Introduction and Background of the Project

Introduction

The leishmaniases comprise a number of diseases caused by obligate intracellular parasites of the genus Leishmania that is transmitted by the bites of infected sandflies. With over 350 million people worldwide at risk of contracting leishmaniasis and, the WHO classifies leishmaniasis as a neglected tropical disease. The leishmanin skin test (LST) was used for decades to determine exposure and immunity to Leishmania infection but the leishmanin antigen used in the LST is no longer available. There are compelling reasons to bring back the LST. Firstly, the LST could identify districts and villages where there is active or past transmission. This information will help to support visceral leishmaniasis elimination programs. Second, since promising vaccines are advancing, the LST would be an effective surrogate marker to help determine vaccine efficacy.

Project objective

The objectives of this proposal are to: 1. Produce and determine the stability of the L. donovani antigen (Leishmanin antigen). 2. Validate the Leishmania donovani LST leishmanin antigen in immune animals. 3. Validate the LST in visceral leishmaniasis cured and asymptomatic infected individuals.

Project design

A protocol for making Leishmanin antigen from L. donovani parasites will be established and GMP grade Leishmanin will be produced for the Leishmanin skin test (LST). Pre-clinical animal models of visceral leishmaniasis in hamsters and cutaneous leishmaniasis in mice will be used to test Leishmanin antigen and validate LST for detection of latent infection and immunity. Immune responses will also be analyzed in these pre-clinical animal models. After completion of animal studies, LST will be validated in visceral leishmaniasis patients as well as asymptomatic individuals from endemic regions.

How can your partnership (project) address global health challenges?

The proposed studies will lead to the reintroduction of the LST which has previously been widely  used effectively for detection of Leishmania infection but is no longer available. There are compelling reasons to bring back the LST. Firstly, the LST could support the visceral leishmaniasis (VL) elimination program in the Indian subcontinent through identifying blocks (subdistricts) and villages with immunity and help to understand why the transmission is highly focal and which blocks are susceptible or resistant to future outbreaks. Second, since promising vaccines are advancing, the LST would be an effective surrogate marker to help determine vaccine efficacy. In summary, the LST will support current ongoing visceral leishmaniasis elimination efforts and future vaccine. development studies.

What sort of innovation are you bringing in your project?

The major drawback of currently available serology and PCR tests is that they only identify acute infection and cannot effectively identify previous exposure or cellular immunity. No tests are currently available to detect asymptomatic Leishmania infection and immunity against the disease. The leishmanin skin test (LST) was used for decades to determine exposure and immunity to Leishmania infection but the leishmanin antigen used in the LST is no longer available. LST will allow enable the detection of individuals exposed to Leishmania as well as undertake surveillance in the community. In addition, LST would be an effective surrogate marker to help determine efficacy vaccines that are being developed against leishmaniasis.

Role and Responsibility of Each Partner

The Ohio State University (OSU) will be the designated grantee for this project and is responsible for coordinating overall project. OSU will also undertake validation of LST using experimental animal models of new world cutaneous leishmaniasis. In addition, OSU will oversee and assist with validation studies in humans in Bihar, India.

Institute of Tropical Medicine at Nagasaki University (NUITM) will be responsible for undertaking safety studies. They will also validate the test using pre-clinical animal models of old world CL

Gennova Biopharmaceuticals Ltd will execute the product development plan and manufacture sufficient quantities of GLP leishmanin material for validation in animal models and humans. They will also complete regulatory formalities in India as needed. They will also undertake clinical studies in Bihar.

US-FDA will undertake validation of cGMP grade LST for detection of latent infection and immunity in pre-clinical animal models using sand fly transmission model of VL in hamsters and CL in mice.

McGill University will establish a protocol for producing leishmanin antigen and determine stability of the antigen. They will also support validation studies of LST in visceral leishmaniasis cured and asymptomatic cases in Bihar, India

Others (including references if necessary)

1.   Reed, S., Badaro, R., Masur, H., Carvalho E., Lorenco R., Lisboa, A., Texeira, R., Johnson W., and Jones, T. Selection of a skin test antigen for American visceral leishmaniasis. Am. J. Trop. Med. Hyg. 35: 79-85, 1986.

