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Periodic Report Summary 2 - MULEVACLIN (Clinical Studies on a Multivalent Vaccine for Human Visceral Leishmaniasis)

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) (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 is 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 represent 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 proposal, Clinical Studies on a Multivalent Vaccine for Human Visceral Leishmaniasis (MuLeVaClin), represents an innovative approach in combining proven technologies and the latest insights on leishmaniasis in order to produce an efficacious vaccine. The purpose of MuLeVaClin is to deliver the most advanced vaccine against human leishmaniasis as shown by clinical trials (phase I/II) confirming the safety and immunogenicity of the candidate vaccine.
To achieve this goal, the MuLeVaClin project aims to the following specific objectives:
1. To test an innovative vaccine for 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 will be combined in an innovative candidate vaccine. The optimal composition of the vaccine formulation and the immune response elicited will be evaluated in preclinical studies in mice, hamsters and dogs. In addition, the immunogenicity of the different proteins will be analysed in asymptomatic and VL cured individuals.
3. To develop a highly stable vaccine to facilitate storage and transportation, suitable for sub-tropical and tropical regions.
4. To establish GMP production technologies for each component and formulation of the vaccine.
5. To evaluate preclinical safety (toxicology studies) in suitable animal models, which will analysed under the scrutiny of the Swiss regulatory authorities.
6. To develop 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. To prepare and submit an IND dossier to the Swiss regulatory authority.
8. To perform phase I/II clinical trials to investigate safety, optimal human dose and regimen and to analyse the immune-response generated after vaccination.

Project Results:
Production of the different antigens, adjuvants and final formulation of the vaccine was carried out to provide enough amounts of the formulated vaccine and its individualized components to complete the preclinical testing assays in mouse and hamster models, and for human antigenicity. 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 corresponding sections) to provide improved reagents to repeat the preclinical assays and go ahead with the planned objectives of the project.

Regarding preclinical testing of vaccine formulations, IBMC confirmmed 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 deserves to be explored, using an adequate infection animal model.

Results obtained at the CBMSO-UAM showed that, 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.

A second efficacy trial was carried out by ISCIII in hamster to test the capability of KMP-11, LJL-143 and F3+ antigens formulated in virosome or alone, with and without GLA-SE adjuvant have been tested. As controls, groups immunized with empty virosome or with PBS were included. Results obtained confirm the high immunogenicity of the complete vaccine formulation (adjuvant + virosome + proteins) in terms of humoral response, inducing antibody production after immunization and experimental challenge, as well as in terms of reduction of the parasite burden in target organs, reduction that was significant in the case of the formulation VPA 5:5:1

The antigenicity of KMP11, LJL143, and F3+ has also been tested in human sera and peripheral blood mononuclear cells (PBMC) from infected individuals. 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+ are 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. 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”.

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. 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-γ and TNFα 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

Potential Impact:
MuLeVaClin’s primary goal is to prove through clinical trials an efficacious, affordable and stable prophylactic vaccine, which combines tried and well evaluated leishmaniasis antigens, implemented in well-established vaccine delivery platforms and analyzed on the basis of the latest understanding of the immunological aspects of leishmaniasis. 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 it continues representing a high burden to the economies of those primarily affected countries. 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. The result being that all efforts in this direction so far have had limited impact.
The clinical research conducted in this proposal will form a significant step forward in our understanding of this disease, and if successful will represent a major step in tackling the global Leishmania problem. Finally, it is also important to add that the potential benefit of a travel vaccine, for both military and general population, obviously has the potential to add significant value for the SME in this proposal.

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