A novel fiber-2-edited live attenuated vaccine candidate against the highly pathogenic serotype 4 fowl adenovirus

Abstract

Recently, the outbreaks of hydropericardium-hepatitis syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry globally. Although several inactivated or subunit vaccines have been developed against FAdV-4, live-attenuated vaccines for FAdV-4 are rarely reported. In this study, a recombinant virus FA4-EGFP expressing EGFP-Fiber-2 fusion protein was generated by the CRISPR/Cas9 technique. Although FA4-EGFP shows slightly lower replication ability than the wild type (WT) FAdV-4, FA4-EGFP was significantly attenuated in vivo compared with the WT FAdV-4. Chickens infected with FA4-EGFP did not show any clinical signs, and all survived to 14 day post-infection (dpi), whereas those infected with FAdV-4 showed severe clinical signs with HHS and all died at 4 dpi. Besides, the inoculation of FA4-EGFP in chickens provided efficient protection against lethal challenge with FAdV-4. Compared with an inactivated vaccine, FA4-EGFP induced neutralizing antibodies with higher titers earlier. All these data not only provide a live-attenuated vaccine candidate against the highly pathogenic FAdV-4 but also give a potential insertion site for developing FAdV-4-based vaccine vectors for delivering foreign antigens.

Immunogenicity of Novel Live Vaccine Based on an Artificial rHN20 Strain against Emerging Fowl Adenovirus 4

“…Antigen-specific neutralizing antibodies in the serum are important indicators of the efficacy of most virus vaccines. The FAdV-4 inactivated vaccine, subunit protein vaccine, and recombinant virus-vectored subunit vaccine induce neutralization activity in the serum [18,19]. Here, all chickens immunized with rHN20 via different routes or with different doses produced robust neutralizing antibodies against FAdV-4.…”

“…Since the outbreak of HHS caused by novel FAdV-4, in 2015, several candidate vaccines have been reported [19]. However, few live attenuated vaccines based on the emerging novel FAdV-4 have been evaluated [17,18]. We previously generated a non-pathogenic chimeric virus rHN20 by replacing the hexon gene of the FAdV-4 virulent strain HLJFAd15 with that of the non-pathogenic strain ON1 [20].…”

“…Live attenuated FAdV-4 have been produced by passaging virulent FAdV-4 in chicken embryos [14] or QT35 cells [15,16], but the protective effect of these attenuated vaccines on the emerging FAdV-4 is also unknown. Recent studies revealed that recombinant viruses generated from novel genotype FAdV-4 strain in which enhanced green fluorescent protein (EGFP) was fused to the N-terminus of fiber-2 gene or in which the N-terminus of fiber-2 was knocked out was not pathogenic to specific pathogen-free (SPF) chickens, but the titers of the recombinant viruses were significantly lower in vitro than those of wild-type FAdV-4 [17,18].…”

A Novel Fiber-1-Edited and Highly Attenuated Recombinant Serotype 4 Fowl Adenovirus Confers Efficient Protection Against Lethal Challenge

“…Recently, we used CRISPR-Cas9 technique to target the virulent gene fiber-2 to generate two live-attenuated fiber-2 -edited vaccine candidates FA4-EGFP and FAV-4_Del ( 7 , 8 ). In this study, we targeted another structural protein Fiber-1 of FAdV-4 to rescue a novel recombinant virus FAdV4-RFP_F1 expressing the fusion protein of RFP and Fiber-1.…”

“…Different from most serotypes of FAdV, FAdV-4 has two Fiber proteins, designated as Fiber-1 and Fiber-2 (5). Recently, Fiber-2 was identified to be closely related with the virulence of FAdV-4, and the fiber-2-edited recombinant viruses FA4-EGFP and FAV-4_Del were highly attenuated to the SPF chickens (6)(7)(8). Notably, Fiber-1, but not Fiber-2, was identified to directly trigger viral infection via its knob and shaft domain through the cellular receptor, the CAR homology (9)(10)(11).…”