In a recent study posted to the bioRxiv* preprint server, researchers studied how messenger ribonucleic acid (mRNA)-based BNT162b2 coronavirus disease 2019 (COVID-19) vaccine altered the biochemical composition of glial and glioma brain cells in vitro.
Study: Decoding COVID-19 mRNA Vaccine Immunometabolism in Central Nervous System: human brain normal glial and glioma cells by Raman imaging. Image Credit: Kateryna Kon/Shutterstock
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Background
Raman spectroscopy imaging enables the examination of the biochemical composition of cell organelles non-invasively, valuable in monitoring molecular interactions in the tumor microenvironment and unraveling mechanisms governing immune response to pathogenic infections.
COVID-19 mRNA vaccine mimics COVID-19 infection, but instead of the whole virus, synthesize only severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein for the immune response, without causing COVID-19 infection.
The harmful effects of mRNA vaccine-produced high levels of S protein are not yet completely understood. Researchers have cautioned that they induce complex reprogramming of innate immune responses; moreover, the vaccine-produced S protein remains near the vaccination site and even circulates in the bloodstream to directly affect the host cells with long-term consequences. Therefore, it is crucial to monitor the biodistribution and location of S protein from mRNA vaccines.
Studies have recovered COVID-19 mRNA from the cerebrospinal fluid of vaccinees, suggesting it can cross the blood-brain barrier (BBB). In addition, even without crossing the BBB, several cytokines induced by COVID-19 infection cross the BBB to affect central nervous system (CNS) function.
In this way, COVID-19 mRNA reaches the brain, infects astrocytes, and triggers neuropathological changes that contribute to the structural and functional alterations in the brain of COVID-19 patients. The researchers have also raised concerns that the lipid nanoparticles (LNPs) can diffuse quickly to the CNS through the olfactory bulb or blood. However, these phenomenons, including the role of innate memory responses to LNPs, need to be further explored in future research.
About the study
In the current study, researchers used Raman spectroscopy to examine several CNS-related symptoms, including loss of taste and smell, twitching, confusion, headaches, impaired consciousness and vision, nerve pain, dizziness, nausea and vomiting, seizures, hemiplegia, stroke, ataxia, and cerebral hemorrhage in human brain glial and glioma cells in vitro. More specifically, they studied normal and tumor brain cells, including normal human astrocytes (NHA), human astrocytoma CCF-STTG1, and human glioblastoma cell line U87-MG.
To this end, they injected the BNT162b2 vaccine and incubated these cells in vitro. Next, they monitored the effect of the vaccine on the biodistribution of different chemical components, particularly alterations in reduction-oxidation (redox) pathways related to cytochrome (cyt) c in cell organelles, including the nucleus, mitochondria, lipid droplets, cytoplasm, and membrane.
Raman imaging helped analyze the vibrational spectra of a sample area (here brain cells), including biodistribution of different biomolecules. Using two-dimensional (2D) spectroscopic data obtained by Raman imaging and cluster analysis algorithm, the researchers created Raman maps to visualize cellular substructures to learn about their composition.
The distinctive coding colors in the K-means cluster analysis represented seven clusters: the blue color represented lipids, including rough endoplasmic reticulum (RER) and lipid droplets filled with retinoids; likewise, orange, magenta, red, green, light grey, and dark grey colors represented lipid droplets filled with triacylglycerols of monounsaturated type (TAG), mitochondria, nucleus, cytoplasm, membrane, and the cell environment, respectively.
They recorded Raman spectra using a confocal Raman microscope that recorded images with a spatial resolution of 1 × 1µm. It was calibrated daily before taking the measurements, using a silica plate with a maximum peak at 520.7 cm-1.
Study findings
Among several study findings, a key one was that the human cells in vitro demonstrated a redox imbalance by downregulation of cyt c, similar to that observed in cancers. They noted that the Raman signal of oxidized cyt c was strongest for astrocytoma control cells and the weakest for the U-87 MG cells, indicating decreased oxidative phosphorylation and apoptosis.
The band intensity (at 1654 cm-1), corresponding to amide I, decreased for glioblastoma U87-MG upon incubation with mRNA, most likely due to deterioration of the adenine nucleotide translocator (ANT), representing about 10% of proteins in mitochondria.
The BNT162b2 vaccine reprogrammed innate immune responses by downregulation of cyt c. Cyt c creates a damage-associated molecular pattern (DAMP) that alarms the immune system of any potential danger in all cell types to help them mount an appropriate immune defense via activation of pattern recognition receptors (PRRs).
At 1584 cm-1, the Raman intensity of the reduced form of cyt c drastically increased upon incubation of U87 MG cells with retinoic acid (RA), indicating that RA is an essential innate immune system molecule with the capability to halt cytokine induction.
It is common knowledge that the mRNA vaccine never enters the cell nucleus, where the deoxyribonucleic acid (DNA) resides, to alter the human genome because - i) they do not have a reverse transcriptase; ii) they cannot travel from cytoplasm to the nucleus; iii) mRNAs are short-lived molecules.
The authors observed no statistically significant changes in cytochrome c activity for NHA and U87-MG at 60 µL/mL dose. In contrast, they observed changes for astrocytoma at the 60 µL/mL dose during incubation time of 96 hours and U87-MG glioblastoma cells at a low dose of one µL/mL for 24 hours.
Since the mRNA vaccine does not introduce changes corresponding to DNA, these results indicated post-translational changes in histones of the nucleus upon incubation with the mRNA vaccine, not in DNA.
Furthermore, they noted a decreased cyt c signal at 1584 cm-1 for all types of glial cells and all periods of incubation and dosages, suggesting statistically significant changes in cyt c biochemical concentration in lipid droplets and lipid structures of RER.
Conclusions
The study highlighted how Raman imaging presented exciting new possibilities to understand the associations between pathways of cancer and the immune system and recognize metabolites regulating these pathways.
Taken together, the study findings demonstrated that the mRNA-based BNT162b2 COVID-19 vaccine altered mitochondria by downregulation of cyt c resulting in lowered oxidative phosphorylation and adenosine triphosphate (ATP) production. This led to the lowering of the immune response.
A decrease of amide I concentration in mitochondrial membrane potential suggested functional deterioration of the ANT. Likewise, the BNT162b2 vaccine significantly modified de novo lipids synthesis in lipid droplets; however, the role of signaling function of lipid droplets increased.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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