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Clinical Studies on a Multivalent Vaccine for Human Visceral Leishmaniasis

Final Report Summary - MULEVACLIN (Clinical Studies on a Multivalent Vaccine for Human Visceral Leishmaniasis)

Executive Summary:
Leishmaniasis caused by Leishmania spp is one of the world’s most neglected diseases and poses a serious health risk to an estimated 350 million people across the world. The development of a human vaccine against Leishmania is goal to achieve. In endemic areas, the majority of infected persons do not develop clinical symptoms and past infection leads to robust immunity against reinfection. In the MuLevaClin project, we developed an innovative vaccine which mimics the natural infection cycle of Leishmania. The vaccine generated was composed of 5 main components: Kinetoplastid membrane protein-11 named KMP11 a structural protein, produced by recombinant technology in E.Coli; LJM11 a sand-fly salivary protein produced in HEK mammalian cell; Leish F3 (NH-SMT) a synthetic fused protein produced in E.Coli; all the three antigens were presented in an influenza virosomal form, adjuvanted by a TLR4 synthetic agonist named GLA-SE.
Early preclinical testing have revealed that both LJM11 and LeishF3 had to be replaced by LJL143 and Leish F3+ respectively which showed better protection data in animal models (mice and hamsters).
Extensive preclinical studies in mouse model have been performed in two independent Institutes: IMBC and University Autonoma de Madrid (UAM-CBMSO). Immunogenicity data were very promising and the most advanced formulations were tested in further preclinical studies in hamster model, performed in ISCIII and outsourced to NIH-NIAID (Bethesda, US). The formulations showing protection were finally tested in a Dog model study at Zydus Cadila (Ahmedabad, India). The dog challenge trial has been accomplished on time and the testing of the samples is ongoing at ISCIII.
In order to prepare toxicological studies and the Investigation Brochure for clinical testing, all components have been developed for GMP production. Several GMP lots were produced, characterized and extensively tested.
In parallel we performed the development of assays required for the standardization and analytical determination of the final GMP-grade formulation of the vaccine to be employed in Phase-1 testing involving human volunteers. The intend was to provide to regulators standardized assays to test the GMP vaccine formulation. As basis for purity of the GMP grade material the WHO requirements for recombinant Hepatitis B and recombinant HPV vaccine has been taken.
Although the virosomal formulation showed the best protection in the efficacy studies, the consortium decided to give up this promising approach. This was mainly due to the complexity of the production process which would have considerably increased the production costs, and therefore restricted its use in affected populations in need of such a vaccine.
Importantly before entering into clinical studies we had to show stability of the candidate vaccines. Frozen and lyophilized formulations have been tested and finally the frozen amorphous formulation was selected as it showed excellent stability during this timeframe.
Due to unexpected factors, we were not able to achieve the planned clinical trial. However, all the preparatory work for the phase 1 clinical study, including immunological tests for the measurement of IgG titres against the recombinant vaccine antigens as well as for the analysis of vaccine-induced parasite-binding antibodies have been established.
As success of the project, we can mention that the vaccine against leishmaniosis developed within this project, obtained the orphan drug status by Swissmedic (the Swiss regulators)
Based on the advanced results of this project, we have the clear intention to further develop this vaccine until marketing authorization.

Project Context and Objectives:
Project Context and objective
Leishmaniasis was declared as one of the world’s most neglected diseases at the 60th WHO Assembly (2007). Leishmaniasis can be manifested as a wide range of clinical etiologies including visceral, mucocutaneous, diffuse, and cutaneous leishmaniasis (CL). Visceral leishmanasis (VL), the most severe form of the disease, can be fatal if left untreated. The devastating effects of this disease affect largely the poorest of the poor, mainly in developing countries with a disease burden calculated at 2 090 000 disability adjusted life years. Each year, there are approximately 300,000 cases of visceral leishmaniasis (90% in Bangladesh, Brazil, India, Nepal and Sudan), with an estimate of more than 50 000 deaths. In some cases, due to cultural reasons and lack of access to treatment, the case-fatality rate is three times higher in women than in men. Environmental changes have also led to leishmaniasis outbreaks spreading to parts of southern Europe.
Existing treatments for this neglected disease have severe drawbacks: debilitating side effects, increasing drug-resistance, high cost and long-term treatment. Attempts at vector control policies have been shown to be insufficient, impractical, or difficult to sustain. To date there is no effective vaccine against human leishmaniasis. Several attempts to develop candidate vaccines against leishmaniasis were inconclusive or gave negative results, and very few candidates progressed beyond the experimental phase in model animals.
We believe that the natural infection cycle of Leishmania should be taken into account in order to generate a vaccine with the potential to efficiently block vector transmission and infection of the human host. Hence, our approach is based on the use of a protein (LJL143) present in the sand fly saliva, together with four Leishmania infantum antigens: Kinetoplastid membrane protein-11 (KMP-11), sterol 24-c-methyltransferase (SMT), nucleoside hydrolase (NH) and cysteine protease b (CPB). All the antigens are produced as recombinant proteins, three of them forming a fusion protein called LEISH-F3+ (or NH/SMT/ΔCPB). Another component included in the vaccine composition is the Glucopyranosyl lipid A (GLA), a well-characterized adjuvant, yet tested in clinical studies, that potentiates Th1 responses. This adjuvant is a synthetic derivative of the lipid A tail of LPS with limited cytotoxicity, but strong potential to induce immune responses in mice, guinea pigs, non-human primates, and humans. This adjuvant is formulated in a squalene-based oil-in-water emulsion (SE). (Duthie et al.2012; Goto et al.2009;Goto et al. 2007; Coler et al.2011).
The intention was to establish two lines of protection against natural infection with Leishmania. Additionally, because recombinant proteins alone, as well as peptides, generally induce only weak T cell responses, to facilitate antigen-presentation to immune cells and, therefore, to enhance the immunological response against the target antigens, recombinant proteins are being formulated into virus-like particles (VLP) based on the proven Virosome technology. The partners and the network created around this EU funded project represented some of the world leading experts in each of the research areas needed for generating and testing this candidate vaccine in clinical trials; all of them at the forefront of research into leishmaniasis. This project, Clinical Studies on a Multivalent Vaccine for Human Visceral Leishmaniasis (MuLeVaClin), represented an innovative approach in combining proven technologies and the latest insights on leishmaniasis in order to produce an efficacious vaccine.
We achieve the following goals:
1. To develop produce and test an innovative multivalent vaccine against human visceral leishmaniasis based on multivalent VLP, adjuvanted with a strong TLR4 agonist, containing the recombinant insect salivary gland protein LJL143, the Leishmania promastigote protein KMP11, and the recombinant fusion protein LEISH-F3+, consisting of three additional Leishmania antigens (sterol 24-c-methyltransferase, nucleoside hydrolase and a truncated version of cysteine protease b).
2. All the components have achieved the proof of concept in animals and/or humans individually. For the first time all of these components were combined in an innovative candidate vaccine. The optimal composition of the vaccine formulation and the immune response elicited was evaluated in preclinical studies in mice, hamsters and dogs. In addition, the immunogenicity of the different proteins was analysed in asymptomatic and VL cured individuals.
3. We developed a highly stable vaccine to facilitate storage and transportation, suitable for sub-tropical and tropical regions.
4. We established GMP production technologies for each component and formulation of the vaccine.
5. We designed a preclinical safety study protocol (toxicology studies) in suitable animal model, which will be analysed under the scrutiny of the Swiss regulatory authorities.
6. We developed specific tools and quantitative procedures to evaluate biomarkers of resistance and susceptibility in individuals from endemic areas, and the strength of the immune response in those enrolled in clinical trials
7. We prepared an IND dossier to be submitted the Swiss regulatory authority.

