Publications by authors named "Jesus G Valenzuela"

174 Publications

Towards a Sustainable Vector-Control Strategy in the Post Kala-Azar Elimination Era.

Front Cell Infect Microbiol 2021 9;11:641632. Epub 2021 Mar 9.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, United States.

Visceral leishmaniasis (VL) is a potentially deadly parasitic disease. In the Indian sub-continent, VL is caused by and transmitted the bite of an infected female sand fly, the only competent vector species in the region. The highest disease burden is in the northern part of the Indian sub-continent, especially in the state of Bihar. India, Bangladesh, and Nepal embarked on an initiative, coordinated by World Health Organization, to eliminate VL as a public health problem by the year 2020. The main goal is to reduce VL incidence below one case per 10,000 people through early case-detection, prompt diagnosis and treatment, and reduction of transmission using vector control measures. Indoor residual spraying, a major pillar of the elimination program, is the only vector control strategy used by the government of India. Though India is close to its VL elimination target, important aspects of vector bionomics and sand fly transmission dynamics are yet to be determined. To achieve sustained elimination and to prevent a resurgence of VL, knowledge gaps in vector biology and behavior, and the constraints they may pose to current vector control methods, need to be addressed. Herein, we discuss the successes and failures of previous and current vector-control strategies implemented to combat kala-azar in Bihar, India, and identify gaps in our understanding of vector transmission towards development of innovative tools to ensure sustained vector control in the post-elimination period.
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http://dx.doi.org/10.3389/fcimb.2021.641632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985538PMC
March 2021

Salivary Protease Inhibitors as Potential Anti-Tick Vaccines.

Front Immunol 2020 4;11:611104. Epub 2021 Feb 4.

Laboratory of Physiology of Hematophagous Insects, Department of Parasitology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

is the main tick associated with human bites in Brazil and the main vector of , the causative agent of the most severe form of Brazilian spotted fever. Molecules produced in the salivary glands are directly related to feeding success and vector competence. In the present study, we identified sequences of salivary proteins that may be involved in hematophagy and selected three proteins that underwent functional characterization and evaluation as vaccine antigens. Among the three proteins selected, one contained a Kunitz_bovine pancreatic trypsin inhibitor domain (named AsKunitz) and the other two belonged to the 8.9 kDa and basic tail families of tick salivary proteins (named As8.9kDa and AsBasicTail). Expression of the messenger RNA (mRNA) encoding all three proteins was detected in the larvae, nymphs, and females at basal levels in unfed ticks and the expression levels increased after the start of feeding. Recombinant proteins rAs8.9kDa and rAsBasicTail inhibited the enzymatic activity of factor Xa, thrombin, and trypsin, whereas rAsKunitz inhibited only thrombin activity. All three recombinant proteins inhibited the hemolysis of both the classical and alternative pathways; this is the first description of tick members of the Kunitz and 8.9kDa families being inhibitors of the classical complement pathway. Mice immunization with recombinant proteins caused efficacies against A. females from 59.4% with rAsBasicTail immunization to more than 85% by immunization with rAsKunitz and rAs8.9kDa. The mortality of nymphs fed on immunized mice reached 70-100%. Therefore, all three proteins are potential antigens with the possibility of becoming a new tool in the control of .
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http://dx.doi.org/10.3389/fimmu.2020.611104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901972PMC
February 2021

Engineering a vector-based pan-Leishmania vaccine for humans: proof of principle.

Sci Rep 2020 10 29;10(1):18653. Epub 2020 Oct 29.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Rockville, MD, 20852, USA.

Leishmaniasis is a spectrum of diseases transmitted by sand fly vectors that deposit Leishmania spp. parasites in the host skin during blood feeding. Currently, available treatment options are limited, associated with high toxicity and emerging resistance. Even though a vaccine for human leishmaniasis is considered an achievable goal, to date we still do not have one available, a consequence (amongst other factors) of a lack of pre-clinical to clinical translatability. Pre-exposure to uninfected sand fly bites or immunization with defined sand fly salivary proteins was shown to negatively impact infection. Still, cross-protection reports are rare and dependent on the phylogenetic proximity of the sand fly species, meaning that the applicability of a sand fly saliva-based vaccine will be limited to a defined geography, one parasite species and one form of leishmaniasis. As a proof of principle of a future vector saliva-based pan-Leishmania vaccine, we engineered through a reverse vaccinology approach that maximizes translation to humans, a fusion protein consisting of immunogenic portions of PdSP15 and LJL143, sand fly salivary proteins demonstrated as potential vaccine candidates against cutaneous and visceral leishmaniasis, respectively. The in silico analysis was validated ex vivo, through T cell proliferation experiments, proving that the fusion protein (administered as a DNA vaccine) maintained the immunogenicity of both PdSP15 and LJL143. Additionally, while no significant effect was detected in the context of L. major transmission by P. duboscqi, this DNA vaccine was defined as partially protective, in the context of L. major transmission by L. longipalpis sand flies. Importantly, a high IFNγ response alone was not enough to confer protection, that mainly correlated with low T cell mediated Leishmania-specific IL-4 and IL-10 responses, and consequently with high pro/anti-inflammatory cytokine ratios. Overall our immunogenicity data suggests that to design a potentially safe vector-based pan-Leishmania vaccine, without geographic restrictions and against all forms of leishmaniasis is an achievable goal. This is why we propose our approach as a proof-of principle, perhaps not only applicable to the anti-Leishmania vector-based vaccines' field, but also to other branches of knowledge that require the design of multi-epitope T cell vaccines with a higher potential for translation.
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http://dx.doi.org/10.1038/s41598-020-75410-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596519PMC
October 2020

Heme Oxygenase-1 Induction by Blood-Feeding Arthropods Controls Skin Inflammation and Promotes Disease Tolerance.

