Self-amplifying mRNA vaccines elicit robust immune responses to H5N1 highly pathogenic avian influenza

The importance of having a vaccine against avian H5N1 influenza
The H5N1 highly pathogenic avian influenza virus (HPAIV) is currently circulating widely and has caused the death of millions of wild birds and domestic poultry. H5N1 can also infect humans with 893 reported human cases since 2003 and a fatality rate exceeding 52%, according to WHO. In addition to causing millions of bird deaths each year, interspecies transmission of this virus has recently been increasingly reported, including in cattle [1]. H5N1 infection in cows is rather mild with an impact on milk production and quality. However, the cow-to-cow transmission of H5N1 is a major concern. These recent evolutions further confirm the pandemic potential of HPAIV H5N1 and increased the need for an effective and easy to produce vaccine. A study performed by the EFSA Panel and the European Union Reference Laboratory for Avian Influenza, simulated the impact of a H5N1 vaccine in poultry in France, Italy and The Netherlands and predicted that emergency protective vaccination within a 3-Km radius around infected farms was the most effective solution to halt  H5N1 transmission [2]. In 2024, the European Centre for Disease Prevention and Control (ECDC) and the European Food Safety Authority are considering planning preventive measures, including vaccination of poultry and individuals occupationally exposed to avian influenza virus [3]. This highlights the urgent need of a vaccine to reduce the HPAIV circulation in poultry and other farm animals, thereby decreasing the risk of human infection and reassortments with human influenza [4]. However, influenza viruses represents a challenging target for vaccination, due to the rapid mutation of the hemagglutinin (HA) viral coat protein.
Advancements and scientific significance of this study 
Since 2008 the Laboratory of Gene Therapy led by professor Niek. N. Sanders at Ghent University, focuses on improving the delivery, purification, and expression of mRNA and self-amplifying mRNA (sa-mRNA) to enhance the efficacy of (sa-)mRNA vaccines and therapeutics to tackle infectious diseases, cancer or other diseases. Compared to conventional mRNA vaccines, sa-mRNA vaccines offer several advantages, including requiring lower dosages and achieving higher protein expression. Additionally, like mRNA vaccines, they benefit from a cell free and rapid production. As a quick update of antigens is essential for HPAIV vaccines, sa-mRNA is a promising candidate to meet the requirements of a flexible H5N1 vaccine.To find the most optimal sa-mRNA vaccine candidate for H5N1, we designed 3 different secreted antigens, all based on the HA protein of H5N1 clade 2.3.4, including secreted full-length HA, secreted head domain and secreted stalk domain. We found that the secreted full-length HA induced the highest antibody response, while the head domain induced the highest cellular response, and stalk domain induced the lowest antibody levels and no cellular immunity. Antibody responses were positively correlated with the dosages. Subsequently, we compared the best performing secreted full-length HA with the membrane anchor HA. Our results showed that the membrane anchored outperformed the secreted one, inducing stronger antibody responses and cellular responses. Collectively, the sa-mRNA vaccine encoding the membrane-anchored full-length HA emerged as the best candidate. Based on the hemagglutinin inhibition (HAI) titers in mice, this vaccine can potentially induce sufficient protective antibody levels after a single injection to protect production chickens throughout their lives. For the delivery of these sa-mRNA vaccines we used an in-housed developed lipid nanoparticle (LNP) consisting of commercially available lipids. To test the safety of this vaccine, the biodistribution of the sa-mRNA after intramuscular injection was assessed. The sa-mRNA predominantly remained in the injection site, but also massively migrated  to the draining lymph nodes and spleen. Small traces of sa-mRNA were also detected in other organs like e.g. the lungs. The detection and expression of the sa-mRNA in the spleen and lymph nodes point to a high delivery of the sa-RNA vaccine in lymphoid tissues and immune cells, which is beneficial for vaccination applications. Notably, mice did not have significant body weight loss or other signs of toxicity at all tested sa-mRNA dosages, supporting the good safety profile of our sa-mRNA vaccines.
Conclusion and future perspective 
Overall, our results highlight that the sa-mRNA vaccine encoding membrane-anchored HA is a potential candidate for preventing H5N1 HPAIV. Sa-mRNA vaccines can elicit the same immune response with a much lower dosage, providing an economic advantage. With Japan approving the first use of the sa-mRNA platform against COVID-19 in human [5,6], a sa-mRNA vaccine against HPAIV also has significant potential for application in human and domestic poultry. The sa-mRNA platform appears to be a viable alternative to conventional modified mRNA. 

1. Caserta, L. C. et al. Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle. Nature (2024). https://doi.org:10.1038/s41586-024-07849-4
2. EFSA Panel on Animal Health and Animal Welfare (AHAW) and European Union Reference Laboratory for Avian Influenza. Søren Saxmose Nielsen, S. S. et al. Vaccination of poultry against highly pathogenic avian influenza – part 1. Available vaccines and vaccination strategies. EFSA J 21, e08271 (2023). https://doi.org:10.2903/j.efsa.2023.8271
3. European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC). Melidou, A. et al. Drivers for a pandemic due to avian influenza and options for One Health mitigation measures. EFSA J 22, e8735 (2024). https://doi.org:10.2903/j.efsa.2024.8735
4. Editorial. What is the pandemic potential of avian influenza A(H5N1)? Lancet Infect Dis 24, 437 (2024). https://doi.org:10.1016/S1473-3099(24)00238-X
5. Wayne, C. J. & Blakney, A. K. Self-amplifying RNA COVID-19 vaccine. Cell 187, 1822-1822 e1821 (2024). https://doi.org:10.1016/j.cell.2024.03.018
6. Dolgin, E. Self-copying RNA vaccine wins first full approval: what’s next? Nature 624, 236-237 (2023). https://doi.org:10.1038/d41586-023-03859-w