Project Results:
In the first 18 month, the following main results have been achieved:
1. The three recombinant proteins were produced and purified, obtaining enough amounts to prepare the vaccine formulations and for the preclinical studies in mice and hamsters.
2. SOP protocols describing expression and purification process and the methodology to determine the antigen quality and content were written by the antigen producers and transferred to Etna-Biotech. These SOPs have been transferred to AMVAC for the GMP production.

3. Virosomes were successfully formulated with each one of the recombinant proteins.

Based on antigenicity presented in the Kick-off meeting (hold in Catania, Nov 2013), it was decided to consider the insect salivary protein LJL143 to replace the LJM11 salivary protein. The rationale behind can be summarized as follows. It has been shown by Dr. Valenzuela’s group at NIH that salivary proteins protect against leishmaniasis in rodent models. One of these salivary proteins, LJM11, when inoculated as a recombinant protein in mice conferred protection against cutaneous leishmaniasis. The protection correlates with the ability of the salivary protein to produce a Th1 response, which is sustained over time and can be recalled in the skin after the bite of a sand fly. Having this in mind, Dr. Valenzuela and his group looked for salivary proteins that can produce a Th1 response in large animals, firstly in dogs and more recently in humans. To search for these molecules they developed a novel approach, named RAS (reverse antigen screening) where animals, previously exposed to sand flies, are injected intradermal with DNA plasmids coding for different sand fly salivary proteins (the skin is used as a natural mammalian expression system) and determine if one of these proteins can produce a local immune response at 48 hours with a Th1 profile. Dr. Valenzuela succeeded in identifying two proteins that were able to elicit a Th1 immune response in dogs, LJM17 and LJL143. Only recently he used a similar approach in humans, but instead of using DNA plasmids, he stimulated PBMC of individuals previously exposed to sand fly bites with different sand fly salivary recombinant proteins and this resulted in the identification of LJL143 as a protein that can produce a Th1 response in humans (more than 90% of individuals tested produced higher levels of interferon gamma as compared to other proteins or controls). In the same study LJM11 protein was not as strong as LJL143, and it was therefore concluded that LJL143 is the most suitable candidate for a Phase I Clinical trial. Furthermore, the proof of concept is the same, the use of a sand fly salivary protein; second, this protein is immunogenic in humans and the study will be conducted in humans; third, this is the first insect protein, discovered to date, that has the ability to produce a Th1 response in humans (and also in an independent study, in dogs) and the best candidate so far to be included together with other Leishmania antigens for the development of the first protective Leishmania vaccine.
Another minor deviation from the initial proposal was the expression of Leishmania KMP11 without any tag to avoid undesired effects of the tag. In the original proposal, it was planned using His-tagged KMP11.







The purity of the three proteins were analysed by SDS-PAGE (see figures below).










Purification of LJL143 protein from supernatant of P. pastoris culture. The protein is obtained as a monomer either using reduced (lane 1) or non-reduced (lane 2) conditions during the purification process. M: molecular weight marker.









Purification of L. infantum KMP11 from E. coli cells expressing the recombinant protein without added tags. For storage and shipment purposes, the protein was lyophylized. In the picture, it is shown that lyophylization does not affect the monomeric nature of the protein. Lanes 1 and 2 shows the protein after and before lyophylization. M: molecular weight marker.

The purified protein was analyzed by SDS-PAGE and its identity was confirmed by immunoblotting with antibodies raised against the NH and SMT components of LEISH-F3. Endotoxin levels were determined to be <100 endotoxin units per mg by the Limulus amoebocyte lysate assay.













Purification of NH-SMT fusion protein (LEISH-F3) LJL143 protein from E. coli cells expressing the recombinant protein. The protein is obtained as a monomer when purified in reduced conditions (lane 1) but forms aggregates when non-reduced conditions (lane 2) are used. M: molecular weight marker.
















Characterization of KMP11-formulated virosomes. Panel A shows SDS-PAGE analysis of KMP11-formulated virosomes (V-KMP11). Different amounts of V-KMP11 (lanes 1-3), KMP11 protein (lanes 4-7) or Empty-virus (lanes 8-9) were analyzed. M: molecular weight marker. Panel B shows the mean size (in nm) of the KMP11-virosome particles from three different preparations (red, blue and green lines).


























Characterization of LJL143-formulated virosomes. Panel A (on the left hand side) shows SDS-PAGE analysis of LJL143-formulated virosomes (VLP-LJL143). Different amounts of LJL143-virosomes (lanes 1-3), LJL143 protein (lanes 4-7) or Empty-virus (lanes 8-9) were analyzed. M: molecular weight marker. Panel B (on the right hand side) shows the mean size (in nm) of the LJL143-virosome particles from three different preparations (lines red, blue and green).


















Characterization of NH-SMT-formulated virosomes. Panel A shows SDS-PAGE analysis of NH-SMT -formulated virosomes (VLP-NH-SMT). Different amounts of VLP- NH-SMT (lanes 1-5), NH-SMT protein (lanes 6-9) or Empty-virus (lanes 10-11) were analyzed. M: molecular weight marker. Panel B shows the mean size (in nm) of the NH-SMT-virosomes.



Immunogenicity and challenge studies in a hamster model.
Previous to immunization and challenge trial in hamster model, a trial was carried out to assess infectivity of the L. infantum strain proposed for challenge, NIH strain, provided by J. Valenzuela lab (NIH) and used in the mice model experiments.
Nine hamsters were inoculated by intracardiac route with 20 x 106 promastigotes of the NIH strain and another 9 hamster were infected with 20 x 106 promastigotes of the JPC strain (the own laboratory strain isolated in Madrid from the spleen of a dog with canine leishmaniasis). Both NIH and JPC promastigotes were cultured and expanded in NNN medium and washed several times in PBS before inoculation. Animals were sacrificed at month 1, 2 and 3 after experimental infection and parasite burden in spleen, liver and bone marrow was analyzed by real time quantitative PCR. Mean values obtained confirmed that hamsters infected with the JPC strain presented a higher parasite burden in all the organs analyzed at month 3, while NIH strain becomes undetectable in the hamster bone in liver and bone marrow three months after infection (parasite burden decreases over time). Furthermore, parasite burden in spleen was also lower, comparing the NIH strain with our own strain. Therefore, JPC strain was used to challenge hamster in the efficacy trial

Standardization of Leishmania sub-unit antigens and antigen formulation
Goal was to set the validation of assays for standardization of each purified sub-unit antigen and final bulk formulation, as required by European Pharmacopeia, for recombinant antigens and virosomal vaccines, in order to make available procedures for Quality, Purity and Identity control of this novel category of vaccine antigens
Total protein assay
The used method was the Bradford assay performed on flat-bottom ELISA plates using Bovine Serum Albumine protein standard and Quick Start Dye Reagent (Biorad). Antigen samples were run in duplicate. The detected protein concentrations were (slightly) different from what reported: antigen KMP-11– Batch 3 = 0.756 mg; antigen LJL143-HIS = 0.797 mg; NH-SMT-Leish F3 = 0.855 mg. The empty virosome sample had a total protein concentration of 0.293 mg/ml.

