Final Report Summary - LEISHDNAVAX (Development of a DNA vaccine for visceral leishmaniasis)
Executive summary:
The LEISHDNAVAX project focused on the preclinical development of a safe and efficacious prophylactic and therapeutic DNA vaccine against both visceral and cutaneous forms of leishmaniasis. The vaccine is based on the minimalistic immunogenically defined gene expression (MIDGE) vector developed for efficient induction of T-cellular immune responses. A double stem loop immunomodulator (dSLIM) adjuvant was used in some studies. Based on preset criteria, five antigens from a pre-defined shortlist were selected and their genes incorporated into MIDGE vectors. The candidate vaccine was evaluated for immunogenicity in both animal models and human cell culture systems, and for efficacy in rodent models of visceral (VL) and cutaneous leishmaniasis (CL). The safety of the candidate vaccine was determined in animal models as required by regulatory authorities. To ensure the preclinical candidate possessed broad potential, immunogenicity and safety studies were conducted for both prophylactic and therapeutic indications. We now have a defined vaccine that has been tested ex-vivo in human cells and in animal models, and has been shown to be safe and capable of producing the desired effect and immune responses.
Following successful completion of the LEISHDNAVAX project, the candidate vaccine, produced under GMP conditions, will enter clinical phase I trials for safety and immunogenicity studies in European and disease endemic countries. In preparation of these trials, clinical sites have been identified in disease endemic regions of both VL and CL and initial preparations made to ensure proposed clinical trials would be conducted at ICH-GCP standards. This preparation included training in preclinical and clinical development, and the establishment of components and infrastructure to conduct trials.
The LEISHDNAVAX project was built upon three phases of study. The initial phase identified the immunogenic antigens for selection and inclusion within expression constructs in work packages (WPs) 1 and 2. This was followed by phases to determine the immunogenicity and efficacy of the DNA vaccine constructs in rodent models (WPs 3 and 5) and pre-clinical safety studies (WPs 4 and 6). Many of the studies were undertaken in parallel. Recent Leishmania isolates from endemic target regions were obtained, propagated and distributed between partners for use in all WPs. In WP1, seven candidate antigens were pre-selected based on specified criteria. Selection of vaccine antigens was established and immunogenicity of candidate antigens was tested on PBMCs from individuals of target populations using human cell culture systems. Five antigens were selected that met the criteria set for antigen selection, i.e. sequence conservation and immunogenicity in population in endemic areas. In WP2, MIDGE vectors needed for preclinical immunogenicity and safety studies were produced after constructing suitable expression cassettes with optimised DNA sequences of selected antigens. Expression in vitro was confirmed prior to production. In WP3, recombinant proteins of selected antigens were used in immunogenicity studies in animals in vivo by respective partners. Immunogenicity and protection studies for all the MIDHE-Th1 vectors encoding Leishmania antigens were performed in two animal species in vivo and the vaccine was shown to be immunogenic. Protection was demonstrated in some animal species. Induction of CD4+ and CD8+ T-cell responses were also studied with positive results. WP4, safety studies for a prophylactic vaccine were initiated following discussion with regulatory authorities in Germany (PEI) and London (EMA); studies were refined according to received scientific advice.
The persistence of MIDGE vectors in animal tissue was determined. WP6, safety studies for a therapeutic vaccine were run in conjunction with WP4. In WP5, the therapeutic efficacy of the vaccine was determined and standardisation procedures for animal studies shared with WP3. Therapeutic effects in mouse models of VL and CL were examined, as well as the positive interaction of the vaccine with an anti-leishmanial drug in the VL model. In WP7, SOP's were established in collaboration with WP1 to test the antigenicity and immunogenicity of the vaccine antigens in human populations, and for immunomonitoring future clinical trials. Three workshops were held to train personnel at the test sites in endemic countries. Exchange of co-workers between laboratories for training purposes was undertaken.
Although clinical studies are not part of the LEISHDNAVAX proposal, the project has developed a candidate vaccine for leishmaniasis which is now ready for Phase I clinical studies. The project has analysed and constructed T-cell epitope maps of the vaccine antigens for humans which will provide a unique and important contribution to understanding the outcome in target vaccinated population with diverse genetic backgrounds. In particular, since there are no validated animal models for human leishmaniases, the information gained from LEISHDNAVAX may provide insight to identify markers of efficacy in human population.
The LEISHDNAVAX consortium (see http://www.leishdnavax.org online) was composed of partners with extensive experiences in Leishmania research and vaccine development, including:
List of partners
1. London School of Hygiene and Tropical Medicine United Kingdom (UK) LSHTM
2. MOLOGEN AG Germany MAG
3. Charite Universitatsmedizin Berlin Germany CHARITE
4. Indian Institute for Chemical Biology India IICB
5. Institute Pasteur de Tunis Tunisia IPT
6. Hebrew University of Jerusalem Israel HUJI
7. Rajendra Memorial Research Institute India RMRI
8. Drugs for Neglected Diseases Initiative Switzerland DNDi
Project context and objectives:
Leishmaniasis, a neglected infectious disease with up to 2 million cases per year worldwide, requires new tools for treatment, diagnosis, surveillance and prevention. There is a consensus that an integrated approach using vector control, prompt diagnosis, effective treatment and vaccines are needed for control and prevention of both visceral and cutaneous forms of leishmaniasis. The focus of this project is the delivery of a preclinical candidate for a safe and effective vaccine that will play a critical role in this integrated approach. Currently, there are no approved vaccines to prevent or treat human leishmaniasis though two licensed vaccines have been developed for canine leishmaniasis. Four vaccine candidates (two funded through this European Union (EU) Seventh Framework Programme (FP7) call, and an additional two funded by the Bill and Melinda Gates Foundation and the Wellcome Trust) are in pre-clinical and clinical development. Our knowledge about the stable, highly-conserved Leishmania antigens from many of the species that cause human disease, including antigens with limited or no human homology, and of the immune responses to these antigens in models of infection, has given the research community confidence to undertake the development of a vaccine effective for the prevention and/or treatment of different forms of leishmaniasis.
The LEISHDNAVAX project focused on the preclinical development of a safe and efficacious prophylactic and therapeutic DNA vaccine, ready for clinical trials, against both visceral and cutaneous forms of human leishmaniasis. The vaccine is based on the minimalistic immunogenically defined gene expression (MIDGE) vector developed for efficient induction of T-cellular immune responses and, if required, a double stem loop immunomodulator (dSLIM) adjuvant. Antigens from a predefined shortlist were selected and their genes incorporated into MIDGE vectors. The vaccine is T-cell directed and its immunogenicity was evaluated in animal models and human cell culture systems. Efficacy was determined in animal models according to a target product profile and safety established in preclinical studies. Clinical sites have been identified and prepared to conduct clinical trials at ICH-GCP standards. This includes training in preclinical and clinical development, and establishing all required components and infrastructure to conduct trials at the highest international standard.
The specific objectives of the project, many performed in parallel, were:
- to select antigens that had previously been shown to be protective in animal models;
- to establish the degree of sequence conservation of the selected antigens in recent Leishmania isolates from patients of our target endemic regions;
- to establish an in vitro system to assay immunogenicity of selected antigens and vaccine composition using PBMC of healthy individuals from target populations;
- to establish antigenicity and immunogenicity (in vitro) of the vaccine antigens for induction of T cell responses in the target populations including matching immunogenicity of the antigens and immunogenetics of the populations;
- to assess the stimulatory activities of vaccine components in recovered patients from target population;
-construction of antigen expression cassettes suitable for MIDGE vector production;
- production of MIDGE vectors coding for leishmanial antigens (MIDGE-Leish-Ag) for preclinical immunogenicity and efficacy studies in mice and hamsters, and select appropriate antigen(s) for prophylactic and therapeutic studies;
- production of selected MIDGE-Leish-Ag's under GMP-like conditions, for preclinical safety studies;
- to find the relation, if any, between immunodominance of an antigen and host protection;
- to analyse and compare the efficacy of induction of human CD4+ and CD8+ T cell-mediated immune responses by different MIDGE-Leish-Ag's individually and collectively with or without the immunomodulator dSLIM;
- to study the efficacy of MIDGE-Leish-Ag's as prophylactic vaccine against experimental Leishmania major, and possibly Leishmania tropica infections in mice;
- to determine the efficacy of all selected MIDGE-Leish-Ag's in mice and hamsters against Leishmania donovani infection as therapeutic vaccine administered at time points after infection;
- to determine the requirement of an adjuvant, in particular dSLIM, in the efficacy of the candidates as therapeutic vaccines at time points after infection;
- to determine the interaction between the selected candidate vaccine and standard anti-leishmanial drugs through measurements of liver and spleen load;
- to analyse and compare CD4+ and CD8+ T cell-mediated immune responses to the vaccine candidate with and without drugs in therapeutic models;
- to identify site(s) and establish all prerequisites for conducting clinical trials of LEISHDNAVAX at current ICH-GCP standards from preclinical development to registration;
- to build expertise in various steps from preclinical to clinical development and registration of LEISHDNAVAX for prophylactic and therapeutic indications through learning by doing concept.
As described in the second periodic report below all objectives were completely or partially met. The results have been reviewed by an external scientific advisory board (see periodic report, WP8) who confirmed that the requirements for a clinical candidate for human leishmaniasis have been met, as well as making some specific recommendations for further studies.
Project results:
The success of LEISHDNAVAX project is built upon three phases of study that have come together in the final months of the project and have delivered a clinical vaccine candidate for both visceral and cutaneous forms of leishmaniasis. The initial phase identified the immunogenic antigens for selection and inclusion within constructs (WPs 1 and 2). This was followed by phases to determine the efficacy in rodent models (WPs 3 and 5) and pre-clinical safety (WPs 4 and 6) of the DNA vaccine constructs, undertaken in parallel.
WP1: Definition of the vaccine antigens
Research over past few years has shown that immunity against leishmaniasis is possible and is mediated by the cellular arm of the immune system, T lymphocytes together with the innate immune macrophages and dendritic cells. This has prompted the development of vaccines that specifically trigger responses of Leishmania parasite-specific T lymphocytes. Such vaccines are different than the established vaccines on the market that are designed to induce humeral immune responses with neutralising antibodies, and they have to be developed in a different way. T lymphocytes do not recognise and react to whole antigens but to small fragments of the antigens, peptides. These molecules are products of proteolytic degradation of proteins inside cells and are bound by special peptide receptors called major histocompatibility complex (MHC) molecules or human leukocyte antigens (HLA) in humans. These HLA are highly polymorphic meaning they occur with a huge number of variants in the population and their composition differs between individuals and populations. In addition, parasites may also have polymorphisms in their antigens that can differ between Leishmania species, isolates and regions. With this background the task in the project was to define vaccine antigens that are conserved in all Leishmania species that induce Leishmania-specific T lymphocytes, and that are immunogenic in the human populations in the endemic regions.
