Food-Grade Saponin Extract as an Emulsifier and Immunostimulant in Emulsion-Based Subunit Vaccine for Pigs

Abstract

Subunit vaccines consisting of highly purified antigens require the presence of adjuvants to create effective and long-lasting protective immunity. Advances on adjuvant research include designing combination adjuvants which incorporate two or more adjuvants to enhance vaccine efficacy. Previously, an oil-in-water emulsion adjuvant (OW-14) composed of mineral oil and an inexpensive gum Arabic emulsifier has been reported demonstrating enhanced and robust immune responses when used as an adjuvant in swine subunit vaccines. This study presents a modified version of OW-14 prepared with food-grade Quillaja saponin extract (OWq). In new OWq emulsion, saponin extract served as an emulsifier for stabilization of emulsion droplets and as an immunoactive compound. The use of saponins allowed to reduce the required amount of emulsifier in the original OW-14. However, emulsion stabilized with saponins demonstrated extended physical stability even at elevated temperature (37°C). The two-dose vaccination with a classical swine fever virus (CSFV) glycoprotein E2-based vaccine formulated with OWq produced higher levels of E2-specific IgG and virus neutralizing antibodies in pigs in contrast with animals that received the vaccine adjuvanted with oil only. In addition, new OWq adjuvant was safe to use in the vaccination of pigs.

Figure 1

General structure of the saponin molecule reveals the presence of hydrophobic and hydrophilic regions responsible for surface activity of the molecule, while carbohydrate regions, acyl domain, and aldehyde group on triterpene domain demonstrate adjuvant properties. The structure representation is adapted from Yang et al. [14].

Figure 2

Size distribution of emulsion-based adjuvants obtained with dynamic light scattering (DLS). (a) Freshly prepared OWq emulsion had droplets within 90–600 nm size range, while oil-based adjuvant (OBA) mixed with PBS produced emulsion with droplets between 100 and 3000 nm. (b) After 180-day storage at different temperatures, the slight shift of size distributions towards bigger droplets was detected in OWq emulsion; however, no creaming or phase separation was observed in visual appearance of emulsion samples (photograph insertion).

Figure 3

Safety and immunological effect of OWq and OBA injected with E2 antigenic protein. (a) E2 subunit vaccine administered with experimental adjuvants did not impact weight gain in pigs as both one-dose and two-dose animals had grown consistently and at the same level as negative control pigs (PBS only). (b) E2 protein coadministered with OWq adjuvant demonstrated higher E2-specific IgG antibody titer detected on days 21 and 28 in serum samples (dilution 1/5000) of two-dose pigs than subunit vaccine with OBA. (c) Two-dose pigs developed significantly higher IgG titer than animals receiving single vaccination on day 21 of the study (dilution 1/5000). (d) IgG1 and IgG2 titers in serum samples (dilution 1/1000) collected from two-dose pigs on day 21 of the study. Data are presented as a mean ± SEM.