Cell Rep 2020 10;33(4):108317

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA. Electronic address:

Hematophagous vectors lacerate host skin and capillaries to acquire a blood meal, resulting in leakage of red blood cells (RBCs) and inflammation. Here, we show that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes, and ticks. Further, we demonstrate that erythrophagocytosis by macrophages, including a skin-residing CD163CD91 professional iron-recycling subpopulation, produces HO-1 after bites. Importantly, we establish that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number, whereas CO, an end product of the HO-1 enzymatic reaction, suppresses skin inflammation. This indicates that HO-1 induction by blood-feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods, thus promoting early-stage disease tolerance to vector-borne pathogens.
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http://dx.doi.org/10.1016/j.celrep.2020.108317DOI Listing
October 2020

Implicating bites from a leishmaniasis sand fly vector in the loss of tolerance in pemphigus.

JCI Insight 2020 12 3;5(23). Epub 2020 Dec 3.

Laboratory of Transmission, Control and Immunobiology of Infections, LR11IPT02, Pasteur Institut de Tunis, Tunis, Tunisia.

A possible etiological link between the onset of endemic pemphigus in Tunisia and bites of Phlebotomus papatasi, the vector of zoonotic cutaneous leishmaniasis, has been previously suggested. We hypothesized that the immunodominant P. papatasi salivary protein PpSP32 binds to desmogleins 1 and 3 (Dsg1 and Dsg3), triggering loss of tolerance to these pemphigus target autoantigens. Here, we show using far-Western blot that the recombinant PpSP32 protein (rPpSP32) binds to epidermal proteins with a MW of approximately 170 kDa. Coimmunoprecipitation revealed the interaction of rPpSP32 with either Dsg1 or Dsg3. A specific interaction between PpSP32 and Dsg1 and Dsg3 was further demonstrated by ELISA assays. Finally, mice immunized with rPpSP32 twice per week exhibited significantly increased levels of anti-Dsg1 and -Dsg3 antibodies from day 75 to 120. Such antibodies were specific for Dsg1 and Dsg3 and were not the result of cross-reactivity to PpSP32. In this study, we demonstrated for the first time to our knowledge a specific binding between PpSP32 and Dsg1 and Dsg3, which might underlie the triggering of anti-Dsg antibodies in patients exposed to sand fly bites. We also confirmed the development of specific anti-Dsg1 and -Dsg3 antibodies in vivo after PpSP32 immunization in mice. Collectively, our results provide evidence that environmental factors, such as the exposure to P. papatasi bites, can trigger the development of autoimmune antibodies.
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http://dx.doi.org/10.1172/jci.insight.123861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714401PMC
December 2020

Binding of Leishmania infantum Lipophosphoglycan to the Midgut Is Not Sufficient To Define Vector Competence in Sand Flies.

mSphere 2020 09 9;5(5). Epub 2020 Sep 9.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA

The major surface lipophosphoglycan (LPG) of parasites is critical to vector competence in restrictive sand fly vectors in mediating attachment to the midgut epithelium, considered essential to parasite survival and development. However, the relevance of LPG for sand flies that harbor multiple species of remains elusive. We tested binding of wild-type (WT), LPG-defective (Δ mutants), and add-back (Δ+) lines to sand fly midguts and their survival in sand flies WT parasites attached to the midgut , with late-stage parasites binding to midguts in significantly higher numbers than were seen with early-stage promastigotes. Δ mutants did not bind to midguts, and this was rescued in the Δ+ lines, indicating that midgut binding is mediated by LPG. When sand flies were infected with the WT or Δ or Δ+ line of the BH46 or BA262 strains, the BH46 Δ mutant, but not the BA262 Δ mutant, survived and grew to numbers similar to those seen with the WT and Δ+ lines. Exposure of BH46 and BA262 Δ mutants to blood-engorged midgut extracts led to mortality of the BA262 Δ but not the BH46 Δ parasites. These findings suggest that LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, but that it is not necessary to prevent evacuation along with the feces in the permissive vector It is well established that the presence of LPG is sufficient to define the vector competence of restrictive sand fly vectors with respect to parasites. However, the permissiveness of other sand flies with respect to multiple species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of survival and development in its natural vector, We found that LPG-mediated midgut binding persists in late-stage parasites. This observation is of relevance for the understanding of vector-parasite molecular interactions and suggests that only a subset of infective metacyclic-stage parasites (metacyclics) lose their ability to attach to the midgut, with implications for parasite transmission dynamics. However, our data also demonstrate that LPG is not a determining factor in retention in the midgut of , a permissive vector. Rather, LPG appears to be more important in protecting some parasite strains from the toxic environment generated during blood meal digestion in the insect gut. Thus, the relevance of LPG in parasite development in permissive vectors appears to be a complex issue and should be investigated on a strain-specific basis.
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http://dx.doi.org/10.1128/mSphere.00594-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485685PMC
September 2020

Leishmania infection induces a limited differential gene expression in the sand fly midgut.