Seven candidate antigens A2, CPA, CPB, HASPB, KMP-11, p74 and TSA were short listed, because it was known from prior work of consortium members and others that they can induce protective immunity in animal models. The gene sequences of these antigens were established for different recent Leishmania isolates from India, Central Asia, Iran, Near East, Saudi Arabia, Sudan, Ethiopia, Tunisia and Italy, and together with all sequences available in public sequence databases that represent old isolates and New World Leishmania species analysed for sequence conservation. Two antigens, A2 and HASPB, were excluded at this stage because of their high variability. Two antigens, KMP-11 and p74, were almost completely conserved and three, CPA, CPB and TSA, had some variations both between different Leishmania species and also within species.
To establish the immunogenicity of the candidate antigens, overlapping peptides that cover the sequences of the five remaining antigens including all variations, and thus all possible T cell epitopes were designed, synthesised and tested with peripheral blood cells of donors with a recent history of leishmaniasis. Matching the results of the sequence analyses and of the immunogenicity test with the peptides, the sequences of the vaccine antigens were designed to be both as conserved as possible and as immunogenic as possible. With peptides that cover the sequences of the five now redesigned antigens, the antigenicity mapping was further refined with white blood cells from donors in India and Tunisia who had recovered from leishmaniasis. The results prove three key points:
- The five candidate vaccine antigens are indeed processed in the patients to induce Leishmania-specific T lymphocytes.
-All five antigens carry epitopes for T lymphocytes that are suitable for the HLA of the populations in the endemic regions.
-The antigens or the selected conserved variants of the antigens are highly immunogenic with active peptides covering between 43 and 100 % of the antigen sequences.
Such high density of antigenic epitopes has so far not been described for any other antigen or infectious agent. The five selected antigens are thus most suitable as vaccine antigens.
The antigen sequences were retrotranslated into DNA sequences that were optimised for high expression yields, and the optimised gene sequences cloned into a plasmid for production of the MIDGE vaccine vectors. This vector system has been shown to be suitable to carry the genetic information for the antigens into cells for processing and presentation of epitopes on HLA and induction of T lymphocytes. This was confirmed in animal studies as described below.
The outcome of the WP1 is a pentavalent vaccine ready for tests in preclinical animal models and clinical trials. The approach to the definition of vaccine antigens for induction of immunity mediated by T lymphocytes that was established within WP1 is novel. It is modular so that the vaccine can be redesigned easily by reducing or extending the number of antigens, or modifying and exchanging antigens in case more promising candidates are identified.
Vaccines for induction of immunity mediated by T lymphocytes require different protocols for monitoring their immunological efficacies and correlating these with outcome. Serology as for standard vaccines cannot be used. Standard operating procedures (SOPs) were developed for all steps involved in monitoring T lymphocyte responses including sample coding, preparation and cryopreservation of peripheral blood mononuclear cells, and bioassays for antigen-specific T lymphocyte responses. The SOPs were tested and implemented together with protocols for quality management at the test sites in India and Tunisia.
WP2: Design and production of vaccine
In this WP, the vaccine candidate has been generated in substance: The antigens selected in WP1 were combined with the MIDGE-Th1 vector. For this purpose, the antigen encoding sequences were synthesised following optimisation for strong expression in humans. The synthetic DNA sequences were then incorporated individually into plasmids optimised for MIDGE-Th1 production. Thereby, the expression cassettes of the antigen-encoding MIDGE-Th1 vectors were generated.
MIDGE vectors are linear, double stranded DNA molecules covalently closed at both ends by single-stranded loops of DNA. The closed structure protects the vector from rapid degradation by exonucleases. MIDGE-Th1 vectors are MIDGE vectors with a peptide attached. In contrast to conventional plasmids usually applied as DNA vaccines, MIDGE-Th1 vectors only contain the expression cassette, but no potentially detrimental sequences like antibiotic resistance genes, bacterial backbone sequences or an origin of replication. In a number of in vitro and in vivo studies, the efficacy of MIDGE-Th1 vectors for expression of the encoded antigens and for DNA immunisation has been shown previously.
However, a functional check of expression cassettes of the vaccine candidate was required, thus antigen expression in vitro for both plasmids and MIDGE-Th1 vectors was evaluated by expression analysis on mRNA level. Mammalian cells were transfected with the respective vectors and specific mRNA transcription was determined. All antigen expression cassettes passed this check of function in vitro.
Moreover, a dose-response relation between DNA amount used for transfection and resulting mRNA amount was shown in vitro for each expression cassette encoding a Leishmania antigen. This result was important for the establishment of a potency assay as described below.
Expression analysis on protein level was conducted as the second functional check of expression cassettes. Except for one antigen (CPA), the antigen proteins were detected in Western Blot assays when evaluating corresponding MIDGE-Th1 plasmids and MIDGE-Th1 vectors side by side. Efforts to detect the CPA protein after expression in vitro are continued.
Taken together, five MIDGE vector expression cassettes each encoding a leishmanial antigen have been synthesised as consensus sequences and optimised for expression, with proven functionality in cell culture, and incorporated into plasmids serving as starting material for MIDGE vector production.
For preclinical efficacy evaluation of the vaccine, candidate MIDGE-Th1 vectors were produced in a simple standardised procedure. The manufacturing process started with the plasmid containing the expression cassette of the future MIDGE Th1 vector. The expression cassette was flanked by recognitions sites for the restriction enzyme Eco31I enabling an enzymatic digestion resulting in two open-ended DNA molecules: the expression cassette and the plasmid backbone. The open ends of the expression cassette were then covalently closed by enzymatic ligation with specific oligodeoxyribonucleotides (ODN) designed to each form a single-stranded loop. The Th1-peptide was coupled to one ODN before. Finally DNA molecules with open ends, i.e. the plasmid backbone including the antibiotic resistance gene and the origin of replication, were enzymatically degraded, and the MIDGE-Th1 vector containing solution purified, desalted and subsequently concentrated.
The final pentavalent vaccine candidate consists of five MIDGE-Th1 vectors each encoding one antigen. The five vectors were produced separately and underwent full quality control (QC), before they were mixed, the final concentration adjusted and final QC tests performed. The vaccine samples were shipped as ready-to-use compositions to other partners for preclinical immunogenicity and efficacy studies.
The preclinical trials to evaluate the safety of the pentavalent vaccine candidate comprised biodistribution pilot and main studies, and a repeated-dose toxicity study. For these studies, the MIDGE-Th1 vectors encoding leishmanial antigens were produced under GMP-like conditions. The steps of the production comply with those described above. Vaccine samples were manufactured ready-to-use and shipped to the respective contract research organisations (CROs).In total, approximately 1 g of MIDGE-Th1 vector DNA has been produced.
In preparation of the GMP manufacturing process for clinical studies, an essential vaccine batch release test, i.e. the potency assay to determine the biologic activity of the vaccine has been designed and presented to the authorities, adjusted accordingly, established and standardised.
The assay starts with transfection of a human cell culture line with defined amounts of MIDGE-Th1 vectors encoding the Leishmania antigens. Subsequently, expression of antigens is quantified on the mRNA/cDNA base employing a quantitative PCR system. Using the parallel-line method the relative potency of vaccine batches is calculated. The MIDGE-Th1 vector encoding KMP-11 was used as the model vector during initial establishment of the system. Later, the potency assay was extended to analyse the complete mixture of all antigen-encoding vectors using antigen-specific labelled TaqMan probes and compatible antigen-specific PCR primers in the same experimental setup.
Extensive tests have been performed to select an appropriate human cell line, the appropriate transfection method, and the range of DNA amount to be used in transfection. To ensure reproducibility and reliability of the potency assay as a future vaccine specific batch release test, the entire procedure has been established, standardised and defined in standard operating procedures (SOPAs). Thus, the potency assay is ready to enter the next step of development, i.e. test validation with clinical batches of the vaccine and comparison of activity measured in vitro to immune response of humans.
In summary, from the production and quality control perspective, the vaccine candidate can be taken forward to next development steps including the first clinical study.
WP3: Animal and human cell culture studies for prophylactic efficacy
Prophylactic vaccination was tested in animal models of leishmaniasis to check if they were immunogenic and had any influence on disease outcome. Five conserved antigens the KMP-11, TSA, CPA, CPB and p74 were finalised based on their conserved nature and T cell stimulatory capacity. Optimised synthetic sequences of MIDGE-Th1 vectors encoding all five antigens were produced.
Immunogenicity studies:
In animal models the main concern was to detect if all the antigens of the multivalent vaccine are recognised by the host and evaluate the type of immune response elicited upon immunisation. From our initial experiments a three dose immunisation scheme at 14 day intervals followed by parasite challenge was adopted. In both BALB/c and C57BL/6 mice antibodies against all the five antigens were produced. Antibodies against KMP-11, TSA and CPA showed the greatest increase upon immunization, but there was also appreciable increase in antibodies against CPB and P74. We looked at the IgG1 and IgG2a response (IgG2a/IgG1 ratio) to understand the underlying nature of the T cell response. The IgG2a/IgG1 ratio was higher in vaccinated animals compared to the PBS injected animals. In BALB/c, there was an increase in both IgG1 and IgG2a isotypes, but antigen specific IgG2a response was higher. This indicates a mixed Th1-Th2 response with a bias towards Th1. On the other hand, C57BL/6 mice exhibited an extreme Th1 bias compared to the BALB/c mouse. Antibodies to all five antigens in the vaccinated C57BL/6 mice were exclusively of IgG2a isotype. The mixed response in BALB/c mice may be due to the inherent Th2 bias of this strain.
To test the cellular immune response generated by the vaccine, spleen cells from immunised mice were restimulated with antigenic peptides for 24 hours. Assuming that the vaccine induced a cellular immune response, then antigen specific T cells from vaccinated mice should recognise MHC bound antigenic peptides upon in vitro restimulation. Higher IL-2 production in mice receiving vaccine formulation indicates T-cell activation, in contrast to mice that received PBS. This T-cell activation was found to be proportional to the vaccine dose. All the peptide pools were immunogenic in both BALB/c and C57BL/6. The KMP-11 pool, which induced highest IL-2 production in BALB/c, produced the least IL-2 in C57BL/6. This shows that there are subtle variations in immune response to the vaccine between different mouse strains.
