Technical Review: Controlling reactogenicity while preserving immunogenicity from a self-amplifying RNA vaccine by modulating nucleocytoplasmic transport

(A) The Cardiovirus leader protein suppresses innate signaling by dampening nucleocytoplasmic transport (NCT) in infected cells. RNAx refers to an mRNA that encodes for the leader protein. (B) RNAx was expressed from VEEV saRNA in cis from an IRES downstream of GOI or added in trans from a modified nucleoside mRNA. (C) RNAx enhances GOI expression in cells with intact innate signaling. BJ or 293 T cells were transfected with reporter constructs and Luc signal measured 48 h later with or without RNAx in cis. (D) RNAx enhances GOI expression in a dose-responsive fashion in trans. BJ cells were transfected with secreting nLuc-expressing saRNA with increasing amounts of RNAx in trans and 6 days post transfection supernatants were harvested for nLuc activity. (E) RNAx reverses suppression of mRNA reporter activity in the presence of IFN-β or dsRNA (Poly I:C). BJ cells were transfected with a modified nucleoside fLuc-encoding mRNA and co-treated with either 1 ng/mL recombinant IFN-β or 100 ng/mL Poly(I:C). Luc activity was measured 48 h post transfection from cell lysates. Mean ± SEM, n = 3–4. p < 0.05, *p < 0.01, ****p < 0.0001 Ratio paired t-test. Numbers above bars indicate average fold change enhancement of RNAx containing groups relative to no RNAx.

Authors: Jason A. Wojcechowskyj, Robyn M. Jong, Imre Mäger, Britta Flach, Paul V. Munson, Progya P. Mukherjee, Barbara Mertins, Katherine R. Barcay & Thomas Folliard

Affiliation: ExcepGen Inc.

Background Introduction

This paper contributes to the field of RNA vaccinology, specifically addressing a key challenge in self-amplifying RNA (saRNA) vaccine development. While saRNA vaccines offer advantages over conventional mRNA platforms by providing longer-lasting protection at lower doses, they also trigger undesirable side effects at protective doses. These side effects stem from excessive innate immune activation driven by double-stranded RNA intermediates generated during self-replication. Current approaches to reduce reactogenicity include modifying nucleosides, altering delivery systems, or targeting innate signaling pathways. However, a universal strategy combining low doses with effective suppression of saRNA-induced reactogenicity has remained elusive.

Materials and Methodology

The researchers leveraged the Cardiovirus leader protein, which dampens innate immune signaling by interfering with nucleocytoplasmic transport (NCT). They created "RNAx," a leader-protein-encoding mRNA that broadly suppresses multiple innate signaling pathways simultaneously. RNAx was tested in two configurations: "in cis" (single RNA molecule with RNAx and vaccine antigen) and "in trans" (separate RNAx mRNA co-delivered with vaccine RNA).

The study used multiple experimental systems:

1. Cell lines (BJ fibroblasts and 293T cells)

2. Primary human immune cells (PBMCs)

3. Mouse models (C57BL/6 and BALB/c)

   
   

For in vitro studies, cells were transfected with saRNA constructs encoding reporter proteins or Influenza hemagglutinin (HA), with or without RNAx. For in vivo studies, mice received intramuscular injections of saRNA-LNPs with or without RNAx.

The team measured:

• Gene expression (luciferase reporters and HA)

• Cytokine production (multiplex assays and ELISAs)

• Antibody responses (binding and neutralizing)

• Cellular immune responses (ELISPOTs for IFN-γ and IL-4)

  
  

Results

Enhancement of Antigen Expression

RNAx significantly enhanced expression of various reporter proteins in BJ cells (up to 62-fold with RNAx in cis and 11-fold with RNAx in trans). In vivo, RNAx in trans enhanced nLuc expression 170-fold one day post-injection compared to saRNA alone.