BMC Genomics 2020 Sep 4;21(1):608. Epub 2020 Sep 4.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.

Background: Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages.

Results: The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (< 32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics.

Conclusion: Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.
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http://dx.doi.org/10.1186/s12864-020-07025-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487717PMC
September 2020

A second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing.

Nat Commun 2020 07 10;11(1):3461. Epub 2020 Jul 10.

Division of Emerging and Transfusion Transmitted Diseases, CBER, FDA, Silver Spring, MD, 20993, USA.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen). Notably, LmCen is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen immunization results in protection and an immune response comparable to leishmanization. LmCen is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.
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http://dx.doi.org/10.1038/s41467-020-17154-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351751PMC
July 2020

Safety and immunogenicity of a mosquito saliva peptide-based vaccine: a randomised, placebo-controlled, double-blind, phase 1 trial.

Lancet 2020 06 11;395(10242):1998-2007. Epub 2020 Jun 11.

LID Clinical Studies Unit, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

Background: In animal models, immunity to mosquito salivary proteins protects animals against mosquito-borne disease. These findings provide a rationale to vaccinate against mosquito saliva instead of the pathogen itself. To our knowledge, no vector salivary protein-based vaccine has been tested for safety and immunogenicity in humans. We aimed to assess the safety and immunogenicity of Anopheles gambiae saliva vaccine (AGS-v), a peptide-based vaccine derived from four A gambiae salivary proteins, in humans.

Methods: In this randomised, placebo-controlled, double-blind, phase 1 trial, participants were enrolled at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Participants were eligible if they were healthy adults, aged 18-50 years with no history of severe allergic reactions to mosquito bites. Participants were randomly assigned (1:1:1), using block randomisation and a computer-generated randomisation sequence, to treatment with either 200 nmol of AGS-v vaccine alone, 200 nmol of AGS-v with adjuvant (Montanide ISA 51), or sterile water as placebo. Participants and clinicians were masked to treatment assignment. Participants were given a subcutaneous injection of their allocated treatment at day 0 and day 21, followed by exposure to feeding by an uninfected Aedes aegypti mosquito at day 42 to assess subsequent risk to mosquito bites in a controlled setting. The primary endpoints were safety and immunogenicity at day 42 after the first immunisation. Participants who were given at least one dose of assigned treatment were assessed for the primary endpoints and analysis was by intention to treat. The trial was registered with ClinicalTrials.gov, NCT03055000, and is closed for accrual.

Findings: Between Feb 15 and Sept 10, 2017, we enrolled and randomly assigned 49 healthy adult participants to the adjuvanted vaccine (n=17), vaccine alone (n=16), or placebo group (n=16). Five participants did not complete the two-injection regimen with mosquito feeding at day 42, but were included in the safety analyses. No systemic safety concerns were identified; however, one participant in the adjuvanted vaccine group developed a grade 3 erythematous rash at the injection site. Pain, swelling, erythema, and itching were the most commonly reported local symptoms and were significantly increased in the adjuvanted vaccine group compared with both other treatment groups (nine [53%] of 17 participants in the adjuvanted vaccine group, two [13%] of 16 in the vaccine only group, and one [6%] of 16 in the placebo group; p=0·004). By day 42, participants who were given the adjuvanted vaccine had a significant increase in vaccine-specific total IgG antibodies compared with at baseline than did participants who were give vaccine only (absolute difference of log-fold change of 0·64 [95% CI 0·39 to 0·89]; p=0·0002) and who were given placebo (0·62 [0·34 to 0·91]; p=0·0001). We saw a significant increase in IFN-γ production by peripheral blood mononuclear cells at day 42 in the adjuvanted vaccine group compared with in the placebo group (absolute difference of log ratio of vaccine peptide-stimulated vs negative control 0·17 [95% CI 0·061 to 0·27]; p=0·009) but we saw no difference between the IFN-γ production in the vaccine only group compared with the placebo group (0·022 [-0·072 to 0·116]; p=0·63).

Interpretation: AGS-v was well tolerated, and, when adjuvanted, immunogenic. These findings suggest that vector-targeted vaccine administration in humans is safe and could be a viable option for the increasing burden of vector-borne disease.

Funding: Office of the Director and the Division of Intramural Research at the National Institute of Allergy and Infectious Diseases, and National Institutes of Health.
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http://dx.doi.org/10.1016/S0140-6736(20)31048-5DOI Listing
June 2020

Immunity to vector saliva is compromised by short sand fly seasons in endemic regions with temperate climates.

Sci Rep 2020 05 14;10(1):7990. Epub 2020 May 14.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, 20852, USA.

Individuals exposed to sand fly bites develop humoral and cellular immune responses to sand fly salivary proteins. Moreover, cellular immunity to saliva or distinct salivary proteins protects against leishmaniasis in various animal models. In Tbilisi, Georgia, an endemic area for visceral leishmaniasis (VL), sand flies are abundant for a short period of ≤3 months. Here, we demonstrate that humans and dogs residing in Tbilisi have little immunological memory to saliva of P. kandelakii, the principal vector of VL. Only 30% of humans and 50% of dogs displayed a weak antibody response to saliva after the end of the sand fly season. Likewise, their peripheral blood mononuclear cells mounted a negligible cellular immune response after stimulation with saliva. RNA seq analysis of wild-caught P. kandelakii salivary glands established the presence of a typical salivary repertoire that included proteins commonly found in other sand fly species such as the yellow, SP15 and apyrase protein families. This indicates that the absence of immunity to P. kandelakii saliva in humans and dogs from Tbilisi is probably caused by insufficient exposure to sand fly bites. This absence of immunity to vector saliva will influence the dynamics of VL transmission in Tbilisi and other endemic areas with brief sand fly seasons.
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http://dx.doi.org/10.1038/s41598-020-64820-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224377PMC
May 2020

A Lutzomyia longipalpis Salivary Protein Induces Cross-Reactive Antibodies to Pemphigus Autoantigen Desmoglein 1.