In the hamster model, an initial study with MIDGE-KMP-11 demonstrated that the three dose immunization scheme produced the best immunogenicity. In a separate experiment where three antigen coding constructs (KMP-11, TSA. P74) were administered separately, and also as a mixture, the KMP-11 and p74 specific antibody responses were higher in the mixture compared to animals immunised with MIDGE-KMP-11 or MIDGE-p74 alone. Antibodies were of the IgG2 isotype, again indicating that vaccination generates a Th1 bias. When all the antigen coding constructs (MIDGE-CPA, -CPB, -P74, TSA and KMP-11) were administered as a mixture to hamsters, antibody responses were observed to all five antigens.
From the immunogenicity studies, it is evident that all five vaccine constructs are immunogenic in two strains of mice, and also in hamsters. The immune response generated by this vaccine is Th1 biased as suggested by the IgG1 and IgG2a isotype ELISA. In addition, the degree of immune response demonstrates a clear dose response with the vaccine dose.
Efficacy studies:
We have used the susceptible BALB/c model to assess the efficacy of the five antigen containing vaccine formulation upon challenge with L. donovani (VL). The prophylactic efficacy of the vaccine formulation showed a clear dose response inversely correlated with the parasite burden. The highest dose of 20 g/antigen provides more than 90 % protection against experimental parasite challenge. The level of protection decreases with the reduction in vaccine dose. Reduction in liver parasite burden at 21 days post infection also corresponds to a similar reduction in spleen parasite burden at a later stage of infection. Spleen cells from these protected animals were found to produce significantly high amounts of IFN-I and TNF-I ± upon antigen recall with peptide pools. Control animals did not have such high levels of pro-inflammatory cytokines. Some degree of protection was observed when resistant C57BL/6 mice were immunised with the pentavalent vaccine and challenged with L. major (CL).
Despite immunogenicity of the component antigens in hamsters, the final formulation failed to show any efficacy against an experimental L. donovani challenge in this animal model. The three-antigen mixture in one experiment exacerbated disease. But immunisation with either MIDGE-TSA or MIDGE-p74 individually, or MIDGE-KMP-11+dSLIM leads to reduction in parasite burden. Unlike the three-antigen mix, the final five antigen containing DNA formulation did not exacerbate or protect against disease. Interestingly, the final formulation when administered along with the immunomodulator dSLIM gave 88 % protection in hamsters. An additional experiment comparing immunization with the pentavalent vaccine + dSLIM versus dSLIM alone is needed to confirm this finding, and is underway.
WP3 has proved that the concept that this vaccine design indeed works in the animal models. All five candidate antigens are immunogenic when DNA encoding them is administered as a mixture. The vaccine induces both humoral and cellular responses, and the cellular response is Th1 biased. The vaccine, in the absence of an adjuvant, does not protect hamsters from experimental parasite challenge, but this may be due to the inherent extreme susceptibility of hamsters towards visceral leishmaniasis. The five antigen formulation of vaccine provides protection against experimental visceral leishmaniasis in BALB/c mice. An almost similar level of protection was observed with a three antigen vaccine formulation in this animal model. Since the human target population for the vaccine will have a large HLA polymorphism, it is best to keep all five antigens in the formulation. Being a mix of different antigen coding constructs, this MIDGE platform has the flexibility built into it that will enable us to fine tune, as needed, the vaccine components during clinical trials.
WP4 and WP6: Preclinical safety evaluation of LEISHDNAVAX for prophylactic and therapeutic indication
Before administration to humans in clinical studies, a vaccine must be proven safe in preclinical studies according to regulatory guidelines. In order to ensure that the preclinical program of LEISHDNAVAX is deemed relevant by the authority granting, a clinical phase 1 study, the program was first discussed with the German authorities in 2009. Thereafter, the consortium applied for the Article 58 procedure - a procedure to review marketing applications for products intended to prevent or treat diseases of major public health interest, and to be used only outside the European Community. This procedure enables the European Medicines Agency (EMA) to give a scientific opinion, in cooperation with the World Health Organization (WHO), for the evaluation of these medicinal products. The resulting Certificate of Pharmaceutical Product issued by the EMA serves as the basis for product approval in other countries. The LEISHDNAVAX vaccine candidate was eligible for the procedure, and scientific advise on the preclinical program was received from the EMA and WHO.
According to this scientific advise, the preclinical safety study plans were adjusted. The studies of WP4 and WP6 were combined by extending the safety studies in non-infected animals to the drafted treatment schedule for a therapeutic vaccine. Safety studies in infected animals were no more required, but limited data on tolerability of the vaccine during treatment of infected animals (see WP5) should complete the preclinical safety data set.
The preclinical safety program comprised studies on the biodistribution and the toxicity of the vaccine. While toxicity of the vaccine was assessed in standard settings under GLP and completed by evaluation of auto-immune responses against DNA, no standard settings existed for biodistribution studies addressing the persistence of DNA vectors in tissues and organs. Thus a quantitative PCR (qPCR) assay to quantify MIDGE-Th1 vectors in tissues has been established and transferred to Contract Research Organisations (CROs). In addition, methods and procedures preceding the qPCR have been standardised.
Two biodistribution studies in rats, a pilot and a main study, were conducted. The pilot study was performed with the only MIDGE-Th1 antigen (KMP11) vector available at that time, in order to obtain preliminary data on the biodistribution, to check and refine the study plan of the main biodistribution study, as well as to set up and standardise all procedures and methods intended for the main study and to evaluate the performance of the CRO's. The study was performed as a blinded study including one vehicle control and one test item group. With the exception of skin tissue of the injection site, a strong reduction of copy numbers of MIDGE-Th1 vectors from 24 hours to 15 days after two immunisations was noted in essentially every tissue.
In the main study the distribution of the final mixture of five MIDGE-Th1 vectors was evaluated in rats after one or four repeated intradermal injections. In samples collected 24 hours after one or four immunisations, MIDGE-Th1 vectors were detected in quantifiable amounts in all tissues. The highest amount of vector DNA was found in samples of skin of injection site. The second-highest amount of vector DNA was found in lymphatic tissues, indicating that the vector is transported to lymph nodes. There was no significant difference between vector copy numbers of animals injected once or repeatedly with vaccine suggesting that the vaccine does not accumulate inside organs.
DNA vectors were cleared from essentially every tissue within 60 days after the fourth injection, with the exception of skin and inguinal lymph node tissue. Most importantly, even after repeated dosing the vaccine did not persist in critical organs such as reproductive organs or bone marrow. In conclusion, the risk of MIDGE-Th1 vector persistence and integration into host genomic DNA even after repeated dosing is considered to be very limited. However, prior to application for a Clinical Phase 1 study the results of the biodistribution study have to be discussed with the competent authority for advice on conduction and design of an integration study.
To assess the toxicity of the pentavalent vaccine, the vaccine was administered to mice intradermally once or five times once weekly at three dose levels. The reversibility of any effect within two recovery periods was also evaluated. The highest dose given to mice in this study corresponded to 200 fold the highest human dose anticipated when compared on the basis of DNA dose per bodyweight. To ensure that efficacious vaccine samples were used in this test for toxicity, vaccine immunogenicity was proven by detection of antigen specific antibodies.
No local intolerance reactions were noted at the injection sites in the animals given one or four weekly inoculations with vaccine. No systemic intolerance reactions to the vaccine were observed concerning mortality, clinical and neurological signs, body weight and body weight gain, food and water consumption, haematology, ophthalmological and auditory examinations, macroscopic post-mortem findings, organ weights, histopathology and auto-immune reactions including determination of anti-double-stranded DNA. The no-observed-adverse-effect-level (NOAEL) was above the highest dose tested in this study.
The reports of the biodistribution and the toxicity studies are part of the documentation to be filed when applying for permission to conduct a clinical phase 1 study in any country.
A workshop on the preclinical development of a DNA vaccine, applicable in preventive as well as in therapeutic approaches, was held in Berlin in 2011. Four scientists and four PhD students attended of the workshop. An overview was given on the worldwide state of the art and the development status of DNA vaccines, and compared to the LEISHDNAVAX project. Production and quality issues of DNA vectors including GMP and non-GMP procedures were discussed and production sites at MAG visited. A seminar was given on regulatory aspects of pre-clinical programs, collaborations with CRO's and documentation requirements. To connect preclinical and clinical development steps a presentation on clinical trials and regulatory requirements in India was given. In the practical part of the workshop, participants successfully designed preclinical safety studies.
WP5: Animal studies for therapeutic efficacy
In addition to the development of a prophylactic vaccine for leishmaniasis the potential application of selected vaccine candidates as immunotherapy was tested in models of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). The main questions addressed in these studies were tolerability (see also WP6) and efficacy measured as reduction in parasite burden (VL) or clinical disease (CL)
Initial studies were undertaken in BALB/c mice infected with L. donovani (VL) with a monovalent vaccine encoding the antigen p74. Efficacy was evaluated at different doses, number of injections, and time points in liver and spleen of infected animals. Although at first a significant reduction in hepatic parasite burden was seen after two injections this result was not reproducible in a repeat experiment. Notably the parasite burden in untreated control animals was higher in this experiment, which may indicate a dependence of efficacy on parasite burden. Similar conclusions were drawn from studies where an anti-leishmanial drug was sequentially co-administered with the final vaccine candidate as an immune-chemotherapy approach (see below).
Further studies on tolerability and efficacy were carried out with the pentavalent vaccine as candidate vaccine for clinical trials (MIDGE-Th1 vectors encoding the antigens CPA, CPB, KMP-11, p74 and TSA, see WP1). Dose kinetic studies were carried out in BALB/c and C57BL/6 mice infected with L. donovani (VL), and designed to include 1, 2 and 3 injections at weekly intervals and comparison to a PBS treated control group. Tolerability parameters assessed included monitoring of bodyweight, behaviour, fur condition, injection site reaction, evaluation of serum levels of ALT, AST, urea and creatinine, and histopathological examination of disease target and vital organs. The parasite burden was evaluated in liver and spleen. No reduction in parasite burden was seen. Disease exacerbation was excluded in this experimental design. The vaccine was well-tolerated and no adverse reactions observed.
An additional study was carried out in C57BL/6 mice infected with L. major (CL) receiving three injections of the pentavalent vaccine at weekly intervals. Due to a low percentage of animals developing lesions in all groups (vaccine treated, PBS treated or untreated) no definite conclusion on the efficacy in a CL model can be made. However, this result suggests no efficacy of the immunotherapy when used alone. Importantly, no adverse reactions were observed.