Antigen content and identification assays
Critical reagents were assayed by Single Radial Immunodiffusion (SRID) assay, the currently acceptable assay for potency determination of influenza vaccines. The assay was set using in-house glassware equipment, agarose (LONZA) and detergent (Zwittergent, Merck). All antigens were tested with their respective antibodies in separate agarose gel plates, to verify first the presence/absence of precipitation rings. The latter included five antibody anti- KMP-11 concentrations to react with 4 antigen KMP-11 concentrations, with or without detergent; one antibody anti-NH-SMT-Leish F3 concentration to react with 4 antigen NH-SMT-Leish F3 concentrations, with or without detergent; one antibody anti- LJL143-HIS concentration to react with 4 antigen LJL143-HIS concentrations, with or without detergent. In some plates, heterologous vaccine antigens were also tested to evaluate cross-reactivity.


In a first attempt the only positive result was obtained with the NH-SMT-Leish F3 antibody/antigen complex. It strongly suggests that antisera production against the vaccinal antigens is a critical step for future standardization assays on GMP antigens/vaccine.
The adjustment and validation of procedures have been successfully achieved as regards the analytical determination of individual antigens dosage from laboratory-grade vaccine batches - KMP11 plus LJL143 plus LEISH F3+ at the doses of 5 or 10, 1 or 2, and 5 or 10 µg, respectively. The procedures included a number of standardized assays for Total Protein content, Quality and Purity of the batch, and Identity and Potency of the antigens.







In the second reporting period the following main results have been achieved
Antigen production and process development for preclinical studies
The main objective was coordinating the production of the different antigens, adjuvants and final formulation of the vaccine. Also, it was a commitment to provide enough amounts of the formulated vaccine and its individualized components to complete the preclinical testing assays in mouse and hamster models. Unforeseen problems regarding vaccine immunogenicity that arose after the first preclinical studies required the development of alternative approaches in both antigen production and vaccine formulation. Details are provided in the following sections (task descriptions). As a consequence of these facts, the activity of this WP had to be extended beyond the initial delivery dates. Nevertheless, the progress has been proper and the WP has been able to resolve initial problems and to provide improved reagents to repeat the preclinical assays and go ahead with the planned objectives of the project.

Production of the LJL143 recombinant protein
As informed in the first part, a replacement in the insect salivary protein was done, after obtaining the approval from the EC project officer. LJL143 recombinant protein with HIS-tag was initially expressed in HEK-293 cells, but due to problems regarding purity, yield and cost for production, alternative methods were assayed. To overcome these issues, the LJL143 protein has being expressed and purified from the yeast Pichia pastoris X-33 cells without HIS-tag. In this system, high yields of protein with high grade of purity, at lower cost of production were obtained. Before to be tested in animal experiment for the MuLeVaClin vaccine, a side by side study of LJL143 produced in HEK-293 cells line, with LJL143 produced in Pichia pastoris X-33 was performed at NIH, in order to assess the equivalent immunogenicity. The fig. below shows IFN-gamma production of human Peripheral Blood Mononuclear Cells after stimulation with media, LJL143 (mammalian) and LJL143P (Pichia) or positive control SGE (salivary gland extract). These individuals were previously exposed to sand fly bites multiple times before purifying PBMCs from their blood.










After having observed the equivalence of the two LJL143 produced in different hosts, LJ143 produced in yeast has been selected as salivary antigen, and enough material was produced in order to be tested either alone or in VLP-formulated preparations in the second set of preclinical studies (WP3). This new LJL143 preparation was also sent to the ISCIII for preclinical studies of antigenicity in humans; identification of biomarkers of resistance and immunization (WP6); and for the validation of assays for standardization of each purified sub-unit antigen (WP4).
This task has been coordinated by Etna Biotech.

Production of the KMP11 recombinant protein
The procedure for production of this protein has not changed, KMP11 has been expressed in Escherichia coli and recovered in soluble form by chromatographic procedures, as reported previously. Nevertheless, three additional protein lots (thirty mg each) have been produced after that. This material was used for vaccine formulations (Etna-Biotech) and also distributed to other project partners for preclinical studies, analysis of antigenicity in human samples and for setting the validation assays for standardization of each purified sub-unit antigen.

Production of the NH/SMT fusion protein
The NH-SMT fusion protein (also known as Leish-F3), which is formed by the tandem linkage of the Leishmania nucleoside hydrolase (NH) and sterol 24-c-methyltransferase (SMT), was provided by IDRI (subcontractor of Amvac) and used for the first preclinical studies in mice and hamsters as mentioned in the previous. During this time IDRI has been keeping the development of such antigen and now has recently developed a superior version based on the same NH-SMT fused with Delta Cysteine Protease B (CPB), an antigen well recognized by infected humans. Noteworthy, previous assays in mice indicated that immunization with CPB elicits a substantial protection against L. infantum (Dumonteil et al (2003). Vaccine 21, 2161; Rafati et al (2005) Vaccine 23, 3716). The so called LEISH F3+ is a superior candidate, in which the downstream process has been improved, as compared with its parental LEISH F3. Besides, this new antigen is able to elicit an efficacious and strong immune-response in hamster challenged with infected sand-fly, as was assessed by NIAID/NIH. Furthermore, the tri-fusion protein (NH-SMT-CPB or Leish-F3+) was found to be more stable and to yield protein preparations with higher purity than the Leish-F3 construct. After a discussion in an ad hoc project meeting, it was decided to replace LeishF3 by Leish-F3+ as no alteration in the antigen content of the proposed vaccine would be introduced.
The procedure for Leish-F3+ production and purification was the same as that used for Leish-F3 (depicted in the First report).
This protein has been used either alone or VLP-formulated in the second set of preclinical studies (WP3). This new Leish F3+ preparation was also sent to the ISCIII for preclinical studies of antigenicity in humans and identification of biomarkers of resistance and immunization (WP6) and to ISS for setting the validation assays for standardization of each purified sub-unit antigen (WP4).
This task has been accomplished by Etna Biotech.



Production of the TLR4 agonist adjuvant
GLA is a synthetic monophosphoryl lipid A (MPL)-like molecule, which acts as a Toll-like receptor 4 (TLR4) agonist. GLA is admixed with squalene oil, excipients, and sterile water to produce a stable emulsion, which is named GLA-SE. This adjuvant developed by IDRI was provided to the consortium in enough amount to fulfill all the preclinical studies. This compound has been provided by IDRI (subcontractor of AMVAC). It was sent to Etna-Biotech and to the laboratories involved in preclinical studies.

