arthropod saliva There are 12 000 species of blood-feeding arthropods [11] and it is thought that this trait evolved independently at least 20 times [12]. Fewer than 1000 arthropod species transmit infectious pathogens to humans [13],

Glossary Antigen: any foreign substance that activates an immune response. Delayed-type hypersensitivity (DTH): also known as a type IV hypersensitivity reaction. A hypersensitivity reaction that is delayed, typically taking 2–3 days to develop. Most DTH responses are mediated by CD4+ T helper cells that secrete interferon (IFN)- g, but eosinophils and basophils have been implicated in some instances. Reaction manifests with dermal inflammation resulting in localized edema and erythema. Pathology can last for several weeks. Erythema: redness of the skin. Hematopoiesis: formation of all types of blood cells, including development, differentiation, and maturation of white blood cells. In adults, this process typically occurs in the bone marrow. Hematophagous: the practice of feeding on blood. Hypersensitivity: exaggerated immune responses to innocuous antigens that lead to damaging reactions in individuals upon re-exposure. Immediate hypersensitivity reactions (types I, II, and III) manifest within minutes to hours and are mediated by antibodies. Type IV or delayed-type hypersensitivity reactions typically manifest after several days and are cell mediated. Salivary gland homogenate (SGH): contains lysed salivary glands. The mixture contains membrane proteins as well as secreted salivary proteins. Sialome: the repertoire of salivary molecules. T helper 1 (Th1) immunity: characterized by the secretion of IFN-g by CD4+ T cells. IFN-g is thought to activate macrophages for elimination of intracellular pathogens. Th1 immunity is known to downregulate Th2 responses. T helper 2 (Th2) immunity: characterized by the secretion of interleukin (IL)-4, IL-5, and IL-13 by CD4+ T cells. This response is commonly associated with an enhanced antibody response and extracellular pathogens. Th2 immunity is known to downregulate Th1 responses. Type 1 hypersensitivity: involves IgE antibody triggering of mast cells and is induced by Th2 immune responses. This hypersensitivity is associated with allergy and is the predominant hypersensitivity exhibited upon mosquito bite, giving the typical redness and swelling at the bite site.

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Opinion with mosquitoes being the most devastating by transmitting malaria, several encephalitic agents, lymphatic filariasis, and river blindness. For the five Plasmodium species known to cause human malaria, nearly 60 different anopheline mosquito vectors have been identified [14]. Thirty different sand fly species have been implicated as vectors of leishmaniasis [15] and at least 77 tick species are known to vector pathogens that cause human illness, including viral, bacterial, and protozoan pathogens [16]. Triatomes, lice, tsetse flies, black flies, and fleas also transmit infections to humans (http://apps.who.int/ iris/bitstream/10665/111008/1/WHO_DCO_WHD_2014. 1_eng.pdf?ua=1). In all cases, the vectors modulate human blood clotting and inflammation through secretion of saliva to facilitate blood feeding [17]. These vectors can be divided into transient and long-term feeders. Transient-feeding arthropods, like mosquitoes and sand flies, take minutes to engorge, whereas ticks may feed for an hour (Argasidae) or may stay attached and feeding for more than a week (Ixodidae) [18]. To avoid rejection from the host, the longer attachment of ticks necessitates a different repertoire of salivary molecules compared to transient feeders and requires the modulation of the secreted cocktail during the course of a single feeding [18]. Additional complexity is added by the fact that tick juvenile forms are also hematophagous. Consequently, ticks express >3500 tick-putative salivary proteins compared to the 1280 identified for transient feeders (sand flies, black flies, biting midges, and mosquitoes) [17]. Five hundred putative secreted proteins are expressed by a single tick species (Ixodes scapularis) [19] compared to 49 for a single sand fly (Phlebotomus papatasi) [20] and 55 for a single mosquito (Aedes aegypti) [21]. Furthermore, vector saliva can contain lipids, nucleic acids, and nucleosides [22]. Although in smaller numbers in transient feeders, these numerous salivary molecules provide a rich source of novel antigen candidates for vaccine development. Rationale for targeting vector saliva in vaccines: inhibit feeding, neutralize enhancement or bystander effect Inhibit feeding Many, but not all, salivary molecules are immunogenic and elicit host immune responses after repeated exposure that reduce the feeding efficiency [23] and fecundity of arthropod vectors [24–28], consequently influencing pathogen transmission. The concept of exploiting this natural response through vaccination to block arthropod infestation has been circulating for >75 years [29] (Figure 1A) and anti-tick vaccines targeting a midgut protein are commercially available. This strategy is only a realistic solution for long-term feeders like ticks, where the majority of associated pathogens require >24 h of arthropod attachment for successful transmission [30]. Identification of suitable salivary antigens present at the tick–host interface has accelerated in recent years [31] and success has been demonstrated for at least one cement antigen [32]. Neutralize enhancement In addition to facilitating blood feeding, injection of salivary molecules in the skin modifies the bite site, favoring 2

