The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory

Abstract

Vaccines based on mRNA-containing lipid nanoparticles (LNPs) are a promising new platform used by two leading vaccines against coronavirus disease in 2019 (COVID-19). Clinical trials and ongoing vaccinations present with very high protection levels and varying degrees of side effects. However, the nature of the reported side effects remains poorly defined. Here we present evidence that LNPs used in many preclinical studies are highly inflammatory in mice. Intradermal injection of these LNPs led to rapid and robust inflammatory responses, characterized by massive neutrophil infiltration, activation of diverse inflammatory pathways, and production of various inflammatory cytokines and chemokines. The same dose of LNP delivered intranasally led to similar inflammatory responses in the lung and resulted in a high mortality rate. In summary, here we show that the LNPs used for many preclinical studies are highly inflammatory. Thus, their potent adjuvant activity and reported superiority comparing to other adjuvants in supporting the induction of adaptive immune responses likely stem from their inflammatory nature. Furthermore, the preclinical LNPs are similar to the ones used for human vaccines, which could also explain the observed side effects in humans using this platform.

Keywords: LNP; SARS-CoV-2 vaccine; inflammation; mRNA-LNP vaccine; side-effects.

Figure 1.. Intradermal inoculation with LNPs induces robust inflammation.

A. Intradermal inoculation with LNP induced visible levels of inflammation. Pictures were taken 24 hours post PBS or LNP injection. B. Skin samples from the mice injected with PBS or LNP were harvested at the indicated time points, analyzed by flow cytometry, and displayed as cell percentages. C. As in A, but LNPs with (iLNP) or without (nLNP) ionizable lipids were used. Unlike iLNPs the nLNPs induced no visible signs of inflammation. D. Skin samples from C were analyzed for leukocytic infiltration 24 hours post inoculation. For all the charts the data were pooled from two separate experiments and displayed as percent ± SD. Each dot represents a separate animal. Student’s two-tailed t-test was used to determine the significance between naïve and the experimental samples. ****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05, ns = not significant. No differences were observed between samples harvested from naïve or PBS treated animals and are used interchangeably throughout the manuscript.

Figure 2.. Intradermal inoculation with LNPs complexed with non-coding poly-cytosine mRNA leads to an inflammatory milieu.

A. Experimental design. The mice were treated as indicated, and 24 hours later, the skin samples were prepared for Luminex^® and bulk RNA-seq analyses. B. and C. Luminex^® data summarizing inflammatory chemokines and cytokines induced by the LNPs. D. Heatmap of gene expression changes triggered by the LNPs (FDR < 0.05, log2 FC > 1 – 4091 genes). E. Volcano plot summarizing the up and downregulated genes upon LNP injection. F. GSEA analyses of the KEGG pathways and displayed as normalized enrichment score (NES). FDR<0.05. Pathways with NES less than ±2 are not displayed. N=4.

Figure 3.. Intranasal LNP delivery induces robust lung inflammation and animal death.

A. Animals were inoculated with the indicated doses of LNP and the survival rate, weight (B), and clinical scores (C) recorded daily for up to 8 days. Data was pooled from two independent experiments. N=9 for each group except PBS/Naïve where n=5. D. Lungs harvested at the indicated time points from PBS, and the 10 μg group were harvested and photographed. E. Animals injected with 10 μg of LNP were sacrificed 9 hours post inoculation and their lungs’ leukocytic composition determined by flow cytometry following a published gating strategy (Yu et al., 2016). Neut.= neutrophils, Eosi. = eosinophils, DC = dendritic cells, NK = natural killer, aMac. = alveolar macrophages, iMac. = interstitial macrophages, iMon. = inflammatory monocytes, rMon. = resident monocytes. F. LNP injection leads to fast and homogenous dispersion in the lung. Animals were inoculated with PBS or 10 μg of DiI-labeled LNP. Six hours later the lungs were harvested, prepared for histology, stained with DAPI and imaged using a confocal microscope. One representative image is shown. For all the charts the data were pooled from at least two separate experiments and displayed as percent ± SD. Each dot represents a separate animal. Student’s two-tailed t-test was used to determine the significance between naïve and the experimental samples. ***p<0.0005, **p<0.005, *p<0.05, ns = not significant.

Figure 4.. Potential mechanism of side effects.

The side effects observed with the SARS-CoV-2 vaccine’s first dose are likely associated with the LNPs’ inflammatory properties. LNPs activate different inflammatory pathways that will lead to the production of inflammatory cytokines, such as IL-1β and IL-6 that can initiate and sustain local and systemic inflammations and side effects. LNPs might also diffuse from the periphery and reach any organs in the body, including CNS (hypothalamus) where they could directly induce side effects (dashed line). PEG is widely used as a food and medicine additive, and many of us develop antibodies to PEG. Therefore, the LNPs’ PEGylated lipids can induce CARP in humans with preexisting PEG-specific antibodies. Humans often experience more severe side effects with the second dose. Here we posit that might be due to multiple reasons. Firstly, innate immune memory against the LNPs might form after the first vaccination, and that could lead to even more robust inflammatory responses upon the second vaccination. Secondly, after the first vaccination adaptive immune responses are formed targeting the viral protein coded by the mRNA. As such, cells (shown as red shape) expressing the viral protein derived peptides or protein itself can become the target of CD8⁺ T or NK cell-mediated killing (ADCC), respectively. Since the LNPs could diffuse throughout the body and transfect any cell in their path with the mRNA, and the mRNA could also be further distributed through extracellular vesicles (Maugeri et al., 2019), the target population could potentially be vast and diverse.