Cysteine-free proteins in the immunobiology of arthropod-borne diseases.

One approach to identify epitopes that could be used in the design of vaccines to control several arthropod-borne diseases simultaneously is to look for common structural features in the secretome of the pathogens that cause them. Using a novel bioinformatics technique, cysteine-abundance and distribution analysis, we found that many different proteins secreted by several arthropod-borne pathogens, including Plasmodium falciparum, Borrelia burgdorferi, and eight species of Proteobacteria, are devoid of cysteine residues. The identification of three cysteine-abundance and distribution patterns in several families of proteins secreted by pathogenic and nonpathogenic Proteobacteria, and not found when the amino acid analyzed was tryptophan, provides evidence of forces restricting the content of cysteine residues in microbial proteins during evolution. We discuss these findings in the context of protein structure and function, antigenicity and immunogenicity, and host-parasite relationships.

Cross-protection against Leishmania donovani but not L. Braziliensis caused by vaccination with L. Major soluble promastigote exogenous antigens in BALB/c mice.

Vaccinating with soluble Leishmania major promastigote exogenous antigens (LmSEAgs) protects mice against challenge with L. major. To explore the potential of LmSEAgs to cross-protect against infection with other species of Leishmania, BALB/c mice were immunized with LmSEAgs prior to challenge with either L. donovani or L. braziliensis promastigotes. Such mice were protected against L. donovani but not L. braziliensis infection. Leishmania braziliensis-infected mice developed lesions that were not significantly different from those of controls and that contained 13-fold more parasites. In contrast, immunized mice infected with L. donovani were protected as illustrated by low splenic parasite loads (as much as 4,913-fold fewer parasites). This protection corresponded to significant increases in gamma interferon and low production of interleukin-4 (IL-4) IL-4 or IL-10, which suggested an enhanced type 1 response.

Protection of susceptible BALB/c mice from challenge with Leishmania major by nucleoside hydrolase, a soluble exo-antigen of Leishmania.

Leishmania major culture-derived, soluble, exogenous antigens have been shown to be a source of vaccine targets for the parasite. We have previously reported that L. major culture-derived, soluble, exogenous antigens can immunize BALB/c mice against challenge with L. major. However, the molecule(s) involved in this protection was not known. We describe the potential of one component of soluble exogenous antigens (recombinant nucleoside hydrolase) to vaccinate mice against challenge with L. major. We found that recombinant nucleoside hydrolase vaccinated BALB/c mice against a subsequent challenge with L. major. Protection was manifested by a significant decrease in lesion size (as much as a 30-fold reduction) and parasite burden (as much as a 71-fold reduction). Protection was achieved whether recombinant nucleoside hydrolase was administered to mice in the presence or absence of adjuvant (interleukin-12). Finally, protection was accompanied by an increase in interferon-gamma production but a decrease in interleukin-10 production by vaccinated animals in response to challenge with L. major.

Effect of Lutzomyia longipalpis salivary gland extracts on leukocyte migration induced by Leishmania major.

The mechanism by which the salivary gland lysate (SGL) of Lutzomyia longipalpis enables Leishmania infection remains under investigation. One possibility is that saliva promotes cellular recruitment leading to development of skin lesions. In this study, we investigated leukocyte recruitment induced by L. major, L. major + SGL, or SGL alone into the peritoneal cavity of BALB/c mice. The administration of L. major with or without SGL induced neutrophil migration six hours after infection. Interestingly, after seven days, the BALB/c mice still had eosinophils and mononuclear cells in their peritoneal cavities. Flow cytometric analysis showed an increase in the CD4(+) CD45RB(low) T cell subset (effector or memory cells) compared with the CD4(+) CD45RB(high) subset (naive cells). Moreover, the co-injection of L. major with SGL enhanced production of interleukin-10. These results suggest that SGL can facilitate Leishmania infection by modulating leukocyte recruitment and Th2 cytokine production at the inflammatory focus.

Immunization against full-length protein and peptides from the Lutzomyia longipalpis sand fly salivary component maxadilan protects against Leishmania major infection in a murine model.