J Invest Dermatol 2020 12 29;140(12):2332-2342.e10. Epub 2020 Apr 29.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA.

Fogo selvagem (FS) is a blistering skin disease caused by pathogenic IgG4 autoantibodies to desmoglein 1 (DSG1). Preclinical FS and leishmaniasis are endemic to certain regions of Brazil and exhibit nonpathogenic anti-DSG1 antibodies. Recurring bites from Lutzomyia longipalpis, the sand fly vector of leishmaniasis, immunize individuals with L. longipalpis salivary antigens LJM17 and LJM11. We measured the antibody responses to LJM17, LJM11, and DSG1 in normal settlers and patients with FS from an endemic focus of FS and nonendemic control populations. We also immunized mice with these antigens and assessed the IgG response. Healthy individuals and patients with FS from endemic areas had significantly higher values of IgG4 anti-LJM17 antibodies than nonendemic controls (P < 0.001 for both). The levels of IgG anti-DSG1 and IgG4 anti-LJM17 and anti-LJM11 antibodies correlated positively in normal settlers and patients with FS. Mice immunized with recombinant LJM17 produced IgG1 antibodies (human IgG4 homolog) that strongly cross-reacted with recombinant DSG1; these IgG1 antibodies were inhibited by LJM17, LJM11, and DSG1 in a dose-dependent manner. However, they did not bind human or mouse epidermis by indirect immunofluorescence. Lastly, we identified short-sequence homologies of surface-exposed residues within the human DSG1 ectodomain and LJM17. Inoculation by LJM17 from L. longipalpis-elicited DSG1-cross-reactive IgG4 antibodies may lead to FS in genetically predisposed individuals.
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http://dx.doi.org/10.1016/j.jid.2020.02.041DOI Listing
December 2020

Exploring Lutzomyia longipalpis Sand Fly Vector Competence for Leishmania major Parasites.

J Infect Dis 2020 09;222(7):1199-1203

Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA.

Lutzomyia longipalpis sand flies are the major natural vector of Leishmania infantum parasites, responsible for transmission of visceral leishmaniasis in the New World. Several experimental studies have demonstrated the ability of Lu. longipalpis to sustain development of different Leishmania species. However, no study had explored in depth the potential vector competence of Lu. longipalpis for Leishmania species other than L. infantum. Here, we show that Lu. longipalpis is a competent vector of L. major parasites, being able to acquire parasites from active cutaneous leishmaniasis lesions, sustain mature infections, and transmit them to naive hosts, causing disease.
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http://dx.doi.org/10.1093/infdis/jiaa203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459136PMC
September 2020

Agaphelin modulates the activation of human bronchial epithelial cells induced by lipopolysaccharide and IL-4.

Immunobiology 2020 05 18;225(3):151937. Epub 2020 Mar 18.

Institute of Health Sciences, Department of Clinical Medicine, Laboratory of Experimental Immunopharmacology, Federal University of Triângulo Mineiro, Uberaba, MG, 38025-350, Brazil. Electronic address:

Sand fly saliva presents molecules with potential to development of compounds for treatment of inflammatory diseases. Agaphelin, isolated from the saliva of the mosquito Anopheles gambiae, demonstrates anti-inflammatory properties such as neutrophils chemotaxis inhibition. Here, we extend these results and evaluated the role of agaphelin (0.1-100 nM) in an in vitro model consisting in the activation of human bronchial epithelial cells (BEAS-2B) by IL-4 (50 ng/mL) or lipopolysaccharide (LPS; 10 ng/mL). Agaphelin is non-cytotoxic for BEAS-2B cells. Notably, agaphelin markedly reduces CCL2 and IL-8 production induced by IL-4 or LPS, without altering the IL-10 production. The TLR4 expression and STAT1 phosphorylation induced by LPS were inhibited by agaphlin. In addition, agaphelin decreased the phosphorylation of STAT6 induce by IL-4, whose effect was independent of IL-4-binding activity. Taken together, these findings identify agaphelin as a potential anti-inflammatory therapeutic agent for airway inflammations.
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http://dx.doi.org/10.1016/j.imbio.2020.151937DOI Listing
May 2020

Distinct gene expression patterns in vector-residing Leishmania infantum identify parasite stage-enriched markers.

PLoS Negl Trop Dis 2020 03 3;14(3):e0008014. Epub 2020 Mar 3.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America.

Background: Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking.

Methodology/principal Findings: We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum.

Conclusions: Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies.
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http://dx.doi.org/10.1371/journal.pntd.0008014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053709PMC
March 2020

Single-cell RNA sequencing of from tsetse salivary glands unveils metacyclogenesis and identifies potential transmission blocking antigens.

