# Influence of the administration route and dose on the expression and antibody responses of a reporter and avian influenza self-amplifying mRNA vaccine in poultry

## TL;DR

This study shows that self-amplifying RNA vaccines can effectively induce immune responses in poultry when administered via specific routes and doses.

## Contribution

The study demonstrates the effectiveness of self-amplifying RNA vaccines in poultry using different administration routes and doses.

## Key findings

- High luciferase expression and anti-luciferase antibodies were observed after intramuscular, subcutaneous, and in ovo administration.
- A single injection of saRNA encoding H5N1 avian influenza hemagglutinin head-domain induced protective immune responses lasting six weeks.
- Boosting the vaccine increased hemagglutinin inhibition titers four-fold.

## Abstract

Vaccination is routinely used in industrial poultry to control infectious diseases. Vaccines based on mRNA and self-amplifying RNA (saRNA) are approved for human use, but research on their application in poultry is limited. In this study the saRNA vaccine platform is evaluated in poultry. First, a luciferase-encoding saRNA (luc-saRNA) was tested as a model vaccine across different administration routes and doses in broilers. High luciferase expression, and anti-luciferase antibodies were observed after intramuscular (IM), subcutaneous (SC), and in ovo (IO) administration. After a second Luc-saRNA injection, seroconversion rates and antibody titers increased in the IM and SC group to almost 100%. Higher doses of Luc-saRNA increased luciferase production. However, they did not linearly increase antibody production, as all tested doses (0.20–5.0 µg) elicited an equipotent immune response. A vaccination experiment with saRNA encoding the hemagglutinin head-domain (HA-HD) of H5N1 avian influenza showed hemagglutinin inhibition (HI) titers that are indicative for protection after a single injection and these titers remained above the protective threshold during 6 weeks without boosting. When boosted, the HI titers increased four-fold. This study confirms effective protein translation and immune response induction in chickens with IM or SC administered saRNA-LNPs, even at the lowest dose of 0.20 µg.