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the establishment and disease progression of some pathogens [10]. Insect saliva contains a complex array of biologically active molecules; many that have immunosuppressive effects that could, in theory, potentiate pathogen establishment. For example, maxadilan, found only in Lutzomyia sand flies, is a potent vasodilatory polypeptide [33] that mediates the enhancement of Leishmania infection by Lutzomyia saliva [34]. Maxadilan exhibits a range of immunomodulatory activities including inhibition of innate and adaptive immune functions [34–36] and also stimulates host hematopoiesis [37]. Salp15, a salivary protein of Ixodes ticks, is immunosuppressive, impairing dendritic cell function and T cell activation [38] and protects the Lymes’ disease agent Borrelia burgdorferi from complement- and antibody-mediated lysis [39,40]. For a more complete catalogue of arthropod salivary proteins refer to recent reviews [5,22,41]. One logical approach to targeting vector saliva in vaccines would be to elicit antibodies that neutralize the proteins associated with infection enhancement (Figure 1B). Indeed, vaccination with synthetic maxadilan abrogates the enhancement of Leishmania infection by Lutzomyia saliva and elicits anti-maxadilan antibodies in animal models [42]. Although there have been no reports of vaccinating with tick Salp15, knockdown of this protein in I. scapularis through RNAi reduces transmission of B. burgdorferi in mice [40], suggesting that anti-Salp15 neutralizing antibodies might reduce establishment of these spriochetes. Bystander effect Insect bites induce immediate, delayed, and systemic hypersensitivity reactions in hosts [43,44]. Repeated exposure to sand fly bites causes a particularly strong delayed type hypersensitivity (DTH) response in humans [45] and mice [46]. Initiation of this response has been suggested to be an evolutionary advantage for sand flies to increase blood flow at the bite site, therefore decreasing the amount of time it takes for a sand fly to take a full blood meal [47]. Although advantageous for sand fly feeding, the DTH response elicited by repeated exposure to sand fly bites [46] or salivary gland homogenates (SGHs) [48] inhibits Leishmania infection in murine models. It is possible that the immunity elicited to repeated saliva exposure could induce leishmanicidal mechanisms by macrophages at the inoculation site, particularly induction of interferon (IFN)- g by a T helper (Th)1-mediated DTH response [46]. During the establishment of Leishmania infection, saliva-induced immunity would induce resistance to Leishmania parasites as a ‘bystander effect’, through macrophage activation. In addition, IFN-g produced by anti-saliva memory T cells could influence priming of naı¨ve T cells specific for leishmanial antigens (Figure 1C). If a biteexposed individual encountered Leishmania along with saliva (an integral part of natural transmission) the bystander effect would induce Th1 immunity towards Leishmania. A similar Th1-mediated mechanism has been proposed for malaria [49,50]. Although targeting non-pathogen antigens is a novel strategy, evidence is emerging that concomitant infections and exposure history can modulate immune responses to unrelated pathogens [51–55] and may not require direct

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(A) Inhibit feeding Naive

Vaccinated

4 1

physical association between infectious agents [52,55]. A successful bystander effect would require memory T cells specific for salivary antigens induced by vaccination to be elicited to the infectious bite site, a behavior that occurs in animal models in response to multiple sand fly bites [41]. If this recruitment occurs naturally in response to fewer bites remains to be determined.

