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Principal Investigator: Jesus Valenzuela
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2024
Award: $1,126,987
Funding agency: National Institute of Allergy and Infectious Diseases
The accomplishment of the section are:
1. In this study, we focused on host factors that mediate Leishmania genetic exchange and explore whether host blood ingested by sand flies provides some elements absent in culture medium that promotes cell fusion and the formation of hybrid parasites. Leishmania parasites are considered mostly asexual, reproducing through clonal propagation. However, the presence of natural hybrids and the demonstration of genetic exchange in the sand fly vector indicate that genetic exchange occurs but is facultative, as seen in other eukaryotic microorganisms. Genetic exchange is a powerful evolutionary route that facilitates the development of pathogenicity in microorganisms and offers an experimental tool for dissecting complex phenotypes that underlie virulence.
We demonstrated that natural IgM (IgMn) antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite–host–vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.
2. In collaboration with the Infectious Disease Division, Department of Medicine, Uniformed Services University of the Health Sciences, we characterized the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector. We repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.
3. We tested 563 serum samples from a longitudinal pediatric cohort study previously conducted in Cambodia. Children enrolled in the study were dengue-naive at baseline and were followed biannually for dengue incidence for two years. We used Western blotting and enzyme-linked immunosorbent assays to identify immunogenic Aedes aegypti salivary proteins and measure total anti-Ae. aegypti IgG. We found a correlation (rs=0.86) between IgG responses against AeD7L1 and AeD7L2 recombinant proteins and those to whole salivary gland homogenate. We observed seasonal fluctuations of AeD7L1+2 IgG responses and no cross-reactivity with Culex quinquefasciatus and Anopheles dirus mosquitoes. The baseline median AeD7L1+2 IgG responses for young children were higher in those who developed asymptomatic versus symptomatic dengue. The IgG response against AeD7L1+2 recombinant proteins is a highly sensitive and Aedes specific marker of human exposure to Aedes bites that can facilitate standardization of future serosurveys and epidemiological studies by its ability to provide a robust estimation of human-mosquito contact in a high-throughput fashion.
4. In collaboration with the FDA and McGill University, we established a Leishmania donovani strain and described the production, under Good Laboratory Practice conditions, of leishmanin soluble antigen used to induce the leishmanin skin test in animal models of infection and vaccination. Using a mouse model of cutaneous leishmaniasis both soluble antigen induced a leishmanin skin test response following infection and vaccination with live attenuated Leishmania major (LmCen-/-). Both the CD4+ and CD8+ T-cells were necessary for the leishmanin skin test response. This study demonstrates the feasibility of large-scale production of leishmanin antigen addressing a major bottleneck for performing the leishmanin skin test in future surveillance and vaccine clinical trials.
5 . We reported the detrimental interaction between sand flies and Tyrophagus sp. and S. scimitus in a closed laboratory sand fly colony, discussed their impact on sand fly production and provided guidelines for limiting the mite population size in a closed laboratory colony leading to improved sand fly yields. Mites were collected from sand fly larval rearing pots and morphologically identified using taxonomic keys. Upon identification, they were photographed with a scanning electron microscope. Several mite control measures were adopted in two different laboratories, one at the Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases-National Institutes of Health (Rockville, MD, USA), and the other at the University of Calgary (Calgary, AB, Canada). The mite species associated with sand fly colonies were morphologically identified as Tyrophagus sp. and Stratiolaelaps scimitus. While complete eradication of mites in sand fly colonies is considered unrealistic, drastically reducing their population has been associated with higher sand fly productivity.
6. In collaboration with the Department of Parasitology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil,select transcripts corresponding to putative immunogenic proteins in the tick Amblyoma sculptum gut epithelial membrane, produce recombinant proteins and evaluate them as antigens against A. sculptum tick infestations. Three gut proteins - AsMucin, AsAPP, and AsLAMP - and a chimeric protein (rAsChimera) based on 22 peptides containing putative B cell epitopes from seven different gut proteins were evaluated as anti-A. sculptum antigens. Mice immunizations revealed that all recombinant targets elicited humoral response with significantly increased IgG levels compared to controls. For rAsChimera, IgG levels remained significantly higher than controls up to 75 days after the end of the immunization. Challenge trials revealed that vaccination with the chimeric protein was the most effective against the tick A. sculptum, inducing 100 % nymph mortality and reaching 80.8 % efficacy against females.
