Flow cytometry: a critical technique in combating leishmaniasis
by Professor Paul Kaye
Leishmaniasis is classified as a neglected tropical disease. It is the cause of a huge health burden and is common in Asia, Africa, South and Central America, and even southern Europe. This article discusses how flow cytometry can help to evaluate diagnosis, monitor the effects of therapy and help in the creation of a vaccine.
The leishmaniases are a family of devastating diseases, affecting a great many people across the globe and presenting a significant risk to both public health and socioeconomic development. The leishmaniases are vector-borne diseases, caused by infection with one of 20 species of the parasitic protozoan Leishmania (Fig. 1), transmitted through the bite of the infected female phlebotomine sand fly.
They can be broadly classified as tegumentary leishmaniases (TLs), affecting the skin and mucosa, and visceral leishmaniasis (VL), affecting internal organs. Whereas VL is responsible for over 20¦000 deaths per year, TL are non-life-threatening, chronic and potentially disfiguring, and account for around two-thirds of the global disease burden.
Within TL, there are three subtypes: self-healing lesions at the location of sand fly bite (cutaneous leishmaniasis; CL), lesions that spread from the original skin lesion to the mucosae (mucosal leishmaniasis; ML), and those which spread uncontrolled across the body (disseminated or diffuse cutaneous leishmaniasis; DCL). VL, also known as kala azar, involves major organs including the spleen, liver and bone marrow. In addition, patients recovering from VL after drug treatment often develop post kala-azar dermal leishmaniasis (PKDL), a chronic skin condition, characterized by nodular or macular lesions beginning on the face and spreading to the trunk and arms. As it may develop in up to half of patients previously treated and apparently cured from VL, it is thought that PKDL plays a central role in community transmission of VL.
The World Health Organization designates leishmaniasis as a neglected tropical disease (NTD), which together affect more than one|billion people across 149 countries worldwide; true prevalence may be even higher. Disproportionately, NTDs affect the poorest, malnourished individuals, and contribute to a vicious circle of poverty and disease. The significant physical marks, including ulcers, often left in the wake of the TLs may have an impact on mental health and perpetuate social stigma associated with the diseases . There are over 1|million new cases of TL and 0.5|million new cases of VL each year, which together account for the loss of approximately 2.4|million disability-adjusted life years.
Leishmaniasis treatment can be quite difficult since at-risk populations may lack access to healthcare, and the limited battery of drugs has been increasingly compromised by resistance. Additionally, because the parasites in question are eukaryotic, they are not dissimilar from human cells, so the medication is also liable to be harmful – even fatal – to host as well as to pathogen.
Although the burden of VL in South Asia has been reduced with single-dose liposomal amphotericin B, the drug is less effective in other geographic locations, namely East Africa. Various drug combinations have been tested, unsuccessfully, and new chemical entities and immune-modulators are in early stages of development and as yet untested in the field. Unfortunately, little has changed in the treatment for CL for the past 50|years.
No vaccines are currently approved for any form of human leishmaniasis, although vaccines for canine VL have reached the market. Barriers to vaccine development include the limited investment in leishmaniases R&D and the high costs involved in bringing new products to those that need them.
My work on leishmaniasis has taken a holistic view, rooted in the immunology of the host-parasite interaction, but employing tools and approaches that span many disciplines: mathematics, ecology, vector biology and most recently neuroscience. Thirty years of discovery science has led to the development of a candidate for a therapeutic vaccine for PKDL, the mysterious sequela to VL . ‘Therapeutic’ vaccines are given after an individual is infected with a pathogen and are designed to enhance our immune system and help eliminate the infection.
With colleagues from Sudan, we are in the midst of a phase IIb clinical trial funded by the Wellcome Trust, evaluating the efficacy of this therapeutic vaccine in Sudanese patients with persistent PKDL.
However, the research has been a long time in the making and has a long way to go. To continue to make progress, we linked with colleagues in Ethiopia, Kenya and Uganda and at the European Vaccine Initiative (http://www.euvaccine.eu/) in Germany, to develop a new research consortium to evaluate the immune status of people suffering from leishmaniasis. For example, using flow cytometry for blood and multiplexed immunohistochemistry for tissue biopsies, we can enumerate the proportions of lymphocytes, monocytes and neutrophils based on surface marker expression (e.g. CD3, CD19, CD14, CD16), and characterize their function, for instance by expression of cytokines (e.g. interferon-gamma) or other cell surface proteins that define function state. To support this endeavour, we recently received a grant from the European & Developing Countries Clinical Trials Partnership (EDCTP) that will allow us to not only extend our vaccine programme in Sudan  but also to address other important research challenges.