To evaluate the potential of the pentavalent vaccine in an immune-chemotherapy approach L. donovani infected C57BL/6 mice (VL) were sequentially treated with a single (suboptimal) dose of AmBisome followed by a single dose of pentavalent vaccine two weeks later. Another 10-12 days later mice were killed, and parasite burden in liver and spleen evaluated. Respective controls (untreated, AmBisome treated, pentavalent vaccine treated, vector treated, AmBisome plus vector treated) were included. Prior to these experiments the dose response of single dose AmBisome was assessed to chose the best dose level for co-administration experiments. These studies confirmed the potential of the sequential co-administration of drug and vaccine approach for treatment of VL, as this group showed the highest reduction in parasite burden. Reduction was more pronounced in the spleen than the liver, although the same trend was seen in both organs. Two different doses of the pentavalent vaccine (20 μg per antigen and 40 μg per antigen) were used in a pilot experiment, which indicated that 20 μg per antigen was sufficient to induce the highest parasite reduction, and 40 μg per antigen does not provide additional benefit. Synergism was not seen when single dose AmBisome was followed by administration of a non-expressing MIDGE vector containing the human IL-2 gene. Thus, it can be concluded that the synergistic effect is due to at least one or more of the antigens. ELISAs on antigen specific IgG2a/IgG1 responses are in progress, and will provide direct proof as to which antigen is responsible for the synergistic protective effect, and the nature of the immune response based on Th1-Th2 bias. In addition, histopathology and frequency of CD3 positive cells have been determined. An experiment to demonstrate the effect of the pentavalent vaccine on a dose response of AmBisome treatment is completed.
In conclusion, studies in WP5 have demonstrated that selected vaccine candidates including the final pentavalent vaccine are well tolerated in animal models of leishmaniasis. When used as monotherapy no consistent anti-leishmanial effect was seen. However, the potential of the pentavalent vaccine in an immune-chemotherapy approach was demonstrated by sequentially administering a single dose AmBisome followed by the pentavalent vaccine candidate. It is hypothesised that the drug reduces the parasite burden, which restores CD4 and CD8 immune responses and allows the immunotherapy to work more effectively than when given alone. Demonstration of a dose sparing effect is ongoing.
In addition to the research work of WPs 1-6 that produced the results that constitute the project foreground, the project also undertook activities in training and site preparation for proposed clinical trials.
WP7: Preparation for clinical trials
The specific objectives of WP 7 were:
(a) to identify sites and establish all prerequisites for conducting clinical trials of LEISHDNAVAX at current ICH-GCP standards from preclinical development for registration, and
(b) to build expertise in various steps from preclinical to clinical development and registration of LEISHDNAVAX for prophylactic and therapeutic indications through learning by doing concept.
It was decided to conduct the first phase 1 clinical trial in Germany. Once satisfactory data is available an additional phase 1 trial will be conducted in endemic districts of India (Bihar, RMRI) for Visceral Leishmaniasis and Tunisia (IPT) for Cutaneous Leishmaniasis.
The reviews of potential clinical trial sites and capability strengthening areas were identified by 24 months of the project. Out of 33 endemic districts in Bihar, India, seven highly endemic districts were identified reporting more than 1500 cases consistently over the past five years; two districts were selected for future vaccine trial. These two districts, Marhaura PHC of Saran and Simri-Bakhtiyarpur PHC from Saharsa, were selected on the basis of logistic support and number of cases. Two workshops and continuous training have been held in these PHCs and both sites are ready to monitor immune responses after vaccination. Required equipment, expertise and laboratory facilities are in place to conduct the above activities at RMRIMS (partner 7). Similarly Sidi-Bouzid city in Tunisia was selected for LEISHDNAVAX vaccine trials for zoonotic CL. Sidi-Bouzid is an endemic region for the disease. About 8558 cases have been reported in the past three years. The most likely spatial cluster of high incidence rates contains regions located close to a dam. Two workshops and continuous training have been completed. With extensive previous experience in immunological and CL studies in the same field particularly clinical trials, the availability of technical expertise and infrastructural facilities, IPT (partner 5) is well situated for conducting clinical trials on CL at ICH-GCP standards.
To ensure trained staff were in place, clinical site adapted SOPs for immunomonitoring vaccine induced T-cell responses were prepared and wet lab training workshops held on:
(a) ex-vivo activity of human cells to different antigens with measurements of various cytokines at Kolkata, India from 4 to 8 November 2009,
(b) in-vitro induction of immune response to various peptides of selected antigens used in the vaccine held at Charite, Berlin, Germany from 20 to 27 October 2010, and
(c) development of SOPs and review of protocols for immunomonitoring during clinical vaccine trials based on ICH was held at RMRIMS, Patna, INDIA from 12 to 14 April 2012.
It was also necessary to ensure training and awareness of staff in project management, protocol development, ICH-GCP guidelines, CRF development, data management, and training monitors. A workshop on target product profiles (TPP) and on the clinical development plan was held in Tunisia from 10 to 13 May 2012 in place of MS project, protocol development ICH- GCP guidelines, CRF development, and data management training.
At this workshop three target product profiles were developed for:
(i) CL prophylaxis,
(ii) CL therapeutic, and
(iii) VL prophylactic indications of LEISHDNAVAX project vaccines.
Additional training on preclinical development was conducted at Mologen, Berlin at another workshop from 15 to 19 November 2011. In summary, preparations for clinical studies have been completed. The future steps will depend on the outcome of the Phase 1 study to be carried out in Germany in 2013. If the data are satisfactory then pilot phase I studies are intended to be conducted in endemic regions of Bihar, India and Sidi Bouzid, Tunisia.
Potential impact:
Over the past decade there has been increased funding available for the development of new drugs, diagnostics, vaccines and vector control methods for leishmaniasis (see http://www.g-finder.policycures.org online). In addition, to the aims to improve treatment, surveillance and control, the potential of elimination of some forms of the disease is now firmly on the agenda (see http://www.who.int/tdr/news/2011/vl-elimination online). The possibility for more effective control programmes and elimination is supported by efforts of WHO to improve knowledge of disease burden, prevalence and distribution (see http://www.who.int/leishmaniasis/burden/magnitude online). As with the control and prevention other infectious diseases, an integrated approach using vector control, prompt diagnosis, effective treatment and vaccines is needed for both visceral and cutaneous forms of leishmaniasis.
The major gap in the armamentarium required for leishmaniasis control is the absence of approved vaccines to prevent or treat human leishmaniasis. The development of a single vaccine for leishmaniasis will have a major impact on the control and development of this disease. The LEISHDNAVAX project has developed a vaccine candidate ready for use in clinical trials for both the visceral and cutaneous forms of leishmaniasis. If successful in clinical trials, this vaccine could have a major impact on disease control and public health in countries in Asia, Africa and Central / South America.
Within the LEISHDNAVAX project, we have already started to engage with experts in India (for VL) and Tunisia (for CL) who are connected to the health authorities. To ensure successful delivery and access of the vaccine it is now essential to fully engage with the regulatory and health authorities in selected disease endemic countries.
This project has also demonstrated that a rational approach, based upon molecular, genetic, and immunogenicity criteria, can be used to select antigens for incorporation into vaccines, and that important information on the immunogenicity and efficacy of candidate vaccines can be provided by rodent models of infection. Further studies in humans are now required to confirm our experimental results showing that immune responses to the specific antigens incorporated in the vaccine are correlated with protection in animal models. This pathway of vaccine development could have an impact on refinement or development of vaccines for other infectious diseases.
Dissemination
Publications
As a vaccine development project LEISHDNAVAX incorporated into the consortium agreement (CA), a clear publication policy and an intellectual property (IP) policy. It was agreed that scientific publications would follow the submission and publication of a patent derived from the Foreground work of the project. A patent application entitled Pharmaceutical for treatment of leishmaniasis was submitted by Mologen AG on behalf of all partners of the consortium, at the UK Patent Office (Patent Application No. UK 1207476.1 date of filing 30 April 2012).
At the sixth steering committee meeting held in June 2012, following publication of the patent, a plan for the submission of eight scientific publications (including one review) from the project was made and approved.
List of subject areas for proposed articles included:
- concept and design of a DNA vaccine against leishmaniasis
-high antigenicity and epitope density of leishmanial antigens
- protocols for monitoring T cell-mediated immune responses in leishmaniasis
- tolerability and efficacy (immunogenicity) of a DNA vaccine in mouse models of visceral leishmaniasis
- immuno-chemotherapy of L. donovani infection in C57BL/6 mice using low dose AmBisome and a DNA vaccine
- preclinical safety of a DNA vaccine against leishmaniasis.
Presentations at meetings in 2012 and 2013
Presentations at the International Symposium on Leishmaniasis Vaccines, Ouro Preto, Brazil, 1 - 6 September 2012:
- Dendritic Cells and Vaccines for Leishmaniasis by PW, Charite, Berlin, Germany
- DNA as Delivery System for Leishmania Antigens by SR, IICB, Kolkata, India
Presentation at the 4th Annual Meeting of COST ACTION CM0801, Crete, Greece, 19 - 21 September 2012:
- Development of a DNA vaccine for leishmaniasis by KS, LSHTM, UK
Meetings / symposia
We propose to organise and coordinate sessions on vaccines for Leishmaniasis at two prestigious international meetings to be held in 2013:
I: Fifth World Leishmaniasis Congress (WorldLeish5), Brazil, 13 to 17 May 2013
II: 62nd Annual Meeting of the American Society for Tropical Medicine and Hygiene, Washington DC, 15 to 19 November 2013
Exploitation
The exploitation strategy of the consortium is based on the IP strategy which has culminated in the filing of a UK patent application in April 2012. The consortium plans to expand this national priority application via international applications to cover all relevant geographical regions. The IP strategy aims to cover both therapeutic and prophylactic treatment approaches and includes all relevant experimental data obtained during the course of the project. International IP protection is regarded as prerequisite for further development and subsequent marketing of any product derived from the project results.
For an economic exploitation of the product, clinical studies covering safety and efficacy of the vaccine are necessary prior to market entry. Currently, the consortium is identifying and contacting potential partners to supply all or part of the necessary financial, technical, regulatory, and logistical resources needed for the entry into the clinical phases. Potential partners can be large pharmaceutical companies (Big Pharma), non-governmental agencies, or national or supra-national public institutions. After a successful completion of clinical studies, the vaccine is intended for marketing in endemic countries.