VLP formulation of the vaccine components
Formulations of antigens in Virus like particles (VLP or virosomes) were prepared by Etna-Biotech following the procedure outlined in the first part. For the preparation of VLP, GMP grade Influenza H1N1 virus (A/H1N1/California strain) was used.
Five different formulations were prepared:
✓ Virosomes loaded with recombinant LJL143;
✓ Virosomes loaded with recombinant KMP11 protein;
✓ Virosomes loaded with recombinant Leish-F3+;
✓ Empty Virosome containing only influenza proteins;
✓ Virosomes loaded simultaneously with LJL143, KMP11 and Leish-F3+ (VLP-MIX).

As described in the previous report particle size determination and SDS PAGE Coomassie Staining were performed to characterize the formulations. Examples of the characterization of MuLeVaClin formulations are shown in the below pictures.








































In addition, in order to exclude any potential drawback related to stability, fresh VLP-formulations were prepared just before each inoculation dose. This material (refrigerated) was sent to the laboratories involved in preclinical studies with mice and hamsters.



Pre-Clinical Evaluation and optimisation of vaccine formulation
Immunogenicity results in mouse model
IBMC data
It deserves to be stated that a significant specific cell proliferation against KMP-11 and TLA was only verified in the animals pre-immunized with Pre-LJL PA(1+1+1). This result indicates that previous administration of the sandfly-saliva derived antigen may be beneficial for the generation of a better response against some of the parasite-derived antigens. Nevertheless, the levels of cytokines detected in the cell proliferation assays (less IFN-γ and more IL-10 in Pre-PA(1+1+1) versus PA(1+1+1) groups), along with the antibody response (dominantly IgG1 versus mixed IgG1/IgG2a antibodies, comparing Pre-LJL PA(1+1+1) with PA(1+1+1) groups), raises the question of which type of immune response is being induced, and what are the final implications in terms of protection. This question had to be explored, using an adequate infection animal model.

CBMSO-UAM data
In general, no significant differences have been observed in lymphoid or myeloid splenocytes in the different groups of immunization regarding the control group (saline). However, lower numbers of splenocytes were detected in mice immunized with single-antigen formulated VLPs containing 5 µg of F3+ and 5 µg of KMP-11 (VPA-5), but this effect must be associated with the amount of inoculated virosomes as similar numbers were determined in the mice inoculated with only empty virosomes (the same volumes of empty virosomes and VLP-formulated particles were used for groups VPA-5 and VA). The decrease in the number of cells was not associated with changes in spleen weight or the percentage of lymphoid or myeloid populations, which were not altered by vaccination.
All vaccine formulations elicited antigen-specific immunological responses, which were detected after in vitro stimulation of splenocytes with either individual antigens or total Soluble Leishmania Antigen (SLA); both antigen-specific proliferation and IFN-γ production were observed in the vaccinated mice. In particular, significant CD4 cell proliferation was observed in response to LJL-143 and F3+. Also, significant humoral IgG responses against LJL-143 and F3+ were detected in the sera of all vaccinated animals.

CONCLUSIONS
These studies of the MuLeVaClin vaccine candidate have allowed evaluating different formulations in terms of immunogenicity.
In general, no significant difference has been observed in lymphoid or myeloid splenocytes in the different groups of immunization regarding the control group (saline). However, lower numbers of splenocytes were detected in mice immunized with single-antigen formulated VLPs containing 5 µg of F3+ and 5 µg of KMP-11 (VPA-5), but this effect must be associated with the amount of inoculated virosomes as similar numbers were determined in the mice inoculated with the empty virosome (the same volumes of empty virosomes and VLP-formulated particles were used for groups VPA-5 and VA). The decrease in the number of cells was not associated with changes in spleen weight or the percentage of lymphoid or myeloid populations, which were not altered by vaccination. in vitro
All vaccine formulations elicited antigen-specific immunological responses, which were detected after stimulation of splenocytes with either individual antigens or total Soluble Leishmania Antigen (SLA); both antigen-specific proliferation and IFN-γ production were observed in the vaccinated mice. In particular, significant CD4 cell proliferation was observed in response to LJL-143 and F3+. Also, significant humoral IgG responses against LJL-143 and F3+ were detected in the sera of all vaccinated animals.
In conclusion, this study has shown that replacing of F3 by F3+ has led to a clear improvement of the anti-leishmanial immune response elicited by the MuLeVaClin vaccine. In addition, this study has shown that both vaccine formulations (antigens combined with GLA-SE and VLP-formulated antigens + GLA-SE) are similarly immunogenic, and, therefore, both vaccines may be suitable for further analysis.

Immunogenicity and challenge studies in hamster model.
The main objective of the project is to test the safety and efficacy of an innovative vaccine for human visceral leishmaniasis. The vaccine candidate is based on Virus-Like Particles loaded with 3 different antigens (2 from the parasite and 1 from the sandfly saliva), adjuvanted with a TLR4 agonist.

Efficacy assays in hamster: experiment layout
We have done a new experiment for testing the safety of the vaccine F3+, KMP-11 and LJL-143 formulated in virosomes with GLA-SE as adjuvant. Animals were immunized three times (separated by four weeks each) intramuscularly (the route to be used in human clinical trials), infected by intracardiac route with Leishmania infantum JPC strain and euthanized three months after infection. Before infection, a blood sample (and sera) was obtained by vena cava venipuncture.
Data were tested for normality using the Shapiro-Wilk test and comparisons between groups were performed with Mann-Whitney test. Significance was set at p<0.05.

Results of the immunization
Hematological parameters:
In general, all the hematological parameters were included within normal limits, indicating the safety of all of the vaccine candidate components.

Humoral response by ELISA assay:
Total IgG levels were determined by ELISA. Immunization produced specific anti- IgG antibodies (Figure 3).
Most of the groups induced a specific statistically significant increase on anti-F3+ and anti-LJL-143 IgG antibodies, more potent with adjuvant and/or virosomes. Only P 5:5:1 and PA:5:5:1 had anti-KMP-11 IgG antibodies. Statistical significances were *p<0.05 **p<0.01 and ***p<0.001.














Results 3 months after infection
Humoral response by ELISA assay:
VPA 5:5:1 and VPA 1:1:1 had significant lower SLA-anti IgG antibodies. Most of the groups induced a specific increase on anti-F3+, anti-KMP-11 and anti-LJL-143 IgG antibodies. It was more potent with adjuvant and/or virosomes. Most hamsters had anti-KMP-11 IgG antibodies (previous to infection, only in P 5:5:1 and PA 5:5:1 immunized hamsters). Statistical significances were represented as *p<0.05 and **p<0.01.



Parasite burden by real-time PCR:
Regarding to PBS control, parasites/ug DNA significantly decreased in spleen of VPA 5:5:1 group. Parasite burden was also decreased in P 5:5:1 group but no in PA 5:5:1 group. There was also a slight decrease in VPA 1:1:1 group.








No changes were found in liver for VPA 5:5:1 or P 5:5:1 groups (Figure 9). There was a significant increase of parasites/µg DNA in PA 5:5:1.








Conclusions
• Tested formulations induced immunological response with specific antibody production, in hamsters.
• After L.infantum challenge, some slight reduction in the parasite burden of visceral tissues was found in P 5:5:1 and VPA 1:1:1 groups.
• Formulation VPA 5:5:1 was efficient decreasing L.infantum burden in hamsters.




Preclinical studies of antigenicity in humans and identification of biomarkers of resistance and immunization
The specific objective was to test the antigenicity, both humoral and cellular, of the recombinant and fusion proteins in both cured and asymptomatic L. infantum infected individuals.

