Biology subjects

Kamhawi, S.

Publications and source records attributed to Kamhawi, S..

14 recordsLinked to original sources

bioRxiv2026

The PEARL toolkit: Using sand flies to identify leishmaniasis animal reservoirs

Animal reservoirs remain unknown in emerging and most endemic leishmaniasis foci hindering control efforts. Here, we developed a field-applicable PERL (Phlebotomines Establish Reservoirs of Leishmania) toolkit using individual blood fed sand flies (IBF) to identify leishmaniasis reservoirs. Using IBF, we optimized DNA and RNA co-extraction and parasite detection of [≥]1 parasite/s by kDNA qPCR and ssu rRNA RT-qPCR. We then screened IBF for expression of two parasite genes, sherp and HPB, identified by RNAseq as having low-to-absent expression in IBF given a first Leishmania donovani-infected blood meal (IBF-iBM1) and high expression in specimens provided subsequent uninfected blood meals (IBF-BMS+). Linear discriminant analysis of target gene expression classified iBM1 parasites with a predictive accuracy of ~87% and ~82% in membrane- or naturally-fed on hamsters sand flies, respectively. By determining the blood source in specimens determined as IBF-iBM1, the PERL toolkit provides an innovative and practical approach to identification of leishmaniasis reservoirs.

Molecular Biology↗

bioRxiv2025

Discovery of an Adaptive Neuroimmune Response Driving Itch and Fast Tick Removal with Implications for Preventing Pathogen Transmission

Acquired tick resistance (ATR) is well characterized in tick-exposed animals, compromising tick fitness through antibody-mediated activation of basophils. Yet, anti-tick vaccines inducing ATR have had limited success. Here, we describe a neuroimmune event preceding ATR that leads to rapid host-mediated tick removal. Tick-sensitized guinea pigs mechanically remove ticks within 3-6 hours via an acquired neuroimmune-induced itch response that correlates with increased dermal expression of itch-associated genes like OSM and skin infiltration by T cells and macrophages, independently of IgG and IgE antibodies. When we expose humans to ticks, a similar immune response is observed. Blocking T cells before tick sensitization prevents immune cell infiltration to bite sites and abrogates scratching and tick removal. This neuroimmune response is independent of Trpv1 as tick-sensitized guinea pigs treated with resiniferatoxin remove ticks effectively. Itch-induced tick removal or IITR offers a novel approach to tick-borne disease prevention through early tick detection and removal.

neuroscience↗

bioRxiv2025

Comprehensive evaluation of antibody responses to mosquitoes and mosquito-borne arboviruses using highly multiplexed serology

Vector-borne diseases (VBDs) are a leading cause of morbidity and mortality worldwide, and their future impact may increase due to climate change. Antigens driving host immune responses to pathogens and vectors can serve as vaccine candidates and biomarkers of previous exposure, but the immunogenicity of the relevant proteomes remains undercharacterized. To comprehensively profile antibody responses to VBDs and to the vectors themselves, we developed a highly multiplexed phage display library (VectorScan) containing over 250,000 peptides derived from diverse arthropod vectors and prevalent vector-borne pathogens. We used phage immunoprecipitation sequencing (PhIP-Seq) to screen VectorScan against blood samples from non-human primates and humans with experimental and natural exposures to arboviruses and mosquitoes (Anopheles gambiae and Aedes aegypti). We analyzed quantitative measurements of peptide seroreactivity to identify epitopes driving viral serotype-level exposure signatures, as well as novel mosquito sialome antigens. Mosquito-directed antibody responses were associated with natural viral exposure, but highly heterogeneous at an individual level. However, recurrent responses to insect-derived cuticular proteins, mucin-like proteins, and fibrinogen-like proteins will inform development of future serosurveillance tools for vector exposure and vector-based vaccines.

immunology↗

bioRxiv2025

Lutzomyia longipalpis salivary proteins elicit human innate and adaptive immune responses detrimental to Leishmania parasites

