Opportunities and Challenges in Developing a Cryptosporidium Controlled Human infection Model For Testing antiparasitic agents

Cryptosporidiosis is a leading cause of moderate-to-severe diarrhea in low- and middle-income countries, responsible for high mortality in children younger than two years of age, and it is also strongly associated with childhood malnutrition and growth stunting. There is no vaccine for cryptosporidiosis and existing therapeutic options are suboptimal to prevent morbidity and mortality in young children. Recently, novel therapeutic agents have been discovered through high-throughput phenotypic and target-based screening strategies, repurposing malaria hits, etc., and these agents have a promising preclinical in vitro and in vivo anti

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage; Plasmodium falciparum; and; Cryptosporidium parvum; in cell-culture studies. Target deconvolution in; P. falciparum; has shown that cladosporin inhibits lysyl-tRNA synthetase (; Pf; KRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both; Pf; KRS1 and; C. parvum; KRS (; Cp; KRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED; 90; = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between; Pf; KRS1 and; Cp; KRS. This series of compounds inhibit; Cp; KRS and; C. parvum; and; Cryptosporidium hominis; in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for; Pf; KRS1 and; Cp; KRS vs. (human); Hs; KRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis

The Glycosylated Rv1860 Protein of <i>Mycobacterium tuberculosis</i> Inhibits Dendritic Cell Mediated TH1 and TH17 Polarization of T Cells and Abrogates Protective Immunity Conferred by BCG

<div><p>We previously reported interferon gamma secretion by human CD4⁺ and CD8⁺ T cells in response to recombinant <i>E. coli</i>-expressed Rv1860 protein of <i>Mycobacterium tuberculosis</i> (MTB) as well as protection of guinea pigs against a challenge with virulent MTB following prime-boost immunization with DNA vaccine and poxvirus expressing Rv1860. In contrast, a Statens Serum Institute <i>Mycobacterium bovis</i> BCG (BCG-SSI) recombinant expressing MTB Rv1860 (BCG-TB1860) showed loss of protective ability compared to the parent BCG strain expressing the control GFP protein (BCG-GFP). Since Rv1860 is a secreted mannosylated protein of MTB and BCG, we investigated the effect of BCG-TB1860 on innate immunity. Relative to BCG-GFP, BCG-TB1860 effected a significant near total reduction both in secretion of cytokines IL-2, IL-12p40, IL-12p70, TNF-α, IL-6 and IL-10, and up regulation of co-stimulatory molecules MHC-II, CD40, CD54, CD80 and CD86 by infected bone marrow derived dendritic cells (BMDC), while leaving secreted levels of TGF-β unchanged. These effects were mimicked by BCG-TB1860His which carried a 6-Histidine tag at the C-terminus of Rv1860, killed sonicated preparations of BCG-TB1860 and purified H37Rv-derived Rv1860 glycoprotein added to BCG-GFP, but not by <i>E. coli</i>-expressed recombinant Rv1860. Most importantly, BMDC exposed to BCG-TB1860 failed to polarize allogeneic as well as syngeneic T cells to secrete IFN-γ and IL-17 relative to BCG-GFP. Splenocytes from mice infected with BCG-SSI showed significantly less proliferation and secretion of IL-2, IFN-γ and IL-17, but secreted higher levels of IL-10 in response to <i>in vitro</i> restimulation with BCG-TB1860 compared to BCG-GFP. Spleens from mice infected with BCG-TB1860 also harboured significantly fewer DC expressing MHC-II, IL-12, IL-2 and TNF-α compared to mice infected with BCG-GFP. Glycoproteins of MTB, through their deleterious effects on DC may thus contribute to suppress the generation of a TH1- and TH17-dominated adaptive immune response that is vital for protection against tuberculosis.</p></div

Analysis of spleen DC <i>ex vivo</i> from infected mice.