Suppression of Inflammatory Cytokines

(A) Unmodified Influenza HA-nLuc expressing saRNA was co-formulated with modified nucleoside RNAx mRNA (in trans) or co-expressed downstream of an IRES (in cis) and packaged into LNPs. ‘In trans’ co-formulations consisted of at 36% (weight/weight) RNAx mRNA and 64% saRNA i.e., 0.72 µg of RNAx and 1.28 µg saRNA. (B) Flow cytometry of Influenza HA surface expression in 293 T cells treated for 24 h with HA-nLuc saRNA-LNPs (no RNAx) at the indicated concentrations. (C) C57BL/6 mice (n = 5 per group) were intramuscularly injected with 2 µg Influenza HA-nLuc expressing saRNA-LNPs and nLuc activity at the injection site quantified with IVIS on the indicated days post injection. (D) Total flux of Luc signal is represented by photons per sec (p/s). Quantification of nLuc activity at the injection site quantified with IVIS on the indicated days post injection or the area under the curve (AUC). Geometric mean ± Geometric SD, p < 0.01, *p < 0.001, Kruskal-Wallis test with Dunn’s multiple comparisons. Numbers above bars indicate average fold change enhancement of RNAx containing groups relative to saRNA only. Created with BioRender.com.

In human PBMCs, RNAx in trans potently suppressed 14 out of 15 saRNA-induced cytokines, including IFN-α. In mouse models, RNAx suppressed key inflammatory cytokines linked to vaccine reactogenicity, including IFN-α (27-fold), IL-6 (5-fold), and MCP-1 (6-fold) following prime, and additionally suppressed IFN-γ (11-fold) and IL-10 (7-fold) following boost.

Preservation of Immune Responses

Despite suppressing innate signaling, RNAx did not negatively affect the cellular immune response to vaccination. The Th1-biased nature and magnitude of cellular responses remained unchanged. For antibody responses, RNAx in trans consistently enhanced binding antibody titers after a single injection (3-7 fold increases). Following boost, RNAx occasionally enhanced binding antibody and neutralization titers, though this varied across mouse strains and vaccine vectors.

Evaluation

This study presents a comprehensive approach to addressing a significant challenge in RNA vaccine development. The experimental design shows rigor through:

1. Multiple experimental systems (cell lines, primary cells, and in vivo models)

2. Careful controls, including zinc-finger mutants to validate mechanism

3. Comprehensive readouts measuring both mechanistic aspects and functional outcomes

The data analysis is sound, using appropriate statistical methods and acknowledging limitations. The researchers recognize that mice can underestimate the beneficial effects of modulating innate signaling from RNA vaccines, as demonstrated by previous clinical experiences with modified versus unmodified mRNA vaccines.

The study makes several notable contributions:

1. Demonstrates that innate signaling can be modulated without sacrificing immunogenicity

2. Establishes RNAx as a potentially universal approach applicable to various RNA platforms

3. Shows that delivering RNAx in trans provides greater flexibility than in cis approaches

4. Provides mechanistic insights into how targeting NCT broadly can suppress multiple innate signaling pathways

   
   

Conclusion

This paper presents RNAx as a promising platform approach for improving tolerability of saRNA vaccines while preserving or enhancing immunogenicity. It demonstrated that innate signaling could be modulated without sacrificing immunogenicity, on occasion even enhancing the antibody response. Beyond these findings, the discrete and modular nature of RNAx offers flexibility in dosing and application across various RNA-based vaccines and therapies, further expanding the scope of this technology.

While mouse studies suggest potential benefits, the authors appropriately note that larger animal studies or human trials will be needed to fully assess RNAx's impact on vaccine immunogenicity. This is especially the case due to mouse studies' tendencies to underestimate the positive effects of immunogenicity.

For applications such as vaccine development, high uniformity and encapsulation efficiency are essential to ensure reliable interpretations of results. The mRNA-LNPs in this paper were prepared using the PreciGenome NanoGenerator Flex-M, which permitted accurate and controlled flow rate conditions.

If you're interested in learning more, dive into the original paper at:

https://www.nature.com/articles/s41541-025-01135-8