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Characterization of a new Leishmania major strain for use in a controlled human infection model.

ABSTRACT: Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical strain of Leishmania major. Two fresh strains of L. major from Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.

SUBMITTER: Ashwin H

PROVIDER: S-EPMC7801518 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Characterization of a new Leishmania major strain for use in a controlled human infection model.

Ashwin Helen H Sadlova Jovana J Vojtkova Barbora B Becvar Tomas T Lypaczewski Patrick P Schwartz Eli E Greensted Elizabeth E Van Bocxlaer Katrien K Pasin Marion M Lipinski Kai S KS Parkash Vivak V Matlashewski Greg G Layton Alison M AM Lacey Charles J CJ Jaffe Charles L CL Volf Petr P Kaye Paul M PM

Nature communications 20210111 1

Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the i ...[more]

PMID: 33431825

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Project description:BackgroundIn vitro assays are widely used in studies on pathogen infectivity, immune responses, drug and vaccine discovery. However, most in vitro assays display significant differences to the in vivo situation and limited predictive properties. We applied medium perfusion methods to mimic interstitial fluid flow to establish a novel infection model of Leishmania parasites.MethodsLeishmania major infection of mouse peritoneal macrophages was studied within the Quasi Vivo QV900 macro-perfusion system. Under a constant flow of culture media at a rate of 360μl/min, L. major infected macrophages were cultured either at the base of a perfusion chamber or raised on 9mm high inserts. Mathematical and computational modelling was conducted to estimate medium flow speed, shear stress and oxygen concentration. The effects of medium flow on infection rate, intracellular amastigote division, macrophage phagocytosis and macropinocytosis were measured.ResultsMean fluid speeds at the macrophage cell surface were estimated to be 1.45 x 10-9 m/s and 1.23 x 10-7 m/s for cells at the base of the chamber and cells on an insert, respectively. L. major macrophage infection was significantly reduced under both media perfusion conditions compared to cells maintained under static conditions; a 85±3% infection rate of macrophages at 72 hours in static cultures compared to 62±5% for cultures under slow medium flow and 55±3% under fast medium flow. Media perfusion also decreased amastigote replication and both macrophage phagocytosis (by 44±4% under slow flow and 57±5% under fast flow compared with the static condition) and macropinocytosis (by 40±4% under slow flow and 62±5% under fast flow compared with the static condition) as measured by uptake of latex beads and pHrodo Red dextran.ConclusionsPerfusion of culture medium in an in vitro L. major macrophage infection model (simulating in vivo lymphatic flow) reduced the infection rate of macrophages, the replication of the intracellular parasite, macrophage phagocytosis and macropinocytosis with greater reductions achieved under faster flow speeds.

Project description:BackgroundLeishmania (L.) are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide non-coding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts.Methodology/principal findingsWe report in this study a comprehensive analysis of miRNA expression profiles in L. major-infected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24 h). We show that expression of 64 out of 365 analyzed miRNAs was consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1?-dependent manner, though it did not contribute to inhibiting anti-apoptotic pathways through pro-apoptotic caspase-3 regulation.Conclusions/significanceOur data suggest that alteration in miRNA levels likely plays an important role in regulating macrophage functions following L. major infection. These results could contribute to better understanding of the dynamics of gene expression in host cells during leishmaniasis.

Project description:Leishmania (L.) are obligated intracellular protozoan parasites that develop electively in macrophages. These cells that are acting as a safe shelter for the pathogens but also as their ultimate killer, making them the alpha and the omega during leishmaniasis diseases. Macrophages are able to secrete a remarkably diverse set of regulators known to influence the physiological functions and differentiation of neighboring cells to trigger an adaptive immune response of protective Th1-type cells, whereas parasites have developed a wide range of mechanisms to circumvent the hostâs immune responses. Most of our understanding of this host-parasite conflict, in the context of macrophage invasion by L. major metacyclic promastigotes, has been gleaned from studies investigating the macrophage responses at late and unique time points after infection. To investigate the dynamics of this duel, we have analyzed the transciptomic profile of monocyte-derived human macrophages at different time points during the first 24h upon in vitro infection using high throughput microarray platform. The gene expression profile of 17,838 genes showed high expression variability between the three human donors at different time points post infection. Cross comparison between the three donors allowed the identification of a common set of expressed genes coding for inflammatory and chemotactic molecules, transcription factors, apoptosis inhibition, glucose synthesis and heme metabolism. The findings presented in this work suggest that transcriptome dynamics of macrophages early during the first 24h post infection enable to identify novel key pathways deregulated upon L. major invasion. Our reported set of expressed genes will be useful in future rounds of data mining and functional analyses. In total 27 samples from three different donors were analyzed. For each donor, samples at 0h, 3h, 6h, 12h and 24h of culture were used as controls. For each donor, samples of cells infected with metacyclic Leishmania major parasites at 3h, 6h, 12h and 24h post-infection were analyzed.