Project website: http://www.leishdnavax.org
The LEISHDNAVAX project focused on the preclinical development of a safe and efficacious prophylactic and therapeutic DNA vaccine against both visceral and cutaneous forms of leishmaniasis. The vaccine is based on the minimalistic immunogenically defined gene expression (MIDGE) vector developed for efficient induction of T-cellular immune responses. A double stem loop immunomodulator (dSLIM) adjuvant was used in some studies. Based on preset criteria, five antigens from a pre-defined shortlist were selected and their genes incorporated into MIDGE vectors. The candidate vaccine was evaluated for immunogenicity in both animal models and human cell culture systems, and for efficacy in rodent models of visceral (VL) and cutaneous leishmaniasis (CL). The safety of the candidate vaccine was determined in animal models as required by regulatory authorities. To ensure the preclinical candidate possessed broad potential, immunogenicity and safety studies were conducted for both prophylactic and therapeutic indications. We now have a defined vaccine that has been tested ex-vivo in human cells and in animal models, and has been shown to be safe and capable of producing the desired effect and immune responses.
Following successful completion of the LEISHDNAVAX project, the candidate vaccine, produced under GMP conditions, will enter clinical phase I trials for safety and immunogenicity studies in European and disease endemic countries. In preparation of these trials, clinical sites have been identified in disease endemic regions of both VL and CL and initial preparations made to ensure proposed clinical trials would be conducted at ICH-GCP standards. This preparation included training in preclinical and clinical development, and the establishment of components and infrastructure to conduct trials.
The LEISHDNAVAX project was built upon three phases of study. The initial phase identified the immunogenic antigens for selection and inclusion within expression constructs in work packages (WPs) 1 and 2. This was followed by phases to determine the immunogenicity and efficacy of the DNA vaccine constructs in rodent models (WPs 3 and 5) and pre-clinical safety studies (WPs 4 and 6). Many of the studies were undertaken in parallel. Recent Leishmania isolates from endemic target regions were obtained, propagated and distributed between partners for use in all WPs. In WP1, seven candidate antigens were pre-selected based on specified criteria. Selection of vaccine antigens was established and immunogenicity of candidate antigens was tested on PBMCs from individuals of target populations using human cell culture systems. Five antigens were selected that met the criteria set for antigen selection, i.e. sequence conservation and immunogenicity in population in endemic areas. In WP2, MIDGE vectors needed for preclinical immunogenicity and safety studies were produced after constructing suitable expression cassettes with optimised DNA sequences of selected antigens. Expression in vitro was confirmed prior to production. In WP3, recombinant proteins of selected antigens were used in immunogenicity studies in animals in vivo by respective partners. Immunogenicity and protection studies for all the MIDHE-Th1 vectors encoding Leishmania antigens were performed in two animal species in vivo and the vaccine was shown to be immunogenic. Protection was demonstrated in some animal species. Induction of CD4+ and CD8+ T-cell responses were also studied with positive results. WP4, safety studies for a prophylactic vaccine were initiated following discussion with regulatory authorities in Germany (PEI) and London (EMA); studies were refined according to received scientific advice.
The persistence of MIDGE vectors in animal tissue was determined. WP6, safety studies for a therapeutic vaccine were run in conjunction with WP4. In WP5, the therapeutic efficacy of the vaccine was determined and standardisation procedures for animal studies shared with WP3. Therapeutic effects in mouse models of VL and CL were examined, as well as the positive interaction of the vaccine with an anti-leishmanial drug in the VL model. In WP7, SOP's were established in collaboration with WP1 to test the antigenicity and immunogenicity of the vaccine antigens in human populations, and for immunomonitoring future clinical trials. Three workshops were held to train personnel at the test sites in endemic countries. Exchange of co-workers between laboratories for training purposes was undertaken.
Although clinical studies are not part of the LEISHDNAVAX proposal, the project has developed a candidate vaccine for leishmaniasis which is now ready for Phase I clinical studies. The project has analysed and constructed T-cell epitope maps of the vaccine antigens for humans which will provide a unique and important contribution to understanding the outcome in target vaccinated population with diverse genetic backgrounds. In particular, since there are no validated animal models for human leishmaniases, the information gained from LEISHDNAVAX may provide insight to identify markers of efficacy in human population.
The LEISHDNAVAX consortium (see http://www.leishdnavax.org online) was composed of partners with extensive experiences in Leishmania research and vaccine development, including:
List of partners
1. London School of Hygiene and Tropical Medicine United Kingdom (UK) LSHTM
2. MOLOGEN AG Germany MAG
3. Charite Universitatsmedizin Berlin Germany CHARITE
4. Indian Institute for Chemical Biology India IICB
5. Institute Pasteur de Tunis Tunisia IPT
6. Hebrew University of Jerusalem Israel HUJI
7. Rajendra Memorial Research Institute India RMRI
8. Drugs for Neglected Diseases Initiative Switzerland DNDi
Project context and objectives:
Leishmaniasis, a neglected infectious disease with up to 2 million cases per year worldwide, requires new tools for treatment, diagnosis, surveillance and prevention. There is a consensus that an integrated approach using vector control, prompt diagnosis, effective treatment and vaccines are needed for control and prevention of both visceral and cutaneous forms of leishmaniasis. The focus of this project is the delivery of a preclinical candidate for a safe and effective vaccine that will play a critical role in this integrated approach. Currently, there are no approved vaccines to prevent or treat human leishmaniasis though two licensed vaccines have been developed for canine leishmaniasis. Four vaccine candidates (two funded through this European Union (EU) Seventh Framework Programme (FP7) call, and an additional two funded by the Bill and Melinda Gates Foundation and the Wellcome Trust) are in pre-clinical and clinical development. Our knowledge about the stable, highly-conserved Leishmania antigens from many of the species that cause human disease, including antigens with limited or no human homology, and of the immune responses to these antigens in models of infection, has given the research community confidence to undertake the development of a vaccine effective for the prevention and/or treatment of different forms of leishmaniasis.
The LEISHDNAVAX project focused on the preclinical development of a safe and efficacious prophylactic and therapeutic DNA vaccine, ready for clinical trials, against both visceral and cutaneous forms of human leishmaniasis. The vaccine is based on the minimalistic immunogenically defined gene expression (MIDGE) vector developed for efficient induction of T-cellular immune responses and, if required, a double stem loop immunomodulator (dSLIM) adjuvant. Antigens from a predefined shortlist were selected and their genes incorporated into MIDGE vectors. The vaccine is T-cell directed and its immunogenicity was evaluated in animal models and human cell culture systems. Efficacy was determined in animal models according to a target product profile and safety established in preclinical studies. Clinical sites have been identified and prepared to conduct clinical trials at ICH-GCP standards. This includes training in preclinical and clinical development, and establishing all required components and infrastructure to conduct trials at the highest international standard.
The specific objectives of the project, many performed in parallel, were:
- to select antigens that had previously been shown to be protective in animal models;
- to establish the degree of sequence conservation of the selected antigens in recent Leishmania isolates from patients of our target endemic regions;
- to establish an in vitro system to assay immunogenicity of selected antigens and vaccine composition using PBMC of healthy individuals from target populations;
- to establish antigenicity and immunogenicity (in vitro) of the vaccine antigens for induction of T cell responses in the target populations including matching immunogenicity of the antigens and immunogenetics of the populations;
- to assess the stimulatory activities of vaccine components in recovered patients from target population;
-construction of antigen expression cassettes suitable for MIDGE vector production;
- production of MIDGE vectors coding for leishmanial antigens (MIDGE-Leish-Ag) for preclinical immunogenicity and efficacy studies in mice and hamsters, and select appropriate antigen(s) for prophylactic and therapeutic studies;
- production of selected MIDGE-Leish-Ag's under GMP-like conditions, for preclinical safety studies;
- to find the relation, if any, between immunodominance of an antigen and host protection;
- to analyse and compare the efficacy of induction of human CD4+ and CD8+ T cell-mediated immune responses by different MIDGE-Leish-Ag's individually and collectively with or without the immunomodulator dSLIM;
- to study the efficacy of MIDGE-Leish-Ag's as prophylactic vaccine against experimental Leishmania major, and possibly Leishmania tropica infections in mice;
- to determine the efficacy of all selected MIDGE-Leish-Ag's in mice and hamsters against Leishmania donovani infection as therapeutic vaccine administered at time points after infection;
- to determine the requirement of an adjuvant, in particular dSLIM, in the efficacy of the candidates as therapeutic vaccines at time points after infection;
- to determine the interaction between the selected candidate vaccine and standard anti-leishmanial drugs through measurements of liver and spleen load;
- to analyse and compare CD4+ and CD8+ T cell-mediated immune responses to the vaccine candidate with and without drugs in therapeutic models;
- to identify site(s) and establish all prerequisites for conducting clinical trials of LEISHDNAVAX at current ICH-GCP standards from preclinical development to registration;
- to build expertise in various steps from preclinical to clinical development and registration of LEISHDNAVAX for prophylactic and therapeutic indications through learning by doing concept.
As described in the second periodic report below all objectives were completely or partially met. The results have been reviewed by an external scientific advisory board (see periodic report, WP8) who confirmed that the requirements for a clinical candidate for human leishmaniasis have been met, as well as making some specific recommendations for further studies.
Project results:
The success of LEISHDNAVAX project is built upon three phases of study that have come together in the final months of the project and have delivered a clinical vaccine candidate for both visceral and cutaneous forms of leishmaniasis. The initial phase identified the immunogenic antigens for selection and inclusion within constructs (WPs 1 and 2). This was followed by phases to determine the efficacy in rodent models (WPs 3 and 5) and pre-clinical safety (WPs 4 and 6) of the DNA vaccine constructs, undertaken in parallel.
WP1: Definition of the vaccine antigens
Research over past few years has shown that immunity against leishmaniasis is possible and is mediated by the cellular arm of the immune system, T lymphocytes together with the innate immune macrophages and dendritic cells. This has prompted the development of vaccines that specifically trigger responses of Leishmania parasite-specific T lymphocytes. Such vaccines are different than the established vaccines on the market that are designed to induce humeral immune responses with neutralising antibodies, and they have to be developed in a different way. T lymphocytes do not recognise and react to whole antigens but to small fragments of the antigens, peptides. These molecules are products of proteolytic degradation of proteins inside cells and are bound by special peptide receptors called major histocompatibility complex (MHC) molecules or human leukocyte antigens (HLA) in humans. These HLA are highly polymorphic meaning they occur with a huge number of variants in the population and their composition differs between individuals and populations. In addition, parasites may also have polymorphisms in their antigens that can differ between Leishmania species, isolates and regions. With this background the task in the project was to define vaccine antigens that are conserved in all Leishmania species that induce Leishmania-specific T lymphocytes, and that are immunogenic in the human populations in the endemic regions.
