Development of an Enzyme-Mediated, Site-Specific Method to Conjugate Toll-Like Receptor 2 Agonists onto Protein Antigens: Toward a Broadly Protective, Four Component, Group A Streptococcal Self-Adjuvanting Lipoprotein-Fusion Combination Vaccine.

Subunit vaccines composed of protein antigens covalently attached to Toll-like receptor (TLR) agonists elicit superior immune responses compared to mixtures of antigens and TLR agonists. Among different conjugation approaches, enzyme-mediated ligation is one of the few that provides an opportunity for the generation of homogeneous, molecularly defined products in which protein antigens are maintained with native structures, which is most critical to elicit protective immune responses upon vaccination. Four highly conserved protein antigens from Group A (GAS) have the potential to be safe and efficacious vaccine candidates. After a TLR2 agonist fibroblast-stimulating lipopeptide-1 (FSL-1) was successfully attached onto each antigen using sortase A and techniques for their purification were developed, a combination vaccine containing interleukin 8 (IL-8) protease ( cell envelope proteinase [SpyCEP]), Group A Streptococcal C5a peptidase (SCPA), anchorless virulence factor arginine deiminase (ADI), and trigger factor (TF)-TLR2 conjugates was produced. This combination was assessed for immunity in mice and compared with mixtures of the four antigens with FSL-1 or alum. High titer antigen-specific IgG antibodies were detected from all vaccine groups, with antibodies elicited from FSL-1 conjugates around 10-fold higher compared to the FSL-1 mixture group. Furthermore, the FSL-1 conjugates afforded a more balanced T1/T2 immune response than the alum-adjuvanted group, suggesting that this combination vaccine represents a promising candidate for the prevention of GAS diseases. Thus, we established a conjugation platform that allows for the production of defined, site-specific antigen-adjuvant conjugates, which maintain the native three-dimensional structure of antigens and can be potentially applied to a variety of protein antigens.