Innate Inhibiting Proteins Enhance Expression and Immunogenicity of Self-Amplifying RNA
Open accessAbstract
Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting, as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. In this study, we screened a library of saRNA constructs with cis -encoded innate inhibiting proteins (IIPs) and determined the effect on protein expression and immunogenicity. We observed that the PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a proteins enhance protein expression 100- to 500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo , and that ruxolitinib, a potent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo . Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the rabies virus ( RABV )-specific immunoglobulin G (IgG) titer and neutralization half-maximal inhibitory concentration (IC 50 ) by ∼10-fold in rabbits, but not in mice or rats. These experiments provide a proof of concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.
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Previous efforts to decrease immunogenicity of saRNA focused on sequence evolution, co-expression of viral inhibitory proteins and optimization of the delivery vehicle.
“Previous efforts to decrease the immunogenicity of saRNA focused on sequence evolution, co-expression of viral inhibitory proteins and optimization of the delivery vehicle16–18.”
From Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency (2025) · cites this paper as [17]
The Introduction describes viral innate-inhibiting proteins as a strategy to dampen the interferon response to RNA, supporting that part of the claim. It does not describe sequence evolution or delivery-vehicle optimization as efforts to decrease saRNA immunogenicity. The Abstract and Discussion instead report that the paper’s inhibitory-protein approach enhanced vaccine immunogenicity in rabbits. (LLM, conf=0.86)
Experimental — accuracy not yet measuredIncorporation of encoded modulators of human PRR or a wider range of potential modifications may positively affect immunogenicity and efficacy of saRNA vaccines.
“Thus, incorporation of encoded modulators of human PRR or of a wider range of potential modifications may positively affect immunogenicity and efficacy of saRNA vaccines.443,444”
From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [444]
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“To ensure that innate inhibiting proteins co-localize with the therapeutic protein or antigen, the genes can be encoded within the same transcript, although in the case of saRNAs, this will increase manufacturing complications (47).”
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- Authors
- Blakney, A. K., McKay, P. F., Bouton, C. R., Hu, K., Samnuan, K., & Shattock, R. J.
- Affiliations
- Imperial College London (GB)
- Corresponding author
- Blakney, Anna K.
- Shattock, Robin J.
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2021-03
- Volume
- 29
- Issue
- 3
- Pages
- 1174-1185
- ISSN
- 1525-0016 (ISSN-L)
- PMC
- PMC7935664
- OpenAlex
- W3115814127
- Cited by
- 105
- References
- 60
- Citations by year
- 2026: 23 · 2025: 22 · 2024: 23 · 2023: 15 · 2022: 16 · 2021: 6
- FWCI
- 3.00
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Spiral (Imperial College London) (submittedVersion, cc-by-nc-nd)
- Funders
- H2020 Marie Skłodowska-Curie Actions
- Engineering and Physical Sciences Research Council
- Whitaker International Fellows and Scholars Program
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
- Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
- Keywords
- self-amplifying RNA, rabies virus, interferon, protein synthesis inhibition, ruxolitinib, JAK/STAT inhibition, rabies virus neutralizing antibodies
- MeSH terms
- Immunogenicity, Vaccine, mRNA Vaccines, Animals, Cell Line, Encephalitis Virus, Venezuelan Equine, Fibroblasts, Gene Expression Regulation, HeLa Cells, Humans, Immunoglobulin G, Immunity, Innate, Nitriles, Pyrazoles, Pyrimidines, Rabbits, Rabies virus, Protein Biosynthesis, Vaccines, Synthetic, Viral Envelope Proteins, Signal Transduction, NF-kappa B, Parainfluenza Virus 5, STAT Transcription Factors, Interferon Regulatory Factor-3, Mice, Rats, Janus Kinases, Host-Pathogen Interactions, Middle East Respiratory Syndrome Coronavirus
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