In the race to develop more effective and durable vaccines, scientists are exploring increasingly sophisticated molecular strategies. One of the most promising frontiers in this space involves the use of unnatural amino acids (UAAs)—engineered building blocks of proteins that go beyond nature’s standard 20 amino acids. By leveraging the unique chemistry of UAAs, researchers are now designing next-generation vaccines with improved stability, enhanced immunogenicity, and even built-in adjuvant properties.

In this third blog of the series “Beyond the 20”, we will explore how UAAs optimize antigen design and push the boundaries of vaccine development. For an overview of the fundamentals of UAAs and their potential in research, visit our previous blog.

Unlocking Vaccine Potential with UAAs

Traditional vaccines work by training the immune system to recognize and respond to specific antigens—typically a protein from a known pathogen. However, many antigens are unstable or poorly immunogenic on their own. UAAs offer a suite of tools to overcome these limitations:

Stabilizing antigens for better immune recognition - One of the critical challenges in vaccine design is keeping antigens stable in a form that mimics their structure on a live pathogen. UAAs can be strategically inserted into proteins to enhance their stability or lock them into a conformation that’s most recognizable by the immune system.

Improving antigen presentation to immune cells - UAAs can also be used to fine-tune how antigens are processed and presented by the immune system. By introducing specific chemical handles, researchers can promote the optimal uptake of antigens by antigen-presenting cells (APCs), enhancing T-cell activation and the overall adaptive response.

Designing self-adjuvanting vaccines - One of the most exciting applications of UAAs is the creation of self-adjuvanting vaccine constructs. Typically, vaccines require adjuvants—substances that enhance the immune response to an antigen. UAAs can be used to introduce immunostimulatory motifs directly into the antigen structure, eliminating the need for separate adjuvants.

For a more in-depth examination, we shall discuss some key examples of how UAAs are being investigated and applied in vaccine formulations.

Increasing Tumor-Associated Antigen Stability and Immunogenicity

The glycoprotein Mucin-1 (MUC1) is a tumour-associated antigen which, according to the US National Cancer Institute, should be a priority antigen in cancer vaccine development. In cancer cells ‘glycosylated MUC1 residues display altered O-glycan profiles’ (Gibadulin, 2025), to which cancer patients produce anti-MUC1 antibodies.

Several clinical trials of vaccine candidates using tumor-associated MUC1 have been unsuccessful because:

· MUC1 glycopeptides are self-antigens and so generate insufficient immune responses.

· MUC1 contains L-α-amino acid residues which are vulnerable to proteolytic degradation in biological fluids, thus lowering their bioavailability.

Generating artificial antigens containing UAAs like, β-amino acids, peptoids, oligourease, D-α-amino acids, and thioamides is a viable solution to improve immunogenicity and provide increased resistance against proteolysis.

Figure 1: Basic schematic of the use of MUC1 antigens with β replacements in cancer vaccines.

Gibadulin’s team synthesized ‘MUC1 glycopeptides with single or multiple α-to-β replacements’ and tested their ability to bind to a murine anti-MUC1 antibody, SM3. Antigens that bound SM3 strongly were tested as vaccine candidates by immobilization onto gold nanoparticles (AuNPs). The study found that multiple α-to-β replacements enhanced proteolytic stability of the glycopeptides and AuNP-MUC1 formulations show sufficient immunogenicity and induced cross-reactive antibodies against natural MUC1 on human cancer cells. Overall, the findings support the use of UAAs as a strategy for designing MUC1-based cancer vaccine candidates.

Incorporation of UAAs to Create Self-Adjuvanting Vaccines

A set of UAAs used to create self-adjuvating vaccines is CpG oligodeoxynucleotides (CpG ODNs). CpG ODNs are ‘oligodeoxynucleotides that have been modified by non-methylation based on cytosine-phosphate-guanine (CpG) dinucleotide sequences’ (Wang, 2024). As toll-like receptor 9 (TLR9) agonists, these compounds increase a host’s cellular and humoral responses to antigens and have been used to produce self-adjuvating human vaccines. One key example of a CpG-containing vaccine is HEPLISAV-B®, targeting the hepatitis B virus (HBV). The preceding hepatitis B vaccine was alum-adjuvanted, required three immunizations over 6 months, and 5-10% of immune-competent individuals failed to achieve long-lasting seroprotection (Shirota, 2013). As a next-generation vaccine, CpG-containing HEPLISAV-B®, shows improved immune responses against HBV. In phase III clinical trials it was able to produce greater seroprotective Ab titers with fewer immunizations compared to the previous vaccine.

UAAs in Bacterial Vaccines

Live attenuated vaccines are powerful because they mimic natural infections, eliciting strong and broad immune responses. However, achieving the right balance between safety and immunogenicity is difficult—too little attenuation risks virulence, while too much can reduce effectiveness.

One solution is the generation of live “suicide” vaccine candidates using UAA auxotrophy. The core of this approach is a conditional suicide system where bacteria can only survive if supplied with a synthetic UAA, not found in nature. By inserting a stop codon into antitoxin genes and enabling readthrough only in the presence of an UAA, vaccine strains die off naturally after administration,

ensuring safety (Figure 2).

Figure 2: In the presence of UAA, antitoxin genes are translated, enabling the survival of bacterial strains. In contrast, in the absence of UAA, antitoxin genes are not translated, preventing bacterial growth.

Key advantages of this system include:

· Speed and simplicity – No need for complex genome editing; vaccine strains are created via plasmid transformation.

· Tunable attenuation – Scientists can adjust survival duration and immunogenicity by choosing different toxin-antitoxin systems and UAA setups.

· Environmental safety – Because uAAs don’t exist naturally, the vaccine strains can’t survive outside controlled conditions, reducing risk of spread.

In one study (Pigula, 2024), scientists modified the essential DNA replication protein DnaN (β-sliding clamp) so it could only form functional dimers in the presence of the UAA, p-benzoyl-L-phenylalanine (BzF). The engineered E. coli strain, EV2.BzF.h5, showed extremely low escape rates and could only grow when BzF was added.

Building on this, the same approach could be applied to the human pathogen Pseudomonas aeruginosa—a major cause of hospital-acquired infections and the leading cause of death in cystic fibrosis patients. With rising antibiotic resistance, P. aeruginosa is an urgent target for vaccine development. The success of UAA-dependent live vaccine strains in E. coli suggests this strategy could be applied broadly, especially since DnaN is conserved in many bacterial species.   

Unnatural amino acids (UAAs) are redefining vaccine design by enhancing antigen stability, boosting immune responses, and improving safety. From self-adjuvanting cancer vaccines to escape-resistant live bacterial vaccines, UAAs offer precise control over vaccine function and longevity. As this technology evolves, UAAs are proving essential in the development of smarter, safer, and more effective vaccines.

Look out for the next blog of our UAA series "Beyond the 20” where we discuss how UAAs are enhancing molecular diagnostics.

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