Leishmaniasis is an arthropod vectored disease causing considerable human morbidity and mortality. Vaccination remains the most realistic and practical means to interrupt the growing number and diversity of sand fly vectors and reservoirs of Leishmania. Since transmission of Leishmania is achieved exclusively by sand fly vectors via immune-modulating salivary substances, conventional vaccination requiring an unmodified host immune response for success are potentially destined to fail unless immunomodulatory factors are somehow neutralized. Using cationic liposome DNA complexes (CLDC) as an adjuvant system along with Lu. longipalpis sand fly salivary component maxadilan (MAX) as antigen (Ag), we show that mice are protected from the MAX-induced exacerbation of infection with Leishmania major (Lm). The CLDC adjuvant and alum were comparable in terms of lesion induration and decreased parasite burden, however the alum adjuvant imposed more inflammation at the injection site. BALB/c, C3H and C57BL/6 mice vaccinated with MAX-CLDC containing either the full-length MAX or peptides spanning the N- and C-terminal regions of MAX are protected against footpad challenges with Lm co-injected with MAX. When compared to unvaccinated controls, all strains of mice immunized with CLDC containing either peptides encompassing the first 20 N-terminal AA or those spanning the last 15 AA of the C-terminal domain of MAX demonstrated decreased parasite burden after 9 or 18 weeks post challenge with Lm + MAX. MAX-CLDC immunized mice showed increased IFNγ-secreting and decreased IL-4-secreting CD4+ cells in footpad-draining lymph nodes. Antisera from C-terminal peptide (P11) MAX-CLDC-vaccinated animals was capable of recognizing FL-MAX and its C-terminal domain and also blocked MAX-mediated reprogramming of bone marrow-derived dendritic cells (BM-DC) in vitro. This peptide vaccine targeting sand fly MAX, improves host immunity against MAX-mediated immunomodulation.

Is it possible to develop pan-arthropod vaccines?

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 species-specific vaccines. An alternative is to target cross-reactive 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.

Salivary gland extracts of Culicoides sonorensis inhibit murine lymphocyte proliferation and no production by macrophages.

Culicoides biting midges serve as vectors of pathogens affecting humans and domestic animals. Culicoides sonorensis is a vector of several arboviruses in North American that cause substantial economic losses to the US livestock industry. Previous studies showed that C. sonorensis saliva, like the saliva of many hematophagous arthropods, contains numerous pharmacological agents that affect hemostasis and early events in the inflammatory response, which may enhance the infectivity of Culicoides-borne pathogens. This paper reports on the immunomodulatory properties of C. sonorensis salivary gland extracts on murine immune cells and discusses the possible immunomodulatory role of C. sonorensis saliva in vesicular stomatitis virus infection of vertebrate hosts. Splenocytes treated with C. sonorensis mitogens were significantly affected in their proliferative response, and peritoneal macrophages secreted significantly less NO. A 66-kDa glycoprotein was purified from C. sonorensis salivary gland extract, which may be in part responsible for these observations and may be considered as a vaccine candidate.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces Leishmania major burdens in C57BL/6 mice.

Acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can suppress adaptive immunity. In this study, pre-exposure of Leishmania major-infected mice to TCDD caused a dose-dependent and unexpected decrease in parasite burdens on day 20 after infection. In contrast, TCDD-mediated lymphoid atrophy, suppressed antibody levels, and enhanced interleukin-2 production were observed as expected. These results suggest that TCDD may enhance resistance to L. major in the face of immune suppression.

The surface-mosaic model in host-parasite relationships.

The dynamics of protein adsorption to a microbial surface could be of significance in host-parasite relationships because non-defense proteins might interfere with the binding of defense proteins. A surface mosaic of defense and non-defense proteins formed on the microbial surface could activate one of the tissue reactivity programs via a binary code (help or silence) generated by the adsorbed proteins. Understanding the mechanisms of the mosaic formation and its evolution might help to identify evasion mechanisms used by virulent microorganisms. This also provides a conceptual framework to design new strategies to control the infectious diseases they cause.

Characterization of the early cellular immune response to Leishmania major using peripheral blood mononuclear cells from Leishmania-naive humans.