3 Th2 Th2 Th2

2

(B) Neutralize enhancement Naive

Vaccinated

4 1 3 Th2 Th2 Th2

Th2 Th2 Th2

2

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(C) Bystander effect Naive

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1

IFN-γ IFN-γ IFN-γ IFN-γ IFN-γ IFN-γ IFN-γ IFN-γ

IFN-γ

3 Th2 Th2 Th2

Th1 Th1 Th1

Th1

Th1

Th1

5

2

Key: Virus

Salivary protein

Plasma B-cell

Bacteria

Macrophage

T-helper cell

Protozoan

Dendric cell

Memory T-cell TRENDS in Parasitology

Figure 1. Proposed mechanisms of saliva-vaccine-induced immunity. (A) Neutralization of feeding. (1) Without vaccination, arthropod releases salivary proteins and pathogens into the bite site where DCs take up saliva and pathogen Ags. (2) DCs migrate to the draining LN, present Ag to naı¨ve T cells, generally inducing a Th2 response. (3) Vaccination with salivary Ag induces memory T cells that activate B cells to mature into antibody-secreting plasma cells. (4) In vaccinated individuals, release of salivary proteins by arthropods results in rapid activation and recruitment of memory T cells and plasma B cells to the bite site. Antibody release at the bite site neutralizes salivary proteins necessary for attachment and feeding, resulting in arthropod detachment. (B) Neutralization enhancement. (1) Without vaccination, arthropods release salivary proteins and pathogens into the bite site where DCs take up saliva and pathogen Ag. (2) DCs migrate to the LNs, present Ag to naı¨ve T cells, inducing naı¨ve T cells to release cytokines, generally Th2, that lead to enhancement of infection. (3) Vaccination with salivary Ag induces memory T cells that activate B cells to mature into antibody-secreting plasma cells. (4) In vaccinated individuals, release of salivary proteins by arthropods results in rapid activation and recruitment of memory T cells and plasma B cells to the bite site. Antibody release at the bite site neutralizes salivary proteins that induce the enhancement response. (5) These Ags are not

Controversy: nature versus nurture It has been suggested that, for some vector transmitted diseases, long-time inhabitants of endemic areas carry attenuated infections and are less likely to exhibit severe clinical disease compared to individuals new to an endemic area (e.g., travelers or children) [56,57]. This phenomenon has been attributed to a gradual onset of anti-pathogen immunity in endemic individuals [57,58]; however, more recently recognition of a role for vector saliva in this process has emerged. Plasmodium infection rates of their mosquito vectors range from < 0.1% to 10% in endemic regions [59,60] and the prevalence of Leishmania-infected sand flies in the field also is low [61–64]. Strikingly, individuals can receive 200 mosquito bites/day [65] and >1000 sand fly bites in a single night [64]. Host responses to saliva may create an inhospitable environment for pathogen establishment and growth. That people in endemic regions succumb to vectortransmitted disease despite the high frequency of uninfected bites, however, argues that the protection induced by saliva exposure might be minor or absent in natural settings or even an artifact of experimental models. Exacerbation of experimental leishmaniasis due to saliva exposure from wild-caught sand flies appears less pronounced compared to saliva from colonized flies [66,67], and protection due to pre-exposure is abrogated [68]. This difference is possibly due to genetic heterogeneity of salivary proteins [69] or different age distributions between sand fly populations as senescence has been shown to influence salivary gene expression [70,71]. Moreover, different microbiomes in these vector populations could influence the induced host immune response [72]. It has been suggested that the saliva exposure schemes employed in experimental models do not mimic the continuous exposure of individuals living in endemic regions [73], possibly influencing study results. Furthermore, the presence of extraneous factors in natural settings such as concomitant infections, infection history, and nutritional status that have not been incorporated in experimental models, could explain different outcomes in natural and experimental systems.

presented to T cells in the LNs, thereby no enhancement response is produced. (C) Bystander effect. (1) Without vaccination, arthropods release salivary proteins and pathogens into the bite site where DCs take up saliva and pathogen Ag. (2) DCs migrate to the LNs, present Ag to naı¨ve T cells, inducing naı¨ve T cells to release cytokines, generally Th2, that lead to enhancement of infection. (3) Vaccination with salivary Ag induces memory T cells that produce Th1 cytokines (IFN-g). 4) In vaccinated individuals, release of salivary proteins by arthropods results in rapid activation and recruitment of saliva-specific memory Th1 cells. IFN-g at the bite site activates infected macrophages to induce killing mechanisms for intracellular pathogens. (5) Memory saliva-specific Th1 cells in the LNs produce IFN-g that influences differentiation of naı¨ve pathogen-specific T cells towards a Th1 phenotype. Abbreviations: Ag, antigen; DC, dendritic cell; IFN, interferon; LN, lymph node; Th, T helper.

