Poster V4 - Nitration of a foreign antigen can elicit a stronger immune response

Introduction: Some bacterial pathogens remain elusive to vaccinate against, often due to antigenic variation between serotypes and an inability to direct immune responses towards highly conserved though weakly immunogenic candidate antigens. As more bacterial pathogens grow resistant to current antibiotic treatments, novel prophylactic measures are needed. Advancements in chemical biology and synthetic biology offer the potential to genetically encode non-standard amino acids (nsAAs) that possess functional groups that differ from the twenty standard amino acids. One such nsAA, para-nitro-L-phenylalanine (pN-Phe), was shown previously to terminate immune tolerance for self-proteins upon substitution of a single surface residue. Here, we explore whether site-specific incorporation of pN-Phe within a foreign protein could increase its immunogenicity, using an antigen from Shigella as a model.

Materials and

Methods: We performed heterologous expression of wild-type and nitrated antigens using recombinant E. coli. We engineered strains to produce the nitrated antigen with a single instance of pN-Phe at a surface site encoded by the UAG codon by co-expressing orthogonal translation machinery that achieved amber stop codon suppression. We then performed endotoxin removal and quantified endotoxin levels using a toll-like receptor 4 (TLR4) activation assay. We assessed immunogenicity via pulmonary delivery of nitrated antigen, wild-type antigen, or a PBS control to mice. Studies lasted 28 days, and groups of n = 5 C57BL/6 mice were immunized via orotracheal instillation on days 1 and 14 with 10 µg of antigen. On day 28, mice were euthanized and serum and bronchoalveolar lavage fluid (BALF) were collected for analysis. Indirect ELISA was used to characterize antibody response to antigens, and ELISA plates were coated with wild-type antigen at a concentration of 1 µg/mL. We used indirect ELISA to measure serum IgG and BALF IgA, which are representative of systemic and mucosal immune responses, respectively, as well as to analyze serum IgG subclass antibodies: IgG1, IgG2b, IgG2c, and IgG3.

Results, Conclusions, and Discussions: Indirect ELISA results using serum and BALF from our animal studies show statistically significant (p < 0.05) increases in the following antibodies in mice immunized with nitrated Shigella antigen relative to both wild-type antigen and PBS control: IgG, IgA, IgG1, IgG2b, and IgG2c. Our results indicate that the ability of pN-Phe to serve as an immunogenic amino acid that elevates humoral immune response is not limited to applications in self-proteins. Overall, this strategy may offer a transformative method for enhancing vaccine design and a strategy to combat antimicrobial resistance.

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