Infectious clone of a contemporary Tembusu virus and replicons expressing reporter genes or heterologous antigens from poultry viruses.

The novel mosquito-borne Tembusu virus (TMUV, family Flaviviridae) was discovered as the cause of a severe outbreak of egg-drop syndrome affecting ducks in Southeast Asia in 2010. TMUV infection can also lead to high mortality in various additional avian species such as geese, pigeons, and chickens. This study describes the construction of an infectious cDNA clone of a contemporary duck-isolate (TMUV WU2016). The virus recovered after transfection of BHK-21 cells shows enhanced virus replication compared to the mosquito-derived MM1775 strain. Next, the WU2016 cDNA clone was modified to create a SP6 promoter-driven, self-amplifying mRNA (replicon) capable of expressing a range of different reporter genes (Renilla luciferase, mScarlet, mCherry, and GFP) and viral (glyco)proteins of avian influenza virus (AIV; family Orthomyxoviridae), infectious bursal disease virus (IDBV; family Bunyaviridae) and infectious bronchitis virus (IBV; family Coronaviridae). The current study demonstrates the flexibility of the TMUV replicon system, to produce different heterologous proteins over an extended period of time and its potential use as a platform technology for novel poultry vaccines.

Evaluation of bird-adapted self-amplifying mRNA vaccine formulations in chickens.

Each year, millions of poultry succumb to highly pathogenic avian influenza A virus (AIV) and infectious bursal disease virus (IBDV) infections. Conventional vaccines based on inactivated or live-attenuated viruses are useful tools for disease prevention and control, yet, they often fall short in terms of safety, efficacy, and development times. Therefore, versatile vaccine platforms are crucial to protect poultry from emerging viral pathogens. Self-amplifying (replicon) RNA vaccines offer a well-defined and scalable option for the protection of both animals and humans. The best-studied replicon platform, based on the Venezuelan equine encephalitis virus (VEEV; family Togaviridae) TC-83 vaccine strain, however, displays limited efficacy in poultry, warranting the exploration of alternative, avian-adapted, replicon platforms. In this study, we engineered two Tembusu virus (TMUV; family Flaviviridae) replicons encoding varying capsid gene lengths and compared these to the benchmark VEEV replicon in vitro. The TMUV replicon system exhibited a robust and prolonged transgene expression compared to the VEEV replicon system in both avian and mammalian cells. Moreover, the TMUV replicon induced a lesser cytopathic effect compared to the VEEV replicon RNA in vitro. DNA-launched versions of the TMUV and VEEV replicons (DREP) were also developed. The replicons successfully expressed the AIV haemagglutinin (HA) glycoproteins and the IBDV capsid protein (pVP2). To assess the immune responses elicited by the TMUV replicon system in chickens, a prime-boost vaccination trial was conducted using lipid nanoparticle (LNP)-formulated replicon RNA and DREP encoding the viral (glyco)proteins of AIV or IBDV. Both TMUV and VEEV replicon RNAs were unable to induce a humoral response against AIV. However, TMUV replicon RNA induced IBDV-specific seroconversion in vaccinated chickens, in contrast to VEEV replicon RNA, which showed no significant humoral response. In both AIV and IBDV immunization studies, VEEV DREP generated the highest (neutralizing) antibody responses, which underscores the potential for self-amplifying mRNA vaccine technology to combat emerging poultry diseases.

Rise of the RNA machines - self-amplification in mRNA vaccine design.

mRNA vaccines have won the race for early COVID-19 vaccine approval, yet improvements are necessary to retain this leading role in combating infectious diseases. A next generation of self-amplifying mRNAs, also known as replicons, form an ideal vaccine platform. Replicons induce potent humoral and cellular responses with few adverse effects upon a minimal, single-dose immunization. Delivery of replicons is achieved with virus-like replicon particles (VRPs), or in nonviral vehicles such as liposomes or lipid nanoparticles. Here, we discuss innovative advances, including multivalent, mucosal, and therapeutic replicon vaccines, and highlight novelties in replicon design. As soon as essential safety evaluations have been resolved, this promising vaccine concept can transform into a widely applied clinical platform technology taking center stage in pandemic preparedness.