Episode 275: SARS-CoV-2 infection or vaccination, risk of reverse transcription in relation to auto-immune and (hypothetical) geno-toxic phenomena with focus on the liver.
A long title, of which I hope that it triggers your attention. It is based on two papers (Ep 275-1 A and B) that try to show a relation of SARS-CoV-2 infection resp mRNA vaccination with reverse transcription into DNA, based on the activity very common cellular retrotransposon (LINE-1) elements. If this is true it has several potential implications such as the risk to elicit auto-immunity or insertion in human DNA. The latter could be at the basis of long COVID or, by accident, trigger oncogenic mechanisms.
I will first summarize both papers, then review the evidence of liver damage, including by auto-immune and other mechanism during COVID disease and after vaccination.
Next, I will quote the EMA report on Pfizer vaccine, come back with criticism by Merchant on the first papers and propose a conclusion.
Ep 275-1 A: Liguo Zhang PNAS May 2021 SARS-CoV-2 infection can lead to reverse transcription and integrate into the genome of cultured human cells and can be expressed in patient-derived tissues
o Reverse transcribed SARS-CoV-2 DNA was directly detected in infected cell lines (HEK293-T and Calu-3): mainly nucleocapsid sequences and disproportionately in coding parts (exons).
O It was also shown via RNAseq analysis of published data on cell lines or “organoids”: mainly human-viral chimeric DNA sequences (evidence of integration) with again mainly parts of the nucleocapsid (technical artefacts are considered unlikely)
o Finally, they also found in patient material (mainly autopsies) that “a large fraction of the viral RNAs could have been transcribed from SARS-CoV-2 sequences integrated into the host genome”.
While these partial host-viral chimeric DNA sequences cannot possibly produce living virus, they could:
- Make PCR reactions positive for a long time after symptoms have disappeared, which is diagnostically confusing;
- Such can also complicate interpretation of effectiveness of anti-viral therapy (you will never see complete PCR suppression even though there is no more replicating virus).
- When translated into peptides, they can induce immune responses (antibodies and T cells). The T cells can contribute to protective immunity, but possibly also to autoimmune reactions.
- Finally, it could also have a pathogenic role in long COVID: expression of viral fragments could trigger inflammatory reactions, with functional consequences according to the organ (GI, neurological, cardiovascular…..)
Ep 275-1 B: Alden Curr Issues Mol Biol Feb 2022: Intracellular Reverse Transcription of Pfizer BioNTech COVID-19 mRNA Vaccine BNT162b2 In Vitro in Human Liver Cell Line
In this paper, the authors provide evidence of massive accumulation of mRNA in lever cell line Huh-7, activation of LINE-1 and reverse transcription into Spike DNA, but without evidence provided for integration.
Unsurprisingly, this paper is used by the anti-vaxxers to warn against potential “auto-immune” “genetic” and “cancer” risks of the mRNA vaccines. The reasoning for oncogenic potential is that inserting of reverse transcribed RNA into the “wrong” place (e.g. tumor suppressor gene) could result in uncontrolled cell growth.
- Effects of SARS-COV-2 infection on the liver are common
Ep 275-2 A: Mengzhen Dong Biomedicine Pharmacotherapy reviews expression of ACE-2 and TMPRSS2 outside the lungs. Both receptors are found in the liver as mRNA: ACE-2 rather low in hepatocytes, but mainly in cholangiocytes while TMPRSS2 is moderately expressed in hepatocytes and cholangiocytes.
Ep 275-2 B: Yijin Wang J Hepatol Oct 2020 SARS-CoV-2 infection of the liver directly contributes to
hepatic impairment in patients with COVID-19
Investigation in hospitalized patients with severe and fatal SARS-CoV-2
- Liver enzyme abnormalities are common in patients with COVID-19 and associated with disease severity.
- SARS-CoV-2 is able to infect the liver and cause conspicuous hepatic cytopathy.
Arrows indicate Corona particles in hepatocytes of deceased COVID patients
Ep 275-3: Reviews on liver COVID-associated liver disease
- Zulal Ozkurt WJCC Feb 2022: hepatitis usually mild. Nice overview of potential pathogenic mechanisms
Cytotoxic T cells, potentially triggered by hepatocyte infection is one of several immune mechanisms.
- Mingjia Luo Hepat Inter May 2022: Patients with pre-existing (cirrhotic) liver disease are at increased risk of liver failure after COVID-19 infection.
Risk on severe COVID and mortality very high in patients with liver disease
Key A, B, C are gradations of severity of cirrhosis; ALD = alcoholic liver disease; AILD = auto-immune liver disease; HBV = chronic hepatitis B; HCV = chronic hepatitis C.
Ep 275-3 C: Singh Eur J Case Rep Int Med March 2021: A very rare case of genuine auto-immune hepatitis during COVID
A 57-year-old man with a medical history of hypertension, prediabetes and beta thalassemia minor, who was diagnosed with COVID-19 and subsequently developed fatigue and arthralgias, and whose blood work showed hyperferritinemia, elevated liver enzymes (AST/ALT/GGT), hypergammaglobulinemia, anti-smooth muscle antibody, anti-mitochondrial antibody, and anti-double-stranded DNA antibodies.
The patient was diagnosed with autoimmune hepatitis–primary biliary cholangitis overlap syndrome triggered by COVID-19.