IBMC data
The study of human humoral reactivity against the MuLeVaClin vaccine candidate individual antigens was done by IBMC. Antigen specific immunoglobulins (IgG) against F3+, KMP-11 and LJL-143 were determined in a total of 234 human sera samples collected in Spain, divided, in negative (74), asymptomatic (41), symptomatic (active VL + active CL) (47) and healed (cured from VL + cured from CL)(72). Reactivity against each of the antigens of the MuLeVaClin formulation was individually tested, using a coating antigen concentration of 1 µg/ml. Cut off values were extrapolated from the analysis of the ROC curves constructed for both KMP-11 and Leish F3+, but not for LJL-143, once there is no way to separate the sera of the “positives” (sandfly bite exposed) from the sera of the negatives (unexposed).
Reactivity was detected against each of the antigens tested, in an extent significantly higher in the group of people with active disease (symptomatics). However, particularly the sensitivity, but also the specificity obtained for both KMP-11 and Leish F3+ were not very high, and because of that, the existence of a non-specific reactivity may not be excluded. In the case of the reactivity against LJL-143 it was not expected. LJL-143 is a salivary protein from the New World vector of visceral leishmaniasis (L. infantum), Lutzomyia longipalpis. However, a LJL-143 Phlebotomus perniciosus salivary protein homologue, PpeSP06 shares 46.8% identity with LJL-143. Therefore, the existence of cross-reactivity between the exposed-population circulatory antibodies against PpeSP06, and LJL-143 is a possible explanation of the verified reactivity.
The reactivity against the different antigens is comparable between the individuals healed from cutaneous leishmaniasis and from visceral leishmaniasis (either immunodepressed or immunocompromised). The only significant difference observed was within the asymptomatic group: sera from immunocompromised individuals significantly reacts more against KMP-11 and LJL-143 (also seen as tendency for Leish F3+) than sera from immunodepressed individuals.

ISCIII data
Cellular specific recognition of the vaccine candidate proteins LJL-143, F3+ and KMP-11, KMP11 was assessed at the ISCIII by in vitro cell proliferation assay using PBMCs isolated from infected humans. Samples from active visceral leishmaniasis (symptomatic) patients, cured patients (from visceral and cutaneous leishmaniasis, CVL/CCL respectively), asymptomatic subjects (AS) and healthy endemic subjects as negative control (EC) were collected in a L. infantum post outbreak area (Fuenlabrada, Spain). Cellular antigenicity was evaluated in PBMCs measuring the specific cytokine profile on supernatants after five days culture with different antigens. KMP-11 induced a significant secretion of IFN-γ in PBMCs from CVL, CCL and AS subjects in comparison to EC individuals, whereas F3+ induced the production of IFN-γ and TNF-α in PBMCs from CCL and AS. Finally, LJL-143 was recognized by PBMCs of some individual from all groups, including some endemic healthy control, as expected from individuals exposed to sandfly bytes.

Conclusions
In conclusion, some reactivity against each of the three antigens was detected in human sera from individuals living in a Leishmania infantum endemic area, in all groups tested. The supposed negative individuals whose sera reacted against both KMP-11 and LJL-143, alerts either for the possible existence of non-specific cross-reactivity, or for their potential “misdiagnose”.
F3+ and KMP-11 antigens were recognized by PBMCs isolated from asymptomatic individuals and cured patients from leishmaniasis caused by L.infantum and are able to induce the production of cytokines as IFN-g and TNFa associated with the Th1-type response.
LJL-143 was recognized by PBMCs from both asymptomatic and cured individuals but also of individuals living in an endemic area of leishmaniasis without a specific cell memory response against the parasite but exposed to sandfly bytes

Significant results achieved
Antigenicity of vaccine proteins have been tested successfully in the human context, both for humoral and cellular mediated immune response. Tools and biomarkers to measure cell immunity to Leishmania and to recombinant antigens have been established


The following main results have been achieved during the third reporting period


Pre-clinical evaluation of prototypes in dogs.
Dogs constitute an excellent model to study vaccine candidates for visceral leishmaniasis. This specie is susceptible to natural infection by L. infantum and develop clinical diseases, as domestic reservoir of the parasite is also a target for control and a good model for human visceral leishmaniasis because the symptoms in dogs are similar to those developed in humans. Therefore, the laboratory model of canine leishmaniasis represents a helpful preclinical approach for vaccine development. Several antigens and approaches for immunization against Leishmania have been tested in dogs. We have previously studied the specific cytokine profile induced by KMP-11 in PBMCs from asymptomatic and oligosymptomatic L. infantum-infected dogs and compared to the cytokine profile induced by SLA, confirming that KMP-11 stimulation produced an upregulation of the IFN-γexpression.

OBJECTIVES OF THE STUDIES

To determine the immunogenicity, safety and efficacy of Leishmania multivalent vaccine (MuLeVaClin vaccine) in Beagle dogs.
Twenty-four animals have been immunized on day 0, day 28 and day 56 by subcutaneous route of administration. After each immunization animals have been observed for any side effect like fever, swelling, erythema, nodule etc.
Animals have been challenged by virulent Leishmania infantum promastigote by intravenous injection. After challenge animals have been observed for general health, clinical pathology, humoral immune response (serology) and cell mediated immune response. Bone marrow biopsy (aspiration) have been carried out before challenge, i.e after third immunization and will be done six month after challenge and at the time of termination of the study.
In addition, the immune response induced by the vaccine have also been assessed by dosing side-effects, if any. The rationale of this study was to identify and characterize effectiveness of the vaccine to conclude that it is reasonably safe to proceed for clinical investigation and to support throughout the clinical development.


Observations
• There was no abnormality observed at the site of injection of vaccine formulation and placebo in any of the animal groups.

• No clinical symptoms related to Leishmania were observed post 6 months of challenge in any of the animal groups.

Significant results achieved
No complete results have been achieved so far, as the experiment is ongoing. However, preliminary results showed that, 6 months after challenge, some animals are positive in the bone marrow to Leishmania by PCR while other dogs are negative.


Preclinical studies of antigenicity in humans and identification of biomarkers of resistance and immunization
Demonstration of the antigenicity of the recombinant antigens included in the formulation of the vaccine, carried out in the previous period of reporting. During this second reporting period we have focused in the development of quantitative test to determine levels of immunity and to distinguish vaccinated individuals from cured VL patients and asymptomatic subjects. Since, up to the moment, no vaccinated individual are available because the Phase I/II trial has not started, we have made different assays for quantification and differentiation on vaccinated individuals. These assays have been done by testing cell and humoral responses in individuals with Leishmania-specific immunity like asymptomatic and VL cured patients, who are a very good approach for that studies. Results obtained will be helpful to develop the corresponding test in vaccinated individual when available.
Since the nature of the specific protection against the parasite is cell mediated, quantitative assessment of immunity induced by the vaccine must be based in a cellular test, while the test to distinguish vaccinated from asymptomatic/cured individuals should be based in the different humoral response elicited by the vaccine or by natural infection. Leishmania-specific serum antibody levels in asymptomatic individuals are usually very low and therefore, high sensible antigens or combinations or antigens are needed to detect such individuals. These aspects has been studied in detail as follows

Development of quantitative test to evaluate level of immunity induced by the vaccine
We have developed new immunoassays with quantifiable biomarkers indicative of the resistant immune response to Leishmania infection. We have found that WBA assay and F3+ antigen with the biomarker IFN-γ is a potential test to evaluate the level of immunity induced by the MuLeVaClin vaccine. In addition, WBA and KMP-11 with the biomarker TNF-α also seems to be a good test to assess the onset and duration of immunity of the vaccine candidates during the clinical phase I/II.

