Strong evidence exists for applying signal peptide engineering to design more efficient vaccines for various infectious viral diseases, including Ebola virus, SARS-Cov-2, and Zika virus.

Signal peptides: Enablers for enhanced immune protection

For any therapeutic protein that needs to be secreted or displayed on the cell surface, the function of a signal peptide is indispensable. The short, N-terminal amino acid peptide sequences direct nascent proteins to the endoplasmic reticulum, which is the starting point of the protein secretion pathway and an essential cellular site for the production of nearly all protein-based therapies. Without an appropriate signal peptide, the nascent protein will remain in the cytosol, which prevents secretion and folding of the protein.

In the design of mRNA therapeutics, the choice of the signal peptide sequence in the protein coding region can dramatically enhance antigen expression and enable broad immune protection.

The following studies provide concrete examples of the importance of signal peptide choice:

COVID-19. McCallum (2022) studied the capability of SARS-Cov-2 Epsilon variant to bypass immune defenses. The group uncovered a new mechanism: signal peptide modifications in the spike glycoprotein (1). Also Moet al. (2024) studied the spike (S) protein of SARS-CoV-2, the main target of mRNA vaccine design, and found that choosing the correct signal peptide sequence was essential to induce effective antibody responses (2).

Zika virus. Several studies, including Richner et al. (2017) and Bollman et al. (2023), have found that Zika virus (ZIKV) RNA vaccines encoding the viral premembrane-envelope (prM-E) glycoprotein, in combination with a signal peptide from Japanese encephalitis virus (JEV), improved vaccine efficacy at lower doses (3-4). This work forms the basis of a discovery patent by Moderna.

Ebola virus. Building on the work of Richner et al. on the Zika virus, a study by Meyer et al. (2018) created two mRNA vaccines based on the EBOV envelope glycoprotein that differed by signal peptides for improved glycoprotein post-translational translocation. The study discovered that an engineered version elicited significantly stronger immune responses (5). Despite challenges in producing an mRNA vaccine for Ebola, the study contributed much-needed evidence to support future preclinical development.

Producing more stable, long-lasting proteins from mRNA therapies

By altering signal peptides in different mRNA constructs, Cheng et al. (2023) demonstrated efficacy against psoriasiform dermatitis and melanoma. With a platform for in situ production of therapeutic proteins in the bloodstream, they enabled the expression of proteins that are typically restricted to the intracellular space, effectively optimizing their secretion and systemic availability. This approach could be used for improving drug stability, reducing dosing frequency, and boosting therapeutic effectiveness (6).

Because signal peptides are important modulators of protein folding, optimizing signal peptides in mRNA design can prevent misfolding or aggregation, reduce the dwell time of the nascent chain in the cytosol, and reduce its degradation by proteases.

Due to these mechanisms, secreted proteins produced from mRNA can have enhanced stability in the body.

The next wave of progress

Large-scale signal peptide screening is reshaping what’s possible in mRNA therapeutics: At long last, signal peptide optimization is evolving from a trial-and-error endeavor into a systematic high throughput strategy.

The volume of screening is particularly important, because signal peptides are highly variable in sequence, lack clear consensus motifs, and are specific in promoting expression of different proteins of interest. Hundreds of thousands of signal peptide sequences exist in nature, and while one signal peptide might boost expression for one protein, it might suppress it for a different one (7).

This requires high volume screening to discover optimal matching signal peptides for different individual target proteins.

> Get to know our technology for simultaneous screening of 5,500 signal peptides for your protein of interest. Ask us to contact you.

Sources:

1. Matthew McCallum et al. SARS-CoV-2 immune evasion by the B.1.427/B.1.429 variant of concern. Science 373,648-654(2021). Doi:10.1126/science.abi7994
2. Mo C, et al. SARS-CoV-2 mRNA vaccine requires signal peptide to induce antibody responses. Vaccine. 2023. Doi:10.1016/j.vaccine.2023.09.059
3. Richner, et al. Modified mRNA Vaccines Protect against Zika Virus Infection, Cell, Volume 168, Issue 6, 2017. Doi: 10.1016/j.cell.2017.02.017
4. Bollman, B., Nunna, N., Bahl, K. et al. An optimized messenger RNA vaccine candidate protects non-human primates from Zika virus infection. npj Vaccines 8, 58 (2023). Doi: 10.1038/s41541-023-00656-4
5. Meyer et al. Modified mRNA-Based Vaccines Elicit Robust Immune Responses and Protect Guinea Pigs From Ebola Virus Disease, The Journal of Infectious Diseases, 2018. Doi: 10.1093/infdis/jix592
6. Cheng et al. In situ production and secretion of proteins endow therapeutic benefit against psoriasiform dermatitis and melanoma, Proc. Natl. Acad. Sci. 2023. Doi: 10.1073/pnas.2313009120
7. Wilkinson et al. A new mRNA structure prediction-based approach to identifying improved signal peptides for bone morphogenetic protein 2. BMC Biotechnol. 2024. Doi: 10.1186/s12896-024-00858-1  

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Image credit: Alex Shuper