- Liver damage after vaccination is rare
A few case reports after Adenoviral vaccines (Covishield = Astra-Zeneca)
Ep 275-4 A: Shorabhi Resp Med Case Rep Dec 2021: A 34 years old HCW developed an acute thrombosis of hepatic artery after 1st dose of Covishield, acute liver failure and death
Ep 275-4 B: Rela J Auto-Immunity 2022: Two cases of acute hepatitis after Covishield. One with clear-cut auto-antibodies responded well to prednisone therapy. The second without auto-antibodies but with massive cholestasis and inflammation died despite several therapies (including corticosteroids, plasma exchange etc…)
A number of cases after mRNA vaccination
Ep 275-5 A: Bril J Hepatol March 2021: first case of AI hepatitis post Pfizer vaccine in a 35 yrs postpartum women (with gestational hypertension) developed hepatitis with auto-antibodies, which responded to prednisone.
Ep 275-5 B: Garrido J Auto-Immunity Set 2021: AI hepatitis in 65 yrs woman after Moderna vaccine with polycytemia under peginterferon therapy: clear auto-antibodies and favorable evolution with prednisolone
Ep 275-5 C: Ghielmetti J Auto-Immunity July 2021: Acute autoimmune-like hepatitis with atypical anti-mitochondrial antibody after mRNA COVID-19 vaccination: A novel clinical entity?
Acute severe autoimmune-like hepatitis in a 63-year old male without a history of autoimmune diseases, with symptom onset one week after the first dose of mRNA-1273 vaccine. The patient responded well to prednisone treatment over two weeks.
As for other similar cases recently reported in the literature a causal link with the vaccine cannot be proven, but it is suggested by the temporal relationship in absence of other potential triggers or risk factors for AIH,
Evaluation of Pfizer vaccine by EMA (Ep 275-6)
Pharmacokinetics in rats after intramuscular injection of 50 µg mRNA in lipo-nanoparticles
Total recovery (% of injected dose) of radiolabeled LNP+modRNA outside the injection site was greatest in the liver (up to 21.5%) and was much less in spleen (≤1.1%), adrenal glands (≤0.1%) and ovaries (≤0.1%).(p.47)
No genotoxicity nor carcinogenicity studies have been provided. The components of the vaccine formulation are lipids and RNA that are not expected to have genotoxic potential (p. 55).
… As per guidance, no genotoxicity nor carcinogenicity studies were performed. The components of the vaccine (lipids and mRNA) are not expected to have genotoxic potential. This is acceptable to the CHMP. (p. 56)
Ep 275-7: Merchant comments in the same journal Curr Issues Mol Biol on the Alden paper (Ep 275-1 B) and Zhang (275-1 A)
- Huh-7 is a cell line, without the in vivo environment of the liver, including the immune cells, which will clear off Spike expressing hepatocytes, thus limiting their possibility to reverse transcribe mRNA.
- Vaccine dose for the in vitro experiment is much higher than what could by reached in the liver
- Huh-7 is a cancer cell with dysregulated (activated) LINE-1 expression, which in normal cells is strongly suppressed by epigenetic mechanisms
- SARS-CoV-3 only partly integrates and cannot result in productive infection.
Thus: the post-injection mRNA distribution and transfection to hepatocytes is not impossible but will trigger an immune response (cytotoxic T cells and anti-spike anti bodies) against the vaccine-transfected hepatocytes.
= transient and very specific towards ‘abnormal hepatocytes’, leading to the clearance of transfected hepatocytes by the immune cells; therefore, the reverse transcription of mRNA may not be possible in vivo.
- The liver can be a secondary target organ for COVID infection. The immune reaction against infected hepatocytes contributes to hepatitis, but it is usually mild. Hepatic failure is seen in the context of pre-existing cirrhotic liver disease. Genuine auto-immune hepatitis, in the context of COVID is very rare.
- mRNA LNP (representative of mRNA vaccine) are temporarily concentrated in the liver (see EMA report Ep 275-6). Auto-immune hepatitis upon vaccination is rare, often associated with pre-existing conditions and usually self-limiting, with good response to prednisone therapy.
- The paper by Liguo Zhang in PNAS (Ep 275-1B) provides evidence that upon infection parts of the SARS-CoV-2 RNA could be reverse transcribed and inserted by cellular mechanisms into genomic DNA, also in vivo, with resultant chimeric human-viral transcripts. These data need to be confirmed, but, as the authors speculate, this phenomenon could have diagnostic, therapeutic, but also pathogenic consequences (e.g. long COVID).
- The evidence from the Alden paper (Ep 275-1A) that vaccine Spike mRNA can be reverse transcribed is limited to purely in vitro observations in a rather artificial system, that is not sufficiently representative to extrapolate to the in vivo situation, as argued by Merchant (Ep275-7).
- Obviously, there is no direct indication that either SARS-CoV-2 infection or vaccination could pre-dispose to cancer, but that is rather expected on the long run and it will be difficult to ever prove or disprove it, because the entire world population has been exposed to the virus and/or the vaccines. So, where to find the controls? Previous trials with mRNA vaccines also did not suggest any oncogenic potential, but those were small and often in untreatable cancer patients or HIV patients.
- Until now, human oncogenic viruses are either DNA viruses (EBV, Kaposi Herpes, HPV, hepatitis B…) or retroviruses (HTLV-1 and -indirectly- HIV), with the possible exception of hepatitis C, which is an positive RNA virus. All of these viruses provoke chronic infections, while evidence for true chronicity of SARS-CoV-2 infection is limited to heavily immune suppressed subjects.
So, clearly, while it is not impossible that partial reverse transcription could happen upon infection, with some consequences, it seems very unlikely after vaccination. Nevertheless, since mRNA vaccination will tend to expand to other infectious diseases, further in vivo animal studies are warranted to ascertain the safety.
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