While the response to Leishmania major is well characterized in mice, there is much less known about the human immune response, particularly early after exposure to the parasite. Therefore, we developed a primary in vitro (PIV) system that allowed us to address these questions. We co-cultured peripheral blood mononuclear cells from Leishmania-naive donors with L. major parasites and found that the responding PIV cells produced interferon-gamma and interleukin-12 (IL-12). When restimulated, these PIV cells also occasionally produced IL-5. Both CD4 and CD8 cells and both HLA class I and II cell activation pathways appeared to play a role in the PIV system, and cell activation was dependent upon the presence of antigen-presenting cells. Moreover, PIV cells generated with L. major showed considerable cross-reactivity with other species of Leishmania. Finally, the PIV cells augmented intracellular killing of L. major when they were co-cultured with macrophages infected with the parasite.

Short report: requirement of b cells for delayed type hypersensitivity-like pathology after secondary infection with Leishmania major in resistant C57BL/6 mice.

B cell-deficient C57B1/6 (microMT) mice were resistant to Leishmania major after both primary and secondary parasite challenge. However, unlike in wild-type mice, secondary infection in microMT mice was not accompanied by a marked delayed type hypersensitivity-like response, and interferon-gamma (IFN-gamma) levels were approximately half of those in wild-type mice. These results suggest that B cells are involved in IFN-gamma production and the pathology of secondary infection.

Type 1 and type 2 responses to Leishmania major.

Leishmania major is a protozoan parasite that is transmitted to the mammalian host by its sand fly vector when the fly probes in the host's skin for a blood meal and injects the parasite within its saliva. In mice experimentally infected with L. major, outgrowth of CD4 type 1 (Th1) cells leads to resolution of the infection, but outgrowth of type 2 (Th2) cells exacerbates disease. To design an effective vaccine against the parasite (and other pathogens that induce polarized Th1 and Th2 responses), we must determine the mechanism underlying this phenomenon so that we can design the vaccine to elicit the appropriate (i.e., protective) Th cell. Recent work indicates that Th bias is influenced by a number of signals delivered by antigen-presenting cells, including cytokines and co-stimulatory molecules. Moreover, recent work also suggests that sand fly saliva influences the immune response to L. major and Th polarization. Determining the mechanisms that lead to polarized Th responses should expand our knowledge regarding immunity to L. major, and should add to our understanding of immunoregulation in general.

The human cytokine response to Leishmania major early after exposure to the parasite in vitro.

Leishmaniasis is caused by the protozoan parasite Leishmania spp. In murine leishmaniasis, a T helper cell type-I (Th1) response, characterized by the secretion of interferon (IFN)-gamma is necessary for clearing the infection. whereas a Th2 response, accompanied by the production of interleukin (IL)-5, can exacerbate the disease. Moreover, the early cytokine milieu is thought to play an important role in determining the outcome of infection. In human leishmaniasis little is known about this early cytokine response. Because of this, we cocultured human peripheral blood mononuclear cells (PBMC) with Leishmania major in vitro and measured the production of IFN-gamma, IL-5, and IL-10. We also treated PBMC cultures with various cytokines and neutralizing anticytokines. We found that the principal cytokine produced was IFN-gamma and that its production was regulated by IL-10 and IL-12. In contrast, only low levels of Th2 cytokines such as IL-5 were produced. Therefore, the Th1-Th2 dichotomy that exists in inbred strains of mice does not appear to apply to the response of humans to L. major. Rather, Th2 cytokines may play a role in regulating IFN-gamma production.

2,3,7,8-tetrachlorodibenzo-p-dioxin slows the progression of experimental cutaneous Leishmaniasis in susceptible BALB/c and SCID mice.

In a model of experimental cutaneous leishmaniasis, pre-exposure of Leishmania major-resistant mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor agonist, causes suppression of the protective anti-parasite T helper 1 response while paradoxically also reducing parasite burdens in those animals. In this study, we examined if TCDD exposure could also reduce parasite burdens in L. major-susceptible BALB/c mice. In the highest dose group (160 µg/Kg), TCDD treatment caused a significant reduction of parasite burdens by 10-fold after three weeks while also causing a significant lymphoid atrophy indicating suppression of the non-protective T helper 2 response. A dose-dependent delay of foot lesion progression was also observed such that lesion size in the highest dose group was less than half that of controls after 35 days of infection. Importantly, although TCDD exposure initially reduced disease severity and prolonged the course of disease by as much as three fold in some animals, this effect was transitory and TCDD did not induce resistance to L. major infection. Because TCDD exposure reduced L. major burdens in both resistant and susceptible mice, we hypothesized that TCDD reduces L. major burdens in mice by a mechanism that does not involve adaptive immunity. To test this, severe combined immunodeficient (SCID) mice were used. In mice infected with a moderate number of L. major (10,000), TCDD treatment caused a time- and dose-dependent decrease of parasite burdens by nearly 100-fold after six weeks in the highest dose group (200 µg/Kg). A significant and dose-dependent delay of foot lesion progression was also observed in these animals. These results indicate that TCDD exposure can reduce the severity of leishmanial disease in mice independent of adaptive immunity.

