Monitoring mRNA vaccine antigen expression in vivo using PET/CT

Abstract

Noninvasive visualization of the distribution and persistence of mRNA vaccine antigen expression in mammalian systems has implications for the development and evaluation of future mRNA vaccines. Here, we genetically fuse E. coli dihydrofolate reductase (eDHFR) to the delta furin diproline modified SARS-CoV-2 spike glycoprotein (S2P^∆f) mRNA vaccine and image its expression in female mice and male non-human primates using [¹⁸F]fluoropropyl-trimethoprim ([¹⁸F]FP-TMP). Whole body positron emission tomography (PET) imaging revealed transient expression of the vaccine antigen in the injection site and draining lymph nodes (dLNs). Fusion of eDHFR did not impact S2P immunogenicity and no humoral or cellular immune response was detected against eDHFR in either species. In this work, we show that eDHFR can be used as an mRNA-encoded PET reporter gene to monitor the spatiotemporal dynamics of mRNA vaccine antigen expression in vivo. This technique could be applied in clinical translation of future mRNA vaccines or therapeutics.

Fig. 1. Expression of S2P^Δf-eDHFR and specific uptake of [¹⁸F]FP-TMP in mammalian cells.

a N1Ψ-modified mRNA encoding the delta furin diproline modified SARS-CoV-2 spike glycoprotein (S2P^∆f) was genetically fused to eDHFR and formulated in LNPs containing cholesterol, an ionizable lipid, a PEG-lipid, and DSPC as a zwitterionic lipid (not to scale). b LNPs transfect antigen-presenting cells (APCs) and mRNA is released and translated into S2P^Δf-eDHFR. Cells expressing the fusion antigen can be imaged using [¹⁸F]FP-TMP. c Confirmation of S2P^Δf-eDHFR fusion by Western blot with an anti-spike antibody. eDHFR adds an 18 kDa shift to the molecular weight. Recombinant spike S2 protein produced in insect cells was used as a positive control, 59 kDa. COX IV was used as a loading control, 18 kDa. Representative data from n = 3 independent experiments. d Mean fluorescence intensity of HEK293T and DC2.4 cells expressing S2P^Δf-eDHFR or S2P and labeled with an anti-spike AF647 conjugated antibody. Data shown is n = 3 technical replicates representative of three independent experiments. e Uptake of [¹⁸F]FP-TMP in HEK293T cells transfected with S2P^Δf-eDHFR or S2P mRNA. Cold TMP (10 µM) was used as a blocking agent. Data is shown as %ID per 1e6 cells. Data shown is n = 3 technical replicates representative of two independent experiments. Data presented as mean. a, b Created in BioRender. Sellmyer, M. (2025) https://BioRender.com/x00q954. Source data for panel (c–e) are provided as a Source Data file.

Fig. 2. Longitudinal PET imaging in vaccinated mice.

a Experimental timeline. Balb/c mice were IM injected with eLNP or S2P^Δf-eDHFR mRNA-LNPs in the right hindlimb on day 0. On day 1 and 3 mice were IV administered [¹⁸F]FP-TMP and PET/CT images were acquired. b Representative PET/CT images of mice on day 1 and 3. Red arrow indicates [¹⁸F]FP-TMP signal in the ipsilateral popliteal LN of the S2P^Δf-eDHFR vaccinated mouse on day 1. c [¹⁸F]FP-TMP SUV_max quantification in the ipsilateral LN and injected muscle on day 1 (n = 6 mice per group) and day 3 (n = 4 mice per group). d Experimental timeline of follow up experiment. Balb/c mice were vaccinated with S2P or S2P^Δf-eDHFR mRNA-LNPs and imaged on day 1. e Representative axial PET/CT slices through the popliteal LNs. Red arrow indicates [¹⁸F]FP-TMP signal in the ipsilateral popliteal LN of the S2P^Δf-eDHFR vaccinated mouse. f [¹⁸F]FP-TMP SUV_max quantification in the ipsilateral LN and injected muscle on day 1 (S2P, n = 4; S2P^Δf-eDHFR, n = 6 mice per group). The ALC-307 lipid was used in these experiments. Groups were compared using an unpaired, two-tailed t test. Only significant pairwise comparisons are shown. Data presented as mean ± SD. (c, f) p < 0.0001 unless otherwise stated. day 1: muscle, p = 0.0002. a, d Created in BioRender. Sellmyer, M. (2025) https://BioRender.com/d90y830. Source data for panel (c, f) are provided as a Source Data file.

Fig. 3. Quantification of antibodies against spike and eDHFR in mouse serum at prime and boost.

a Experimental timeline. Balb/c mice were vaccinated with eDHFR, S2P^Δf-eDHFR, or S2P mRNA-LNPs on day 0 and 21 (n = 5 mice per group). On day 21 and 35 serum was collected for analysis. The ALC-0315 lipid was used in this experiment. b Total IgG antibodies against full length spike and c eDHFR protein in serum of vaccinated mice at day 21 (prime) and 35 (boost). Data presented as mean. a Created in BioRender. Sellmyer, M. (2025) https://BioRender.com/w43k765. Source data for panel (b, c) are provided as a Source Data file.