Development of immunoassays to distinguish vaccinated from asymptomatic and naïve individuals

Due to the delay in the start of the phase I trial and in order to advance the experimental trials, we have carried out in recent months a series of studies on the humoral response of cured and asymptomatic patients that will finally be completed when we have vaccinated individuals
The main objective of the work was to test the safety and efficacy of an innovative vaccine for human visceral leishmaniasis. Results concerning specific humoral responses of humans from an endemic area against a panel of different antigens were obtained. The analysis of human humoral responses was important to characterize the vaccine target population, allowing in theory to distinguish non-infected from infected individuals (including asymptomatics), alone or together with human cellular responses. Such studies will help us to predict the used-antigens value as biomarkers of vaccination/exposure and to hypothesize a potential DIVA (Differentiating Infected from Vaccinated Animals) strategy.

Significant results achieved
Main results obtained were related to the identification of rK28 as the best antigens to establish a test to identify asymptomatic individuals. Further, studies on specific cell response to the vaccine antigens confirmed that expression of IFNγ after stimulation of lymphocytes is a good marker of immunity that could be used to establish the test to distinguish vaccinated from asymptomatic individuals

Preparation of the Clinical Trial Applications, and Ethics Commission approval.
An application for recognition of orphan disease status of the polyvalent vaccine against Leishmaniasis was submitted to the Swiss drug authorities (Swissmedic) on 11 May 2017. A list of questions was received on 18 August and responded to on 18 September.
On 23rd January 2018 the status as an important medicinal product for the rare disease “prophylaxis of leishmaniasis at high risk of infection" was obtained by order from Swissmedic. This status will facilitate the conduct of the Phase I trials in Switzerland, as the sponsor and consortium will have free access to scientific advice and a waiver of the costs for registration of the product under the “Marketing Authorization for Global Health Products scheme” of Swissmedic.
An agreement that Swiss TPH will formally represent the sponsor of the Phase I trial, Etna Biotech S.r.l. in Switzerland was signed on 6 April 2018.
Awaiting the results of the toxicological studies (WP5) by January 2019, the preparation of the clinical trial application has started. The list of documents needed to be established for the clinical trial application (Swissmedic and Ethics Committee) in Switzerland has been shared with the consortium in December 2017. An ICH-GCP compliant template for the Investigator’s Brochure as well as the Swissethics formal study synopsis and protocol templates were shared with the consortium in January 2018. Swiss TPH has revised the trial synopsis and adapted it to results obtained during the preclinical profiling of the vaccine. The revised version was circulated between all partners, further modified along with the comments received and is now approved.
To analyze immune-responses elicited by the candidate vaccines in humans.
Within this task, Swiss TPH had the responsibility to analyse the vaccine-induced humoral immune responses. For the establishment of the immunological tests required for the clinical study, Swiss TPH has received the recombinant Leishmania proteins (LJL143, LEISH-F3+, and KMP11) from Etna Biotech and rabbit antisera to all three antigens from IDRI. These reagents are now being used to develop an Enzyme-Linked Immunosorbent Assay (ELISA). The ELISA will be validated and optimized for the analysis of human sera by testing serum samples from laboratory-confirmed Leishmaniasis patients and from control individuals.


Significant results achieved
The orphan drug status as an important medicinal product for the rare disease “prophylaxis of leishmaniasis at high risk of infection" was obtained by order from Swissmedic.
An agreement that Swiss TPH will formally represent the sponsor of the Phase I trial, Etna Biotech S.r.l. in Switzerland was signed.
The list of documents needed to be established for the clinical trial application (Swissmedic and Ethics Committee) in Switzerland has been shared with the consortium members.
An ICH-GCP compliant template for the Investigator’s Brochure as well as the Swissethics formal study synopsis and protocol templates were shared with the consortium members.
The trial synopsis has been adapted to results obtained during the preclinical profiling of the vaccine.



In the fourth reporting period the main results are:

Pre-clinical evaluation of prototypes in dogs.
At the end of the trial (end of October), dogs have been sacrified and testing of all samples are ongoing.

GMP production. and Toxicological studies
The main objectives were to develop a highly stable vaccine suitable for transportation and storage in sub-tropical and tropical regions and to establish GMP production technologies for each antigen and adjuvant component of the vaccine and for the final vaccine formulation.
The combined efforts resulting into strong processes to obtain valid candidate vaccines to be used in preclinical testing dog trial, toxicological testing and for Clinical trial (data are on file at Zydus Cadila).
To this end, frozen formulations of vaccine antigens without virosomes were prepared for a repeated dose toxicology study. This study was planned at MediTox s.r.o. using the three purified Leishmania antigens derived from E. coli (KMP11 and LEISH-F3+) and Pichia pastoris (LJL143) and the adjuvant GLA-SE. The frozen formulation and two additional lyophilized formulations of the antigens without virosomes were undergoing stability testing by IDRI (Infectious Disease Research Institute) in order to select a final formulation for GMP manufacturing.
It was intended to perform a similar stability study with the vaccine antigens in virosomes in order to identify a final formulation for GMP production. However, instability of the bulk virosome material after production led the consortium to decide not to pursue the virosome vaccine further.
GMP production and testing of: LJM11, KMP11, and NH-SMT.
Three lead formulations (two lyophilized and one frozen) were identified, and a stability study was initiated with the non-virosome vaccine to evaluate the formulations under accelerated and real-time conditions in order to select a lead formulation for cGMP production. The antigens were prepared in each formulation at a concentration of 40 μg/mL KMP11, 40 μg/mL LeishF3+, and 8 μg/mL LJL143 (which is the target vial concentration for the final vaccine), and vials were filled with 0.5 mL of antigen formulation. The frozen/liquid formulation was prepared and then stored at the temperatures listed in. The formulations were prepared and then lyophilized using an appropriate lyophilization cycle for each type of formulation (i.e. an amorphous lyophilization cycle was used for Formulation 2 because it contains 10% sucrose). After lyophilization, the vials were stored at the temperatures indicated. Duplicate samples were prepared for each formulation at each condition (temperature and time point). At each time point, the formulations were evaluated using the following metrics: visual assessment, pH, aggregation by optical density at 350 nm, and individual antigen content by SDS-PAGE and densitometry analysis. Currently, three months of stability data have been collected for this study.
Clinical Evaluation of candidate vaccines
In preparation for this we continued to work on the clinical trial documentation. The trial design needed to be adapted due to formulation changes (formulation without virosomes) and the synopsis was modified accordingly and approved by all partners. Assays to analyze the humoral immune response of study participants to the vaccine antigens required for the clinical study were established.

