COVID-19 Vaccine-Induced Pro-thrombotic Immune Thrombocytopenia (VIPIT): State of the Art

Abstract

In 2020, as the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic spread rapidly throughout the world, scientists worked relentlessly to develop and test the safety and effectiveness of potential vaccines. Usually, the vaccine development process involves years of investigation and testing prior to gaining approval for use in practice. A pathogenic PF4-dependent syndrome, unrelated to the use of heparin therapy, may be manifested following the administration of viral vector vaccines. It leads to severe clot formation at unusual sites approximately in 1 out of 110.000 vaccinated persons. This side effect, although rare, represents a newly devastating clotting phenomenon manifested in otherwise healthy young adults, who are often female. An in-depth description of the specific biological mechanisms implicated in the syndrome is here summarized.

1. INTRODUCTION

Following the development of the first vaccine in 1796 by Edward Jenner, vaccinations have achieved a high degree of success in preventing, or at times completely eradicating infectious diseases. Conversely, achieving herd immunity through natural infection takes a lot of time [1, 2].

In 1890, the scientist Emil von Behring, who won the Nobel Prize, used horse-derived antiserum as a therapy against diphtheria, thus sowing the seeds for larger use of passive immunotherapy [3].

Steady progress in clinical immunology led to using immune globulins and convalescent plasma, provided by affected people with high levels of antibodies, as prophylaxis for viral infection, such as the recent COVID-19 pandemic [1, 4].

In 2020, as the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic spread rapidly throughout the world, scientists worked relentlessly to develop and test potential vaccines. Usually, the process requires years prior to gaining approval for use in practice [5].

Human clinical trials of approximately 100 vaccines are currently ongoing worldwide, but approval will ultimately be granted to very few [6]. Vaccines elicit an immune response by triggering the development of antibodies and memory cells that prevent future infection from the same illness. A series of different methods are applied in the vaccine development process. Traditionally, a vaccine based on a harmless modified virus, an adenovirus incapable of reproducing in humans, was developed as an adjuvant to deliver viral genes into the body cells. A relatively new approach involves the development of a vaccine containing the genetic instructions required to make viral proteins in the form of mRNA. Once inside the body, the genetic code causes the body cells to produce distinctive proteins normally present in the virus. These proteins trigger an immune response, thus creating an immunological imprint and potential side effects [6-8].

2. GENETIC VACCINES

The new mRNA technology represented a dramatic change in the traditional approach to vaccine development. Vaccines with a non-viral vector developed to combat the coronavirus pandemic encode a stabilized ectodomain version of the immunogenic Spike protein specific to the coronavirus genes derived from the Wuhan-Hu-1 variant isolated in December 2019 in the host cell cytoplasm, displaying an efficacy exceeding 94% in preventing COVID-19 disease and eliciting a nAbs immune response [9].

In January 2020, BioNTech researchers embarked on engineering a genetic molecule-named messenger RNA (mRNA), creating the required genetic instructions for building a coronavirus protein known as Spike. Following injection of the resulting vaccine, spike proteins were formed and subsequently released into the body, thus eliciting an immune response. In November 2020, in a collaborative effort, the pharmaceutical giant Pfizer and the Germany-based company BioNTech published results highlighting for the first time an efficacy rate for their jointly developed coronavirus vaccine of more than 90 percent. In December 2020, a week after granting authorization for the use of the Pfizer/BioNTech vaccine, the US Food and Drug Administration (FDA) approved the emergency use of another mRNA-based vaccine developed by the Boston-based company Moderna [10].

3. VIRAL VECTOR VACCINES

A conventional vaccine developed in 2020 by the Oxford Vaccine Group in association with the Astra Zeneca pharmaceutical company entailed the genetic engineering of an antigenic component (spike protein) of the virus combined with an adjuvant adenovirus known to infect chimpanzees. When administered to monkeys, the vaccine was found to protect the animals against disease. Recombinant adeno-associated virus is a gene delivery tool used extensively in the field of research and clinical applications [11-13].

The ChAdOx1 nCoV-19 vaccine is made up of the replication-deficient simian adenovirus vector ChAdOx1, having the full-length structural surface glycoprotein (spike protein) of SARS-CoV-2, with a tissue plasminogen activator leader sequence. ChAdOx1 nCoV-19 expresses a codon-optimised coding sequence for the spike protein (GenBank accession number MN908947). In rhesus macaques, a single vaccination with ChAdOx1 nCoV-19 gives rise to humoral and cellular immune system responses. Protection against lower respiratory tract infection was noted in vaccinated non-human primates after a high-dose SARS-CoV-2 challenge. https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)31604-4/fulltext - bib9The low cost and ease of storage made the vaccine a particularly attractive solution. The vaccine was first approved for emergency use in the United Kingdom and Argentine on December 30^th 2020, with other countries rapidly following suit. Covishield, a version of the same vaccine manufactured by the Serum Institute of India, obtained approval on January 3^rd 2021. The same month, the European Medicines Agency (EMA) authorized the use of the AstraZeneca/Oxford vaccine [12, 13].

On February 16^th, the World Health Organization (WHO) granted approval for emergency use of the vaccine in adults over the age of 18 years. Brazil authorized the use of the vaccine on March 13^th. The Covax facility, with an expected annual manufacturing capacity of two million doses, commenced the delivery of millions of doses of the vaccine to low- and middle-income countries in March 2021. On February 27^th, the FDA granted authorization for emergency use of the Johnson & Johnson vaccine, the third coronavirus vaccine to be approved in the United States and the fourth in the EU [14]. The Johnson & Johnson vaccine differed slightly from the previously approved vaccines, as it required the administration of a single dose rather than two. Research work resulting in the development of the vaccine started almost ten years ago at the Beth Israel Deaconess Medical Center in Boston, based on the use of the human Adenovirus 26 (Ad26). Johnson & Johnson had previously adopted Ad26 in the development of Ebola and other types of vaccines [11].