Seven candidate antigens A2, CPA, CPB, HASPB, KMP-11, p74 and TSA were short listed, because it was known from prior work of consortium members and others that they can induce protective immunity in animal models. The gene sequences of these antigens were established for different recent Leishmania isolates from India, Central Asia, Iran, Near East, Saudi Arabia, Sudan, Ethiopia, Tunisia and Italy, and together with all sequences available in public sequence databases that represent old isolates and New World Leishmania species analysed for sequence conservation. Two antigens, A2 and HASPB, were excluded at this stage because of their high variability. Two antigens, KMP-11 and p74, were almost completely conserved and three, CPA, CPB and TSA, had some variations both between different Leishmania species and also within species.
To establish the immunogenicity of the candidate antigens, overlapping peptides that cover the sequences of the five remaining antigens including all variations, and thus all possible T cell epitopes were designed, synthesised and tested with peripheral blood cells of donors with a recent history of leishmaniasis. Matching the results of the sequence analyses and of the immunogenicity test with the peptides, the sequences of the vaccine antigens were designed to be both as conserved as possible and as immunogenic as possible. With peptides that cover the sequences of the five now redesigned antigens, the antigenicity mapping was further refined with white blood cells from donors in India and Tunisia who had recovered from leishmaniasis. The results prove three key points:
- The five candidate vaccine antigens are indeed processed in the patients to induce Leishmania-specific T lymphocytes.
-All five antigens carry epitopes for T lymphocytes that are suitable for the HLA of the populations in the endemic regions.
-The antigens or the selected conserved variants of the antigens are highly immunogenic with active peptides covering between 43 and 100 % of the antigen sequences.
Such high density of antigenic epitopes has so far not been described for any other antigen or infectious agent. The five selected antigens are thus most suitable as vaccine antigens.
The antigen sequences were retrotranslated into DNA sequences that were optimised for high expression yields, and the optimised gene sequences cloned into a plasmid for production of the MIDGE vaccine vectors. This vector system has been shown to be suitable to carry the genetic information for the antigens into cells for processing and presentation of epitopes on HLA and induction of T lymphocytes. This was confirmed in animal studies as described below.
The outcome of the WP1 is a pentavalent vaccine ready for tests in preclinical animal models and clinical trials. The approach to the definition of vaccine antigens for induction of immunity mediated by T lymphocytes that was established within WP1 is novel. It is modular so that the vaccine can be redesigned easily by reducing or extending the number of antigens, or modifying and exchanging antigens in case more promising candidates are identified.
Vaccines for induction of immunity mediated by T lymphocytes require different protocols for monitoring their immunological efficacies and correlating these with outcome. Serology as for standard vaccines cannot be used. Standard operating procedures (SOPs) were developed for all steps involved in monitoring T lymphocyte responses including sample coding, preparation and cryopreservation of peripheral blood mononuclear cells, and bioassays for antigen-specific T lymphocyte responses. The SOPs were tested and implemented together with protocols for quality management at the test sites in India and Tunisia.
WP2: Design and production of vaccine
In this WP, the vaccine candidate has been generated in substance: The antigens selected in WP1 were combined with the MIDGE-Th1 vector. For this purpose, the antigen encoding sequences were synthesised following optimisation for strong expression in humans. The synthetic DNA sequences were then incorporated individually into plasmids optimised for MIDGE-Th1 production. Thereby, the expression cassettes of the antigen-encoding MIDGE-Th1 vectors were generated.
MIDGE vectors are linear, double stranded DNA molecules covalently closed at both ends by single-stranded loops of DNA. The closed structure protects the vector from rapid degradation by exonucleases. MIDGE-Th1 vectors are MIDGE vectors with a peptide attached. In contrast to conventional plasmids usually applied as DNA vaccines, MIDGE-Th1 vectors only contain the expression cassette, but no potentially detrimental sequences like antibiotic resistance genes, bacterial backbone sequences or an origin of replication. In a number of in vitro and in vivo studies, the efficacy of MIDGE-Th1 vectors for expression of the encoded antigens and for DNA immunisation has been shown previously.
However, a functional check of expression cassettes of the vaccine candidate was required, thus antigen expression in vitro for both plasmids and MIDGE-Th1 vectors was evaluated by expression analysis on mRNA level. Mammalian cells were transfected with the respective vectors and specific mRNA transcription was determined. All antigen expression cassettes passed this check of function in vitro.
Moreover, a dose-response relation between DNA amount used for transfection and resulting mRNA amount was shown in vitro for each expression cassette encoding a Leishmania antigen. This result was important for the establishment of a potency assay as described below.
Expression analysis on protein level was conducted as the second functional check of expression cassettes. Except for one antigen (CPA), the antigen proteins were detected in Western Blot assays when evaluating corresponding MIDGE-Th1 plasmids and MIDGE-Th1 vectors side by side. Efforts to detect the CPA protein after expression in vitro are continued.
Taken together, five MIDGE vector expression cassettes each encoding a leishmanial antigen have been synthesised as consensus sequences and optimised for expression, with proven functionality in cell culture, and incorporated into plasmids serving as starting material for MIDGE vector production.
For preclinical efficacy evaluation of the vaccine, candidate MIDGE-Th1 vectors were produced in a simple standardised procedure. The manufacturing process started with the plasmid containing the expression cassette of the future MIDGE Th1 vector. The expression cassette was flanked by recognitions sites for the restriction enzyme Eco31I enabling an enzymatic digestion resulting in two open-ended DNA molecules: the expression cassette and the plasmid backbone. The open ends of the expression cassette were then covalently closed by enzymatic ligation with specific oligodeoxyribonucleotides (ODN) designed to each form a single-stranded loop. The Th1-peptide was coupled to one ODN before. Finally DNA molecules with open ends, i.e. the plasmid backbone including the antibiotic resistance gene and the origin of replication, were enzymatically degraded, and the MIDGE-Th1 vector containing solution purified, desalted and subsequently concentrated.
The final pentavalent vaccine candidate consists of five MIDGE-Th1 vectors each encoding one antigen. The five vectors were produced separately and underwent full quality control (QC), before they were mixed, the final concentration adjusted and final QC tests performed. The vaccine samples were shipped as ready-to-use compositions to other partners for preclinical immunogenicity and efficacy studies.
The preclinical trials to evaluate the safety of the pentavalent vaccine candidate comprised biodistribution pilot and main studies, and a repeated-dose toxicity study. For these studies, the MIDGE-Th1 vectors encoding leishmanial antigens were produced under GMP-like conditions. The steps of the production comply with those described above. Vaccine samples were manufactured ready-to-use and shipped to the respective contract research organisations (CROs).In total, approximately 1 g of MIDGE-Th1 vector DNA has been produced.
In preparation of the GMP manufacturing process for clinical studies, an essential vaccine batch release test, i.e. the potency assay to determine the biologic activity of the vaccine has been designed and presented to the authorities, adjusted accordingly, established and standardised.
The assay starts with transfection of a human cell culture line with defined amounts of MIDGE-Th1 vectors encoding the Leishmania antigens. Subsequently, expression of antigens is quantified on the mRNA/cDNA base employing a quantitative PCR system. Using the parallel-line method the relative potency of vaccine batches is calculated. The MIDGE-Th1 vector encoding KMP-11 was used as the model vector during initial establishment of the system. Later, the potency assay was extended to analyse the complete mixture of all antigen-encoding vectors using antigen-specific labelled TaqMan probes and compatible antigen-specific PCR primers in the same experimental setup.
Extensive tests have been performed to select an appropriate human cell line, the appropriate transfection method, and the range of DNA amount to be used in transfection. To ensure reproducibility and reliability of the potency assay as a future vaccine specific batch release test, the entire procedure has been established, standardised and defined in standard operating procedures (SOPAs). Thus, the potency assay is ready to enter the next step of development, i.e. test validation with clinical batches of the vaccine and comparison of activity measured in vitro to immune response of humans.
In summary, from the production and quality control perspective, the vaccine candidate can be taken forward to next development steps including the first clinical study.
WP3: Animal and human cell culture studies for prophylactic efficacy
Prophylactic vaccination was tested in animal models of leishmaniasis to check if they were immunogenic and had any influence on disease outcome. Five conserved antigens the KMP-11, TSA, CPA, CPB and p74 were finalised based on their conserved nature and T cell stimulatory capacity. Optimised synthetic sequences of MIDGE-Th1 vectors encoding all five antigens were produced.
Immunogenicity studies:
In animal models the main concern was to detect if all the antigens of the multivalent vaccine are recognised by the host and evaluate the type of immune response elicited upon immunisation. From our initial experiments a three dose immunisation scheme at 14 day intervals followed by parasite challenge was adopted. In both BALB/c and C57BL/6 mice antibodies against all the five antigens were produced. Antibodies against KMP-11, TSA and CPA showed the greatest increase upon immunization, but there was also appreciable increase in antibodies against CPB and P74. We looked at the IgG1 and IgG2a response (IgG2a/IgG1 ratio) to understand the underlying nature of the T cell response. The IgG2a/IgG1 ratio was higher in vaccinated animals compared to the PBS injected animals. In BALB/c, there was an increase in both IgG1 and IgG2a isotypes, but antigen specific IgG2a response was higher. This indicates a mixed Th1-Th2 response with a bias towards Th1. On the other hand, C57BL/6 mice exhibited an extreme Th1 bias compared to the BALB/c mouse. Antibodies to all five antigens in the vaccinated C57BL/6 mice were exclusively of IgG2a isotype. The mixed response in BALB/c mice may be due to the inherent Th2 bias of this strain.
To test the cellular immune response generated by the vaccine, spleen cells from immunised mice were restimulated with antigenic peptides for 24 hours. Assuming that the vaccine induced a cellular immune response, then antigen specific T cells from vaccinated mice should recognise MHC bound antigenic peptides upon in vitro restimulation. Higher IL-2 production in mice receiving vaccine formulation indicates T-cell activation, in contrast to mice that received PBS. This T-cell activation was found to be proportional to the vaccine dose. All the peptide pools were immunogenic in both BALB/c and C57BL/6. The KMP-11 pool, which induced highest IL-2 production in BALB/c, produced the least IL-2 in C57BL/6. This shows that there are subtle variations in immune response to the vaccine between different mouse strains.