To develop vaccines and indeed new drugs, we often need tools capable of performing in-depth comparisons of how the body’s immune system is coping with the infection when a patient is first admitted to hospital and how it changes as the patient undergoes treatment and is hopefully cured. For example, recent evidence suggests that during infection, T lymphocytes may become ‘exhausted’ and unable to fight infection and the exhausted state can be identified by expression of surface molecules such as programmed cell death protein|1 (PD-1) and lymphocyte activation gene 3 protein (LAG-3). It is important to know if exhaustion can be reversed following treatment or whether we need to stimulate new populations of T lymphocytes. By understanding these nuanced changes in immune cells in our blood, we can design ways to improve how vaccines and drugs work in concert with immune cells, and understand why some patients might relapse from their disease or develop PKDL. Flow cytometry is a central tool for immunologists and plays a critical role in uncovering mechanisms of immunity and in assessing how well vaccines work and biomarkers of drug response. It uses antibodies that recognize specific molecules or markers on the surface or inside immune cells, such as those mentioned above, that help us predict their function. These antibodies are fluorescently labelled and the fluorescent signal can be detected by exposing each cell individually to laser light as they pass through a small aperture, the essence of flow cytometry.
For flow cytometry to be beneficial in this project, we needed to purchase five new flow cytometers that could meet exacting standards. They needed to be sufficiently sensitive to identify rare cell populations, often with low levels of surface marker expression. For multicentre research projects, reproducibility of data between sites is essential. Hence, we needed excellent inter-machine reproducibility and the Figure 2. Initial training course with recently appointed flow managers (Credit: Dr Karen Hogg, University of York) | 10 manufacturer had to be able to provide service support across the region. In our search for the right flow cytometer to support the consortium, we settled upon the CytoFLEX, Beckman Coulter Life Sciences’ research flow cytometer, which uses avalanche photodiode detection to arrive at the required level of sensitivity. With assistance from Beckman Coulter, we devised and have run initial training courses with a group of recently appointed flow managers from each partner country, to share standard operating procedures, develop high-level data analysis strategies as well as to provide instruction in routine instrument maintenance.
Beckman Coulter also provides another important aid to reducing errors in flow cytometry for multisite projects such as this, namely freeze-dried antibody cocktails (DURAClone panels) , that allow highly multiplexed phenotyping of small volumes of blood added directly to a single tube. Particularly for investigators in remote locations, the use of dry, preformulated reagents, rather than liquid (‘wet’) antibodies, removes the need for a cold chain. Equally importantly, staining of cells when manual mixing of 15 or 16 antibodies is required can introduce data inconsistencies when conducted by different individuals and at different locations.
Together, these innovations have allowed us to establish a new network for flow cytometry in East Africa that will allow us to identify and functionally characterize and identify the types of immune cells present in the blood during these devastating diseases. We will match this data with similar multiplexed techniques in pathology to compare blood immune cell profiles with those of cells found in the skin, to give a more complete picture of the host response to infection before and after treatment or vaccination.
As mentioned, we are currently in the midst of an efficacy trial of our therapeutic vaccine, ChAd63-KH. The technology we are using is similar to that being used by researchers at the university of Oxford to develop a coronavirus vaccine. In short, we introduce two genes from Leishmania parasites (KMP-11 and HASPB1) into a well-studied chimpanzee adenovirus (ChAd63 viral vector). After vaccination with this vaccine, host cells become infected with the virus and express the Leishmania proteins in a way that can be recognized efficiently by the immune system. We are particularly interested in how well this vaccine can generate T|cells to fight the infection.
With the first of our clinical objectives now well underway – the ongoing therapeutic clinical trial in patients with PKDL will be completed in mid-2021 – we have two additional goals. The next, funded by EDCTP, is to start a new clinical trial to determine whether the vaccine can prevent progression from VL to PKDL. And finally, we hope to develop a human challenge model of leishmaniasis to test the vaccine for its ability to protect against infection by different forms of parasite. This would open the way to the development of a cost-effective prophylactic vaccine to prevent these diseases occurring in vulnerable populations across the world.
Our research also has larger ambitions for the long term. Our East African partners are also linked together through their work on leishmaniasis in drug development, as members of the Leishmaniasis East Africa Platform group, established to help coordinate drug development activities in the region by the Drugs for Neglected Diseases Partnership. Central questions about why the disease varies between countries are being addressed, and the increased capacity for flow cytometry will additionally support patient monitoring during drug trials conducted by DNDi or other groups. Indeed, through the capacity building this project provides, we hope this project will extend its reach beyond leishmaniasis, providing muchneeded support for research on other neglected diseases of poverty that affect people in the region, including bacterial, fungal, other parasitic and viral diseases. By continuing to demonstrate the analytical power of flow cytometry and its role in helping design much-needed therapies, we hope to open up additional discovery research possibilities for colleagues in Africa and around the world.
The research described in this article is part of the EDCTP2 programme supported by the European Union (grant number RIA2016V-1640; PREV_PKDL; https://www.prevpkdl.eu).
Paul Kaye PhD, FRCPath, FMedSci
Hull York Medical School, University of York, York, UK