Terms: <0-11 years old><19S Gamma Globulin><7S Gamma Globulin><Address><Adenosine Aminohydrolase><Adopted><Aedes><Animal Model><Animal Models and Related Studies><Animals><Anopheles><Anopheles Genus><Anophelines><Antibodies><Antibody Response><Antigens><Attenuated><B burgdorferi><B-Cell Epitopes><B-Lymphocyte Epitopes><B. burgdorferi><Binding><Bio-Informatics><Biochemical><Bioinformatics><Biological Markers><Biopsy><Bite><Blood><Blood Eosinophil><Blood Plasma><Blood Reticuloendothelial System><Blood Serum><Borrelia burgdorferi><Borrelia burgdorferi sensu stricto><Borreliella burgdorferi><Brazil><CD8 Cell><CD8 T cells><CD8 lymphocyte><CD8+ T cell><CD8+ T-Lymphocyte><CD8-Positive Lymphocytes><CD8-Positive T-Lymphocytes><CSIF><CSIF-10><Cambodia><Canada><Cell Mediated Immunology><Cell fusion><Cell-Mediated Immunity><Cellular Immunity><Child><Child Youth><Children (0-21)><Chimera Protein><Chimeric Proteins><Clinical Protocols><Cohort Studies><Collaborations><Communicable Diseases><Complex><Concurrent Studies><Culex><Culex (Genus)><Culicidae><Cutaneous Leishmaniasis><Cytokine Synthesis Inhibitory Factor><DNA><DNA Molecular Biology><Dengue><Deoxyribonucleic Acid><Development><ELISA><Electron Microscope><Elements><Enrollment><Enzyme-Linked Immunosorbent Assay><Eosinophilic Granulocyte><Eosinophilic Leukocyte><Epidemiologic Research><Epidemiologic Studies><Epidemiological Studies><Epidemiology Research><Event><Exposure to><Female><Fusion Protein><Future><General Taxonomy><Genetic><Glycohydrolases><Glycosidases><Glycoside Hydrolases><Goals><Guidelines><Gut Epithelium><H and E><Health Sciences><Hematoxylin and Eosin><Hematoxylin and Eosin Staining Method><Host Factor><Host Factor Protein><Human><Human Volunteers><Hybrids><Hypermelanoses><Hypermelanosis><Hyperpigmentation><Hypersensitivity skin testing><IFN-Gamma><IFN-g><IFN-γ><IFNG><IFNγ><IL-10><IL10><IL10A><IgG><IgM><Immune><Immune Interferon><Immune response><Immunes><Immunization><Immunoblotting><Immunochemical Immunologic><Immunoglobulin G><Immunoglobulin M><Immunologic><Immunological><Immunological response><Immunologically><Immunologics><In Vitro><Incidence><Infection><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Infiltration><Ingestion><Insect Bites><Insect Vectors><Integration Host Factors><Interferon Gamma><Interferon Type II><Interleukin 10 Precursor><Interleukin-10><Ixodida><Kampuchea><Khmer Republic><Knowledge><L donovani><L. donovani><Laboratories><Leishmania><Leishmania (Leishmania) donovani><Leishmania (Leishmania) major><Leishmania donovani><Leishmania major><Leishmania tropica major><Leishmaniasis><Lyme Borreliosis><Lyme Disease><Lyme Disease Spirochete><Malaria><Marrow Eosinophil><Measures><Mediating><Medicine><Membrane><Mice><Mice Mammals><Midgut><Mite Controls><Mites><Modern Man><Molecular><Molecular Biology><Molecular Interaction><Mononuclear><Montenegro antigen><Morphology><Mosquitoes><Murine><Mus><NIAID><NIH><National Institute of Allergy and Infectious Disease><National Institutes of Health><Nymph><PBMC><Paludism><Parasites><Parasitic Diseases><Parasitology><Partner in relationship><Pathogenicity><Pattern><Pediatric cohort><Peptides><Peripheral Blood Mononuclear Cell><Phenotype><Phlebotominae><Phlebotomus><Plasma><Plasma Serum><Plasmodium Infections><Play><Population><Population Sizes><Production><Productivity><Proteins><Punch Biopsy><RNA immunization><RNA vaccination><Reaction><Recombinant Proteins><Recombinants><Reporting><Research><Resistance><Reticuloendothelial System, Serum, Plasma><Role><Route><Saliva><Salivary><Salivary Gland Proteins><Salivary Glands><Salivary Glands Head and Neck><Salivary Proteins><Sampling><Scanning><Seasons><Serum><Site><Skin><Skin Tests><Staining method><Stains><Standardization><Surface><T8 Cells><T8 Lymphocytes><Taxonomy><Testing><Tick Infestations><Tick-Borne Diseases><Ticks><Time><Transcript><Translating><Transmission><United States National Institutes of Health><Universities><University Health Services><Vaccination><Vaccine Clinical Trial><Vaccines><Virulence><Western Blotting><Western Immunoblotting><adenosine deaminase><anti-tick immunization><anti-tick vaccine><arm><asexual><attenuate><attenuates><bio-markers><biologic marker><biomarker><communicable disease transmission><compare to control><comparison control><cross reactivity><dermal leishmaniasis><developmental><disease control><disease transmission><disorder control><enroll><entire genome><enzyme linked immunoassay><eosinophil><epidemiologic investigation><epidemiology study><exposed human population><fitness><full genome><full scale manufacturing><gastrointestinal epithelium><good laboratory practice><host response><human exposure><hypersensitivity test><immune system response><immunization against ticks><immunogen><immunogenic><immunologic skin test><immunoresponse><improved><in vivo><infectious disease transmission><infested with ticks><ingest><kids><lFN-Gamma><large scale manufacturing><large scale production><leishmanin><lipophosphoglycan><lipophosphonoglycan><lyme spirochete><mRNA immunization><mRNA vaccination><macula><macular><mass production><mate><membrane structure><microorganism><model of animal><molecular vector><mortality><mouse model><murine model><natural antibodies><neglect><pathogen><protein blotting><resistant><response><sandfly><serology survey><serosurvey><social role><success><tick bite><tick infested><tick-borne illness><tick-borne pathogen><tickborne disease><tickborne illness><tickborne pathogen><tool><transmission process><vaccine against ticks><vector><vector-borne><vectorborne><volunteer><whole genome><youngster>