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

Abstract

Self-amplifying RNA (saRNA)-based vaccines have emerged as a potent and durable RNA vaccine platform relative to first generation mRNA vaccines. However, RNA vaccine platforms trigger undesirable side effects at protective doses, underscoring the need for improved tolerability. To address this, we leveraged the Cardiovirus leader protein, which is well-characterized to dampen host innate signaling by modulating nucleocytoplasmic transport (NCT). Co-administration of a leader-protein-encoding mRNA (which we have named "RNAx") delivered alongside vaccine cargo saRNA reduced interferon production while enhancing Influenza hemagglutinin (HA) expression in human primary cells and murine models. RNAx potently decreased serum biomarkers of reactogenicity after immunizations with an HA-expressing saRNA-LNP vaccine while maintaining the magnitude of the antibody and cellular response. RNAx also consistently enhanced binding antibody titers after a single injection and in some conditions enhanced binding antibody and neutralization titers post-boost. These findings support RNAx as a promising platform approach for improving tolerability of saRNA-LNP vaccines while preserving or enhancing immunogenicity.

Fig. 1. RNAx enhances GOI expression from saRNA in vitro.

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.

Fig. 2. RNAx enhances GOI expression from saRNA in vivo.

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.

Fig. 3. RNAx suppresses the expression of proinflammatory cytokines and enhances GOI expression in human PBMC.

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 Human PBMC (n = 4 donors) were treated with HA-nLuc saRNA-LNPs for 24 h and supernatants harvested for cytokines and cell lysates for nLuc activity. C The induction of 48 cytokines was measured from supernatants of PBMC treated with 5 µg/mL saRNA-LNPs. Cytokines induced by saRNA alone were determined by comparing the relative levels (ratio of geometric means) between saRNA without RNAx and untreated PBMC. Dotted lines = p < 0.05 (ratio paired t-test) and fold change = 2. D Among saRNA-LNP-responsive cytokines, the relative impact (ratio of geometric means) of RNAx in cis or trans with saRNA LNPs was determined with the ratio paired t-test (*p < 0.05, **p < 0.01, ***p < 0.001). E Supernatants from the same experiment were independently analyzed for IFN-α by ELISA and F lysates for nLuc activity. Mean ± SEM, n = 4. donors, *p < 0.05, **p < 0.01 Ratio paired t-test. Dotted lines = upper or lower limit of quantification. Numbers above bars indicate average fold change enhancement or inhibition of RNAx containing groups relative to saRNA only. Created with BioRender.com.

Fig. 4. RNAx suppresses proinflammatory cytokines in mice following vaccination.

A 5mC-modified Influenza HA expressing saRNA was co-formulated with modified nucleoside RNAx mRNA in trans at 1% and 10% (weight/weight) into LNPs. B Flow cytometry of Influenza HA surface expression in 293 T cells treated for 24 h with HA saRNA-LNPs (no RNAx) at the indicated concentrations. C C57BL/6 mice (n = 5 per group) were intramuscularly injected with 0.5 µg of Influenza HA saRNA-LNPs and were analyzed for serum cytokines 6 h post injection following (D–F) the prime on day 1 or (G–I) the boost on day 28. D, G Cytokines induced by saRNA only containing LNPs were determined by comparing the relative levels (ratio of geometric means) between saRNA alone and vehicle treated animals. Dotted line = p < 0.05 (Mann–Whitney t-test). E, H Among saRNA-LNP-responsive cytokines, the relative impact (ratio of geometric means) of co-formulating 1% or 10% (w/w) RNAx with saRNA LNPs was determined with Kruskal-Wallis test with Dunn’s multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001) following the prime and boost, respectively. F, I Absolute IFN-α abundance levels for all treatment groups following the prime and boost, respectively. Created with BioRender.com.

Fig. 5. Impact of RNAx on immunogenicity from an Influenza HA saRNA-LNP vaccine.

A C57BL/6 mice (n = 5 per group) were intramuscularly injected (prime and boost) with 0.05 µg or 0.5 µg of HA saRNA-LNPs (see Fig. 4A for details on RNA co-formulations). Serum was collected 6 h after the boost on day 28 (post prime measurement) and serum and splenocytes collected on day 42 (post boost measurement). Splenocytes were treated with 1 µg/mL Influenza HA peptide pools and analyzed for B IFN-γ and C IL-4 secreting cells by ELISPOT. Spot forming cells (SFC) were subtracted by untreated samples and normalized by 10⁶ splenocytes. Mean ± SEM. Total anti-HA IgG in the serum (EC50) was quantified by ELISA at D post prime and E post boost. F Neutralization titers were quantified by HAI assay post boost. Dotted line = lower limit of quantification, Geometric mean ± Geometric SD, *p < 0.05, Kruskal-Wallis test with Dunn’s multiple comparisons. Numbers above bars indicate fold enhancement of RNAx containing groups relative to saRNA only. Created with BioRender.com.