Type I Interferons Interfere with the Capacity of mRNA Lipoplex Vaccines to Elicit Cytolytic T Cell Responses

Purpose: mRNA vaccines are promising for inducing cytolytic CD8⁺ T cell responses, and lipoplex carriers improve mRNA delivery to dendritic cells. However, the innate factors that regulate T cell induction by mRNA lipoplex vaccines remain unresolved. In particular, type I interferons (IFNs) are induced by mRNA but can either promote or inhibit T cell immunity depending on context.

Hypothesis: If mRNA lipoplexes induce type I IFN responses in vivo, then type I IFN signaling will interfere with the generation of potent cytolytic T cell responses and antitumor efficacy, and blocking IFNAR at the site of immunization will enhance vaccine-induced tumor control.

Aims: Determine whether mRNA lipoplexes induce type I IFN responses after subcutaneous, intradermal, and intranodal immunization. - Assess the impact of type I IFN signaling on the magnitude, proliferation, and cytolytic function of vaccine-elicited CD8⁺ T cells. - Evaluate whether type I IFNs limit prophylactic and therapeutic antitumor efficacy of mRNA lipoplex vaccines in the B16.OVA melanoma model. - Test whether antibody-mediated IFNAR blockade at the immunization site improves vaccine-elicited T cell responses and tumor control.

Delivery system: Carrier: Cationic liposomes composed of DOTAP and DOPE. - Payload: In vitro transcribed mRNA encoding ovalbumin (OVA), luciferase, or EGFP. - Formulation: mRNA lipoplexes at nitrogen/phosphate (N/P) ratio 1, selected because it yielded high mRNA expression and proper CD8⁺/CD4⁺ T cell induction. - Physicochemical properties: N/P1 lipoplexes were ~300–400 nm and negatively charged (zeta potential −18 mV); N/P10 lipoplexes were positively charged (+32 mV). - Targeting ligand: None. - Immunization routes: Subcutaneous, intradermal, and intranodal. - Adjuvant/blocking agent: Anti-mouse IFNAR blocking antibody MAR1-5A3 or isotype control, coadministered at the immunization site.

Approach: Mice: Female C57BL/6 wild-type mice, IFNAR1-deficient (Ifnar1⁻/⁻) mice, IFN-β reporter mice, and OT-I transgenic CD8⁺ T cell receptor mice. - Tumor model: Highly aggressive B16.OVA melanoma. - Prophylactic vaccination: Prime at week 0, boost at week 2, challenge with 100,000 B16.OVA cells at week 4; n = 12–16 mice/group. - Therapeutic vaccination: Inoculate 75,000 B16.OVA cells, then immunize on day 4 and day 6 or day 9; n = 5–8 mice/group. - Doses: 10 or 20 µg OVA mRNA lipoplexes; 20 µg IFNAR blocking antibody or isotype control. - Controls: PBS, naked mRNA, DOTAP/DOPE liposomes without mRNA, isotype antibody, and untreated mice.

Key methods: In vivo bioluminescence imaging for IFN-β promoter induction and luciferase expression. - Flow cytometry for OT-I proliferation, OVA-specific CD8⁺ T cell quantification, and dextramer staining. - ELISPOT for IFN-γ-secreting OVA-specific T cells. - In vivo killing assay with CFSE-labeled OVA peptide-pulsed target cells. - Tumor survival analysis in B16.OVA melanoma model. - Antibody-mediated IFNAR blockade at the immunization site.

Key results: mRNA lipoplexes induced potent type I IFN responses after subcutaneous, intradermal, and intranodal injection. Naked mRNA also induced IFN-β, whereas liposomes alone did not, indicating IFN induction is inherent to mRNA rather than the lipoplex formulation. - In Ifnar1⁻/⁻ mice, OT-I proliferation was strongly elevated; OVA-specific CD8⁺ T cells reached up to 3% of all CD8⁺ T cells in blood, whereas they were hardly detectable in wild-type mice. ELISPOT showed increased IFN-γ-secreting T cells, and in vivo killing was nearly complete in Ifnar1⁻/⁻ mice versus limited killing in wild-type mice. - Prophylactic vaccination increased median survival from 17 to 29 days in wild-type mice and from 14 to 40 days in Ifnar1⁻/⁻ mice. - Therapeutic vaccination increased median survival from 34 to 47 days in wild-type mice (not significant) and from 20 to 35 days in Ifnar1⁻/⁻ mice (significant). - IFNAR blocking antibody coadministered with mRNA lipoplexes increased OT-I proliferation and significantly improved survival in both prophylactic and therapeutic vaccination settings. - Luciferase expression after subcutaneous mRNA lipoplex injection was only slightly elevated in Ifnar1⁻/⁻ mice and did not reach significance, suggesting the enhanced T cell responses were not simply due to increased antigen expression.

Interpretation: The authors conclude that type I IFNs induced by mRNA lipoplex vaccines negatively regulate the generation of cytolytic T cell responses and antitumor immunity, regardless of subcutaneous, intradermal, or intranodal route. Because type I IFN induction is inherent to in vitro transcribed mRNA, preventing type I IFN induction and/or IFNAR signaling at the immunization site may be a broadly applicable strategy to improve mRNA vaccine potency.

Limitations: All experiments were performed in mouse models; no human or large-animal validation is reported. - The B16.OVA melanoma model is highly aggressive and uses a model antigen, which may not fully reflect clinical tumor antigen settings. - Genetic Ifnar1⁻/⁻ mice lack spontaneous antitumor immune responses, complicating direct comparison with wild-type mice; the authors addressed this partly by using local antibody blockade. - The study does not fully resolve the downstream mechanism by which type I IFNs impair T cell priming, though it rules out a major effect on mRNA expression. - Only unmodified in vitro transcribed mRNA was tested; strategies such as nucleoside modification to reduce IFN induction were not evaluated. - Long-term safety, repeat dosing, and translation to clinical mRNA vaccine formulations remain to be established.