BluePort: a platform to study the eosinophilic response of mice to the bite of a vector of Leishmania parasites, Lutzomyia longipalpis sand flies.

BACKGROUND: Visceral Leishmaniasis is a serious human disease transmitted, in the New World, by Lutzomyia longipalpis sand flies. Natural resistance to Leishmania transmission in residents of endemic areas has been attributed to the acquisition of immunity to sand fly salivary proteins. One theoretical way to accelerate the acquisition of this immunity is to increase the density of antigen-presenting cells at the sand fly bite site. Here we describe a novel tissue platform that can be used for this purpose. METHODOLOGY/PRINCIPAL FINDINGS: BluePort is a well-vascularized and macrophage-rich compartment induced in the subcutaneous tissue of mice via injection of agarose beads covered with Cibacron blue. We describe the sequence of inflammatory events leading to its formation and how it can be used to study the dermal response to the bite of L. longipalpis sand flies. Results presented indicate that a shift in the inflammatory response, from neutrophilic to eosinophilic, is the main histopathological feature associated with the immunity acquired through repeated exposure to the bite of sand flies, and that the BluePort tissue compartment could be used to accelerate this process. In addition, changes observed inside the BluePort parenchyma indicate that it could be used to study complex immunobiological processes, and to develop ectopic secondary lymphoid structures. CONCLUSIONS/SIGNIFICANCE: Understanding the characteristics of the dermal response to the bite of sand flies is a critical element of strategies to control leishmaniasis using vaccines that target salivary proteins. Finding that dermal eosinophilia is such a prominent component of the anti-salivary immunity induced by repeated exposure to sand fly bites raises one important consideration: how to avoid the immunological conflict derived from a protective Th2-driven immunity directed to sand fly saliva with a protective Th1-driven immunity directed to the parasite. The BluePort platform is an ideal tool to address experimentally this conundrum.

Local suppression of T cell responses by arginase-induced L-arginine depletion in nonhealing leishmaniasis.

The balance between T helper (Th) 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. Here we show that high arginase activity, a hallmark of nonhealing disease, is primarily expressed locally at the site of pathology. The high arginase activity causes local depletion of L-arginine, which impairs the capacity of T cells in the lesion to proliferate and to produce interferon-gamma, while T cells in the local draining lymph nodes respond normally. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, competitive inhibition of arginase as well as supplementation with L-arginine restored T cell effector functions and reduced pathology and parasite growth at the site of lesions. These results demonstrate that in nonhealing leishmaniasis, arginase-induced L-arginine depletion results in impaired T cell responses. Our results identify a novel mechanism in leishmaniasis that contributes to the failure to heal persistent lesions and suggest new approaches to therapy.

Lutzomyia longipalpis salivary peptide maxadilan alters murine dendritic cell expression of CD80/86, CCR7, and cytokine secretion and reprograms dendritic cell-mediated cytokine release from cultures containing allogeneic T cells.