In Leishmania-endemic areas, humans are constantly exposed to sand fly bites. To explore the immune consequences of this chronic vector exposure, we performed a human challenge study with the sand fly Lutzomyia longipalpis. Peripheral blood mononuclear cells were collected from fifteen healthy volunteers who underwent multiple controlled exposures to sand fly bites. We identified two Lu. longipalpis salivary proteins, LJM19 and LJL143, which elicited type 1 cytokine responses in cells from exposed individuals and which correlated with enhanced killing of Leishmania parasites in co-cultured macrophages. Interestingly, LJM19 also exerted this parasite-killing effect in cells from unexposed individuals, consistent with innate immune activation. In support of this, both LJM19 and LJL143 stimulated the production of the innate cytokines IL-1{beta} and IFN-. Our results demonstrate that repeated exposure to sand fly bites induces innate and adaptive cytokine responses to vector salivary proteins that can be co-opted to protect humans against Leishmania infection.

immunology↗

bioRxiv2022

Genomic Analysis of Two Phlebotomine Sand Fly Vectors of Leishmania from the New and Old World

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the devastating kinetoplastid parasites of the genus Leishmania, the causative agents of diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. As no approved efficacious vaccine exists, available drugs are expensive and/or toxic, and resistance is emerging, management of sand fly populations to break transmission is currently the most effective disease control strategy. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two of the most important sand fly species: Phlebotomus papatasi, a cutaneous leishmaniasis vector, (distributed in the Middle East and North Africa) and Lutzomyia longipalpis, a visceral leishmaniasis vector (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites. Author SummaryThe leishmaniases are a group of neglected tropical diseases caused by protist parasites from the Genus Leishmania. Different Leishmania species present a wide clinical profile, ranging from mild, often self-resolving cutaneous lesions that can lead to protective immunity, to severe metastatic mucosal disease, to visceral disease that is ultimately fatal. Leishmania parasites are transmitted by the bites of sand flies, and as no approved vaccine exists, available drugs are toxic and/or expensive and resistance is emerging, new dual control strategies to combat these diseases must be developed, combining interventions on human infections and integrated sand fly population management. Effective vector control requires a good understanding of the biology of sand flies. To this end, we sequenced and annotated the genomes of two sand fly species that are important leishmaniasis vectors from the Old and New Worlds. These genomes allow us to better understand, at the genetic level, processes important in the vector biology of these species, such as finding hosts, blood-feeding, immunity, and detoxification. These genomic resources highlight the driving forces of evolution of two major Leishmania vectors and provide foundations for future research on how to better prevent leishmaniasis by control of the sand fly vectors.

genomics↗

bioRxiv2022

Centrin-deficient Leishmania mexicana confers protection against Old World visceral leishmaniasis

Leishmaniasis is one of the top neglected tropical diseases with significant morbidity and mortality in low and middle-income countries (LMIC). However, this disease is also spreading in the developed world. Currently, there is a lack of effective strategies to control this disease. Vaccination can be an effective measure to control leishmaniasis and has the potential to achieve disease elimination. Recently, we have generated centrin gene-deleted new world L. mexicana (LmexCen-/-) parasites using CRISPR/Cas9 and showed that they protect mice against a homologous L. mexicana infection that causes cutaneous disease. In this study, we tested whether LmexCen-/- parasites can also protect against visceral leishmaniasis caused by L. donovani in a hamster model. We show that immunization with LmexCen-/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver and lack of mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the Th2 response as indicated by a significant reduction in the anti-inflammatory cytokines such as IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-{gamma} resulting in higher IFN-{gamma}/IL-10 and IFN-{gamma}/IL4 ratios compared to non-immunized animals. This contrasts with our studies with L. major centrin deletion mutants that showed a dominant Th1 response compared to L. major wild-type infection suggesting the divergent mechanisms of protection in the two mutant parasites. LmexCen-/- immunization resulted in long-lasting protection against L. donovani infection. Further, since the efficacy of LmCen-/- has not been determined against Leishmania strains prevalent in the Americas, LmexCen-/- may be a viable alternative. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against old world visceral leishmaniasis.

