The immunomodulatory factors of arthropod saliva and the potential for these factors to serve as vaccine targets to prevent pathogen transmission.

In general, attempts to develop vaccines for pathogens transmitted by arthropods have met with little or no success. It has been widely observed that the saliva of arthropods that transmit disease enhances the infectivity of pathogens the arthropod transmits to the vertebrate host. Indeed, it has been observed that vaccinating against components of the saliva of arthropods or against antigens expressed in the gut of arthropods can protect the host from infection and decrease the viability of the arthropod. These results suggest that multi-subunit vaccines that target the pathogen itself as well as arthropod salivary gland components and arthropod gut antigens may be the most effective at controlling arthropod-borne pathogens as these vaccines would target several facets of the lifecycle of the pathogen. This review covers known immunomodulators in arthropod salivary glands, instances when arthropod saliva has been shown to enhance infection and a limited number of examples of antiarthropod vaccines, with emphasis on three arthropods: sandflies, mosquitoes and hard ticks.

Infection with Leishmania major stimulates haematopoiesis in susceptible BALB/c mice and suppresses haematopoiesis in resistant CBA mice.

Cytokine responses to Leishmania infection begin very early in infection, and differ between susceptible and resistant mice. Susceptibility to chronic Leishmania infection has been associated with increased haematopoiesis. To analyse the effect that acute infection with L. major has on bone-marrow haematopoiesis in susceptible (BALB/c) and resistant (CBA) mice, we enumerated erythroid progenitors and granulocyte-monocyte progenitors 3 days after infection. We found that haematopoiesis was stimulated in BALB/c mice infected with L. major, while haematopoiesis was inhibited in CBA mice. We found that this effect could be partially explained by cytokine production: interleukin-4 was involved in stimulation of BALB/c haematopoiesis and tumour necrosis factor-alpha was involved in inhibition of CBA haematopoiesis. Our conclusions are that haematopoietic changes occur shortly after L. major infection, and may be related to disease outcome.

Sandfly maxadilan exacerbates infection with Leishmania major and vaccinating against it protects against L. major infection.

Bloodfeeding arthropods transmit many of the world's most serious infectious diseases. Leishmania are transmitted to their mammalian hosts when an infected sandfly probes in the skin for a bloodmeal and injects the parasite mixed with its saliva. Arthropod saliva contains molecules that affect blood flow and modulate the immune response of the host. Indeed, sandfly saliva markedly enhances the infectivity of L. major for its host. If the salivary molecule(s) responsible for this phenomenon was identified, it might be possible to vaccinate the host against this molecule and thereby protect the host against infection with Leishmania. Such an approach represents a novel means of controlling arthropod-borne disease transmission. Here, we report that a single molecule, maxadilan, in sandfly saliva can exacerbate infection with L. major to the same degree as whole saliva, and that vaccinating against maxadilan protects mice against infection with L. major.

Leishmania major induces differential expression of costimulatory molecules on mouse epidermal cells.

Levels of expression of costimulatory molecules have been proposed to influence the outcome of antigen-specific T cell priming. We found that Leishmania major selectively modulated the expression of costimulatory molecules on various populations of epidermal cells. B7.2 expression was down-regulated on Thy1.2+ epidermal cells (keratinocytes) from disease-resistant C3H mice, but not from disease-susceptible BALB/c mice. In addition, epidermal cells from BALB/c mice showed a down-regulation of B7.1 expression on NLDC 145+ Langerhans cells. In vitro T cell priming experiments, using syngeneic epidermal cells as antigen-presenting cells (APC), showed that the production of IFN-gamma was inhibited when either B7.1 or B7.2 signaling pathways were blocked. Blockade of B7.2, but not B7.1, significantly inhibited the ability of epidermal cells to induce IL-4 production from CD4+ T cells. In addition, C3H CD4+ T cells, which were unable to secrete detectable levels of IL-4 in cultures with syngeneic APC, were now able to secrete IL-4 following presentation of L. major antigens by congenic BALB/K epidermal cells. Conversely, C3H epidermal cells supported the priming of BALB/K CD4+ T cells for IL-4 production in vitro. Thus, the differential expression of B7 molecules on epidermal cells may not represent the sole factor governing the polarization of L. major-specific CD4+ T cells in vitro.

Interleukin-6 deficiency influences cytokine expression in susceptible BALB mice infected with Leishmania major but does not alter the outcome of disease.

Since interleukin-6 (IL-6) may promote Th2 responses, we infected BALB IL-6-deficient (IL-6(-/-)) mice with Leishmania major. There was not a significant difference between the courses of infection (lesion size and parasite burden) in IL-6(-/-) and wild-type mice, but IL-6(-/-) mice expressed lower levels of Th2- and Th1-associated cytokines.

