all received the AstraZeneca-Oxford vaccine published papers in The New England Journal of Medicine

On April 9, two teams of researchers who studied 11 patients in Germany and Austria and five in Norway who had all received the AstraZeneca-Oxford vaccine published papers in The New England Journal of Medicine.

Both teams of scientists found that the patients had unusual antibodies that trigger clotting reactions.

Of the 11 patients in Germany and Austria, nine were women, with a median age of 36 years.

Between five and 16 days after vaccination, ten of the patients presented with one or more thrombotic events. One patient had a fatal intracranial haemorrhage. All the patients had concomitant thrombocytopenia. None of the patients had received heparin before symptom onset.

Of the patients with one or more thrombotic events, nine had cerebral venous thrombosis, three had splanchnic vein thrombosis, three had pulmonary embolism, and four had other thromboses. Six of the patients died. Five patients had disseminated intravascular coagulation.

The researchers who studied the German and Austrian patients were led by Andreas Greinacher, who heads the Institute of Immunology and Transfusion Medicine at the Greifswald University Hospital in Germany.

Greinacher et al. suggested that some of the virus particles in the vaccine dose might break apart and release their DNA, triggering the production of antibodies.

They wrote that interactions between the vaccine and platelets or between the vaccine and platelet factor 4 (PF4) could play a role. The vaccine recipients who had clotting reactions had antibodies to PF4, the researchers found.

“One possible trigger of these PF4-reactive antibodies could be free DNA in the vaccine,” Greinacher et al. wrote.

As Chongxu Shi et al. point out in an article published in Frontiers in Immunology on October 7, 2020, extracellular DNA has been shown to contribute to the process of immunothrombosis.

Alternatively, Greinacher et al. said, antibodies might already be present in the patients and the vaccine may boost them.

“Whether these antibodies are autoantibodies against PF4 induced by the strong inflammatory stimulus of vaccination or antibodies induced by the vaccine that cross-react with PF4 and platelets requires further study,” Greinacher et al. wrote.

The researchers suggested naming “this novel entity” vaccine-induced immune thrombotic thrombocytopenia (VITT) to avoid confusion with heparin-induced thrombocytopenia.

Nina H. Schultz et al. in Norway studied five patients who presented with venous thrombosis and thrombocytopenia seven to ten days after receiving a first dose of the AstraZeneca-Oxford vaccine.

The patients were health care workers aged 32 to 54 years. All of them had high levels of antibodies to platelet factor 4-polyanion complexes, Schultz et al. said. Four of the patients had severe cerebral venous thrombosis with intracranial haemorrhage and three of them died. None of the patients had previous exposure to heparin.

One of the patients – a 32-year-old man – had severe thrombocytopenia and thrombosis of several branches of the portal vein and in the splenic vein, the azygos vein, and the hemiazygos vein. After treatment, his platelet count returned to normal and an abdominal CT scan indicated partial resolution of the thrombosis. He was discharged from hospital on day 12.

Greinacher and 23 other scientists from Germany published a preprint on Research Square on April 20 in which they state clearly that, rarely, the AstraZeneca-Oxford vaccine causes VITT that – like autoimmune heparin-induced thrombocytopenia – “is mediated by platelet-activating anti-platelet factor 4 (PF4) antibodies”.

They say their research has shown that AstraZeneca-Oxford vaccine constituents form antigenic complexes with PF4, that the constituent ethylenediaminetetraacetic acid EDTA, which is a calcium-binding agent and stabiliser, increases microvascular permeability, and that components of the vaccine cause acute inflammatory reactions.

“Antigen formation in a proinflammatory milieu offers an explanation for anti-PF4 antibody production,” Greinacher et al. wrote. “High-titer anti-PF4 antibodies activate platelets and induce neutrophil activation and NETs [Neutrophil extracellular traps] formation, fuelling the VITT prothrombotic response.”

NETs are networks of extracellular fibres, primarily composed of DNA from neutrophils, which bind pathogens.

“We have provided evidence that VITT is not a consequence of antibodies directed against the SARS-CoV-2 spike protein (produced by all vaccines) cross-reacting with PF4,” Greinacher et al. wrote.

The scientists say their findings indicate that it is the adenovirus vector-based vaccines that are at risk of inducing VITT through adenovirus and/or other PF4-DNA interactions.

“The degree of acute inflammatory response induced by the vaccine components appears as an important – potentially remediable – co-factor that could be diminished by reducing impurities and omitting EDTA,” they wrote.

Greinacher et al. say their biophysical analyses showed “formation of complexes between PF4 and vaccine constituents, including virus proteins that were recognised by VITT antibodies”.

They say their research showed that EDTA increased microvascular leakage in mice allowing for the circulation of virus and virus-producing cell culture-derived proteins.

“Antibodies in normal sera cross-reacted with human proteins in the vaccine and likely contribute to commonly observed acute ChAdOx1 nCov-19 post-vaccination inflammatory reactions,” the scientists wrote.

