Introduction and Background of the Project

1.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.

2.Project objective

The objectives of this continuation proposal are to: 1. Produce and characterize cGMP-grade Leishmania antigen (liquid or lyophilized) from L. donovani. 2. Test cGMP leishmanin antigen formulations (liquid and lyophilized) by performing LST in animal models. 3. Perform pre-clinical toxicology studies with cGMP leishmanin antigen formulation that is selected for further advancement based on results from LST studies in animals. 4. Analyze cytokine production in PBMCs or whole blood isolated from healthy individuals and cured VL patients following in vitro stimulation with the selected formulation of cGMP leishmanin antigen. 5. Prepare an IND package for clinical trials.

3.Project design

During the previous funding period, the project team successfully accomplished goals of the project by 1) optimizing the protocol to produce and scale up production of leishmanin antigen from L. donovani parasites using a cost-effective, scalable and industry suitable osmotic shocklysis technique; 2) performed stability studies; 3) validated GLP leishmanin using experimental animal models, and 4) completed manufacturing of cGMP cell bank of L. donovani at ATCC. In this project, three different formulations of leishmanin antigen will be produced from cGMP L. donovani cell bank. The formulations will be validated for safety and potency using preclinical animal models of vaccination and cured VL. One formulation will be selected for further advancement and scale-up cGMP production on the basis of on results from stability studies and LST studies in animals, and pre-clinical toxicology studies will be performed as per regulatory guidelines. To assess the immunogenicity of the product, cytokine production in PBMCs or whole blood isolated from healthy individuals and cured VL patients following in vitro stimulation with the selected formulation of cGMP leishmanin antigen will also be analyzed. IND package will be prepared for the submission to regulators for clinical trials.

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

The proposed studies will lead to the reintroduction of LST which has been already used effectively in the past for detection of Leishmania infection and immunity in CL and VL. 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 to help 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 VL 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 latent 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 us to detect 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 lyophilized formulation of leishmanin experimental vaccination animal models of CL and/or VL. 

Institute of Tropical Medicine at Nagasaki University (NUITM) will be responsible for undertaking safety studies. They will also validate the liquid leishmanin antigen formulation test using pre-clinical animal models of old-world CL. They will also assist with sample analysis for studies in Bangladesh.

Gennova Biopharmaceuticals Ltd will execute the product development plan and manufacture sufficient quantities of GLP leishmanin antigen products (both liquid and lyophilized) for pre-clinical validation. They will produce sufficient quantities for selected leishmanin antigen product, undertake pre-clinical toxicology testing and provide material for studies proposed in Bangladesh.  They will also be involved in compiling IND package.

US-FDA will ensure cGMP manufacturing as per FDA guidelines, undertake validation of liquid formulation of leishmanin antigen for LST using LmCen^-/- vaccination model VL in hamsters and CL in mice.

McGill University will assist with planning, execution and data analysis of studies in Bangladesh. They will also assist with compilation of IND, data analysis, and overall planning and execution of the project.

Icddr, b will conduct studies to analyze immune response of PBMCs and whole blood from healthy individuals and healed VL patients to selected cGMP leishmanin antigen formulation produced by Gennova.

Others (including references if necessary)

Dey R, Alshaweesh J, Singh KP, Lypaczewski P, Karmakar S, Klenow L, Paulini K, Kaviraj S, Kamhawi S, Valenzuela JG, Singh S, Hamano S, Satoskar AR, Gannavaram S, Nakhasi HL, Matlashewski G.: Production of leishmanin skin test antigen from Leishmania donovani for future reintroduction in the field. Nat Commun. 2023; 14(1): 7028. doi: 10.1038/s41467-023-42732-2.