immunology↗

bioRxiv2022

Genetic exchange in Leishmania is facilitated by IgM natural antibodies

Host factors mediating Leishmania genetic exchange are not well defined. Here, we demonstrate that IgM antibodies, but not IgG or IgA, facilitate parasite genetic hybridization in vitro and in vivo. IgM induces the gradual and transient formation of a structured parasite clump in a process essential for L. major and L. tropica hybridization in vitro. Parasite hybrids and 3-nucleated parasites were observed inside this structure, named the Leishmania mating clump. IgM was also required for or significantly increased Leishmania hybrid formation in vivo. At minimum, we observed a 12-fold increase in the proportion of hybrids recovered from sand flies provided a second blood meal containing IgM compared to controls. Notably, genetic backcross events in sand flies were only observed in the presence of IgM, and were reproducibly recovered, reinforcing the relevance of IgM for Leishmania genetic exchange in vivo. The in vitro and in vivo Leishmania crosses from these studies resulted in full genome hybrids. Leishmania co-option of a host antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector coevolution that promotes parasite diversity and fitness through genetic exchange.

microbiology↗

bioRxiv2021

Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism

BackgroundDogs are the primary reservoir for human visceral leishmaniasis due to Leishmania infantum. Phlebotomine sand flies maintain zoonotic transmission of parasites between dogs and humans. A subset of dogs is infected transplacentally during gestation, but at what stage of the clinical spectrum vertically infected dogs contribute to the infected sand fly pool is unknown. Methodology/Principal FindingsWe examined infectiousness of dogs vertically infected with L. infantum from multiple clinical states to the vector Lutzomyia longipalpis using xenodiagnosis and found that vertically infected dogs were infectious to sand flies at differing rates. Dogs with moderate disease showed significantly higher transmission to the vector than dogs with very mild or severe disease. We documented a substantial parasite burden in the skin of vertically infected dogs by RT-qPCR, despite these dogs not having received intradermal parasites via sand flies. There was a highly significant correlation between skin parasite burden at the feeding site and sand fly parasite uptake. This suggests dogs with high skin parasite burden contribute the most to the infected sand fly pool. Although skin parasite load and parasitemia correlated with one another, the average parasite number detected in skin was significantly higher compared to blood in matched subjects. Thus, dermal resident parasites were infectious to sand flies from dogs without detectable parasitemia. Conclusions/SignificanceTogether, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies. AUTHOR SUMMARYSand flies transmit Leishmania parasites between infected dogs and humans leading to the life-threatening tropical disease Visceral Leishmaniasis (VL). Identifying which dogs transmit parasites well to sand flies is important to curb disease spread. The offspring of both dogs and humans can also be infected vertically while in utero. Despite this, the infectiousness of dogs that receive parasites in utero to sand flies has not been thoroughly investigated. Thus, we allowed sand flies to feed on a group of vertically infected dogs at varying stages of VL disease severity and measured sand fly parasite uptake. We found vertically infected dogs were readily able to transmit parasites to the sand flies. Dogs that were most infectious had moderate clinical disease and relatively high levels of parasite infection in their blood and skin. However, the level of skin infection was significantly higher than that observed in the blood, and the skin parasite load had the strongest correlation with sand fly parasite uptake. This implicates the skin may be an underappreciated driver of canine infectiousness to the sand fly vector. In addition, this work highlights that vertically infected dogs are very important parts of the transmission cycle and must be considered in all public health efforts addressing VL.

microbiology↗

bioRxiv2020

Preclinical validation of a second generation leishmanization vaccine against vector transmitted fatal visceral leishmaniasis

