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Development of a Next-Generation, Dual-Target Rabies/Flavivirus Infectious DNA (iDNA) Vaccine

Periodic Reporting for period 2 - RABYD-VAX (Development of a Next-Generation, Dual-Target Rabies/Flavivirus Infectious DNA (iDNA) Vaccine)

Reporting period: 2018-07-01 to 2019-12-31

Rabies is a devastating and massively neglected disease, for which the mortality rate and burden per capita falls disproportionately upon the poorest regions of the world. Rabies is a viral disease that is largely transmitted to humans via a bite by infected animals. Once the first clinical symptoms (such as hydrophobia) have developed, the disease is uniformly lethal, and patients, if not put into an artificial coma, die in great agony. Worldwide, rabies causes approximately 59.000 deaths annually, from which the majority are young children. A vaccine exists but is associated with several drawbacks such as the need for a cold chain (kept at cool temperature), high costs, multiple dosing regimen with concomitant visits to health care facilities etc., due to which the coverage remains unacceptably low.

The yellow fever virus (YFV), a mosquito-borne flavivirus, causes severe and life-threatening infections with jaundice, systemic bleeding, shock and multi-organ failure. An estimated 900 million people living in 45 endemic countries of Africa and Latin America are at high risk of infection. Although safe and highly efficient live-attenuated prophylactic vaccines [YFV-17D, Stamaril® and YF-Vax®] are available, an estimated 200,000 cases of yellow fever still occur annually, resulting in ~30,000 deaths (www.who.int) because of inadequate supplies, the need for trained staff for administration and a cold chain.

The Japanese encephalitis virus (JEV), also a mosquito-borne flavivirus, causes viral encephalitis in many countries of Asia, with an estimated 68,000 clinical cases every year. The case-fatality rate among the patients that develop encephalitis is up to 30%, permanent neurologic or psychiatric sequelae is reported in 30-50%. As for rabies, it is primarily a children’s disease. More than 3 billion people are at risk of infection.

During the course of the RABYD-VAX project, five European research institutes are combining their expertise to develop and validate a new vaccine that protects against both rabies and yellow fever/Japanese encephalitis virus. This vaccine will be based on a novel proprietary vaccine technology (i.e. PLLAV, plasmid-launched live-attenuated viral vaccine platform), which has been developed by the team of the project coordinator (KU Leuven) and which has several advantages over the currently used commercial vaccines. Ultimately, this vaccine could be given as a prophylactic childhood vaccine after incorporation in the standard childhood vaccination schedule of endemic regions and this, side-by-side, with domestic and wildlife animal vaccination programs to eradicate rabies. At the end of the project, this vaccine candidate should be ready to enter the non-clinical development phase.
The RABYD-VAX project started in January 2017, during the last 3 years, the following has been achieved: (i) Several cloning efforts led to the development of one prefered construct (cfr. stability and immune profile) expressing the rabies antigen from the yellow fever virus (YFV) backbone. A dual and specific anti-YFV and anti-RABV humoral immune response could be demonstrated. (ii) Moreover, three PLLAV vaccine constructs that carry the G proteins from more divergent (other than phylogroup I) lyssaviruses were constructed. (iii) An optimized rabies/JEV PLLAV vaccine construct was generated. A potent immune response against JEV, RABV and YFV could be observed for the cell-culture derived viral vaccine (LAV). (iv) Likewise, an optimized rabies/ZIKV PLLAV vaccine construct was generated. A potent immune response against ZIKV, RABV and YFV could be observed for the cell-culture derived viral vaccine (LAV). (v) Several MHC class I and II epitopes of the RabG-protein, against which cellular immune responses are induced in mice, were identified. (vi) Evidence was obtained for successful immunization in the presence of pre-existing vector immunity. (vii) The superior thermal stability of PLLAV vaccines has been confirmed by head-to-head comparison with the licensed YFV vaccine Stamaril®. (viii) The production process of PLLAV in fermenters has been further optimized to be able to generate standardized batches. (ix) The PLLAV-based vaccines show no neurovirulence in mice as compared to the commercial vaccine Stamaril ®. (x) Moreover, the viral particles derived from PLLAV-YF/RabG are not pseudotyped with RabG protein. (xi) Proof-of-Concept has been reached for the protective effect of PLLAV cell-culture derived YF/RabG virus against lethal challenge with either RABV or YFV, all animals survived lethal challenge after vaccination. (xii) A literature study, has been concluded, summarizing the regulatory guidelines which may apply to this novel platform technology; it sums up approved RABV, YFV and DNA vaccines; and outlines a nonclinical development plan. (xiii) A target product profile has been concluded. This document defines the characteristics of the RABV-YF vaccine and how the product will be used by the end user. (xiv) Scientific Technical Advice was sought at two regulatory authorities, FAMHP and PEI. (xv) Several dissemination activities have been established, i.e. a project website, press releases, 8 publications, IP, a MOOC on vaccines and a webinar on rabies. Our data have been presented at a number of international meetings.
The team of the project coordinator developed a novel vaccine platform technology, named PLLAV,[patent WO/2014/174078, inventors K. Dallmeier & J. Neyts]. In essence, the technology consists of a proprietary Bacterial Artificial Chromosome shuttle vector in which the genome of the YFV vaccine YFV-17D, which is a live-attenuated vaccine (LAV), has been cloned. Once this recombinant DNA plasmid is introduced in an eukaryotic cell, the LAV is expressed and immunity triggered. Hence, this approach combines, (i) the high efficacy of LAVs for inducing protective immunity and (ii) the physical and genetic stability of classical DNA vaccines, bypassing the manufacturing problems and need for a cold chain. Moreover, this approach allows to conveniently design and rapidly produce vaccines against other pathogens (i.e. rabies) as well. The PLLAV technology presents key advantages over the currently available vaccines, i.e. (i) it can be produced at high quantities in fermenters without the need for cell cultures or embryonated chicken eggs; (ii) it’s genetically stable; (iii) no cold-chain is needed and (iv) it can be administered needle-free.
In fact, the PLLAV technology addresses the need for good vaccines for the developing world as articulated by MSF “developing vaccines that are better adapted to reach children in remote or unstable locations – vaccines that do not require refrigeration, do not require needles, and that can be given in fewer doses […], paying enough attention to getting vaccine prices down”.
Current rabies vaccines need multiple dosing in order to induce virus neutralising antibodies that confer protection in individuals following exposure to RABV. In developing countries, where both YF and rabies cases are endemic, vaccination is often unaffordable. The results from this study have supported the feasibility of making a dual YF/RABV vaccine and the effect of the vaccine in protecting mice from lethal rabies challenge after one dose of vaccination. The new vaccine has the potential to make vaccination more accessible and easier to administer in resource-limited countries.
Logo of the RABYD-VAX project