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Opinion Experimental studies can be confounded by the use of salivary gland homogenates (SGH) for saliva exposure rather than bites, as it has been reported that injections of SGH may not elicit the same immune response as exposure to insect bites [49,74]. Most salivary proteins are secreted in sub-nanogram amounts [41], some require activation during feeding [75–77] and other proteins present in SGH are not deposited into a host [74], so the large amounts of SGH utilized in experimental systems could explain differences from natural exposure. Nonetheless, some studies have reproduced SGH results via bites in animal models [41,49] and in human volunteers [78], suggesting that SGH can be used as a proxy for bites in some cases. Only a handful of studies have assessed human immunity to natural arthropod bite exposure and these reports do not coalesce into a model for either disease protection or immune response. Some studies suggest that saliva exposure induces T helper (Th)2-type immune responses [79– 82] and other studies indicate a more mixed response in the population [50,83,84] or even within individuals [78,83– 85]. Not surprisingly, exposure to mosquito [86–88] or sand fly [89] bites, as assessed by serum antibody levels, correlates with active disease; however, increased exposure may influence severity of disease [89], at least for cutaneous leishmaniasis in some areas. Clearly, additional studies in human populations will be required before anti-salivary vaccines can be realized. Although targeting vector saliva as a component of antivector-borne pathogen vaccines is an attractive idea, the general utility of saliva-based vaccines remains controversial and requires further exploration. Even for the most well-studied vector–pathogen pair, Leishmania and sand flies, studies are relatively scarce and involve only a few research groups [90]. Natural exposure to vector bites may or may not influence resistance to the pathogens they transmit in endemic settings. However, because saliva exposure always occurs during the natural transmission process, there is motivation to capitalize on this distinctive aspect of vector-borne infection. Public health vaccines need not provide sterile immunity to all individuals, even semi-immunity can decrease the disease burden [91]. Pan-vector vaccine: panacea or pipedream? Given that vertebrate hemostasis and inflammation are highly complex, it is no surprise that hematophagous arthropods have adapted many strategies to overcome these obstacles. The diversity of such strategies makes it difficult to identify one salivary antigen that is adequately conserved across vector species that can be elaborated into a pan anti-vector vaccine. Even development of diseasespecific anti-saliva vaccines remains challenging due to complex disease pathogenesis, the host specificity of immune responses, evolutionary pressures on salivary proteins and environmental modulation of saliva expression. Diversity of vector–pathogen pairs The potentiation of disease by saliva extends to many vector–pathogen pairs (Table 1), that include sand flies, mosquitoes, tsetse flies, and kissing bugs with a variety of different pathogen types. Moreover, infection enhancement 4

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and increased pathogenesis has been demonstrated for several sand fly–Leishmania pairs [90]. Yet, in other species combinations, sand fly saliva has no enhancement effect [92–94] or inhibits disease [95]. Even less uniformity is observed for saliva-induced protection of disease (Table 2). The protective effect due to sand fly saliva exposure is well established for many, but not all, experimental leishmaniasis models [90], and has been demonstrated for other vector–pathogen pairs [24,49,96–99]. For other combinations, repeated exposure to saliva has no effect [100] or enhances infection [101–105]. Host specificity Murine models historically have been the mainstay of vaccine research [106]. However, the extent to which such models accurately represent pathology and identify protective antigens for human disease is not clear [106,107]. While murine models have been used to validate the influence of arthropod saliva in the modulation of vectortransmitted disease [5], these experimental models have not identified salivary proteins that are immunogenic or protective across host species [41]. For example, DNA vaccination with plasmids encoding salivary antigens from Lutzomyia longipalpis elicited different immune responses in hamsters and mice [92]. Furthermore, one of these antigens, LJM19, provides protection against Leishmania infantum chagasi [92] and Leishmania braziliensis [108] in hamsters, but does not elicit immunity in dogs [109]. Immunogenicity of some salivary proteins clearly differs from host to host and will require testing in the target host before a vaccine is formulated, an approach required for most vaccines. Vector diversity Every hematophagous arthropod studied to date contains at least one anti-clotting, one anti-platelet, and one vasodilatory molecule, however, the diversity of mechanisms vectors display to achieve these functions is enormous [22]. Even among a single vector type, the different salivary cocktails contain divergent molecules. Salivary glands of phlebotomine sand flies, for instance, contain a complex array of biologically active molecules that diverge widely among sand fly species. For example, maxadilan is present only in the new world sand flies (Lutzomyia spp.); the old word sand flies, P. papatasi (76) and Phlebotomus argentipes (77), secrete adenosine for vasodilation instead. Furthermore, sand fly species of the same genus can differ in salivary antigens [110]. Pre-exposure of mice to P. papatasi SGH [48] or uninfected bites [46] protects against Leishmania major infection. DNA vaccination with a gene encoding a P. papatasi salivary protein, PpSP15, recapitulates the protective response [111] and induces a Th1-type DTH response [112]. This response seems to be limited to P. papatasi, as the PpSP15 homolog in Phlebotomus ariasi, a vector of L. infantum, does not induce a significant DTH response in mice [113]. Assessment of other vectors of L. major is of paramount importance. Recent evidence is promising, indicating that protection induced by P. papatasi bites can induce protection against L. major challenged with Phlebotomus duboscqi SGH in a murine model [114]. While not