Visceral Leishmaniasis (VL) is fatal if untreated. There is no licensed vaccine available against human leishmaniasis. We recently demonstrated protection in mice against L. major infection using a CRISPR genome edited attenuated Leishmania major strain (LmCen-/-). Here, as a pre-clinical step, we evaluated the protective efficacy of LmCen-/- against VL induced by sand fly transmitted Leishmania donovani in hamsters. Intradermal immunization of hamsters with LmCen-/- did not develop any lesion; while still priming a pro-inflammatory immune response. When challenged with L. donovani either by intradermal needle injection or by infected sand flies, LmCen-/--immunized hamsters were protected, not showing spleen or liver pathology averting VL fatality compared to control animals. Spleen cells from LmCen-/- immunized and infected sand fly challenged hamsters produced significantly higher Th1-associated cytokines and chemokines including IFN-{gamma} and TNF-, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized challenged animals. We further developed a GLP-grade LmCen-/- which showed equal protection as laboratory-grade LmCen-/- parasites in hamsters. Importantly, GLP-grade LmCen-/- parasites also induced a proinflammatory immune response in the PBMCs isolated from healthy people living in non-endemic and endemic for VL as well as cured VL people living in endemic region. Together, this study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and it is a strong candidate vaccine to be tested in a human clinical trial.

immunology↗

bioRxiv2020

Binding of Leishmania infantum LPG to the midgut is not sufficient to define vector competence in Lutzomyia longipalpis sand flies

The major surface lipophosphoglycan (LPG) of Leishmania parasites is critical to vector competence in restrictive sand fly vectors by mediating Leishmania 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 Leishmania remains elusive. We tested binding of Leishmania infantum wild type (WT), LPG-defective ({Delta}lpg1 mutants) and add-back lines ({Delta}lpg1 + LPG1) to sand fly midguts in vitro and their survival in Lutzomyia longipalpis sand flies in vivo. Le. infantum WT parasites attached to the Lu. longipalpis midgut in vitro with late-stage parasites binding to midguts in significantly higher numbers compared to early-stage stage promastigotes. {Delta}lpg1 mutants did not bind to Lu. longipalpis midguts, and this was rescued in the {Delta}lpg1 + LPG1 lines, indicating that midgut binding is mediated by LPG. When Lu. longipalpis sand flies were infected with either Le. infantum WT, {Delta}lpg1, or {Delta}lpg1 + LPG1 of the BH46 or BA262 strains, the BH46 {Delta}lpg1 mutant, but not the BA262 {Delta}lpg1 mutant, survived and grew to similar numbers as the WT and {Delta}lpg1 + LPG1 lines. Exposure of BH46 and BA262 {Delta}lpg1 mutants to blood engorged midgut extracts led to the mortality of the BA262 {Delta}lpg1 but not the BH46 {Delta}lpg1 parasites. These findings suggest that Le. infantum LPG protects parasites on a strain-specific basis early in infection, likely against toxic components of blood digestion, however, it is not necessary to prevent Le. infantum evacuation along with the feces in the permissive vector Lu. longipalpis. IMPORTANCEIt is well established that LPG is sufficient to define the vector competence of restrictive sand fly vectors to Leishmania parasites. However, the permissiveness of other sand flies to multiple Leishmania species suggests that other factors might define vector competence for these vectors. In this study, we investigated the underpinnings of Leishmania infantum survival and development in its natural vector Lutzomyia longipalpis. We found out that LPG-mediated midgut binding persists in late-stage parasites. This observation is paradigm-changing and suggests that only a subset of infective 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 Leishmania infantum retention in the midgut of Lutzomyia longipalpis, 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 complex and should be investigated on a strain-specific basis.

microbiology↗

bioRxiv2020

A Second Generation Leishmanization Vaccine with a Markerless Attenuated Leishmania major Strain using CRISPR gene editing

Leishmaniasis is a debilitating and often fatal 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 unacceptable skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is the first genetically engineered gene deleted Leishmania strain that is antibiotic resistant marker free and does not have any off-target mutations. Mice immunized with LmCen-/- had virtually no visible lesions following challenge with L. major-infected sand flies while non-immunized animals developed large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization showed protection and an immune response comparable to leishmanization. LmCen-/- is safe since it was unable to cause disease even 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.

