Preclinical and preparation of early clinical testing of a new vaccine candidate against cutaneous leishmaniasis
Project Completed
Please click to see the final report.
  • RFP Year
  • Awarded Amount
  • Disease
    NTD (Leishmaniasis)
  • Intervention
  • Development Stage
    Preclinical Development
  • Collaboration Partners
    Nagasaki University ,  Charité –Universitätsmedizin Berlin ,  London School of Hygiene and Tropical Medicine(LSHTM) ,  Mologen ,  European Vaccine Initiative e.V. (EVI e.V.)

Introduction and Background of the Project


Leishmaniases are vector-borne protozoal diseases with clinical manifestations ranging from self-healing or chronic cutaneous leishmaniasis (CL), disfiguring mucocutaneous leishmaniasis (MCL) to fatal visceral leishmaniasis (VL). Causative agents are parasites of the genus Leishmania that are transmitted by female sand flies to mammalian hosts where they infect and proliferate inside phagocytes, especially macrophages. Treatment options are limited to a few drugs, by high costs, significant adverse effects and, in some areas, increasing parasite drug resistance. So far, preventative measures are restricted to vector control with bed nets and indoor residual spraying, of limited effect, and insecticides, which raises environmental issues.

The use of vaccines in therapy of leishmaniasis has been a long-term aim, however, there is no licensed vaccine against human leishmaniasis. Several have been proposed and are at different stages of development. We previously developed a pentavalent DNA vaccine coding for optimized and T cell epitope-enriched antigens of Leishmania. This DNA vaccine was tested in ex vivo human T cell stimulation studies for antigenicity and immunogenicity in CL and VL, and in a mouse model for immunogenicity and effectiveness against VL. It is the basis for the present application.

The present partnership seeks to establish a new vaccine principle for leishmaniasis with a special focus on the induction of cell-mediated immunity, including the potential of the LEISHDNAVAX vaccine candidate to protect against cutaneous leishmaniasis in a preclinical animal model. Moreover, we will prepare a Phase I clinical trial for the evaluation of the safety and immunogenicity of the vaccine candidate.


Project objective

LEISHDNAVAX is a candidate DNA vaccine against leishmaniasis that has been successfully tested for antigenicity in humans in ex vivo studies, and for efficacy in a mouse model for visceral leishmaniasis. We aim to complete the preclinical development with tests for cutaneous leishmaniasis and to prepare a clinical Phase I trial. At the end of the project we will have fulfilled the prerequisites for a clinical Phase I trial to test safety and immunogenicity of the vaccine.


Project design

The project comprises the preclinical evaluation of the immunogenicity, prophylactic and therapeutic efficacy of the DNA leishmaniasis vaccine LEISHDNAVAX in mouse models of cutaneous leishmaniasis (CL). In addition, we will prepare the evaluation of the vaccine candidate in a clinical Phase I trial. To achieve this goal, the project is divided in four specific objectives:

Objective 1: To test the preclinical efficacy of a preventive vaccine, we will examine T cell immune responses in mice, before and after infection. Several Leishmania species that are endemic in different regions of the world will be used and different immunization schemes will be tested.

Objective 2: Based on previous experience with L. donovani infection models in mice, we will study the therapeutic effect of the vaccine candidate alone and in combination with known anti-leishmanial drugs against CL.

Objective 3: The vaccine production process will be established at the site of a Contract Manufacturing Organisation (CMO) and the vaccine material for the clinical Phase I trial will be produced.

Objective 4: Under this objective we will prepare a future clinical Phase I trial to evaluate safety, tolerability and immunogenicity of the vaccine candidate in human volunteers.

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

Leishmaniases are neglected, vector-borne diseases, affecting large populations in all tropical and subtropical regions, and adjacent areas like the Mediterranean basin, Middle East and Central Asia. Through development of a preventive/therapeutic vaccine, this project supports the overarching strategic goal provided by the World Health Organization “Sustaining the drive to overcome the global impact of neglected tropical diseases”. It is also aligned with the London declaration on neglected tropical diseases, aimed to ensure the availability of necessary medicines and other interventions for preventing, diagnosing and controlling neglected tropical diseases (NTDs), including leishmaniasis.

The development of therapeutic and preventive vaccines against leishmaniasis would contribute to:

a)     Control of CL where the disease has reached epidemic proportions in areas of conflict and public health breakdown, for example Syria, Afghanistan and Iraq, and consistent exposure and transmission in zoonotic endemic areas, for example Central and South America.

b)    The potential for treatment using a drug plus vaccine immunotherapy approach, especially for the management of forms of CL that fail to respond to currently available drugs. Here, a vaccine could shorten the courses of treatment, prevent relapses and reduce toxic side effects.

c)     The sustainability of VL elimination programs in India, Nepal, Bangladesh and Bhutan (ISC). To sustain the elimination target beyond the VL post-elimination phase is a major consideration for health authorities in the ISC countries where the effect of a long-term and broad application of drugs and insecticides is questionable. A vaccine with proven ability to prevent transmission and that can be used in targeted populations in endemic areas is the ideal tool for sustained elimination.  

What sort of innovation are you bringing in your project?

T cell-directed vaccines need different designs than conventional vaccines, due to the antigen processing requirements. Many different approaches have been explored including attenuated or recombinant viruses or bacteria, antigen-encoding DNA or RNA, proteins and peptides, each of which has their advantages and disadvantages.

