(24d) Nitration of a Foreign Antigen Via Genetic Code Expansion Increases In Vivo Immune Response

Some bacterial pathogens remain elusive to vaccinate against, often due to antigenic variation and an inability to direct immune responses towards highly conserved candidate antigens. As more bacterial pathogens grow resistant to 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. However, this has yet to be applied to nonself proteins. Here, we site-specifically incorporate pN-Phe within a foreign protein to increase its immunogenicity, using Shigella invasion plasmid antigen D (IpaD) as our model antigen.

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 pN-Phe residue at a surface site encoded by the UAG codon via amber 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. Immunization studies lasted 28 days, and groups of female 6-week old C57BL/6 mice (n = 5) were immunized on days 0 and 14 with 10 µg of antigen. In addition, an 18h in vivo study was completed to assess inflammatory response and antigen-presenting cell (APC) surface marker expression. After each study, mice were euthanized and serum and bronchoalveolar lavage fluid (BALF) were collected for ELISA analyses of antibody and cytokine responses. Lungs were collected from mice at the end of the 18h study, and cell suspensions were analyzed via flow cytometry. To gain more precise information regarding the effects of pN-Phe on APC phenotype and marker expression, we performed 24h in vitro experiments in which RAW264.7 cells were dosed with antigen at 15 µg/mL and analyzed via flow cytometry.

Indirect ELISA results show statistically significant (p < 0.05) increases in the following antibodies in mice immunized with nitrated Shigella antigen: IgG, IgA, IgG1, IgG2b, and IgG2c. Cytokine analyses of BALF samples show substantial increases in both B-cell activating factor (BAFF) and TNF-a for mice immunized with nitrated IpaD. Flow cytometry data from both in vivo and in vitro studies suggests that nitrated antigen increases expression of costimulatory molecules on APCs, which play a direct role in the activation and proliferation of T cells. These findings show that pN-Phe is an immunogenic amino acid that elevates humoral and cellular immune responses, and its application is not limited to self-proteins. Overall, this strategy may offer a transformative method for enhancing vaccine design and combatting antimicrobial resistance.