microbiology↗

bioRxiv2019

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

BackgroundPhlebotomine sand flies are the vectors of Leishmania worldwide. To develop in the sand fly midgut, Leishmania multiplies and undergoes multiple stage differentiations leading to the infective form, the metacyclic promastigotes. To gain a better understanding of the influence of Leishmania infection in midgut gene expression, we performed RNA-Seq comparing uninfected Lutzomyia longipalpis midguts and Leishmania infantum-infected Lutzomyia longipalpis midguts at seven time points which cover the various developmental Leishmania stages including early time points when blood digestion is taking place and late time points when the parasites are undergoing metacyclogenesis. ResultsOut of over 13,841 transcripts assembled de novo, only 113 sand fly transcripts, about 1%, were differentially expressed. Further, we observed a low overlap of differentially expressed sand fly transcripts across different time points suggesting a specific influence of each Leishmania stage on midgut gene expression. Two main patterns of sand fly gene expression modulation were noticed. 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 4 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 small fold changes (< 32 fold), and the molecular function of such genes are associated with the metabolism of lipids and detoxification of xenobiotics (P450). ConclusionOverall, it appears that Leishmania modulates sand fly gene expression early on in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points, when modest midgut gene expression changes correlate with a massive amount of parasites in the anterior midgut.

microbiology↗

bioRxiv2019

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

Promastigotes of Leishmania infantum undergo a series of extracellular developmental stages inside the natural sand fly vector Lutzomyia longipalpis 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. 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 mapped the sequences corresponding to the procyclic, nectomonad, leptomonad or metacyclic promastigote stage into distinct positions, with the procyclic stage being the most divergent population. Transcriptional levels across genes varied on average between 10- to 100-fold. Comparison between procyclic and nectomonad promastigotes resulted in 836 differentially expressed (DE) genes; between nectomonad and leptomonad promastigotes in 113 DE genes; and between leptomonad and metacyclic promastigotes in 302 DE genes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each 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 stage-specific markers for L. Infantum. Leading stage-specific marker candidates include genes encoding a zinc transporter in procyclics, a beta-fructofuranidase in nectomonads, a surface antigen-like protein in leptomonads, and an amastin-like surface protein in metacyclics. Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of stage-specific markers for further development as molecular tools for epidemiological studies.

microbiology↗

bioRxiv2019

Phlebotomus papatasi sand fly salivary protein diversity and immune response potential in Egypt and Jordan populations

Phlebotomus papatasi sand flies inject their hosts with a myriad of pharmacologically active salivary proteins to assist with blood feeding and to modulate host defenses. These salivary proteins have been studied for their role in cutaneous leishmaniasis disease outcome with different salivary proteins attenuating or exacerbating lesion size. Studies have shown that while co-administered sand fly saliva exacerbates Leishmania major infections in naive mice, animals pre-exposed to saliva are protected, with the infection attenuated via a delayed-type hypersensitivity immune reaction. These studies highlight the potential of the salivary components to be used as a vaccine. One protein in particular, P. papatasi salivary protein 15 (PpSP15) has been intensively studied because of its ability to protect mice against Le. major challenge. The number of antigenic molecules included in vaccines is restricted thus emphasizing the role of population genetics to identify molecules, like PpSP15, that are functionally significant, conserved across populations and do not experience selection. Three distinct ecotope study sites, one in Egypt (Aswan) and two in Jordan (Swaimeh and Malka), were chosen based on their elevation, rainfall, vegetation, differing reservoir species, and the presence or absence of Le. major. The objective of this work was to analyze the genetic variability of nine of the most abundantly expressed salivary proteins including PpSP12, PpSP14, PpSP28, PpSP29, PpSP30, PpSP32, PpSP36, PpSP42, and PpSP44 and to predict their ability to elicit an immune response. Two proteins, PpSP12 and PpSP14, demonstrated low genetic variability across the three sand fly populations represented in this study, with multiple predicted MHCII epitope binding sites, identified by alleles present in the human populations from the study sites. The other seven salivary proteins revealed greater allelic variation across the same sand fly populations indicating that their use as vaccine targets may prove to be challenging.

genetics↗