Chitosan nanoparticle based ΔgE IBRV Vaccine: A Novel DIVA compatible strategy for enhanced immunity in guinea pigs

Infectious Bovine Rhinotracheitis (IBR), caused by Bovine Herpesvirus-1, is a significant disease in cattle that results in substantial economic losses worldwide. Vaccination remains the primary control strategy, with inactivated vaccines offering a safer alternative to live-attenuated counterparts. However, their reduced immunogenicity necessitates the use of adjuvants to enhance protective immune responses. Conventional adjuvants, such as aluminium salts and oil emulsions, are constrained by their Th2-biased immune stimulation, limited ability to elicit strong cellular or mucosal immunity, absence of controlled antigen release, and association with local and systemic reactogenicity. In some instances, they have also been linked to rare adverse events, further underscoring the need for safer and more versatile adjuvant platforms. This Study evaluated chitosan nanoparticles (CNPs) as an adjuvant-delivery system for an inactivated glycoprotein E-deleted (ΔgE) IBR virus to achieve sustained antigen release, robust immune activation, and Differentiating Infected from Vaccinated Animals (DIVA) capability. CNPs, prepared by ionic gelation (Chitosan: Sodium Tripolyphosphate 2:1), exhibited a mean size of 410 nm, +15 mV zeta potential, PDI < 0.5, and > 95% encapsulation efficiency. Release kinetics showed a biphasic profile with an initial burst followed by sustained release. In guinea pigs, intramuscular vaccination with CNP- ΔgE IBRV resulted in mean virus neutralization titers of 1:64 on day 30 and 1:256 on day 60, accompanied by elevated expression of IL-2, IL-4, IL-10, and IFN-γ, significant PBMC proliferation, and gB antibody positivity, with the absence of gE antibodies, confirming DIVA compatibility. Intranasal delivery produced moderate responses. The Study also successfully demonstrated the encapsulation of live ΔgE IBR virus during formulation optimization, validating the platform's applicability for both live and inactivated antigens. These findings indicate that CNP-based vaccines can overcome the limitations of traditional adjuvants, combining high antigen loading, biocompatibility, and prolonged release to elicit potent and durable immune responses.