Leishmania protozoan parasites, the etiologic agent of leishmaniasis, are transmitted exclusively by phlebotomine sand flies of the genera Phlebotomus and Lutzomyia. In addition to parasites, the infectious bite inoculum contains arthropod salivary components. One well-characterized salivary component from Lutzomyia longipalpis is maxadilan (MAX), a vasodilator acting via the type I receptor for the pituitary cyclic AMP activating peptide. MAX has been shown to elicit immunomodulatory effects potentially dictating immune responses to Leishmania parasites. When exposed to MAX, both resting and LPS-stimulated dendritic cells (DCs) show reduced CD80 and CD86 expression on most DCs in vitro. However, CD86 expression is increased significantly on a subpopulation of DCs. Furthermore, MAX treatment promoted secretion of type 2 cytokines (IL-6 and IL-10) while reducing production of type 1 cytokines (IL-12p40, TNF-alpha, and IFN-gamma) by LPS-stimulated DCs. A similar trend was observed in cultures of MAX-treated DCs containing naive allogeneic CD4(+) T cells: type 2 cytokines (IL-6 and IL-13) increased while type 1 cytokines (TNF-alpha and IFN-gamma) decreased. Additionally, the proinflammatory cytokine IL-1beta was increased in cultures containing MAX-treated mature DCs. MAX treatment of LPS-stimulated DCs also prevented optimal surface expression of CCR7 in vitro. These MAX-dependent effects were evident in DCs from both Leishmania major-susceptible (BALB/c) and -resistant (C3H/HeN) murine strains. These data suggest that modification of DC phenotype and function by MAX likely affects crucial cellular components that determine the pathological response to infection with Leishmania.

Objective evaluation of skin prick test reactions using digital photography.

BACKGROUND: The skin prick test has been used worldwide to determine IgE-mediated hypersensitivity. However, the most current method to record this reaction has problems with accuracy and precision. OBJECTIVE: To demonstrate a new approach to measure the skin prick reaction and its kinetics with precision. METHODS: The skin prick test was induced using histamine or Dermatophagoides pteronyssinus antigen in 80 volunteers aged 4 and 67 years who had different skin colors. Digital photographs were obtained at 0, 3, 5, 10, 15 and 20 min. The mean wheal and erythema area as well as the erythema intensity were determined using Adobe Photoshop software. The accuracy and precision of this approach were also evaluated. RESULTS: The digital photographic analysis measured the wheal and erythema sizes independent of the antigen or skin color with precision. In addition, a new variable of this test, the skin erythema intensity, could be determined objectively using the chromaticity of reflected light. CONCLUSIONS: Digital photographic analysis is a precise and objective method to evaluate the skin prick test reaction, which can be used independent of the patient's skin color in clinical or research settings.

Immunomodulatory effects of the Lutzomyia longipalpis salivary gland protein maxadilan on mouse macrophages.

Infection with Leishmania major is enhanced when the sand fly Lutzomyia longipalpis salivary peptide maxadilan (MAX) is injected along with the parasite. Here we determined the effect that MAX has on the secretion of cytokines and nitric oxide (NO) and on parasite survival in macrophages (MPhis). The cytokines produced by MPhis can enhance a type 1 response, which will increase NO and the killing of intracellular pathogens such as L. major, or a type 2 response, leading to antibody production that is ineffective against intracellular pathogens such as L. major. A mouse macrophage cell line (RAW 264.7) was stimulated with various concentrations of MAX and lipopolysaccharide (LPS), and the supernatants were collected after 1, 2, and 3 days. Supernatants were assayed for interleukin-12p70 (IL-12p70), IL-10, IL-6, transforming growth factor beta (TGF-beta), NO, and tumor necrosis factor alpha (TNF-alpha). Our results indicate that the addition of MAX upregulates the cytokines associated with a type 2 response (IL-10, IL-6, and TGF-beta) but downregulates type 1 cytokines (IL-12p70 and TNF-alpha) and NO. MAX was also added to L. major-infected mouse peritoneal exudate cells (PECs), and the parasite load increased significantly. The enhanced parasite load correlated with decreased NO production by PECs that were stimulated with LPS and gamma interferon in the presence of MAX. The ability of MAX to foster a type 2 response, to enhance parasite survival, and to decrease NO argues that MAX may be crucial for the early survival of Leishmania in the vertebrate host, and therefore, MAX holds considerable promise as an antigenic component for a vaccine against Leishmania.

Novel compounds active against Leishmania major.

Leishmania major is an important trypanosomatid pathogen that causes leishmaniasis, which is a serious disease in much of the Old World. Current treatments include a small number of antimony compounds that, while somewhat effective, are limited by serious side effects. We have screened a small portion of a unique chemical library and have found at least three novel compounds that are effective against L. tarentolae and L. major in vitro and in a murine macrophage model of L. major infection. These compounds were effective in both assays at doses significantly lower than those of sodium stibogluconate (Pentostam) and represent possible candidates for drug development.