Regulation of differentiation to the infective stage of the protozoan parasite Leishmania major by tetrahydrobiopterin.

A critical step in the infectious cycle of Leishmania is the differentiation of parasites within the sand fly vector to the highly infective metacyclic promastigote stage. Here, we establish tetrahydrobiopterin (H4B) levels as an important factor controlling the extent of metacyclogenesis. H4B levels decline substantially during normal development, and genetic or nutritional manipulations showed that low H4B caused elevated metacyclogenesis. Mutants lacking pteridine reductase 1 (PTR1) had low levels of H4B, remained infectious to mice, and induced larger cutaneous lesions (hypervirulence). Thus, the control of pteridine metabolism has relevance to the mechanism of Leishmania differentiation and the limitation of virulence during evolution.

Major histocompatibility complex class II-independent generation of neutralizing antibodies against T-cell-dependent Borrelia burgdorferi antigens presented by dendritic cells: regulation by NK and gammadelta T cells.

We previously showed that adoptive transfer of Borrelia burgdorferi-pulsed dendritic cells (DCs) into syngeneic mice protects animals from challenge with tick-transmitted spirochetes. Here, we demonstrate that the protective immune response is antibody (Ab) dependent and does not require the presence of major histocompatibility complex (MHC) class II molecules on DCs. Mice sensitized with B. burgdorferi-pulsed MHC class II-deficient (MHC class II(-/-)) DCs mounted a humoral response against protective antigens, including B. burgdorferi outer surface protein A (OspA) and OspC. B-cell help for the generation of neutralizing anti-OspC immunoglobulin G Abs could be provided by gammadelta T cells. In contrast, anti-OspA Ab production required the presence of alphabeta T cells, although this pathway could be independent of MHC class II molecules on antigen-presenting cells. Moreover, depletion of NK cells prior to transfer of antigen-pulsed MHC class II(-/-) DCs resulted in significant increases in the levels of neutralizing Abs induced by DCs. Altogether, these data suggest that the initial interactions between DCs and innate immune cells, such as gammadelta and NK cells, can influence the generation of a protective humoral response against B. burgdorferi antigens.

Identification of an IL-2 binding protein in the saliva of the Lyme disease vector tick, Ixodes scapularis.

A potent inhibitor of mitogen-stimulated T cell proliferation exists in the saliva of several species of hard ticks, including the Lyme disease vector tick, Ixodes scapularis. Our characterization of this phenomenon has led to the identification of a possible mechanism for the T cell inhibitory activity of I. scapularis saliva. The T cell inhibitor can overcome stimulation of mouse spleen cells with anti-CD3 mAb; however, a direct and avid interaction with T cells does not appear to be necessary. Tick saliva inhibits a mouse IL-2 capture ELISA, suggesting that a soluble IL-2 binding factor is present in the saliva. This hypothesis was verified by using a direct binding assay in which plate-immobilized tick saliva was shown to bind both mouse and human IL-2. Elimination of the IL-2 binding capacity of saliva in the in vitro assays by trypsin digestion demonstrated that the IL-2 binding factor is a protein. These experiments comprise the first demonstration of the existence of such a secreted IL-2 binding protein from any parasite or pathogen. This arthropod salivary IL-2 binding capacity provides a simple mechanism for the suppression of T cell proliferation as well as for the activity of other immune effector cells that are responsive to IL-2 stimulation. Relevance of the tick T cell inhibitory activity to the human immune system is demonstrated by the ability of tick saliva to inhibit proliferation of human T cells and CTLL-2 cells grown in the presence of human IL-2.

Influence of costimulatory molecules on immune response to Leishmania major by human cells in vitro.

The importance of CD40, CD80, and CD86 costimulatory molecules in anti-Leishmania immune responses has been established in murine models. A role for these costimulatory molecules in human anti-Leishmania immune responses was investigated in this study. Autologous macrophages and peripheral blood leukocytes (PBL) were prepared from peripheral blood mononuclear cells of Leishmania-naive donors and cultured with or without Leishmania major in various combinations. After 7 days of culture, high levels of CD40 and CD86 were expressed on macrophages in the presence or absence of L. major. When macrophages were cultured for an additional 7 days with PBL, expression of all three costimulatory molecules was detected. When L. major was present in these cultures, the expression of CD80, and to a lesser extent CD40, on macrophages was enhanced. Blockade of CD80, CD86, or both molecules (in the order of greatest effect) in cultures containing macrophages, PBL, and L. major significantly inhibited the production of gamma interferon, interleukin-5 (IL-5), and IL-12. Blockade of CD40-CD154 interactions also significantly inhibited production of these cytokines in response to L. major. Production of IL-10 was unaltered by the blockade of these costimulatory molecules. Thus, these data suggest that CD40, CD80, and CD86 expression and regulation may significantly impact anti-Leishmania immune responses in humans.