DNA and RNA vaccines have the proven ability to induce CD8+ T cell responses. They are safe and especially suitable for long-term vaccination programs and repeated applications as they lack vector immunogenicity. Moreover, they can be adjusted rapidly to new antigen sequences of emerging pathogen variants. Production and distribution, especially of DNA vaccines, are easy.

Recent progress in DNA vaccines has led to improved immunogenicity, and increasing numbers of registered clinical trials (, which testifies to their increasing acceptance. The DNA vaccine used in this project is based on a Minimalistic Immunogenically Defined Gene Expression (MIDGE) vector, with one of the most advanced DNA vector designs. MIDGE vectors are linear double-stranded DNA molecules, only containing the antigen-encoding sequences, promoter and poly-adenylation site, but no bacterial plasmid backbone sequences that may have detrimental effects. Biodistribution and toxicity data for MIDGE vectors have recently been published, and document an excellent safety profile.

Role and Responsibility of Each Partner

European Vaccine Initiative (EVI) will be responsible for the overall coordination and management of the programme of activities that will be conducted within the project.

Nagasaki University will be responsible for preclinical animal experiments (Objectives 1 and 2), principally of the preclinical evaluation of the vaccine candidate´s prophylactic and therapeutic potential against CL. Efficacy data derived from this work, together with future clinical safety data, are essential to proceed to clinical Phase II studies in target countries. With established CL immunotherapeutic models in place, Nagasaki University will be a key partner to perform additional preclinical experiments, potentially requested by local authorities during further clinical vaccine development.

Mologen will be responsible for the manufacturing of the vaccine at the CMO, lead the preparation of the Phase I clinical trial to establish the safety and, interacting closely with Charité Berlin, prepare the evaluation of the immunogenicity of the vaccine candidate.

Charité will be responsible for the human immunology relating to future testing the vaccine in the clinical Phase I trial. This involves establishing and supervising the implementation of standard operating procedures (SOPs) for the immune monitoring procedures, and design, production and quality control of the synthetic peptides for human and murine assays.

LSHTM will be responsible for the transfer of recent clinical isolates of Leishmania species that cause CL and have been established in inbred mouse models of infection. This will be accompanied by work on establishing the models in Nagasaki University, and methodologies to measure parasite burden in skin nodules and ulcers.

Final Report

1. Project objective

The objective of this project was to complete the preclinical development of LEISHDNAVAX, a candidate DNA vaccine that has been successfully tested for antigenicity in humans in ex vivo studies, and for efficacy in a mouse model for visceral leishmaniasis, by assessing LEISHDNAVAX efficacy against cutaneous leishmaniasis (CL), as recommended by the German regulatory agency. The project encompassed three specific goals: preclinical evaluation of LEISHDNAVAX to study its (i) prophylactic and (ii) therapeutic potential against CL; and (iii) preparation of a phase I clinical trial.


2. Project design

LEISHDNAVAX, a T cell-directed DNA vaccine with minimal expression cassettes for 5 Leishmania antigens, vaccination schemes were optimized to be tested against two Leishmania species endemic in different regions of the world, L. major and L. mexicana.

Prophylactic and therapeutic vaccine efficacy were evaluated in resistant C57BL/6 and susceptible BALB/c mouse rump infection CL models. Vaccine efficacy assessment was based on immunological criteria (T-cell response, cytokine and antibody production), lesion size and parasite burden. Production and quality control of pre-GMP level vaccine, synthetic peptides, peptide libraries and antigens as well as preparation of phase I clinical trial activities were initiated.


3. Results, lessons learned

Synthetic peptides, which cover the five LEISHDNAVAX antigens sequence were designed to assess the vaccine-induced T-cell responses. Quality control by mass spectrometry identified 232 out of 242 synthesised peptides with a purity of >90%. Three expression systems were developed to produce the vaccine antigens, pLEXSY Leishmania, E. coli K12 and human cells.

Upon optimisation of the preclinical experimental setting, the immunization protocol was set to 100 μg vaccine, 3-times with 2-week intervals. Two versions of the vaccine (L-MIDGE and MIDGE-Th1) were initially tested using C57BL/6 mice. Both versions were immunogenic, however MIDGE-Th1 induced stronger IFN-g production in response to most antigenic peptide pools and had previously demonstrated protective effect on challenge infection with L. donovani in a mouse model for visceral leishmaniasis, thus, further studies were carried using MIDGE-Th1 LEISHDNAVAX.

Prophylactic effects of LEISHDNAVAX vaccine on L. major infection, assessed by immunogenicity and challenge experiments revealed no significant difference in parasite burden and lesion size between immunized and control group in both C57BL/6 and BALB/c mice model. Immunization induced antibody production to Leishmania antigens and IFN-g production in spleen T-cells in response to most antigen peptide pools. The prophylactic effects with L. mexicana challenge were similar to those observed with L. major.

LEISHDNAVAX’s therapeutic effects were also evaluated, alone or in combination with standard treatment drug, paromomycin. Parasite burden was reduced in paromomycin treated group, in a dose dependent manner, however no difference was seen when the mice were immunized. It was concluded that MIDGE-Th1 vaccine has no significant therapeutic effect on BALB/c mice that were previously infected with L. major.

As in the previously published study in mouse models for visceral leishmaniasis no adverse effects such as toxicity or disease exacerbation were observed in the cutaneous leishmaniasis models. 

Two immune monitoring marker panels for clinical phase Ia were designed, for mass flow cytometry analysis of antigen-specific T-cell responses from unfractionated peripheral blood mononuclear cells, and for fluorescence flow cytometry analysis of innate immune cells from blood.