Proc Natl Acad Sci U S A 2020 02 21;117(5):2613-2621. Epub 2020 Jan 21.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale University, New Haven, CT 06520;

Tsetse-transmitted African trypanosomes must develop into mammalian-infectious metacyclic cells in the fly's salivary glands (SGs) before transmission to a new host. The molecular mechanisms that underlie this developmental process, known as metacyclogenesis, are poorly understood. Blocking the few metacyclic parasites deposited in saliva from further development in the mammal could prevent disease. To obtain an in-depth perspective of metacyclogenesis, we performed single-cell RNA sequencing (scRNA-seq) from a pool of 2,045 parasites collected from infected tsetse SGs. Our data revealed three major cell clusters that represent the epimastigote, and pre- and mature metacyclic trypanosome developmental stages. Individual cell level data also confirm that the metacyclic pool is diverse, and that each parasite expresses only one of the unique metacyclic variant surface glycoprotein (mVSG) coat protein transcripts identified. Further clustering of cells revealed a dynamic transcriptomic and metabolic landscape reflective of a developmental program leading to infectious metacyclic forms preadapted to survive in the mammalian host environment. We describe the expression profile of proteins that regulate gene expression and that potentially play a role in metacyclogenesis. We also report on a family of nonvariant surface proteins (Fam10) and demonstrate surface localization of one member (named SGM1.7) on mature metacyclic parasites. Vaccination of mice with recombinant SGM1.7 reduced parasitemia early in the infection. Future studies are warranted to investigate Fam10 family proteins as potential trypanosome transmission blocking vaccine antigens. Our experimental approach is translationally relevant for developing strategies to prevent other insect saliva-transmitted parasites from infecting and causing disease in mammalian hosts.
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http://dx.doi.org/10.1073/pnas.1914423117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007551PMC
February 2020

Amine-binding properties of salivary yellow-related proteins in phlebotomine sand flies.

Insect Biochem Mol Biol 2019 12 8;115:103245. Epub 2019 Oct 8.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

The amine-binding properties of sand fly salivary yellow-related proteins (YRPs) were described only in Lutzomyia longipalpis sand flies. Here, we experimentally confirmed the kratagonist function of YRPs in the genus Phlebotomus. We utilized microscale thermophoresis technique to determine the amine-binding properties of YRPs in saliva of Phlebotomus perniciosus and P. orientalis, the Old-World vectors of visceral leishmaniases causative agents. Expressed and purified YRPs from three different sand fly species were tested for their interactions with various biogenic amines, including serotonin, histamine and catecholamines. Using the L. longipalpis YRP LJM11 as a control, we have demonstrated the comparability of the microscale thermophoresis method with conventional isothermal titration calorimetry described previously. By homology in silico modeling, we predicted the surface charge and both amino acids and hydrogen bonds of the amine-binding motifs to influence the binding affinities between closely related YRPs. All YRPs tested bound at least two biogenic amines, while the affinities differ both among and within species. Low affinity was observed for histamine. The salivary recombinant proteins rSP03B (P. perniciosus) and rPorASP4 (P. orientalis) showed high-affinity binding of serotonin, suggesting their capability to facilitate inhibition of the blood vessel contraction and platelet aggregation.
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http://dx.doi.org/10.1016/j.ibmb.2019.103245DOI Listing
December 2019

RNA-seq analysis of the salivary glands and midgut of the Argasid tick Ornithodoros rostratus.

Sci Rep 2019 05 1;9(1):6764. Epub 2019 May 1.

Section of Vector Biology, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

Ornithodoros rostratus is a South American argasid tick which importance relies on its itchy bite and potential as disease vector. They feed on a wide variety of hosts and secrete different molecules in their saliva and intestinal content that counteract host defences and help to accommodate and metabolize the relatively large quantity of blood upon feeding. The present work describes the transcriptome profile of salivary gland (SG) and midgut (MG) of O. rostratus using Illumina sequencing. A total of 8,031 contigs were assembled and assigned to different functional classes. Secreted proteins were the most abundant in the SG and accounted for ~67% of all expressed transcripts with contigs with identity to lipocalins and acid tail proteins being the most representative. On the other hand, immunity genes were upregulated in MG with a predominance of defensins and lysozymes. Only 10 transcripts in SG and 8 in MG represented ~30% of all RNA expressed in each tissue and one single contig (the acid tail protein ORN-9707) represented ~7% of all expressed contigs in SG. Results highlight the functional difference of each organ and identified the most expressed classes and contigs of O. rostratus SG and MG.
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http://dx.doi.org/10.1038/s41598-019-42899-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494864PMC
May 2019

Functional and structural similarities of D7 proteins in the independently-evolved salivary secretions of sand flies and mosquitoes.

Sci Rep 2019 03 29;9(1):5340. Epub 2019 Mar 29.

The Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Rockville, Maryland, 20852, USA.