The immunomodulatory factors of bloodfeeding arthropod saliva.

There are potent immunomodulators in saliva of the bloodfeeding arthropods which transmit many of the world's most serious diseases that may benefit the arthropod by preventing the vertebrate host from becoming sensitized to the saliva. In addition, saliva can enhance transmission of parasites/pathogens by arthropods. As a result, vaccines that target the arthropod (e.g. salivary immunomodulators) should be considered as one component of multisubunit vaccines against arthropod-borne parasites/pathogens. Indeed, since vaccines against the pathogens themselves are often not fully protective, vaccines that target several facets of the life cycle of the pathogen may be the most effective at controlling disease transmission. This review covers known immunomodulatory factors in arthropod vector saliva, focusing mainly on sandflies and ixodid ticks.

Virulent or avirulent (dhfr-ts-) Leishmania major elicit predominantly a type-1 cytokine response by human cells in vitro.

In this study we have compared the immune response of normal human cells cultured in vitro to two virulent strains of Leishmania major (CC1 and LV39), and to an avirulent vaccine strain (dhfr-ts-) made by targeted deletion of the essential gene DHFR-TS. We utilized an in vitro system in which naive T cells from normal human donors were primed with autologous Leishmania-infected macrophages. All three parasites infected macrophages and transformed into amastigotes within the cells. However, whereas LV39 and CC1 replicated in macrophages, dhfr-ts- did not. When peripheral blood lymphocytes (PBL) were stimulated with autologous macrophages infected with any of the three parasites, the lymphocytes produced a type-1-biased cytokine response. Finally, addition of IL-12 during the first stimulation period increased the production of interferon-gamma but decreased IL-5 secretion. On the other hand, anti-IL-12 resulted in the opposite effect.

Priming of a beta-galactosidase (beta-GAL)-specific type 1 response in BALB/c mice infected with beta-GAL-transfected Leishmania major.

To determine whether an ongoing response to Leishmania major would affect the response to a non-cross-reacting, non-leishmanial antigen, susceptible BALB/c mice and resistant C3H mice were infected with L. major parasites expressing Escherichia coli beta-galactosidase (beta-GAL); this parasite was designated L. major-betaGAL. BALB/c and C3H mice responded to infection with L. major-betaGAL by mounting a CD4 T-cell response to both parasite antigens and to the reporter antigen, beta-GAL. The phenotypes of these T cells were characterized after generating T-cell lines from infected mice. As expected, BALB/c mice responded to infection with L. major-betaGAL by producing interleukin 4 in response to the parasite and C3H mice produced gamma interferon (IFN-gamma) in response to the parasite and beta-GAL. Interestingly, however, BALB/c mice produced IFN-gamma in response to beta-GAL. Taken together, these results demonstrate that priming of IFN-gamma-producing cells can occur in BALB/c mice despite the fact the animals are simultaneously mounting a potent Th2 response to L. major.

Rapid early onset lymphocyte cell death in mice resistant, but not susceptible to Leishmania major infection.

Leishmania major (Lm) infection in mice is a prototypical model for the role of immune deviation in disease resistance. Resistant strains of mice develop a Th1 response to Lm infection, distinguished by secretion of IL-12 and interferon gamma. In contrast, susceptible strains display sustained IL-4 expression characteristic of a Th2 response. However, when mechanisms of cell death are blocked, mice display a susceptible phenotype even in the presence of a strong Th1 response, suggesting that cell death, and not cytokine bias, may be an important factor in disease resistance. Here, we investigated this hypothesis by comparing lymphocyte cellularity, cell death and Fas expression in resistant CBA and susceptible BALB/c mice during the course of Lm infection. We found that delayed onset of cell death and late Fas induction correlated with massive lymphocyte accumulation and susceptibility to leishmaniasis, while early cell death and rapid Fas induction occurred in resistant mice.

A method for the isolation and analysis of leucocytic cells from Leishmanial ear lesions in mice.

The standard model of experimental cutaneous leishmaniasis involves infection of mice with Leishmania major in a single footpad or the rump, and analysis of the subsequent immune response in draining lymph nodes. Relatively few studies have examined the lesion directly. Here, we describe a method for the isolation of cells from established leishmanial lesions in mouse ears. After physical disruption of lesion tissue and isolation of cells on density gradients, a variety of leucocytic cell phenotypes were identified by flow cytometry and cytology. The phenotypes of the viable cells obtained were similar, in proportion, to those observed in histologic sections of ear lesions. This technique may be useful for studying lesion-specific cell function within the first weeks after infection with Leishmania parasites.

