Connection between auto-antibodies and the rare reaction to adenovirus-vector vaccines

As the coronavirus XBB variant wave begins to crest, the World is getting a respite with a moderate increase in infection rates in many areas. Nevertheless Covid-19 still poses a major health hazard, and many questions remain regarding its pathophysiology. One mystery surrounds the rare blood clotting adverse events linked to the AstraZeneca (AZ) and Johnson & Johnson (J&J) adenovirus vector vaccines. Both vaccines have excellent safety records, but in the UK where tens of millions of doses of the AZ vaccine have been administered, a small number of unusual blood clotting events associated with the vaccine began to surface. The incidence rate is very low with a frequency of about 1 in 100,000 and a mortality rate of roughly 1 in 1,000,000. In addition, a better understanding of the disorder has led to faster recognition and improved treatment. In the U.S., use of the J&J vaccine has given rise to 54 cases of the unusual blood clotting leading to 9 deaths which corresponds to an incidence rate of 3 to 4 cases per 1 million vaccinated persons.

Early on, several research groups including the team led by German blood clotting expert Andreas Greinacher connected the dots between these deadly events and another rare clotting syndrome called heparin-induced thrombocytopenia (HIT). Heparin is a widely-used anti-coagulant (prevents blood clotting), but in HIT, heparin has the opposite of its intended effect through an interaction with Platelet Factor 4 (PF4) which together form a large complex. Binding to heparin causes neoepitopes (new binding surfaces) on PF4 to become exposed due to conformational changes, and in certain rare individuals, auto-antibodies (antibodies against self molecules) are induced that bind these neoepitopes resulting in immune clusters of antibodies bound to the PF4-heparin complex. These clusters activate platelets in an intense, self-destructive fashion via the platelet FcγRIIa receptors. The activated platelets produce excess clotting, but they also are quickly used up resulting in thrombocytopenia or the decreased level of platelets. 

The severe blood clotting events observed in a small number of subjects vaccinated with the AZ vaccine showed similarities to HIT. They exhibited out-of-control clotting in conjunction with a low platelet count, and most tellingly, anti-PF4 antibodies. The main difference was that this disorder was not induced by heparin. Dr. Greinacher and others have named the new syndrome vaccine-induced immune thrombotic thrombocytopenia (VITT).

One open question is how do the platelet-activating immune complexes form without heparin in VITT? In a paper that appeared in Nature last August, a research group investigated the binding properties of the anti-PF4 antibodies from VITT patients that provided some hints into a possible mechanism. More specifically, the researchers systematically mutated PF4 to determine where the auto-antibodies bound:

"The objective of this study was to determine the binding site on PF4 of antibodies from patients with VITT. Using alanine scanning mutagenesis, we determined the binding of VITT anti-PF4 antibodies (n=5) was restricted to 8 surface amino acids, all of which were located within the heparin binding site on PF4, and the binding was inhibited by heparin. In contrast, HIT sampled (n=10) bound to amino acids corresponding to 2 different sites on PF4."

The auto-antibodies in the VITT patients bound PF4 where heparin binds in HIT patients, and thus may substitute for heparin when forming the PF4 immune complex aggregates that activate platelets (Figure 1). One can speculate that some foreign antigen from the vaccine is inducing these VITT auto-antibodies that trigger the formation of platelet-activating aggregates even in the absence of heparin.

We do not know yet the identity of the foreign antigen(s) that may induce the anti-PF4 auto-antibodies, but in a previous post, I discussed the possibility that cryptic splice sites in the spike protein gene encoded in the adenovirus vaccine DNA vector might be utilized to create alternatively spliced mRNA which are translated into aberrant protein products, which in rare individuals could elicit these auto-antibodies. This would not be an issue with the mRNA vaccines in which the introns have been removed (VITT is less frequently observed with mRNA vaccines). Other possibilities for the culprit foreign antigen include some other component of the adenovirus particles, or some additive introduced during the manufacturing process.

VITT is very rare; B-cells that produce auto-antibodies tend to be eliminated during immune system maturation. But in some subjects, certain autoreactive B-cell clones can persist and give rise to autoimmune disorders such as rheumatoid arthritis or lupus. In this case, it appears that some very small number of people may have autoreactive B-cells that produce anti-PF4 antibodies that can be induced by the adenovirus vaccines.

Thus, the biochemical data presented in the new work add another piece to the puzzle, explaining how anti-PF4 auto-antibodies can substitute for heparin to form immune complexes that trigger platelets, as scientists continue to try to better understand these rare but severe events that are associated with the adenovirus vaccines.

Figure 1. "Proposed mechanism for VITT antibodies binding to and clustering PF4 tetramers, independent of heparin, and forming platelet-activating immune complexes. a, b, We postulate that VITT antibodies bind the antigen, PF4 (a), which in turn can cluster PF4 tetramers and create platelet-activating immune complexes in the absence of heparin (b). c, d, The immune complexes can be found on the platelet surface and in solution, resulting in the aligning and close proximity of the Fc part of these antibodies, which then are able to cross-link FcγRIIa receptors (c) and lead to platelet activation (d)" (Huynh et al. Nature, 2021).