In the hamster model, an initial study with MIDGE-KMP-11 demonstrated that the three dose immunization scheme produced the best immunogenicity. In a separate experiment where three antigen coding constructs (KMP-11, TSA. P74) were administered separately, and also as a mixture, the KMP-11 and p74 specific antibody responses were higher in the mixture compared to animals immunised with MIDGE-KMP-11 or MIDGE-p74 alone. Antibodies were of the IgG2 isotype, again indicating that vaccination generates a Th1 bias. When all the antigen coding constructs (MIDGE-CPA, -CPB, -P74, TSA and KMP-11) were administered as a mixture to hamsters, antibody responses were observed to all five antigens.
From the immunogenicity studies, it is evident that all five vaccine constructs are immunogenic in two strains of mice, and also in hamsters. The immune response generated by this vaccine is Th1 biased as suggested by the IgG1 and IgG2a isotype ELISA. In addition, the degree of immune response demonstrates a clear dose response with the vaccine dose.
Efficacy studies:
We have used the susceptible BALB/c model to assess the efficacy of the five antigen containing vaccine formulation upon challenge with L. donovani (VL). The prophylactic efficacy of the vaccine formulation showed a clear dose response inversely correlated with the parasite burden. The highest dose of 20 g/antigen provides more than 90 % protection against experimental parasite challenge. The level of protection decreases with the reduction in vaccine dose. Reduction in liver parasite burden at 21 days post infection also corresponds to a similar reduction in spleen parasite burden at a later stage of infection. Spleen cells from these protected animals were found to produce significantly high amounts of IFN-I and TNF-I ± upon antigen recall with peptide pools. Control animals did not have such high levels of pro-inflammatory cytokines. Some degree of protection was observed when resistant C57BL/6 mice were immunised with the pentavalent vaccine and challenged with L. major (CL).
Despite immunogenicity of the component antigens in hamsters, the final formulation failed to show any efficacy against an experimental L. donovani challenge in this animal model. The three-antigen mixture in one experiment exacerbated disease. But immunisation with either MIDGE-TSA or MIDGE-p74 individually, or MIDGE-KMP-11+dSLIM leads to reduction in parasite burden. Unlike the three-antigen mix, the final five antigen containing DNA formulation did not exacerbate or protect against disease. Interestingly, the final formulation when administered along with the immunomodulator dSLIM gave 88 % protection in hamsters. An additional experiment comparing immunization with the pentavalent vaccine + dSLIM versus dSLIM alone is needed to confirm this finding, and is underway.
WP3 has proved that the concept that this vaccine design indeed works in the animal models. All five candidate antigens are immunogenic when DNA encoding them is administered as a mixture. The vaccine induces both humoral and cellular responses, and the cellular response is Th1 biased. The vaccine, in the absence of an adjuvant, does not protect hamsters from experimental parasite challenge, but this may be due to the inherent extreme susceptibility of hamsters towards visceral leishmaniasis. The five antigen formulation of vaccine provides protection against experimental visceral leishmaniasis in BALB/c mice. An almost similar level of protection was observed with a three antigen vaccine formulation in this animal model. Since the human target population for the vaccine will have a large HLA polymorphism, it is best to keep all five antigens in the formulation. Being a mix of different antigen coding constructs, this MIDGE platform has the flexibility built into it that will enable us to fine tune, as needed, the vaccine components during clinical trials.
WP4 and WP6: Preclinical safety evaluation of LEISHDNAVAX for prophylactic and therapeutic indication
Before administration to humans in clinical studies, a vaccine must be proven safe in preclinical studies according to regulatory guidelines. In order to ensure that the preclinical program of LEISHDNAVAX is deemed relevant by the authority granting, a clinical phase 1 study, the program was first discussed with the German authorities in 2009. Thereafter, the consortium applied for the Article 58 procedure - a procedure to review marketing applications for products intended to prevent or treat diseases of major public health interest, and to be used only outside the European Community. This procedure enables the European Medicines Agency (EMA) to give a scientific opinion, in cooperation with the World Health Organization (WHO), for the evaluation of these medicinal products. The resulting Certificate of Pharmaceutical Product issued by the EMA serves as the basis for product approval in other countries. The LEISHDNAVAX vaccine candidate was eligible for the procedure, and scientific advise on the preclinical program was received from the EMA and WHO.
According to this scientific advise, the preclinical safety study plans were adjusted. The studies of WP4 and WP6 were combined by extending the safety studies in non-infected animals to the drafted treatment schedule for a therapeutic vaccine. Safety studies in infected animals were no more required, but limited data on tolerability of the vaccine during treatment of infected animals (see WP5) should complete the preclinical safety data set.
The preclinical safety program comprised studies on the biodistribution and the toxicity of the vaccine. While toxicity of the vaccine was assessed in standard settings under GLP and completed by evaluation of auto-immune responses against DNA, no standard settings existed for biodistribution studies addressing the persistence of DNA vectors in tissues and organs. Thus a quantitative PCR (qPCR) assay to quantify MIDGE-Th1 vectors in tissues has been established and transferred to Contract Research Organisations (CROs). In addition, methods and procedures preceding the qPCR have been standardised.
Two biodistribution studies in rats, a pilot and a main study, were conducted. The pilot study was performed with the only MIDGE-Th1 antigen (KMP11) vector available at that time, in order to obtain preliminary data on the biodistribution, to check and refine the study plan of the main biodistribution study, as well as to set up and standardise all procedures and methods intended for the main study and to evaluate the performance of the CRO's. The study was performed as a blinded study including one vehicle control and one test item group. With the exception of skin tissue of the injection site, a strong reduction of copy numbers of MIDGE-Th1 vectors from 24 hours to 15 days after two immunisations was noted in essentially every tissue.
In the main study the distribution of the final mixture of five MIDGE-Th1 vectors was evaluated in rats after one or four repeated intradermal injections. In samples collected 24 hours after one or four immunisations, MIDGE-Th1 vectors were detected in quantifiable amounts in all tissues. The highest amount of vector DNA was found in samples of skin of injection site. The second-highest amount of vector DNA was found in lymphatic tissues, indicating that the vector is transported to lymph nodes. There was no significant difference between vector copy numbers of animals injected once or repeatedly with vaccine suggesting that the vaccine does not accumulate inside organs.
DNA vectors were cleared from essentially every tissue within 60 days after the fourth injection, with the exception of skin and inguinal lymph node tissue. Most importantly, even after repeated dosing the vaccine did not persist in critical organs such as reproductive organs or bone marrow. In conclusion, the risk of MIDGE-Th1 vector persistence and integration into host genomic DNA even after repeated dosing is considered to be very limited. However, prior to application for a Clinical Phase 1 study the results of the biodistribution study have to be discussed with the competent authority for advice on conduction and design of an integration study.
To assess the toxicity of the pentavalent vaccine, the vaccine was administered to mice intradermally once or five times once weekly at three dose levels. The reversibility of any effect within two recovery periods was also evaluated. The highest dose given to mice in this study corresponded to 200 fold the highest human dose anticipated when compared on the basis of DNA dose per bodyweight. To ensure that efficacious vaccine samples were used in this test for toxicity, vaccine immunogenicity was proven by detection of antigen specific antibodies.
No local intolerance reactions were noted at the injection sites in the animals given one or four weekly inoculations with vaccine. No systemic intolerance reactions to the vaccine were observed concerning mortality, clinical and neurological signs, body weight and body weight gain, food and water consumption, haematology, ophthalmological and auditory examinations, macroscopic post-mortem findings, organ weights, histopathology and auto-immune reactions including determination of anti-double-stranded DNA. The no-observed-adverse-effect-level (NOAEL) was above the highest dose tested in this study.
The reports of the biodistribution and the toxicity studies are part of the documentation to be filed when applying for permission to conduct a clinical phase 1 study in any country.
A workshop on the preclinical development of a DNA vaccine, applicable in preventive as well as in therapeutic approaches, was held in Berlin in 2011. Four scientists and four PhD students attended of the workshop. An overview was given on the worldwide state of the art and the development status of DNA vaccines, and compared to the LEISHDNAVAX project. Production and quality issues of DNA vectors including GMP and non-GMP procedures were discussed and production sites at MAG visited. A seminar was given on regulatory aspects of pre-clinical programs, collaborations with CRO's and documentation requirements. To connect preclinical and clinical development steps a presentation on clinical trials and regulatory requirements in India was given. In the practical part of the workshop, participants successfully designed preclinical safety studies.
WP5: Animal studies for therapeutic efficacy
In addition to the development of a prophylactic vaccine for leishmaniasis the potential application of selected vaccine candidates as immunotherapy was tested in models of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). The main questions addressed in these studies were tolerability (see also WP6) and efficacy measured as reduction in parasite burden (VL) or clinical disease (CL)
Initial studies were undertaken in BALB/c mice infected with L. donovani (VL) with a monovalent vaccine encoding the antigen p74. Efficacy was evaluated at different doses, number of injections, and time points in liver and spleen of infected animals. Although at first a significant reduction in hepatic parasite burden was seen after two injections this result was not reproducible in a repeat experiment. Notably the parasite burden in untreated control animals was higher in this experiment, which may indicate a dependence of efficacy on parasite burden. Similar conclusions were drawn from studies where an anti-leishmanial drug was sequentially co-administered with the final vaccine candidate as an immune-chemotherapy approach (see below).
Further studies on tolerability and efficacy were carried out with the pentavalent vaccine as candidate vaccine for clinical trials (MIDGE-Th1 vectors encoding the antigens CPA, CPB, KMP-11, p74 and TSA, see WP1). Dose kinetic studies were carried out in BALB/c and C57BL/6 mice infected with L. donovani (VL), and designed to include 1, 2 and 3 injections at weekly intervals and comparison to a PBS treated control group. Tolerability parameters assessed included monitoring of bodyweight, behaviour, fur condition, injection site reaction, evaluation of serum levels of ALT, AST, urea and creatinine, and histopathological examination of disease target and vital organs. The parasite burden was evaluated in liver and spleen. No reduction in parasite burden was seen. Disease exacerbation was excluded in this experimental design. The vaccine was well-tolerated and no adverse reactions observed.
An additional study was carried out in C57BL/6 mice infected with L. major (CL) receiving three injections of the pentavalent vaccine at weekly intervals. Due to a low percentage of animals developing lesions in all groups (vaccine treated, PBS treated or untreated) no definite conclusion on the efficacy in a CL model can be made. However, this result suggests no efficacy of the immunotherapy when used alone. Importantly, no adverse reactions were observed.