“In the presence of platelets, PF4 enhanced VITT antibody-driven procoagulant NETs formation, while DNase activity was reduced in VITT sera, with granulocyte-rich cerebral vein thrombosis observed in a VITT patient.”

The scientists laid out the sequence of events that their data suggests is mediating VITT. They say that, in step one, a neo-antigen is generated.

“Following intramuscular injection, vaccine components and platelets come into contact, resulting in platelet activation,” they wrote.

“ChAdOx1 nCov-19 vaccine activates platelet by multiple mechanisms including platelet interaction with adenovirus, cell-culture derived proteins (currently, it is unknown which of the > 1,000 proteins identified in the vaccine are involved in platelet activation), and EDTA.”

Activated platelets then release PF4, the scientists say.

In step 2, they say, an inflammatory co-signal is generated that further stimulates the immune response.

“EDTA in the vaccine increases capillary leakage at the inoculation site, likely by endothelial (VE)-cadherin disassembly.”

Greinacher explained further to Changing Times: “The first signal is the inflammatory signal. The vaccine constituents form antigenic complexes with PF4. This is facilitated by the open junctions and endothelial cells. This allows the immune cells to see the PF4.

“This all happens on day one or two after vaccination. The B cells then start to produce antibodies against PF4, which reach high titer in the blood circulation in the second week after vaccination.

“At that time, the vaccine is gone and the platelets are no longer activated by the vaccine. The primary inflammatory response is also gone.

“However, the resulting anti-PF4 antibodies become auto-antibodies that bind to PF4 on the platelets and activate them.”

The body erroneously thinks it is reacting to massive amounts of pathogens in the body, so the immune system overshoots, Greinacher explains.

The scientists say that proteins found in the vaccine include virus proteins, but also proteins originating from the human kidney-derived production cell line T-REx HEK-293.

“Increased vascular permeability facilitates dissemination of these proteins into the blood,” they wrote

Blood dissemination of vaccine components is not unique to the AstraZeneca-Oxford vaccine, they say.

“A ChAdOx1 vector variant (with a hepatitis B vector insert) was detectable by PCR in multiple organs, including liver, heart, and lymph nodes at days two and 29 after intramuscular injection in mice in preclinical studies reported by others,” they wrote.

The scientists say that, in step three, extracellular DNA in NETs binds PF4 “and resulting DNA/PF4 complexes further recruit anti-PF4 antibodies with lower avidity”.

The scientists say their study does have limitations. “The detailed specifications of the ChAdOx1 nCov-19 vaccine are not publicly available and potential impact of about 20 µg human cell culture proteins per vaccination dose remain to be assessed by the responsible regulatory agencies,” they state.

“Furthermore, we did not analyse the constituents of other adenovirus-based Covid-19 vaccines such as the Covid-19 Vaccine Janssen and the Sputnik V vaccine (these were not available to us). More importantly, quality control of vaccines requires the comprehensive methodological expertise of regulatory agencies.”

In another paper published on Research Square on April 9, Greinacher and a separate group of scientists concluded: “The antibody responses to PF4 in SARS-CoV-2 infection and after vaccination with Covid-19 Vaccine AstraZeneca differ.

“Antibodies against SARS-CoV-2 spike protein do not cross-react with PF4 or PF4/heparin complexes through molecular mimicry. These findings make it very unlikely that the intended vaccine-induced immune response against SARS-CoV-2 spike protein would itself induce VITT.”

Molecular geneticist Roland Baker says many scientists have suspected that the SARS-CoV-2 spike protein was involved in production of antibodies that cross-reacted with PF4.

“At this point we have to look at all the possible suspects and dismiss them as the cause one at a time by a process of elimination. The spike was an important contender.

“The finding by Andreas Greinacher et. al. puts to rest concerns that other vaccines producing a spike would have a similar risk. They clearly do not.

“Another factor was tPA [tissue plasminogen activator], but assuming the mechanism of VITT is identical for the Janssen Biotech and AstraZeneca-Oxford vaccines, then we can rule out tPA because it is used in the AstraZeneca-Oxford vaccines, but not in the Janssen Biotech one. So that leaves the adenovirus vector or the DNA as the likely suspects.”

Baker points out in a tweet, however, that the AstraZeneca-Oxford (ChAdOx1) and Janssen Biotech (Ad26.COV2.S) vaccines use substantially different vectors and spikes.

“Ad26.COV2.S features a human Ad26 vector of species D engaging CD46 as its cellular receptor with coding for a membrane-bound SARS-CoV-2 S protein in the prefusion conformation stabilised by two proline substitutions that does not shed S1 due to a KO furin cleavage site,” Baker tweeted.

“ChAdOx1 features a chimpanzee adenovirus vector of species E engaging Coxsackie and adenovirus receptor (CAR) as its cellular receptor and possibly others with coding for a membrane-bound wild-type S protein in the prefusion conformation which may may shed the S1 subunit.

“Shedding of the S1 subunit occurs during native infection and AstraZeneca may shed the S1 subunit as well.”