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Table 1. Studies assessing saliva-induced potentiation of the disease Vector Mosquitoes Anopheles stephensi

Aedes triseriatus

Aedes aegypti

Culex pipiens Culex tarsalis Tsetse flies Glossina morsitans morsitans Kissing bugs Rhodnius prolixus Sand flies Lutzomyia longipalpis

Lutzomyia flaviscutellata Lutzomyia complexus Phlebotomus papatasi Fleas Xenopsylla cheopis

Pathogen

Response

Refs

Plasmodium berghei Plasmodium berghei Plasmodium yoelii Cache Valley virus La Cross virus Vesicular Stomatitis virus Cache Valley virus Sindbis virus Dengue virus Rift Valley Fever virus Cache Valley virus West Nile virus

Enhancement No Effect No Effect Enhancement Enhancement Enhancement Enhancement Enhancement Enhancement Enhancement Enhancement Enhancement

[125] [100] [100] [126] [127] [128] [126] [127] [129] [130] [126] [131]

Trypanosoma brucei brucei

Enhancement

[103,132]

Trypanosoma cruzi

Enhancement

[133]

Leishmania infantum chagasi Leishmania infantum chagasi Leishmania amazonensis

Enhancement No Effect Enhancement

Leishmania Leishmania Leishmania Leishmania Leishmania Leishmania Leishmania

Enhancement Enhancement Inhibition Inhibition Inhibition Inhibition Enhancement

[134] [92,93] [42,66,67,95,105, 135,136] [137–140] [42,136,141,142] [95] [95] [95] [95] [48,136,143]

No Effect

[94]

braziliensis major amazonensis braziliensis amazonensis braziliensis major

Yersinia pestis

a factor when pre-exposing by bite or with SGH, DNA vaccination with a different P. papatasi salivary protein, PpSP44, results in exacerbation of L. major lesions [112], highlighting the importance of choosing vaccine antigens

carefully. Formulation of these antigens with the correct adjuvant can induce a protective response as has been demonstrated for mosquito saliva and West Nile virus infection [115].

Table 2. Studies assessing saliva-induced protection Vector Mosquitoes Anopheles stephensi Unreported mosquito species Anopheles fluviatilis Aedes aegypti Culex tarsalis Tsetse flies Glossina morsitans morsitans Sand flies Lutzomyia longipalpis Lutzomyia intermedia Phlebotomus papatasi Phlebotomus duboscqi Ticks Dermacentor andersoni Ixodes scapularis Borrelia burgdorferi

Pathogen

Response

Refs

Plasmodium yoelii Plasmodium berghei Plasmodium berghei Plasmodium gallinaceum West Nile virus West Nile virus

Protection No Effect Protection Protection Enhancement Enhancement

[49] [100] [98,144] [99] [101] [102]

Trypanosoma brucei brucei

Enhancement

[103]

Leishmania Leishmania Leishmania Leishmania Leishmania

Enhancement Protection Enhancement Protection Protection

[105] [145,146] [104] [46,48,69,114] [73]

Protection Protection Protection

[96] [97] [147]