To evaluate the potential of the pentavalent vaccine in an immune-chemotherapy approach L. donovani infected C57BL/6 mice (VL) were sequentially treated with a single (suboptimal) dose of AmBisome followed by a single dose of pentavalent vaccine two weeks later. Another 10-12 days later mice were killed, and parasite burden in liver and spleen evaluated. Respective controls (untreated, AmBisome treated, pentavalent vaccine treated, vector treated, AmBisome plus vector treated) were included. Prior to these experiments the dose response of single dose AmBisome was assessed to chose the best dose level for co-administration experiments. These studies confirmed the potential of the sequential co-administration of drug and vaccine approach for treatment of VL, as this group showed the highest reduction in parasite burden. Reduction was more pronounced in the spleen than the liver, although the same trend was seen in both organs. Two different doses of the pentavalent vaccine (20 μg per antigen and 40 μg per antigen) were used in a pilot experiment, which indicated that 20 μg per antigen was sufficient to induce the highest parasite reduction, and 40 μg per antigen does not provide additional benefit. Synergism was not seen when single dose AmBisome was followed by administration of a non-expressing MIDGE vector containing the human IL-2 gene. Thus, it can be concluded that the synergistic effect is due to at least one or more of the antigens. ELISAs on antigen specific IgG2a/IgG1 responses are in progress, and will provide direct proof as to which antigen is responsible for the synergistic protective effect, and the nature of the immune response based on Th1-Th2 bias. In addition, histopathology and frequency of CD3 positive cells have been determined. An experiment to demonstrate the effect of the pentavalent vaccine on a dose response of AmBisome treatment is completed.
In conclusion, studies in WP5 have demonstrated that selected vaccine candidates including the final pentavalent vaccine are well tolerated in animal models of leishmaniasis. When used as monotherapy no consistent anti-leishmanial effect was seen. However, the potential of the pentavalent vaccine in an immune-chemotherapy approach was demonstrated by sequentially administering a single dose AmBisome followed by the pentavalent vaccine candidate. It is hypothesised that the drug reduces the parasite burden, which restores CD4 and CD8 immune responses and allows the immunotherapy to work more effectively than when given alone. Demonstration of a dose sparing effect is ongoing.
In addition to the research work of WPs 1-6 that produced the results that constitute the project foreground, the project also undertook activities in training and site preparation for proposed clinical trials.
WP7: Preparation for clinical trials
The specific objectives of WP 7 were:
(a) to identify sites and establish all prerequisites for conducting clinical trials of LEISHDNAVAX at current ICH-GCP standards from preclinical development for registration, and
(b) to build expertise in various steps from preclinical to clinical development and registration of LEISHDNAVAX for prophylactic and therapeutic indications through learning by doing concept.
It was decided to conduct the first phase 1 clinical trial in Germany. Once satisfactory data is available an additional phase 1 trial will be conducted in endemic districts of India (Bihar, RMRI) for Visceral Leishmaniasis and Tunisia (IPT) for Cutaneous Leishmaniasis.
The reviews of potential clinical trial sites and capability strengthening areas were identified by 24 months of the project. Out of 33 endemic districts in Bihar, India, seven highly endemic districts were identified reporting more than 1500 cases consistently over the past five years; two districts were selected for future vaccine trial. These two districts, Marhaura PHC of Saran and Simri-Bakhtiyarpur PHC from Saharsa, were selected on the basis of logistic support and number of cases. Two workshops and continuous training have been held in these PHCs and both sites are ready to monitor immune responses after vaccination. Required equipment, expertise and laboratory facilities are in place to conduct the above activities at RMRIMS (partner 7). Similarly Sidi-Bouzid city in Tunisia was selected for LEISHDNAVAX vaccine trials for zoonotic CL. Sidi-Bouzid is an endemic region for the disease. About 8558 cases have been reported in the past three years. The most likely spatial cluster of high incidence rates contains regions located close to a dam. Two workshops and continuous training have been completed. With extensive previous experience in immunological and CL studies in the same field particularly clinical trials, the availability of technical expertise and infrastructural facilities, IPT (partner 5) is well situated for conducting clinical trials on CL at ICH-GCP standards.
To ensure trained staff were in place, clinical site adapted SOPs for immunomonitoring vaccine induced T-cell responses were prepared and wet lab training workshops held on:
(a) ex-vivo activity of human cells to different antigens with measurements of various cytokines at Kolkata, India from 4 to 8 November 2009,
(b) in-vitro induction of immune response to various peptides of selected antigens used in the vaccine held at Charite, Berlin, Germany from 20 to 27 October 2010, and
(c) development of SOPs and review of protocols for immunomonitoring during clinical vaccine trials based on ICH was held at RMRIMS, Patna, INDIA from 12 to 14 April 2012.
It was also necessary to ensure training and awareness of staff in project management, protocol development, ICH-GCP guidelines, CRF development, data management, and training monitors. A workshop on target product profiles (TPP) and on the clinical development plan was held in Tunisia from 10 to 13 May 2012 in place of MS project, protocol development ICH- GCP guidelines, CRF development, and data management training.
At this workshop three target product profiles were developed for:
(i) CL prophylaxis,
(ii) CL therapeutic, and
(iii) VL prophylactic indications of LEISHDNAVAX project vaccines.
Additional training on preclinical development was conducted at Mologen, Berlin at another workshop from 15 to 19 November 2011. In summary, preparations for clinical studies have been completed. The future steps will depend on the outcome of the Phase 1 study to be carried out in Germany in 2013. If the data are satisfactory then pilot phase I studies are intended to be conducted in endemic regions of Bihar, India and Sidi Bouzid, Tunisia.
Potential impact:
Over the past decade there has been increased funding available for the development of new drugs, diagnostics, vaccines and vector control methods for leishmaniasis (see http://www.g-finder.policycures.org online). In addition, to the aims to improve treatment, surveillance and control, the potential of elimination of some forms of the disease is now firmly on the agenda (see http://www.who.int/tdr/news/2011/vl-elimination online). The possibility for more effective control programmes and elimination is supported by efforts of WHO to improve knowledge of disease burden, prevalence and distribution (see http://www.who.int/leishmaniasis/burden/magnitude online). As with the control and prevention other infectious diseases, an integrated approach using vector control, prompt diagnosis, effective treatment and vaccines is needed for both visceral and cutaneous forms of leishmaniasis.
The major gap in the armamentarium required for leishmaniasis control is the absence of approved vaccines to prevent or treat human leishmaniasis. The development of a single vaccine for leishmaniasis will have a major impact on the control and development of this disease. The LEISHDNAVAX project has developed a vaccine candidate ready for use in clinical trials for both the visceral and cutaneous forms of leishmaniasis. If successful in clinical trials, this vaccine could have a major impact on disease control and public health in countries in Asia, Africa and Central / South America.
Within the LEISHDNAVAX project, we have already started to engage with experts in India (for VL) and Tunisia (for CL) who are connected to the health authorities. To ensure successful delivery and access of the vaccine it is now essential to fully engage with the regulatory and health authorities in selected disease endemic countries.
This project has also demonstrated that a rational approach, based upon molecular, genetic, and immunogenicity criteria, can be used to select antigens for incorporation into vaccines, and that important information on the immunogenicity and efficacy of candidate vaccines can be provided by rodent models of infection. Further studies in humans are now required to confirm our experimental results showing that immune responses to the specific antigens incorporated in the vaccine are correlated with protection in animal models. This pathway of vaccine development could have an impact on refinement or development of vaccines for other infectious diseases.
Dissemination
Publications
As a vaccine development project LEISHDNAVAX incorporated into the consortium agreement (CA), a clear publication policy and an intellectual property (IP) policy. It was agreed that scientific publications would follow the submission and publication of a patent derived from the Foreground work of the project. A patent application entitled Pharmaceutical for treatment of leishmaniasis was submitted by Mologen AG on behalf of all partners of the consortium, at the UK Patent Office (Patent Application No. UK 1207476.1 date of filing 30 April 2012).
At the sixth steering committee meeting held in June 2012, following publication of the patent, a plan for the submission of eight scientific publications (including one review) from the project was made and approved.
List of subject areas for proposed articles included:
- concept and design of a DNA vaccine against leishmaniasis
-high antigenicity and epitope density of leishmanial antigens
- protocols for monitoring T cell-mediated immune responses in leishmaniasis
- tolerability and efficacy (immunogenicity) of a DNA vaccine in mouse models of visceral leishmaniasis
- immuno-chemotherapy of L. donovani infection in C57BL/6 mice using low dose AmBisome and a DNA vaccine
- preclinical safety of a DNA vaccine against leishmaniasis.
Presentations at meetings in 2012 and 2013
Presentations at the International Symposium on Leishmaniasis Vaccines, Ouro Preto, Brazil, 1 - 6 September 2012:
- Dendritic Cells and Vaccines for Leishmaniasis by PW, Charite, Berlin, Germany
- DNA as Delivery System for Leishmania Antigens by SR, IICB, Kolkata, India
Presentation at the 4th Annual Meeting of COST ACTION CM0801, Crete, Greece, 19 - 21 September 2012:
- Development of a DNA vaccine for leishmaniasis by KS, LSHTM, UK
Meetings / symposia
We propose to organise and coordinate sessions on vaccines for Leishmaniasis at two prestigious international meetings to be held in 2013:
I: Fifth World Leishmaniasis Congress (WorldLeish5), Brazil, 13 to 17 May 2013
II: 62nd Annual Meeting of the American Society for Tropical Medicine and Hygiene, Washington DC, 15 to 19 November 2013
Exploitation
The exploitation strategy of the consortium is based on the IP strategy which has culminated in the filing of a UK patent application in April 2012. The consortium plans to expand this national priority application via international applications to cover all relevant geographical regions. The IP strategy aims to cover both therapeutic and prophylactic treatment approaches and includes all relevant experimental data obtained during the course of the project. International IP protection is regarded as prerequisite for further development and subsequent marketing of any product derived from the project results.
For an economic exploitation of the product, clinical studies covering safety and efficacy of the vaccine are necessary prior to market entry. Currently, the consortium is identifying and contacting potential partners to supply all or part of the necessary financial, technical, regulatory, and logistical resources needed for the entry into the clinical phases. Potential partners can be large pharmaceutical companies (Big Pharma), non-governmental agencies, or national or supra-national public institutions. After a successful completion of clinical studies, the vaccine is intended for marketing in endemic countries.
Project website: http://www.leishdnavax.org