The habit of blood feeding evolved independently in many insect orders of families. Sand flies and mosquitoes belong to separate lineages of blood-feeding Diptera and are thus considered to have evolved the trait independently. Because of this, sand fly salivary proteins differ structurally from those of mosquitoes, and orthologous groups are nearly impossible to define. An exception is the long-form D7-like proteins that show conservation with their mosquito counterparts of numerous residues associated with the N-terminal domain binding pocket. In mosquitoes, this pocket is responsible for the scavenging of proinflammatory cysteinyl leukotrienes and thromboxanes at the feeding site. Here we show that long-form D7 proteins AGE83092 and ABI15936 from the sand fly species, Phlebotomus papatasi and P. duboscqi, respectively, inhibit the activation of platelets by collagen and the thromboxane A analog U46619. Using isothermal titration calorimetry, we also demonstrate direct binding of U46619 and cysteinyl leukotrienes C, D and E to the P. papatasi protein. The crystal structure of P. duboscqi ABI15936 was determined and found to contain two domains oriented similarly to those of the mosquito proteins. The N-terminal domain contains an apparent eicosanoid binding pocket. The C-terminal domain is smaller in overall size than in the mosquito D7s and is missing some helical elements. Consequently, it does not contain an obvious internal binding pocket for small-molecule ligands that bind to many mosquito D7s. Structural similarities indicate that mosquito and sand fly D7 proteins have evolved from similar progenitors, but phylogenetics and differences in intron/exon structure suggest that they may have acquired the ability to bind vertebrate eicosanoids independently, indicating a convergent evolution scenario.
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http://dx.doi.org/10.1038/s41598-019-41848-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440969PMC
March 2019

DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica.

PLoS Negl Trop Dis 2019 01 11;13(1):e0007067. Epub 2019 Jan 11.

Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.

Background: The vector-borne disease leishmaniasis is transmitted to humans by infected female sand flies, which transmits Leishmania parasites together with saliva during blood feeding. In Iran, cutaneous leishmaniasis (CL) is caused by Leishmania (L.) major and L. tropica, and their main vectors are Phlebotomus (Ph.) papatasi and Ph. sergenti, respectively. Previous studies have demonstrated that mice immunized with the salivary gland homogenate (SGH) of Ph. papatasi or subjected to bites from uninfected sand flies are protected against L. major infection.

Methods And Results: In this work we tested the immune response in BALB/c mice to 14 different plasmids coding for the most abundant salivary proteins of Ph. sergenti. The plasmid coding for the salivary protein PsSP9 induced a DTH response in the presence of a significant increase of IFN-γ expression in draining lymph nodes (dLN) as compared to control plasmid and no detectable PsSP9 antibody response. Animals immunized with whole Ph. sergenti SGH developed only a saliva-specific antibody response and no DTH response. Mice immunized with whole Ph. sergenti saliva and challenged intradermally with L. tropica plus Ph. sergenti SGH in their ears, exhibited no protective effect. In contrast, PsSP9-immunized mice showed protection against L. tropica infection resulting in a reduction in nodule size, disease burden and parasite burden compared to controls. Two months post infection, protection was associated with a significant increase in the ratio of IFN-γ to IL-5 expression in the dLN compared to controls.

Conclusion: This study demonstrates that while immunity to the whole Ph. sergenti saliva does not induce a protective response against cutaneous leishmaniasis in BALB/c mice, PsSP9, a member of the PpSP15 family of Ph. sergenti salivary proteins, provides protection against L. tropica infection. These results suggest that this family of proteins in Ph. sergenti, Ph. duboscqi and Ph. papatasi may have similar immunogenic and protective properties against different Leishmania species. Indeed, this anti-saliva immunity may act as an adjuvant to accelerate the cell-mediated immune response to co-administered Leishmania antigens, or even cause the activation of infected macrophages to remove parasites more efficiently. These findings highlight the idea of applying arthropod saliva components in vaccination approaches for diseases caused by vector-borne pathogens.
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http://dx.doi.org/10.1371/journal.pntd.0007067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345478PMC
January 2019

Coinfection With Confers Protection Against Cutaneous Leishmaniasis.

Front Immunol 2018 11;9:2855. Epub 2018 Dec 11.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, United States.

Infection with certain bacteria, parasites, and viruses alters the host immune system to influencing disease outcome. Here, we determined the outcome of a chronic infection with on cutaneous leishmaniasis (CL) caused by . C57BL/6 mice infected with were given a sub-curative treatment with diminazene aceturate then coinfected with by vector bites. Our results revealed that infection with controls CL pathology. Compared to controls, coinfected mice showed a significant decrease in lesion size ( < 0.05) up to 6 weeks post-infection and a significant decrease in parasite burden ( < 0.0001) at 3 weeks post-infection. Protection against resulted from a non-specific activation of T cells by trypanosomes. This induced a strong immune response characterized by IFN-γ production at the site of bites and systemically, creating a hostile inflammatory environment for parasites and conferring protection from CL.
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http://dx.doi.org/10.3389/fimmu.2018.02855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297747PMC
October 2019

The PAGODAS protocol: pediatric assessment group of dengue and Aedes saliva protocol to investigate vector-borne determinants of Aedes-transmitted arboviral infections in Cambodia.

Parasit Vectors 2018 Dec 20;11(1):664. Epub 2018 Dec 20.

National Center of Parasitology, Entomology, and Malaria Control, Phnom Penh, Cambodia.

Background: Mosquito-borne arboviruses, like dengue virus, continue to cause significant global morbidity and mortality, particularly in Southeast Asia. When the infectious mosquitoes probe into human skin for a blood meal, they deposit saliva containing a myriad of pharmacologically active compounds, some of which alter the immune response and influence host receptivity to infection, and consequently, the establishment of the virus. Previous reports have highlighted the complexity of mosquito vector-derived factors and immunity in the success of infection. Cumulative evidence from animal models and limited data from humans have identified various vector-derived components, including salivary components, that are co-delivered with the pathogen and play an important role in the dissemination of infection. Much about the roles and effects of these vector-derived factors remain to be discovered.