Project description:BACKGROUND: Leishmania (L.) species are the causative agent of leishmaniasis. Due to the lack of efficient vaccine candidates, drug therapies are the only option to deal with cutaneous leishmaniasis. Unfortunately, chemotherapeutic interventions show high toxicity in addition to an increased risk of dissemination of drug-resistant parasites. An appropriate laboratory animal based model is still missing which allows testing of new drug strategies in the context of human immune cells in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Humanized mice were infected subcutaneously with stationary phase promastigote L. major into the footpad. The human immune response against the pathogen and the parasite host interactions were analyzed. In addition we proved the versatility of this new model to conduct drug research studies by the inclusion of orally given Miltefosine. We show that inflammatory human macrophages get infected with Leishmania parasites at the site of infection. Furthermore, a Leishmania-specific human-derived T cell response is initiated. However, the human immune system is not able to prevent systemic infection. Thus, we treated the mice with Miltefosine to reduce the parasitic load. Notably, this chemotherapy resulted in a reduction of the parasite load in distinct organs. Comparable to some Miltefosine treated patients, humanized mice developed severe side effects, which are not detectable in the classical murine model of experimental leishmaniasis. CONCLUSIONS/SIGNIFICANCE: This study describes for the first time L. major infection in humanized mice, characterizes the disease development, the induction of human adaptive and innate immune response including cytokine production and the efficiency of Miltefosine treatment in these animals. In summary, humanized mice might be beneficial for future preclinical chemotherapeutic studies in systemic (visceral) leishmaniasis allowing the investigation of human immune response, side effects of the drug due to cytokine production of activated humane immune cells and the efficiency of the treatment to eliminate also not replicating ("hiding") parasites.

Project description:Controlled human infection models (CHIMs) are useful for vaccine development. To improve on existing models, we developed a CHIM using a lyophilized preparation of Shigella sonnei strain 53G produced using current good manufacturing practice (cGMP). Healthy adults were enrolled in an open-label dose-ranging study. Following administration of a dose of rehydrated S. sonnei strain 53G, subjects were monitored for development of disease. The first cohort received 500 CFU of 53G, and dosing of subsequent cohorts was based on results from the previous cohort. Subjects were administered ciprofloxacin on day 5 and discharged home on day 8. Subjects returned as outpatients for clinical checks and sample collection. Attack rates increased as the dose of S. sonnei was increased. Among those receiving the highest dose (1,760 CFU), 70% developed moderate to severe diarrhea, 50% had dysentery, and 40% had fever. Antilipopolysaccharide responses were observed across all cohorts. An S. sonnei CHIM using a lyophilized lot of strain 53G was established. A dose in the range of 1,500 to 2,000 CFU of 53G was selected as the dose for future challenge studies using this product. This model will enable direct comparison of study results between institutions and ensure better consistency over time in the challenge inoculum.IMPORTANCE Controlled human infection models (CHIMs) are invaluable tools utilized to understand the human response to infection, potentially leading to protective immune mechanisms and allowing efficacy testing of enteric countermeasures, including vaccines, antibiotics, and other products. The development of an improved Shigella CHIM for both Shigella sonnei and Shigella flexneri is consistent with international efforts, supported by international donors and the World Health Organization, focused on standardizing Shigella CHIMs and using them to accelerate Shigella vaccine development. The use of lyophilized Shigella challenge strains rather than plate-grown inoculum preparations is considered an important step forward in the standardization process. Furthermore, the results of studies such as this justify the development of lyophilized preparations for additional epidemiologically important S. flexneri serotypes, including S. flexneri 3a and S. flexneri 6.

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Characterization of a new Leishmania major strain for use in a controlled human infection model.

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Characterization of a new Leishmania major strain for use in a controlled human infection model.

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