A new design of the clinical study without inclusion of virosomal formulations has been generated, discussed and agreed within all partners. The trial synopsis was adapted to the new trial design
Assays to analyze the humoral immune response of study participants to the vaccine antigens required for the clinical study were established.

Potential Impact:
Socioeconomic aspects of leishmaniasis control

Leishmaniasis was declared as one of the world’s most neglected diseases at the 60th WHO Assembly (2007). Leishmaniasis can be manifested as a wide range of clinical etiologies including visceral, mucocutaneous, diffuse, and cutaneous leishmaniasis (CL). Visceral leishmanasis (VL), the most severe form of the disease, can be fatal if left untreated. The devastating effects of this disease affect largely the poorest of the poor, mainly in developing countries with a disease burden calculated at 2 090 000 disability adjusted life years. Each year, there are approximately 300,000 cases of visceral leishmaniasis (90% in Bangladesh, Brazil, India, Nepal and Sudan), with an estimate of more than 50 000 deaths. In some cases, due to cultural reasons and lack of access to treatment, the case-fatality rate is three times higher in women than in men. Environmental changes have also led to leishmaniasis outbreaks spreading to parts of southern Europe.



Poverty is associated with ecological factors that increase the risk for infection due to proliferation of the vector or increased human–vector contact. In areas of anthroponotic peridomestic transmission, such as the Indian subcontinent, proliferation of the vector is enhanced by poor housing conditions, such as damp earthen floors, which prolong survival of the vector, and cracked mud walls, which provide the vector with daytime resting places.
Leishmaniasis is becoming a periurban disease, linked to migration of poor rural families to major cities. Poor environmental sanitation in these settings and erratic rubbish collection may increase the risk for leishmaniasis. Poverty also worsens clinical outcomes in leishmaniasis, as malnutrition and anaemia increase the severity of the disease. Leishmaniasis also aggravates poverty. Even when households do not have to pay the direct medical costs of care, such as antileishmanial medicines, the economic impact of the disease includes direct nonmedical costs (e.g. transport) and income loss for patients and their families due to absence from work.
Several studies of the cost or burden of illness have addressed the economic burden of leishmaniasis on households. On the Indian subcontinent, the median total expenditure by a patient on visceral leishmaniasis treatment was 1.2 to 1.4 times the annual per capita income.
Clinical disease in men is reported more frequently than that in women in most endemic countries. Although this difference could be due to more frequent exposure of males than females, it is also due to under-detection of disease in women in traditionally male-dominated societies. Community based studies in Bangladesh showed that the incidence of visceral leishmaniasis was about equal, but the case fatality rate was threefold higher in women than men. The difference was attributed to higher rates of malnutrition and anaemia in women and longer delays in seeking care.
Cost–effectiveness of control measures
Formal analyses have been conducted of cost–effectiveness, based on decision analytical models, to compare alternative options in diagnostics and treatment for VL. It was found that the cost–effectiveness depends more on the cost of treatment than on that of testing. A strategy based on the rK39 rapid test or the direct agglutination test is more efficient than one based on bone-marrow or lymph node aspiration, as the sensitivity of a test is paramount in this fatal disease.
A recent study of the cost of medicines for India based on international drug prices and anthropometric data from a specialized treatment centre in Bihar showed that paromomycin is the cheapest option (US$ 7.4 per patient) and liposomal amphotericin B the most expensive (US$ 162–229 per patient). Treatment with miltefosine would cost US$ 119 per patient at the private company price and US$ 64–75 at the WHO-negotiated price. These calculations do not include other direct or indirect costs and may differ widely from country to country. For example, in Nepal, a full course of treatment with miltefosine can cost up to US$ 150 per patient for medicines only. (In developed countries, the drug price can be 10 to 50 times higher than the preferential price.) Short regimens, including combination therapies, can reduce the cost to the public health system and patients by reducing the duration of treatment.
Access to medicines for the treatment of visceral, cutaneous and mucocutaneous leishmaniases is problematic in the poverty-stricken countries that have the highest burden of cases. Although many efforts have been made by WHO, medical nongovernmental organizations and manufacturers to improve access to medicines for leishmaniasis, problems persist.
Vaccine for neglected disease are usually not attractive for vaccine producer industry, since the market in general is not sufficient lucrative to turn in profit. However, the exceptionally high number of cases and the above reported cost-effectiveness may induce the different local government, with the help of WHO and other no profit organisations, to initiate, once a human vaccine became available, o large vaccination mass to eradicate the disease. Thus for example India has recently signed a Memorandum of Understanding (with Nepal and Bangladesh) pledging to collaborate to eliminate VL from the country.
Such a commitment of the respective governments and population is a favourable ground for the success of the present research project and future developments. Also the European Commission till now has funded several projects to fight neglected diseases and in particular the leishmaniasis within this FP7 framework and within former FPs as showed in the fig below. Another market opportunities for the SME which will use most of the benefits obtained with the successful conclusion of the present project is given by the travellers and by the military Army which might located their forces in area where the Leishmania is an endemic disease.
The innovation brought by MuLeVaClin lay in the development of a combinatorial immunisation strategy which includes protective structure antigens, enzymatic antigens and transmission blocking antigen. All three antigens have been shown to develop a certain degree of productivity alone. In addition using the Virus Like Particle Formulation the highest degree of protection could be achieved. This new vaccine concept would help to reduce the burden of disease that leishmaniasis imposes across Europe and other regions on an annual basis. The development of a vaccine that elicits broad long-lasting defence would allow to eradicate the disease. Furthermore, it would facilitate vaccination campaigns in low and middle-income countries and thereby also confer protection against Leishmaniasis in hitherto untargeted groups with limited health care programmes.
Importantly, the knowledge gathered from manufacturing (i.e. production and purification) and characterising (i.e. quality control) would allow to enter into large scale production and would provide the basis to easily be transferred into pilot- and full-scale cGMP clinical manufacturing in low income Countries which produce the vaccine at a very low price.
As shown in ‘Section A’ of this document, the results of MuLeVaClin have been disseminated at conferences, as poster and oral communications, and in form of scientific peer-reviewed publications. Additional publications (e.g. present the latest pre-clinical data) are in preparation.
The workshop and summer school series organised by the project’s partners were a great opportunity to bring together experts from vaccinology, immunology and leishmaniasis community.
The three summer schools organized in the project raised great interest and have been organized in Catania, Madrid and Porto.
In order to meet the challenges posed by the MuLeVaClin Project, the consortium has been constructed around an integrated assembly of eminent scientific groups with acknowledged complimentary expertise in their own fields: molecular biology, immunology, vaccinology, and clinical evaluation. The consortium included 9 partners, located in Spain, Portugal, Italy, Switzerland Czech Republic and the US. The partners have been selected for their capability to cover all the aspects for this specific vaccine development, and in particular contains the most relevant experts in the field of Leishmaniasis, a renowned center specialized with a extensive experience in clinical trials for tropical disease, and three industrial partners: Etna Biotech with expertise in R&D in the vaccine field with deep knowledge in virosome formulation, from bench to the market and with access to vaccine GMP facilities located in India (Zydus); Meditox s.r.o. a well-known preclinical organisation specialised in toxicology study; AMVAC a young and ambitious company with a state of the art facility for the GMP production, and IDRI a renowned non-profit organization with clinical experience in the design and implementation of leishmania clinical trials and a state of the art for the GMP facility. Some partners are located in the Mediterranean area, an area where the level of attention to the disease is particularly high, with some organizations which are the national centres for the surveillance of the disease and reference centres of the WHO. At the same time to increase the likelihood of success the consortium has also looked at eminent centres located externally from the EU. The collaboration with IDRI, enhanced the high likelihood of success. IDRI is a no profit organisation, dedicated to neglected disease which has already performed clinical trials, with different candidate vaccines, in the field of leishmaniasis. The aim to create such broad consortium was to speed up the vaccine development while reducing the possibility of failure. The collaboration within the consortium included: (i) exchange of essential reagents such as purified antigens and formulations, animal sera for vaccine standardization purposes; (ii) sharing and transfer of skills and expertise, mobility of researchers, with a rapid access to training; (iii) mutual guidance and advice, especially at key decision points, during the progress of the project; (iv) advice and guidance on solving IP issues; (v) organization of a summer school in Catania about the different aspects of leishmaniasis and leishamania vaccine.