Fig. 4. T cell polyfunctionality against spike and eDHFR.

a Experimental timeline. Balb/c mice were vaccinated with eLNP or S2P^Δf-eDHFR mRNA-LNPs on day 0 and 21. On day 10 and 35 spleens were collected for analysis. The ALC-307 lipid was used in this experiment. b Representative flow plots (spike-stimulated) and quantification of CD4⁺ IL2⁺ and c CD8⁺ IFN-γ⁺ antigen-specific T cells after stimulation with eDHFR and spike peptides at prime (eLNP, n = 5; S2P^Δf-eDHFR, n = 5 mice per group) and boost (eLNP, n = 5; S2P^Δf-eDHFR, n = 7 mice per group). Groups were compared using a two-way ANOVA with Sidak’s multiple comparisons test. d Experimental timeline. Balb/c mice were vaccinated with eDHFR, S2P^Δf-eDHFR, or S2P mRNA-LNPs on day 0 and 21 and spleens were collected on day 35 for analysis. The ALC-0315 lipid was used in this experiment. e Quantification of antigen-specific CD4⁺ T cells following stimulation with eDHFR or spike. n = 5 mice per group. Groups were compared using a two-way ANOVA with Tukey’s multiple comparisons test. Only significant pairwise comparisons are shown. Data presented as mean. (b, c, e) p < 0.0001 unless otherwise stated. Anti-spike CD4⁺ TNF-α⁺: eDHFR vs^. S2P^Δf-eDHFR, p = 0.0032. a, d Created in BioRender. Sellmyer, M. (2025) https://BioRender.com/a98s821. Source data for panel (b, c, e) are provided as a Source Data file.

Fig. 5. In vivo cytotoxicity against splenocytes loaded with spike or eDHFR peptides.

a Experimental timeline. Balb/c mice were vaccinated with eLNP or S2P^Δf-eDHFR mRNA-LNPs on day 0 and boosted on day 21. On day 33 mice were IV injected with a 1:1 ratio of unstimulated and peptide-loaded syngeneic splenocytes and sacrificed on day 34 for analysis. The ALC-307 lipid was used in this experiment. b Schematic of peptide-loaded splenocytes. c Flow plot populations of unstimulated (CFSE^lo) and spike-loaded (CFSE^hi) splenocytes and d quantification. e Flow plot populations of unstimulated (CFSE^lo) and eDHFR-loaded (CFSE^hi) splenocytes and f quantification. Position of loaded splenocytes and CFSE^lo and CFSE^hi cell populations are indicated on the S2P^Δf-eDHFR vaccinated flow plots. S2P^Δf-eDHFR, n = 5; eLNP n = 3 mice per group. CFSE^lo and CFSE^hi groups were compared using a two-way ANOVA with Sidak’s multiple comparisons test. Only significant pairwise comparisons are shown. Data presented as mean ± SD. d p < 0.0001 unless otherwise stated. a, b Created in BioRender. Sellmyer, M. (2025) https://BioRender.com/p80q175. Source data for panel (d, f) are provided as a Source Data file.

Fig. 6. Dynamic whole body PET imaging in vaccinated NHPs.

a Experimental timeline. NHP 1 was IM injected with S2P^Δf-eDHFR mRNA-LNPs in the right deltoid on day 0 and PET/CT imaged on day 1, 7, and 35. The NHP was boosted on day 15. Blood was collected on day 7 and 35 for analysis. b Dynamic PET/CT images of NHP 1 at 1, 60, and 120 min post [¹⁸F]FP-TMP administration on day 1. Red arrows indicate [¹⁸F]FP-TMP signal in the injected muscle and ipsilateral axillary LN. c Axial PET/CT slices through the axillary LNs at 60 min post [¹⁸F]FP-TMP administration on day 1, 7, and 35. Red arrow indicates the ipsilateral axillary LN. d Quantification of [¹⁸F]FP-TMP SUV_max and SUV_r in tissues of interest in NHP 1 at 60 min post [¹⁸F]FP-TMP administration on day 1, 7, and 35. Inlet graph of the time activity curve of [¹⁸F]FP-TMP SUV_r in the ipsilateral axillary LN across timepoints. e Experimental timeline. An NHP was vaccinated with a C. diff mRNA-LNP vaccine in the right deltoid muscle on day 0 and PET/CT imaged on day 1. f Full body PET/CT image and g axial PET/CT slice through the axillary LNs at 60 min post [¹⁸F]FP-TMP administration on day 1. h Quantification of [¹⁸F]FP-TMP SUV_max in tissues of interest in S2P^Δf-eDHFR vaccinated NHPs (n = 2) compared to the C. diff vaccinated NHP (n = 1) at 60 min post [¹⁸F]FP-TMP administration on day 1. The ALC-0315 lipid was used in S2P^Δf-eDHFR NHP 1 and the ALC-307 lipid was used in NHP 2. a, e Created in BioRender. Sellmyer, M. (2025) https://BioRender.com/v54j936. Source data for panel (d, h) are provided as a Source Data file.

Fig. 7. Quantification of humoral and cellular immune responses against spike and eDHFR in NHPs.

a Total IgG antibodies against full length spike and eDHFR protein at prime and boost in NHP 1 and 2. n = 2 NHPs. b Representative plate wells with spot counts and c quantification from IFN-γ and IL-2 ELISPOT looking at cellular responses to eDHFR and spike peptides at boost. DMSO and Anti-CD3 were used as a negative and positive control, respectively. Dashed lines indicate maximum background levels for each cytokine. n = 2 NHPs per group. Source data are provided as a Source Data file.