Pre-exposure to mRNA-LNP inhibits adaptive immune responses and alters innate immune fitness in an inheritable fashion

Abstract

Hundreds of millions of SARS-CoV-2 mRNA-LNP vaccine doses have already been administered to humans. However, we lack a comprehensive understanding of the immune effects of this platform. The mRNA-LNP-based SARS-CoV-2 vaccine is highly inflammatory, and its synthetic ionizable lipid component responsible for the induction of inflammation has a long in vivo half-life. Since chronic inflammation can lead to immune exhaustion and non-responsiveness, we sought to determine the effects of pre-exposure to the mRNA-LNP on adaptive immune responses and innate immune fitness. We found that pre-exposure to mRNA-LNPs or LNP alone led to long-term inhibition of the adaptive immune response, which could be overcome using standard adjuvants. On the other hand, we report that after pre-exposure to mRNA-LNPs, the resistance of mice to heterologous infections with influenza virus increased while resistance to Candida albicans decreased. The diminished resistance to Candida albicans correlated with a general decrease in blood neutrophil percentages. Interestingly, mice pre-exposed to the mRNA-LNP platform can pass down the acquired immune traits to their offspring, providing better protection against influenza. In summary, the mRNA-LNP vaccine platform induces long-term unexpected immunological changes affecting both adaptive immune responses and heterologous protection against infections. Thus, our studies highlight the need for more research to determine this platform's true impact on human health.

Fig 1. Pre-exposure to mRNA-LNPs or LNPs significantly inhibits subsequent adaptive immune responses induced by the mRNA-LNP vaccine.

A). Experimental model. Animals were shaved and intradermally inoculated in the left upper spot with either PBS, mRNA-LNP coding for eGFP (eGFP) or empty LNP (eLNP). Two weeks later the same areas were injected with PR8 HA mRNA-LNP (HA). Serum and skin draining lymph nodes were harvested 2 weeks later and the anti-HA serum antibody levels determined using ELISA and the germinal center (GC) B cell responses using flow cytometer (please see Materials and Methods for details on data normalization). B). Serum anti-HA antibody levels detected by ELISA. OD450 readings (top) at different serum dilutions. Summary graph of the relative area under the curve (AUC) for each sample (middle). C). GC B cell responses (CD38^- GL7⁺) from the same mice. Each dot represents a separate mouse. Data from at least two separate experiments pooled and are shown as mean ±SD. One-way ANOVA was used to establish significance. ns = not significant. **p<0.005, ***p<0.0005, ****p<0.0001. Figures were drawn by the authors using BioRender.

Fig 2. Pre-exposure to mRNA-LNPs has systemic effects.

A). Experimental model. As in Fig 1, but one of the groups was injected at a spot distinct from the primary exposure. B). ELISA OD450 readings and summary AUC graph on serum anti-HA antibody levels. C). Relative GC B cell responses from the same mice. Each dot represents a separate mouse. Data from at least two separate experiments pooled and are shown as mean ±SD. One-way ANOVA was used to establish significance. ns = not significant. *p<0.05, **p<0.01, ****p<0.0001. Figures were drawn by the authors using BioRender.

Fig 3. Pre-exposure to mRNA-LNPs has long-lasting effect on adaptive immune responses.

A). Experimental model. Animals at the indicated timepoints post-exposure to PBS or eGFP mRNA-LNP were injected into the same spot with PR8 HA mRNA-LNP and then anti-HA responses assessed as depicted two weeks later. B). Summary AUC graph on serum anti-HA antibody levels. C). Relative GC B cell responses from the same mice. Each dot represents a separate mouse. Data from at least two separate experiments pooled and are shown as mean ±SD. One-way ANOVA was used to establish significance. ns = not significant. *p<0.05, **p<0.01, ****p<0.0001. Figures were drawn by the authors using BioRender.

Fig 4. The general inhibition of adaptive immune responses induced by pre-exposure to mRNA-LNP can be overcome with the use of adjuvants.

A). Experimental model. CFSE-labeled TEα cells were transferred 1 day before (D-1) or 13 days after (D13) exposure to PBS or mRNA-LNP coding for eGFP. Anti-Langerin-Eα peptide was intravenously delivered on D14. The mice were sacrificed and the SDLNs were harvested for TEα cells examination by flow cytometry. B). Representative flow plots of TEα cell responses with/out Eα peptide stimulation (top), and the correspond summary graphs (bottom) on % of TEα cells in live cells and the fold change of expanded TEα cells over the non-immunized controls. C). Experimental model. Two weeks post-exposure to PBS or eGFP mRNA-LNP the animals were injected into the same spot with PR8 HA mRNA-LNP, or HA protein mixed with Alum or AddaVax. The anti-HA responses were assessed as depicted two weeks later. D). Summary AUC graph on serum anti-HA antibody levels. E). Relative GC B cell responses from the same mice. Each dot represents a separate mouse. Data from at least two separate experiments pooled and are shown as mean ±SD. Welch’s t test was used to establish significance. ns = not significant. **p<0.005, ***p<0.0005, ****p<0.0001. Figures were drawn by the authors using BioRender.

Fig 5. Innate immune fitness is altered with pre-exposure to mRNA-LNP.

A). Experimental model. Two weeks post-exposure to PBS or eGFP mRNA-LNP the animals were infected with sublethal doses of PR8 HA influenza or Candida albicans. The weights and other attributes were monitored as depicted. B). Percent of body weight drop after PR8 HA influenza infection, and the corresponding AUC changes. C). Viral copies in the lungs. D). Percent of body weight drop after Candida infection, and the corresponding AUC changes. E). Candida CFU numbers in the kidneys. F). Representative flow plots for neutrophils (gated on live cells/CD45⁺/Ly-6G⁺CD11b⁺) in the PBMCs of PBS or mRNA-LNP exposed mice, and the summary bar graph. G). Candida albicans (CA) killed rate in a candidacidal assay. H). Number of CA killed per neutrophil. Each dot represents a separate mouse. The data were pooled from 2–3 independent experiments. Welch’s t test was used to establish significance. ns = not significant. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. Figures were drawn by the authors using BioRender.

Fig 6. Immune changes induced by the mRNA-LNP platform can be inherited.

A). Experimental model. Adult WT B6 male and female mice were intradermally immunized with 10 μg of mRNA-LNP coding for influenza PR8 HA. Two weeks post-immunization the mice were mated. Non-immunized males mated to non-immunized females served as controls (DMN; dad-mom non-immunized). Immunized males with unimmunized (DI; dad immunized) or immunized females (DMI; dad-mom immunized), and immunized females with unimmunized males (MI; mom immunized). Offspring from 1^st, 2^nd, and 4^th litters at eight-ten weeks of age were intranasally inoculated with a sublethal dose of PR8 influenza and weight monitored for 14 days. B). Percent of body weight drop after influenza infection (upper), and the corresponding AUC changes (lower). The data are from one experiment using litters from 3 separate mating. The number of mice (female/male) from 1^st litters used for influenza challenge was 6/7 (DMN), 8/6 (DI), 3/9 (MI) and 8/7 (DMI); for the 2^nd litters 1/3 (DMN), 6/9 (DI), 7/5 (MI) and 6/2 (DMI); for the 4^th litters 2/9 (DMN), 8/13 (DI), 9/7 (MI) and 7/8 (DMI). One way ANOVA was used to establish significance. ns = not significant. ***p<0.0005, ****p<0.0001. Figures were drawn by the authors using BioRender.