<p>Splenocytes obtained from mice uninfected (UI) or infected 12 hours earlier with 10⁷ cfu of BCG-GFP or BCG-TB1860 were processed for flow cytometry as described in Methods and illustrated in Figure S5 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004176#ppat.1004176.s001" target="_blank">Text S1</a>. The number of CD11c-high DC displaying upregulated expression MHC class II (A), secreting IL-2/TNF-α (B) and secreting IL-12p40 (C) are shown as a percentage of total splenocytes within gate P1 of Figure S5A. Splenocytes simultaneously secreting IL-2/TNF-α along with IL-12p40 (D) or simultaneously secreting IL-2/TNF-α along with expression of MHCII (E) are shown as a percentage of total CD11c-high DC.</p

Expression of MTB Rv1860 in BCG suppressed cytokine secretion from mouse BMDC.

<p>(A) BALB/c bone marrow differentiated for 7 days with GM-CSF into dendritic cells were infected at 5 bacteria per cell of the indicated BCG strains. Culture supernatants were harvested 8 hrs later and used for cytokine measurements. IL-12p70 and IL-10 were not determined for BCG-TB1860His. Values are mean ± SD obtained from five mice. (B) BALB/c BMDC were treated with sonicated preparations of the BCG strains indicated, comparable to infection at 5 MOI. Glycosylated native Rv1860 (g1860) was added at 10 ng/well, estimated to be consistent with to that coming from 5 MOI infection as described in Methods. Cytokines were measured after 8 hrs of incubation. Values are mean ± S.D. from minimum of 3 mice.</p

MTB Rv1860 abrogates the protective efficacy of BCG.

<p>The Rv1860 gene of MTB was inserted into the genome of BCG-SSI to give BCG-TB1860, as described in Methods. Groups of eight guinea pigs immunized once with 10⁶ viable BCG-GFP and BCG-TB1860 strains were challenged by the intramuscular route with 2×10⁵ viable virulent MTB field strain NTI83949. Challenge bacterial burden in the spleens of guinea pigs six weeks post challenge was enumerated. Naïve control animals received saline injection.</p

MTB Rv1860 abrogates the protective efficacy of BCG.

<p>The Rv1860 gene of MTB was inserted into the genome of BCG-SSI to give BCG-TB1860, as described in Methods. Groups of eight guinea pigs immunized once with 10⁶ viable BCG-GFP and BCG-TB1860 strains were challenged by the intramuscular route with 2×10⁵ viable virulent MTB field strain NTI83949. Challenge bacterial burden in the spleens of guinea pigs six weeks post challenge was enumerated. Naïve control animals received saline injection.</p

Polarization of syngeneic splenocytes by infected BMDC.

<p>7 day differentiated BALB/c BMDC were either untreated (NT) or infected with the indicated BCG strains at 5 MOI for 24 hrs, gamma irradiated as described in methods section and co-cultured with splenocytes from BALB/c mice infected with 10⁶ viable bacilli of BCG-GFP or BCG-TB1860 3 weeks previously, at T cell/DC ratios of 20 for 72 hrs. Supernatants were then collected for measurement of IFN-γ (B) and IL-17 (C). Cells were pulsed with 0.5 µCi tritiated thymidine for 16 hrs, harvested and counted. Stimulation indices (A) were obtained by dividing cpm obtained for each stimulation by sum of cpm obtained for corresponding DC plus T cells alone. Values are mean ± S.D. from four mice.</p

Description of strains and reagents used in the study.

<p>Description of strains and reagents used in the study.</p

<i>In vitro</i> recall responses of splenocytes from BCG-immunized mice.

<p>6 week old BALB/c mice were immunized with 10⁷ viable BCG-SSI. Splenocytes recovered 3 weeks later, were stimulated <i>in vitro</i> at an MOI of 20 with BCG-GFP or BCG-TB1860 for 72 hrs. Con-A at 5 µg/ml was included as positive control. Supernatants were collected for the measurement of IFN-γ (A), IL-2 (B), IL-17 (C) and IL-10 (D). (E) Splenocytes stimulated for 36 hrs with the indicated BCG strains were stained for intracellular IL-17. Percentage of cells secreting IL-17 among those gated sequentially on CD3⁺, CD4⁺ and CD25⁻ are displayed. Values are mean ± S.D. from four mice.</p