Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen−/−) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen−/− parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen−/− amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (WT) parasites. We have also determined the safety and efficacy of LmexCen−/− in vivo using experimental murine models of L. mexicana. We demonstrate that LmexCen−/− parasites are safe and do not cause lesions in susceptible mouse models. Immunization with LmexCen−/− is also efficacious against challenge with WT L. mexicana parasites in genetically different BALB/c and C57BL/6 mouse models. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens at the infection site and draining lymph nodes compared to the control group. Overall, we demonstrate that LmexCen−/− parasites are safe and efficacious against New World cutaneous leishmaniasis in pre-clinical models.

Introduction: Leishmaniasis is a major public health problem and the second most lethal parasitic disease in the world due to the lack of effective treatments and vaccines. Even when not lethal, leishmaniasis significantly affects individuals and communities through life-long disabilities, psychosociological trauma, poverty, and gender disparity in treatment.Areas covered: This review discusses the most relevant and recent research available on Pubmed and GoogleScholar highlighting leishmaniasis' global impact, pathogenesis, treatment options, and lack of effective control strategies. An effective vaccine is necessary to prevent morbidity and mortality, lower health care costs, and reduce the economic burden of leishmaniasis for endemic low-and middle-income countries. Since there are several forms of leishmaniasis, a pan-Leishmania vaccine without geographical restrictions is needed. This review also focuses on recent advances and common challenges in developing prophylactic strategies against leishmaniasis. Expert opinion: Despite advances in pre-clinical vaccine research, approval of a human leishmaniasis vaccine still faces major challenges -including manufacturing of candidate vaccines under Good Manufacturing Practices, developing well-designed clinical trials suitable in endemic countries, and defined correlates of protection. In addition, there is a need to explore Challenge Human Infection Model to avoid large trials because of fluctuating incidence and prevalence of leishmanasis.

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Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression in real time. This information can be helpful in prognosis, treatment selection, and stratification of cancer patients. AccuCyte® is a density-based unbiased isolation method that transfers nucleated cells from whole blood to slides for the characterization of CTCs and other rare cells. RarePlex® panel kits are IF staining reagents used on automated slide staining instruments to label cells to differentiate CTCs from white blood cells (WBC). CyteFinder® is a seven-channel automated fluorescent imaging system that rapidly scans microscope slides and applies machine learning algorithms to identify CTCs. Together, these technologies provide an end-to-end solution for CTC characterization. For analysis, blood is drawn into AccuCyte blood collection tubes (BCTs) containing a preservative which maintains cell properties prior to processing onto slides. Once slides are prepared, they can be stored at -20°C without significant biomarker degradation. This flexible workflow allows investigators to bank samples for batch analysis and to begin sample collection prior to validating the IF assay to be used. This study was designed to evaluate: (1) stability time between collection in the AccuCyte BCT and sample processing; (2) performance of an improved version of the AccuCyte kit with higher nucleated cell isolation capacity; and (3) storage time that AccuCyte prepared slides can be banked frozen prior to staining. The study was performed using model CTCs and cancer patient samples. Metrics to determine performance were CTC recovery and mean fluorescence intensity (MFI) of biomarker expression. Our results demonstrate that the AccuCyte BCT preserves blood components for at least 5 days after collection without significant effect on CTC recovery or biomarker expression. The latest version of the AccuCyte kit demonstrated a higher cell isolation capacity and could collect up to 60% more nucleated blood cells than the previous version, increasing CTC recovery. The increased capacity was demonstrated in patients treated with hematopoietic growth factors, whose WBC count was significantly higher than the normal range. Finally, accelerated-aging study results demonstrated that AccuCyte-prepared slides can be stored at -20°C for at least 4 years without significant effect on most biomarkers tested. In conclusion, enhancements to the AccuCyte-CyteFinder platform reported here increase flexibility and performance for analysis of CTCs in global clinical trials by allowing longer periods of time before collected blood samples need to be processed and by extending the length of time processed slides can be banked before they are stained. Citation Format: Arturo B. Ramirez, Lillian Costandy, Brady S. Gardner, Ryan H. Huston, A Anders Larson Tevis, Casey E. Helmicki, Alisa C. Clein, Daniel E. Sabath, Joshua J. Nordberg, Tad C. George. Validation of enhanced performance of the AccuCyte®-CyteFinder® platform for circulating tumor cell characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1952.