Feline herpesvirus type 1 (FHV-1) is a member of the Alphaherpesvirinae subfamily, which includes important pathogens affecting humans and animals, such as herpes simplex virus types 1 and 2, pseudorabies virus, and Marek's disease virus. While herpesviruses show promise as vaccine vectors for delivering foreign antigens, ensuring their safety is a significant concern that must be addressed alongside their efficacy. In this study, we report the development of a replication-defective FHV-1 ΔgL-based vaccine candidate, which is propagated exclusively in a specially modified F81 cell line. Vaccination of cats with the FHV-1 ΔgL-based candidate demonstrated a favorable safety profile and elicited protective immunity against FHV-1 challenge. More importantly, the FHV-1 ΔgL platform exhibits excellent efficacy in delivering the VP2 antigen of the feline panleukopenia virus (FPV), inducing high-titer neutralizing antibodies in cats. Furthermore, we evaluated a trivalent vaccine candidate, rQD1 ΔgL/gI/gE-VP2-VP1, in which an expression cassette for the feline calicivirus (FCV) VP1 was inserted to replace the gI/gE genes. Vaccination of cats with rQD1 ΔgL/gI/gE-VP2-VP1 produced a strong protective effect against challenges from FHV-1, FPV, and FCV. To support clinical manufacturing, we generated a stable cell line through site-specific integration of the gL gene into the Rosa26 safe-harbor locus of F81 cells. In conclusion, the FHV-1 ΔgL platform demonstrates safety, efficacy, and promising potential for future applications and serves as an example for constructing attenuated vaccines in alphaherpesviruses.IMPORTANCEHerpesviruses are highly effective at presenting foreign antigens and serve as excellent viral vectors, yet their safety warrants careful attention. We developed a replication-defective feline herpesvirus type 1 (FHV-1) ΔgL vector platform. Using this platform, a bivalent vaccine (rQD1 ΔgL-VP2) and a trivalent vaccine (rQD1 ΔgL/gI/gE-VP2-VP1) were constructed. Both vaccines demonstrated robust immunoprotection and favorable safety profiles in immunized cats. Furthermore, to facilitate the application of this platform, a stable cell line with site-specific integration, F81-gL-Rosa26, was generated by inserting the gL gene into the Rosa26 safe-harbor locus of F81 cells. The strategy of the FHV-1 ΔgL platform also provides a reference for other members of the herpesvirus family.

Keywords: FCV; FHV-1; FPV; herpesvirus; neutralizing antibody; vaccine safety.