2.   Alimohammadian M., Kojori Z., Darabi H., Malekzadeh M. et al. Soluble Leishmanin as an ideal reagent for skin testing in human leishmaniasis. Iran. Biomed. J. 1: 39-47, 1997.

3.   Gramiccia M., Bettini S., Gradoni L., Verrilli L., Loddo S., and Cicalo C. Leishmaniasis in Sardinia. Leishmanin reaction in the human population of a focus of low endemicity of canine leishmaniasis. Trans. Roy. Soc. Trop. Med. Hyg. 84: 371-374, 1990.

Final Report

1. Project objective

 1: Produce and determine the stability of the Indian isolate of L. donovani antigen (Leishmanin antigen).

 2: Validate the L. donovani LST leishmanin antigen in immune hamsters and mice.

 3: Validate the LST in VL-cured individuals in Bihar, India

2. Project design

Soluble L. donovani leishmanin antigen is produced from laboratory-cultured parasites, and its short- and long-term stability is determined to establish optimized procedures for preparation and storage (Aim 1). Diagnostic performance is then validated in animal models by assessing LST responses in Leishmania-infected, LmCen⁻/⁻-vaccinated, or otherwise immune hosts, together with local immune transcript analysis (Aim 2). Finally, the leishmanin antigen is evaluated by stimulating PBMCs from VL-cured and asymptomatic individuals to measure  immunological responses to confirm its ability to detect prior exposure and cellular immunity, supporting surveillance and future vaccine studies (Aim 3).

3. Results, lessons learned

We achieved following substantial progress:

 1. GLP-grade soluble leishmanin antigen was successfully produced from L. donovani parasites using the conventional freeze–thaw (F-T) method. This established a robust, reproducible upstream and downstream process based on Reed (1986), enabling antigen production from VL-relevant etiologic species. Whole-genome sequencing confirmed the genetic identity of the L. donovani Indian isolate used in manufacturing.

 2. The biological performance of the antigen was validated in multiple animal models. In mice, healed (leishmanized) animals infected with L. major FV9 and animals vaccinated with the live-attenuated L. major LmCen⁻/⁻ strain showed strong delayed-type hypersensitivity (DTH) responses after intradermal administration of the L. donovani antigen, whereas naïve mice showed no reaction.  Immunological analyses of the DTH sites demonstrated that both CD4⁺ and CD8⁺ T cells contribute to the LST response, reinforcing the value of LST as a functional readout of cell-mediated immunity. Similar results were obtained in hamsters, a relevant VL model,  vaccinated with LmCen⁻/⁻ or healed (leishmanized) infected with L. major, exhibited clear LST responses. These findings confirmed that L. donovani–derived antigen can reliably detect prior exposure or vaccine-induced immunity across Leishmania species.

 3. For manufacturing large scale Leishmania antigen to be used in the field, our team developed a novel osmotic-lysis–based antigen extraction method that was lyophilized and demonstrated similar efficacy comparable to the conventional F-T preparations.

 4. To support future cGMP-deliverable antigen production, ATCC generated and qualified Master Cell Banks (MCB) and Working Cell Banks (WCB) for the L. donovani Indian isolate. These cell banks met all release criteria, including sterility, absence of adventitious agents, genomic identity, and consistent growth characteristics, thus establishing the foundation for subsequent cGMP manufacturing which will be validated in preclinical studies and ex vivo in human PBMCs before testing in field studies.

Lessons learned:  Develop a field deployable a well-controlled process of manufacturing lyophilized Leishmania antigen to avoid lot-to-lot inconsistency, to support preclinical validation necessary for future regulatory approval for use in clinical studies to assess immunity to vaccine candidates.   Develop a Leishmania antigen from Leishmania donovani that can detect VL cases, a major cause of mortality, and pan-leishmania cases.