Introduction and Background of the Project

Up to 390 million infections of dengue have been estimated to occur annually of which 96 million have clinical manifestations. Dengue is estimated to cause 22,000 deaths every year, mainly among children. Although mortality is relatively lower compared to other tropical infectious diseases such as malaria, the scale of human suffering and the resources invested in the control of dengue make it a major global health problem. Several vaccine candidates for dengue are currently in pre-clinical or clinical development, the most advanced of which recently finished phase III efficacy trials. Although these studies have provided important insights and have shown that populations can be protected from dengue disease, they have also raised numerous other issues. Importantly, the studies showed a wide disparity in efficacy across the four different dengue serotypes. This might prove as a risk factor due to the dengue viral interference problem: Sequential infections with different Dengue serotypes can increase the risk of developing a severe and potentially lethal disease.

Our proposal has the objective to demonstrate the proof-of concept of the measles- virus-dengue vaccine candidate (MVDVax) in a non-human primate model, with ultimate long-term goal to develop an effective prevention tool for dengue. MVDVax primarily targets population at risk of dengue infection including young children in dengue endemic regions and travellers to affected areas. Unlike other dengue vaccines in development, MVDVax uses a single live dengue vaccine designed to activate both humoral and cellular immune response against the four serotypes of dengue virus simultaneously, hopefully overcoming thereby the viral interference problem. The current project will produce all data required for follow up projects to be ready to directly start GMP production, toxicology studies and phase I clinical trials.

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

The EVI – Institut Pasteur - NEKKEN partnership brings together leading institutions from Germany, France and Japan with long-standing experience in the development of vaccines for dengue and other diseases which have joined their complementary expertise with the objective to accelerate the development of the MVDVax dengue vaccine candidate. All three partners - one public research organisations, one non-profit foundation and one product development partnership - are non-profit organisations which have come together in this proposal thanks to their shared interest and their strong commitment to develop technologies for the control and eradication of diseases of poverty, dengue in particular. Dengue is counted as a major global health problem, with the huge scale of human suffering it induces and the large amount of resources invested in its control.

This project will directly address this major global health problem, as its objective is to advance the clinical development of the MVDVax vaccine candidate with the long-term goal to develop an effective prevention tool for dengue. Preparing the way for following clinical studies of the MVDVax dengue vaccine candidate, this project is thereby the first step towards an affordable, all serotypes encompassing dengue vaccine.

What sort of innovation are you bringing in your project?

So far no vaccine has been licensed against dengue. Several vaccine candidates for dengue are currently in pre-clinical or clinical development, the most advanced of which recently finished phase III efficacy trials. Although these studies have provided important insights and have shown that populations can be protected from dengue disease, they have also raised numerous other issues. Importantly, the studies showed a wide disparity in efficacy across the four different dengue serotypes. Consequently, we have gained a lot of insights from previous studies into improved vaccine design, and fresh approaches for the development of dengue vaccines as the one proposed in this application provide a promising way for the development of a safe and effective dengue vaccine which is expected to be an important tool for an effective way to control dengue diseases in the future. More specifically, unlike other dengue vaccines in development, the MVDVax uses a single live dengue vaccine designed to activate both humoral and cellular immune response against the four serotypes of dengue virus simultaneously, hopefully overcoming the viral interference problem. It furthermore uses the measles virus as a way to express the dengue antigens, thereby offering an affordable production cost on the level of measles vaccine cost, apt for a disease of poverty.

Role and Responsibility of Each Partner

NEKKEN will be responsible for virological and immunological studies of the project. For the virology, the obtained plasma after the challenge infection will be monitored for its virus RNA contents by RT-PCR, viral particles by PFU assay. To see the humoral immunogenicity of MVDVax, the plasma after vaccination will be examined for its specific antibody levels by ELISA and by the neutralizing antibody test. For the T cell immunity monitoring, the supernatants after ex vivo stimulation of peripheral lymphocytes with the antigens will be applied to multiplex cytokine assay to confirm the stimulatory activity of the vaccine containing designed T cell epitopes. Institut Pasteur has demonstrated in previous studies the feasibility of recombinant MV platform in humans by introducing safely two vaccine candidates against HIV and chikungunya in clinical trials. The MVDVax dengue vaccine candidate of the current project showed promising capabilities that encourage further development. In this program, Institut Pasteur will be responsible for evaluating immunogenicity and protective MVDVax in NHP, Rhesus Monkey, and for the EDIII assay development for detection of serotypes specificity. The NHP experiment is essential to process MVDVax to human trial. The EVI-Institut Pasteur-NEKKEN partnership is led by EVI, the designated grantee. EVI will be responsible for the overall coordination of the work programme.

Final Report

1. Project objective

The two objectives of this project were (i) to evaluate the immunogenicity and protective efficacy in non-human primates (NHP) of MVDVax, a measles-virus (MV) based dengue vaccine candidate, and (ii) to develop an assay to quantify the amount of envelope domain III (EDIII) expressed for each of the four dengue virus (DENV) serotypes and demonstrate consistent expression of the vaccine candidate antigens across several batches.

2. Project design

MVDVax is a prototype of a monovalent live dengue vaccine candidate based on replicating measles-virus (MV) vector expressing a tetravalent dengue antigen composed of four EDIII envelope domains (one for each dengue serotype) and a chimeric antigen composed of fragments of non-structural (NS) dengue virus proteins to induce CD8+ T cell immunity. This vaccine candidate demonstrated strong immunogenicity (antibodies and T cells) and efficacy in mice. To achieve the first objective, cynomolgus macaques were immunized with MVDVax, followed for immune responses, and challenged by dengue virus. For the second objective, different vaccine batches were prepared and analyzed for their composition by ELISA and mass spectrometry.

3. Results, lessons learned

Twelve adult cynomolgus macaques negative for anti-flaviviruses and anti-measles antibodies were divided in two groups. One group was immunized with MVDVax on week 0, 12 and 23, and the other group was immunized with empty MV on week 23. The animals were followed for clinical symptoms, body weight, temperature, CBC, anti-DENV antibody binding and neutralizing titers in serum, and anti-DENV and MV specific T-cells in PBMC at several time points.

All animals seroconverted to DENV serotypes and MV after prime. The geometric mean titer increased after each boost and all animals had high titers against the four DENV serotypes and neutralizing antibodies after immunization completion.  No DENV T-cell response was observed along the immunizations while MV response was observed. However, T cell response to the NS antigen was observed in immunized animal after challenge, while control animals had no response.

Upon challenge with DENV1, infection was monitored in plasma by qRT-PCR (RNA viral load) and focus forming assay (infectivity). RNA viral load did not differ between groups aside of a one-day delay in vaccinated animals. However, infectious viremia at day-2 post challenge (peak viremia) revealed low to no infectious virus in vaccinated animals versus controls. If confirmed, this observation would indicate that MVDVax immunization could protect animals from being infectious.

Another important observation concerns the detection of T cell responses only in vaccinated animals after challenge. This reflects the recall of memory anamnestic T cell response, and the role of this response remains to be determined.

For the second objective we set up a sandwich ELISA assay to quantify the level of EDIII antigens in vaccine batches. This assay demonstrated that the amount of EDIII from the four DENV serotypes in non-GMP MVDVax batches is reproducible among batches. This assay can be used for further qualification of future GMP batches. Mass spectrometry was also used to demonstrate the presence of the different EDIII. This test demonstrated the presence of EDIII from DENV1 to 3 but not 4. This needs to be further explored as western blots and ELISA demonstrated the presence of EDIII from serotype 4 in the vaccine batches.