# Antigenic Matching of rHVT-H5 via CRISPR/Cas9 Confers Complete Protection Against Novel H5N1 Clade 2.3.4.4b in Chicken

## TL;DR

A new CRISPR-based vaccine for H5N1 bird flu in chickens offers full protection and reduces virus spread, helping control outbreaks more effectively.

## Contribution

A CRISPR/Cas9-based platform for rapidly developing antigenically matched rHVT-H5 vaccines against emerging H5N1 variants.

## Key findings

- The rHVT-H5 vaccine provided 100% protection against lethal H5N1 infection in chickens.
- The vaccine significantly reduced oropharyngeal and completely inhibited cloacal viral shedding.
- The vaccine induced robust hemagglutination inhibition antibody titers in SPF chickens.

## Abstract

The rapid and continuous evolution of clade 2.3.4.4b H5N1 highly pathogenic avian influenza (HPAI) poses a severe threat to the global poultry industry and public health. While vaccination is a key control strategy, traditional vaccine development often lags behind the rapid antigenic changes in the virus. In this study, we utilized an advanced CRISPR/Cas9 gene-editing platform to rapidly develop a new recombinant turkey herpesvirus (rHVT-H5) vaccine that is precisely matched to the currently circulating 2.3.4.4b strains. Our results demonstrate that this vaccine provides 100% protection against lethal infection in chickens and significantly suppresses the shedding of the virus into the environment. These findings are highly significant as they offer a rapid-response solution to poultry disease management, providing a technological platform for quickly updating vaccines to halt the spread of emerging viral variants and safeguard food security.

The widespread panzootic of clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 necessitates the development of vaccine platforms capable of rapid adaptation to emerging antigenic variants. Although commercial recombinant turkey herpesvirus (rHVT) vaccines are available, they often utilize heterologous inserts that may fail to optimally limit viral shedding of novel field strains. Here, we report the rapid construction of a homologous rHVT-H5 vaccine expressing the hemagglutinin (HA) gene of a representative clade 2.3.4.4b isolate via CRISPR/Cas9-mediated non-homologous end joining (NHEJ). In vitro characterization confirmed stable HA surface expression and growth kinetics comparable to the parental virus. In specific-pathogen-free (SPF) chickens, rHVT-H5 elicited robust hemagglutination inhibition (HI) antibody titers. Following lethal challenge with a homologous clade 2.3.4.4b H5N1 virus, the vaccine conferred 100% protection against mortality and clinical signs while significantly reduced oropharyngeal sheddings and completely inhibited viral shedding in cloacal samples. These findings demonstrate that an antigenically matched rHVT-H5 constitutes a promising strategy for mitigating the ongoing global threat posed by clade 2.3.4.4b HPAI H5N1.