An OspC-specific monoclonal antibody passively protects mice from tick-transmitted infection by Borrelia burgdorferi B31.

A murine monoclonal antibody directed against Borrelia burgdorferi B31 outer surface protein C (OspC) antigen was generated by a method whereby borreliae were inoculated into the mouse via the natural transmission mode of tick feeding. Passive immunization with this antibody resulted in protection of C3H/HeJ and outbred mice from a tick-transmitted challenge infection. Immunofluorescence staining of borrelia cells indicated surface exposure of the OspC epitope reactive with the monoclonal antibody.

Variation in the salivary peptide, maxadilan, from species in the Lutzomyia longipalpis complex.

Maxadilan is an approximately 7kDa peptide that occurs in the saliva of the sand fly Lutzomyia longipalpis. This peptide is a potent vasodilator and may also have immunomodulatory effects related to the pathogenesis of leishmanial infections. Variation in the primary DNA and inferred amino acid sequence of maxadilan is reported. Differences were found within and among natural field populations as well as among sibling species. Extensive amino acid sequence differentiation, up to 23%, was observed among maxadilan from different populations. This is a remarkable degree of polymorphism considering the small size of this peptide. The vasodilatory activity of maxadilan was equivalent among recombinant maxadilan variants. All maxadilan variants induce interleukin-6. Predicted secondary structure and hydrophobicity plots suggest that these characteristics are conserved among variant peptides. However, profiles based on the antigenic index do differ among peptides.

Indomethacin treatment slows disease progression and enhances a Th1 response in susceptible BALB/c mice infected with Leishmania major.

Prostaglandins of the E series inhibit the development of Th1 responses. When infected with Leishmania major, BALB/c mice fail to develop a Th1 response, but instead mount a Th2 response and die of the disease. Therefore, we treated L. major-infected BALB/c mice with indomethacin, which inhibits prostaglandin production. Indomethacin lessened disease severity (parasite burden and pathology), and promoted a Th1 response, but the mice still succumbed to infection. The explanation for these observations may be two-fold: (1) the beneficial effects of indomethacin were predominantly observed later in infection (beyond two weeks), a time at which indomethacin was unable to sufficiently block the development of a Th2 response; (2) indomethacin was unable to induce a Th1 response in BALB/c mice that was of the same magnitude as the Th1 response observed in C57BL/6 mice infected with L. major.

Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania major in BALB/c mice.

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.

Phlebotomus papatasi sand fly salivary gland lysate down-regulates a Th1, but up-regulates a Th2, response in mice infected with Leishmania major.

A vertebrate host becomes infected with Leishmania major when the sand fly vector injects parasites into skin along with saliva. Previous studies showed that salivary gland lysate of the New World sand fly Lutzomyia longipalpis markedly enhanced L. major infection in CBA mice. However, L. major is an Old World parasite transmitted in nature by the Old World sand fly Phlebotomus papatasi. Here we examine the ability of P. papatasi salivary gland lysate to enhance infection (lesion size and parasite burden) by L. major. In addition, we examine the effects of salivary gland lysate on the immune response to L. major by monitoring the levels of cytokine mRNA from the lymph nodes draining cutaneous lesions. We found that P. papatasi salivary gland lysate dramatically exacerbated lesion development in disease-resistant CBA mice. This exacerbation of disease correlated with inhibition of the production of Thl cytokines and associated factors (IFN-gamma, IL-12, and inducible nitric oxide synthase), but with enhancement of the Th2 cytokine IL-4, whereas no changes in the levels of IL-10 and TGF-beta were noted. Importantly, salivary gland lysate directly up-regulated expression of IL-4 mRNA in mice in the absence of infection with L. major.

The PACAP-type I receptor agonist maxadilan from sand fly saliva protects mice against lethal endotoxemia by a mechanism partially dependent on IL-10.

Sand fly saliva contains maxadilan, a peptide that causes vasodilation and modifies the secretion of pro-inflammatory cytokines by macrophages. We show that 1 to 10 microg maxadilan protected BALB/c mice against a lethal dose of LPS. Maxadilan reduced serum levels of TNF-alpha by approximately tenfold, while it caused a threefold increase in IL-6 and IL-10. The protective effect of maxadilan is partially dependent on its ability to induce IL-10 production since maxadilan did not prevent death from endotoxic shock in IL-10(-/-) mice. Finally, maxadilan is a selective agonist of the pituitary adenylate cyclase-activating peptide (PACAP) type I receptor, and we found that the natural ligand of this receptor (PACAP 38) also protected mice against lethal endotoxemia. These results indicate that activation of the PACAP type I receptor may contribute to the control of systemic inflammation by a mechanism that is partially dependent on IL-10.