Methods/design: We describe a longitudinal, pagoda (community)-based pediatric cohort study to evaluate the burden of dengue virus infection and document the immune responses to salivary proteins of Aedes aegypti, the mosquito vector of dengue, Zika, and chikungunya viruses. The study includes community-based seroprevalence assessments in the peri-urban town of Chbar Mon in Kampong Speu Province, Cambodia. The study aims to recruit 771 children between the ages of 2 and 9 years for a three year period of longitudinal follow-up, including twice per year (rainy and dry season) serosurveillance for dengue seroconversion and Ae. aegypti salivary gland homogenate antibody intensity determinations by ELISA assays. Diagnostic tests for acute dengue, Zika and chikungunya viral infections will be performed by RT-PCR.

Discussion: This study will serve as a foundation for further understanding of mosquito saliva immunity and its impact on Aedes-transmitted arboviral diseases endemic to Cambodia.

Trial Registration: NCT03534245 registered on 23 May 2018.
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http://dx.doi.org/10.1186/s13071-018-3224-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300895PMC
December 2018

Saliva Induces Heme Oxygenase-1 Expression at Bite Sites.

Front Immunol 2018 28;9:2779. Epub 2018 Nov 28.

Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Brazil.

Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 . Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24-48 h after bites. Additionally, assays after bites and after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted infections.
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http://dx.doi.org/10.3389/fimmu.2018.02779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279893PMC
October 2019

Immunization of Experimental Dogs With Salivary Proteins From , Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against .

Front Immunol 2018 16;9:2558. Epub 2018 Nov 16.

Fundação Oswaldo Cruz, Instituto Gonçalo Moniz, Salvador, Brazil.

Metacyclic promastigotes are transmitted by sand flies that inject parasites and saliva into the host's skin. Previous studies have demonstrated that DNA plasmids encoding salivary proteins LJM17 and LJL143, when used to immunize dogs, resulted in a systemic and local Th1 cell-mediated immunity that interfered in parasite survival . Here we evaluated the ability of these same salivary antigens to induce anti- immunity and to confer protection by immunizing dogs using a novel vaccination strategy more suitable for use in the field. The strategy consisted of a single dose of plasmid followed by two doses of recombinant () expressing salivary proteins (LJM17 or LJL143). Thirty days after the final immunization, dogs were intradermally challenged with 10 promastigotes in the presence of saliva. We followed the experimentally infected dogs for 10 months to characterize clinical, parasitological, and immunological parameters. Upon vaccination, all immunized dogs presented strong and specific humoral responses with increased serum concentrations of IFN-γ, TNF, IL-7, and IL-15. The serum of dogs immunized with LJM17 also exhibited high levels of IL-2, IL-6, and IL-18. infection was established in all experimental groups as evidenced by the presence of anti- IgG, and by parasite detection in the spleen and skin. Dogs immunized with LJM17-based vaccines presented higher circulating levels of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF, CXCL10, and GM-CSF post-infection when compared with controls. Results demonstrated that relevant -specific immune responses were induced following vaccination of dogs with salivary antigen LJM17 administered in a single priming dose of plasmid DNA, followed by two booster doses of recombinant Canarypox vector. Importantly, a significant increase in pro-inflammatory cytokines and chemokines known to be relevant for protection against leishmaniasis was evidenced after challenging LJM17-vaccinated dogs as compared to controls. Although similar results were observed following immunization with LJL143, the pro-inflammatory response observed after immunization was attenuated following infection. Collectively, these data suggest that the LJM17-based vaccine induced an immune profile consistent with the expected protective immunity against canine leishmaniosis. These results clearly support the need for further evaluation of the LJM17 antigen, using a heterologous prime-boost vaccination strategy against canine visceral leishmaniosis (CVL).
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http://dx.doi.org/10.3389/fimmu.2018.02558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251279PMC
September 2019

Human antibody reaction against recombinant salivary proteins of Phlebotomus orientalis in Eastern Africa.

PLoS Negl Trop Dis 2018 12 4;12(12):e0006981. Epub 2018 Dec 4.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

Background: Phlebotomus orientalis is a vector of Leishmania donovani, the causative agent of life threatening visceral leishmaniasis spread in Eastern Africa. During blood-feeding, sand fly females salivate into the skin of the host. Sand fly saliva contains a large variety of proteins, some of which elicit specific antibody responses in the bitten hosts. To evaluate the exposure to sand fly bites in human populations from disease endemic areas, we tested the antibody reactions of volunteers' sera against recombinant P. orientalis salivary antigens.

Methodology/principal Findings: Recombinant proteins derived from sequence data on P. orientalis secreted salivary proteins, were produced using either bacterial (five proteins) or mammalian (four proteins) expression systems and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Using these recombinant proteins, human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure.

Conclusions/significance: This is the first report of screening human sera for anti-P. orientalis antibodies using recombinant salivary proteins. The recombinant salivary proteins mYEL1 and mAG5 proved to be valid antigens for screening human sera from both Sudan and Ethiopia for exposure to P. orientalis bites. The utilization of equal amounts of these two proteins significantly increased the capability to detect anti-P. orientalis antibody responses.
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http://dx.doi.org/10.1371/journal.pntd.0006981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279015PMC
December 2018

The relapsing fever spirochete Borrelia turicatae persists in the highly oxidative environment of its soft-bodied tick vector.