amazonensis major braziliensis major major

Francisella tularensis Borrelia burgdorferi Borrelia burgdorferi

5

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Opinion Antigen polymorphism Not all vectors are entirely anthropophilic and some feed on a variety of vertebrate hosts. In addition, many insects feed on plants as a source of sugar. The availability of diverse sugar sources could exert selective pressure on salivary proteins of distinct vector populations. Therefore, elucidation of salivary gland protein polymorphisms across populations of a single vector species will be essential prior to incorporation into a vaccine. For example, maxadilan differs genetically by as much as 23% from different sibling species of the Lu. complex [116]. This genetic variability has no effect on their vasodilatory activity [116]; however, bites from sibling species produce different sized erythemas at the bite site and it has been postulated that differences in maxadilan expression contribute to these distinct responses and also to the atypical cutaneous leishmaniasis present in some areas [117]. In spite of this diversity, some salivary proteins have been identified as conserved across species [41], or exhibiting low diversity in field populations [118,119]. In addition to genetic diversity between populations, expression level differences of targeted salivary proteins influenced by age, diet, or climate might impact the effectiveness of an anti-saliva vaccine. Indeed, expression level polymorphisms have been reported in geographically separated P. papatasi populations [120] and are influenced by age and diet [70], highlighting the importance of selecting vaccine antigens that are conserved and expressed in all populations. The lack of a unifying theme for saliva exposure is not surprising given the enormous diversity of salivary proteins, arthropod and pathogen and life-cycles, and the varied pathogenesis of each disease. The concept of a pan-arthropod vaccine is attractive but the heterogeneous nature of vector–pathogen systems makes it unlikely, much as development of a single vaccine against all infectious diseases is implausible. Nonetheless, custom-made vaccines targeting specific vector–pathogen pairs may be justified in some situations or for certain geographical regions (e.g., deadly leishmaniasis on the Indian subcontinent). Obstacles to salivary proteins in transmission (SPIT) vaccine development Because arthropod saliva is intrinsic in the transmission of these infections, we cannot afford to overlook the potential to exploit this unique aspect of the pathogen biology. Incorporation of vector salivary components into multicomponent vaccines, however, could skew immune responses in such a fashion to increase protection. Vaccines containing saliva and pathogen antigens are showing promise [121,122], but require understanding of issues regarding tolerance associated with aging populations, potential cross-reactivity, and adverse reactions before these vaccines will be realized. Tolerance Long-term exposure to insect bites gradually results in desensitization such that eventually a cutaneous reaction is not elicited in humans [43,123]. This waning reactivity to saliva could render an anti-saliva vaccine ineffective 6

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as the natural boosting effect afforded by insect bites could lead to tolerance. The mechanisms that mediate tolerance to arthropod bites are not clearly understood and likely differ for each vector. Desensitization is best studied for mosquito bites where the predominant initial hypersensitivity reactions are Th2 mediated [43]. It is likely that desensitization to mosquito bites involves a reduction in Th2 cytokines, thus promoting a switch to a Th1 environment known to be protective to many mosquito-transmitted pathogens. An encouraging recent study concerning sand flies showed that the anti-saliva DTH response associated with resistance to L. major infection is maintained through childhood [83], when vaccines may have the most effect at reducing mortality and morbidity. Crossreactivity Although the sialomes of hematophagous arthropods are diverse, some ubiquitous protein families are present and a few conserved antigens have been identified [17,18]. Vaccination with a conserved salivary protein could have unintended consequences if antigenic crossreactivity is present in different blood-feeding species, causing allergic reactions or possible increased susceptibility to other vector-transmitted infections. Indeed, some reactivity to sand fly [78,82] and mosquito [124] salivary proteins has been observed in unexposed individuals, suggesting immunoreactivity to conserved, ubiquitous antigens. Adverse reactions People sometimes exhibit severe hypersensitivity to insect bites [44]; for example, repeated exposure to sand fly bites causes a DTH response known by local inhabitants as a skin disease called ‘harara’ [45]. While the boosting effect of natural exposure could be an advantage, the possibility of adverse reactions occurring every time an individual is bitten could hamper the approval of such a vaccine. Identification of antigens that induce immunity in the absence of pathology is a priority. At least for sand flies, such antigens have been identified in hamsters [92] and dogs [109]. In addition to eliciting adverse hypersensitivity reactions, arthropod salivary proteins themselves can affect host physiology, including vasodilation, blood blotting, and inflammation. Careful consideration will be required in developing vaccination formulation if these pharmacologically active compounds are utilized. Concluding remarks Controlling vector-transmitted infections would greatly enhance the human condition. Achieving this goal will require a broad strategy, exploring novel approaches and leaving no stone unturned. Peculiar only to vectortransmitted diseases is the integral role of arthropod saliva in the infection process. Exploiting this unique aspect of the biology may be the key to anti-vector-transmitted disease vaccine development. Although many obstacles remain for developing vaccines that incorporate vector salivary proteins (Box 1), the potential of this approach should not be disregarded as a possible addition to the antipathogen arsenal.

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Opinion Box 1. Outstanding questions  What are the human immune responses to vector salivary proteins through natural and vaccine-induced exposure? Responses of endemic, or naturally exposed, individuals as well as naı¨ve subjects should be explored.  Will salivary-based vaccines be ineffective in older individuals that have been exposed to a lifetime of arthropod bites?  Can protective salivary antigens be identified that do not elicit adverse reactions upon natural exposure?

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