Impact across the European research community
Multidisciplinary and cross-border sharing of research endeavours, ensuring good knowledge sharing was the cornerstone for success in this project. MuLeVaClin brought together experts from across 4 different European countries along with leveraging a lead candidate antigen provided by the Etna.
As can be seen in the partner descriptions, the majority of partners have been involved in former FP funded projects specifically researching Leishmania (including Leish-MED, Leish-EPINET, RAPSODI, and TRICONT). The European teams have strong expertise in immunogenetic and animal model aspects of the disease, the SME has the know-how and capabilities to enable the consortium to deliver viable vaccine, and other partners bring strong competencies from endemic areas that contribute a concrete and practical experience of field research in leishmaniasis. We involved the laboratories of the Italian regulatory authorities responsible for the national surveillance of Leishmania and who are the reference laboratories for the WHO. This reflects the similar set up we have in Spain, albeit it with private laboratories. The Swiss Tropical Institute is a core partner and is one of the world’s foremost institutes in investigating and managing diseases of this kind.
This partnership has increased the level of comprehension of the disease and has served to validate the current understanding of leishmania immunology through a unique collaborative effort. The cross-consortium sharing of knowledge, including training and technology transfers, has been secured by having a dedicated manager, for knowledge dissemination and is integrated as a core part of each milestone. Furthermore, other means to extend all of our findings across the European research community and the developing countries where it will be of help are detailed in our dissemination plan.
Importantly, it is envisioned that MuLeVaClin has significantly contributed to the visibility of European research in this field, especially across the countries most concerned, including India. This had the impact of improving relations and enabling a greater engagement with global scientists and their awareness of both leishmaniasis and also programmes of this kind. Finally, the scientists within our consortium are already engaged with the world’s leading organisations in this space, such as the WHO, and this collaborative sharing of expertise has enhanced these links.
Impact for SME partner
The lead SME partner for MuLeVaClin was AmVac. Their specific expertise in toxicology and the establishment of GMP production and testing procedures means that they are ideally placed to produce all the vaccine components and perform the final formulation under cGMP conditions. AmVac role was to develop the know-how to deliver a production process adapted to a large scale (200L fermentation process) and they should of course directly benefit from future vaccine production and commercialisation. Unfortunately AmVac had to give up operation during the project due to their financial difficulties. Fortunately the consortium was able to replace the activities, so that the project was not blocked. In addition we could replace the role of SME during the MuLeVaClin with IDRI.
It is envisioned that in addition to the large unmet medical need in the most affected countries, there is also the potential for any vaccine to have a secondary market as a travel vaccine. Both the military and private use of such vaccines has proved beneficial to companies in the past and we see no reason why the same route to market strategies and commercial opportunities cannot be exploited here.
Economic impact
As an effort to confront the major threat to public health that leishmania represents, MuLeVaClin presents an innovative solution. Current leishmania control strategies are unfortunately proving to be either ineffective or very expensive and therefore represent a high burden to the economies of those countries most affected. The spread of this disease to previous non-endemic areas is also presenting particular challenges. Moreover, several forms of the disease are anthroponoses or have sylvatic reservoirs, meaning that vector control through insecticide spraying is unfeasible. This result proved that all efforts in this direction so far have had limited impact.
Treatment cost must also be taken into account. The first-line treatment (pentavalent antimonials) is expensive and becoming less effective as drug resistance is developing (up to 60% in Bihar, India). Second line treatments, such as liposomal Amphotericin B) are proving too expensive for mass therapeutic strategies. While the new miltefosine drug may have some impact, the treatment cost is still over 100 Euros per patient, and it is unlikely that drug resistance will not also become a problem.
In addition to the costs of treatment and costs of control, leishmaniasis also has a significant impact on productivity and welfare. In particular, some core areas of society, such as agriculture or industrial rural development programmes, are often the most heavily impacted. The rural populations and labour forces are the most affected and this can present a significant barrier to continued development. On several occasions, as previously mentioned, epidemics of leishmaniasis have delayed or prevented development projects by international and national organisations. It is also unfortunate that those most affected are those with the least influence over policy and a very limited capacity to assume the costs of the disease. These people are therefore the most at risk from the vicious circle of poverty, malnutrition and disease.
It is therefore evident that a prophylactic vaccine is the most viable option. The development of such a vaccine would have the potential to decrease both the direct and indirect health cost burden in the affected developing countries. MuLeVaClin vaccine candidate is now ready to be tested in Clinical Trials. The promising results achieved during this project give a high likelihood of chance of success when tested in clinical trials.
Compliance with European Policies
The MuLeVaClin project goal of developing a human vaccine against leishmaniasis will contribute to the improvement of public health in developing countries, this has positive repercussions on the fight against poverty and the attempts to enhance rural development. An essential part of this collaboration is not only the bringing together leading European and American scientists and institutions in the field of leishmaniasis, but also the inclusion of expertise and insights from the endemic areas. This project has therefore built on our partners’ openness to technology transfers, education of scientists, both within and beyond the consortium, and the sharing of the results for the benefit of the broader scientific community and those most affected by the disease. The activities envisaged has contributed to the awareness of leishmaniasis and added to the knowledge and skill of those in the medical field fighting the disease.
Summary
Leishmaniasis is one of the most neglected tropical diseases, in terms of the few tools available for control and the lack of clear criteria for methods of control. WHO has focused research priorities on control of leishmaniasis, and consequently, recent strategic research has led to the development of rapid and reliable non-invasive diagnostic techniques, new medicines, such as orally-administered miltefosine (now in phase IV trials) or injectable paromomycin (now in phase III/IV trials), drug combinations that reduce the risk of resistance, and immunochemotherapy.
MuLeVaClin had the potential to create a powerful tool in confronting this major threat to public health. Combined with this are the benefits it will bring to furthering the understanding of immunology of kinetoplastid diseases. In addition, there was the significant know-how and value created for the partners. Taken together, these elements indicate that the expected impacts of the project is high.

List of Websites:
MuLeVaClin Please find further information about the MuLeVaClin project at www.mulevaclinc.eu


MuLeVaClin Project Management Team (info@etnabiotech.it)
Etna Biotech Via V. Lancia 57, 95121 Catania Italy