Cell Microbiol 2019 02 4;21(2):e12987. Epub 2019 Jan 4.

Departments of Pediatrics and Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas.

The relapsing fever spirochete Borrelia turicatae possesses a complex life cycle in its soft-bodied tick vector, Ornithodoros turicata. Spirochetes enter the tick midgut during a blood meal, and, during the following weeks, spirochetes disseminate throughout O. turicata. A population persists in the salivary glands allowing for rapid transmission to the mammalian hosts during tick feeding. Little is known about the physiological environment within the salivary glands acini in which B. turicatae persists. In this study, we examined the salivary gland transcriptome of O. turicata ticks and detected the expression of 57 genes involved in oxidant metabolism or antioxidant defences. We confirmed the expression of five of the most highly expressed genes, including glutathione peroxidase (gpx), thioredoxin peroxidase (tpx), manganese superoxide dismutase (sod-1), copper-zinc superoxide dismutase (sod-2), and catalase (cat) by reverse-transcriptase droplet digital polymerase chain reaction (RT-ddPCR). We also found distinct differences in the expression of these genes when comparing the salivary glands and midguts of unfed O. turicata ticks. Our results indicate that the salivary glands of unfed O. turicata nymphs are highly oxidative environments where reactive oxygen species (ROS) predominate, whereas midgut tissues comprise a primarily nitrosative environment where nitric oxide synthase is highly expressed. Additionally, B. turicatae was found to be hyperresistant to ROS compared with the Lyme disease spirochete Borrelia burgdorferi, suggesting it is uniquely adapted to the highly oxidative environment of O. turicata salivary gland acini.
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http://dx.doi.org/10.1111/cmi.12987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6454574PMC
February 2019

Comparative Evolution of Sand Fly Salivary Protein Families and Implications for Biomarkers of Vector Exposure and Salivary Vaccine Candidates.

Front Cell Infect Microbiol 2018 29;8:290. Epub 2018 Aug 29.

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, United States.

Sand fly salivary proteins that produce a specific antibody response in humans and animal reservoirs have been shown to be promising biomarkers of sand fly exposure. Furthermore, immunity to sand fly salivary proteins were shown to protect rodents and non-human primates against infection. We are missing critical information regarding the divergence amongst sand fly salivary proteins from different sand fly vectors, a knowledge that will support the search of broad or specific salivary biomarkers of vector exposure and those for vaccines components against leishmaniasis. Here, we compare the molecular evolution of the salivary protein families in New World and Old World sand flies from 14 different sand fly vectors. We found that the protein families unique to OW sand flies are more conserved than those unique to NW sand flies regarding both sequence polymorphisms and copy number variation. In addition, the protein families unique to OW sand flies do not display as many conserved cysteine residues as the one unique to the NW group (28.5% in OW vs. 62.5% in NW). Moreover, the expression of specific protein families is restricted to the salivary glands of unique sand fly taxon. For instance, the ParSP15 family is unique to the Larroussius subgenus whereas phospholipase A2 is only expressed in member of Larroussius and Adlerius subgenera. The SP2.5-like family is only expressed in members of the Phlebotomus and Paraphlebotomus subgenera. The sequences shared between OW and NW sand flies have diverged at similar rates (38.7 and 45.3% amino acid divergence, respectively), yet differences in gene copy number were evident across protein families and sand fly species. Overall, this comparative analysis sheds light on the different modes of sand fly salivary protein family divergence. Also, it informs which protein families are unique and conserved within taxon for the choice of taxon-specific biomarkers of vector exposure, as well as those families more conserved across taxa to be used as pan-specific vaccines for leishmaniasis.
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http://dx.doi.org/10.3389/fcimb.2018.00290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123390PMC
August 2019

Antibody targeting of a specific region of blocks malaria transmission.

NPJ Vaccines 2018 10;3:26. Epub 2018 Jul 10.

1Laboratory of Malaria and Vector Research, National Insti6tute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852 USA.

Transmission-blocking vaccines are based on eliciting antibody responses in the vertebrate host that disrupt parasite development in the mosquito vector and prevent malaria transmission. The surface protein Pfs47 is present in gametocytes and female gametes. The potential of Pfs47 as a vaccine target was evaluated. Soluble full-length recombinant protein, consisting of three domains, was expressed in as a thioredoxin fusion (T-Pfs47). The protein was immunogenic, and polyclonal and monoclonal antibodies (mAb) were obtained, but they did not confer transmission blocking activity (TBA). All fourteen mAb targeted either domains 1 or 3, but not domain 2 (D2), and immune reactivity to D2 was also very low in polyclonal mouse IgG after T-Pfs47 immunization. Disruption of the predicted disulfide bond in D2, by replacing cysteines for alanines (C230A and C260A), allowed expression of recombinant D2 protein in . A combination of mAbs targeting D2, and deletion proteins from this domain, allowed us to map a central 52 amino acid (aa) region where antibody binding confers strong TBA (78-99%). This 52 aa antigen is immunogenic and well conserved, with only seven haplotypes world-wide that share 96-98% identity. Neither human complement nor the mosquito complement-like system are required for the observed TBA. A dramatic reduction in ookinete numbers and ookinete-specific transcripts was observed, suggesting that the antibodies are interacting with female gametocytes and preventing fertilization.
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http://dx.doi.org/10.1038/s41541-018-0065-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039440PMC
July 2018