4. COVID-19 DISEASE AND VACCINATION

SARS-CoV-2 infection is caused by a viral spike protein (S) comprising an S1 domain in turn containing an N-terminal domain (NTD), a C-terminal domain (CTD), and a receptor-binding domain (RBD), which facilitates binding to the entry angiotensin-converting enzyme 2 (ACE2) receptor [15], together with an S2 domain in which the fusion machinery is located, capable of resulting in systemic multiorgan failure accompanied by a risk of venous and arterial thromboembolic manifestations. Different studies have reported a variable incidence of thrombocytopenia in patients with COVID-19, with reports of mild thrombocytopenia in approximately one-third of patients and higher rates in patients with severe (57.7%) compared to non-severe disease (31.6%) [16, 17].

By the date of May 21^st 2021, the COVID‐19 pandemic had produced more than 145 million infections and surpassed 3 million deaths worldwide. Johns Hopkins University, however, reported that the number of actual infections is thought to be considerably higher than the number of cases confirmed to date [18].

A series of vaccines have been licensed and are currently being used in the European Union and USA to fight the COVID-19 pandemic. More than 534 million people globally are reported to have received at least one dose of a COVID-19 vaccine. Despite the huge efforts underlying the development of the coronavirus vaccines, much uncertainty remains over their degree of efficacy. Our knowledge of the human immune response to the virus and its variant forms is still lacking, and there is a possibility that the vaccines produced may not generate long-term protection and prevent reinfection. Administration of a vaccine may imply a lower probability of contracting the severe disease, although a yearly booster may be required, like for variants of flu vaccination. Though mRNA and Adenovirus vector vaccines both display high clinical efficacy, few serious adverse events have been reported as more people are vaccinated, and follow-up is extended, succeeding in the most extensive vaccination campaign ever set up over such a short period of time [19-21].

Some studies highlighted the onset of severe adverse thrombotic events following administration of ChAdOx1 nCoV-19 (AstraZeneca) [22-26] and then JNJ-78436735 Ad26.COV2.S (Johnson & Johnson/Janssen) vaccines [27-29]. In fact, extremely rare Cerebral Venous Sinus (CVST) and/or Splanchnic Venous Thrombosis (SVT), with thrombocytopenia and subsequent bleeding, were reported in subjects who had received adenovirus vector COVID-19 vaccines. The first vaccine–induced stroke case series was reported by the European Medicines Agency (EMA): 18 cases of CVST, 67% of whom had associated thrombocytopenia [30]. After that report, the Medicines and Healthcare products Regulatory Agency (MHRA) reported another 22 more cases amongst 18.1 million recipients in the United Kingdom [31]. However, further cases from Italy and other countries likely remained unpublished.

After new cases in European countries like Austria, Germany, Denmark, Italy, France, the United Kingdom, and Norway, the ChAdOx1 nCoV-19 vaccine was suspended by EMA but quickly resumed because of favourable benefit/risk ratio. By the end of April, a total of 169 cases of CVST and 53 cases of SVT were finally reported over 34 million vaccinations administered.

Greinacher et al. [23] provided a detailed report of the clinical and laboratory profiles of 11 German and Austrian patients who had developed thrombotic thrombocytopenia following administration of the AstraZeneca vaccine. Nine of these were females aged 20-50 years, 6 of whom presented with CVST 4-16 days after vaccination, and 3 died. Table 1 summarizes the first reports of pro-thrombotic thrombocytopenia syndrome following the administration of recombinant adenovirus vector vaccines.

Table 1.

First reports of pro-thrombotic thrombocytopenia syndrome following the administration of recombinant adenovirus vector vaccines.

Number of Patients	Vaccine	Counties	Age Range	Time from Vaccination to Symptoms	Deaths
11 (9 F) [3]	Astrazeneca	Germany and Austria	22-49 yr	5-16 d	1
5 (4 F) [4]	Astrazeneca	Norway	32-54 yr	7-10 d	none
13 (12 F) [7]	Astrazeneca	Germany	20-63 yr	4-16 d	none
12 (12 F) [10]	Jansen/Johnson&Johnson	USA	18-60 yr	6-15 d	3

Acronyms: F: female; yr: years; d: days.

In Sicily, six fatal cases of acute vein thrombosis, published in local newspaper, occurred within a few hours of ChAdOx1 nCoV-19 vaccination (Fig. 1). The clinical manifestation of thrombotic thrombocytopenic complications was suggestive of a disorder sharing pathophysiological features with heparin-induced thrombocytopenia (HIT), a well-studied prothrombotic condition induced by platelet-activating antibodies that recognize multimolecular complexes between cationic PF4 (platelet factor 4) and anionic heparin [32, 33].

Fig. (1).

Right internal jugular vein associated with CVST in a young Sicilian woman after ChAdOx1 nCoV-19 vaccination.

The condition was named VIPIT, i.e., a vaccine-induced prothrombotic immune thrombocytopenic syndrome, first affecting young women within 1-2 weeks from the vaccine administration [34].

Five more cases of CVST and thrombocytopenia were manifested in health care workers 7–10 days after receiving ChAdOx1 nCoV-19, none of whom had experienced previous exposure to heparin, but everyone displayed high levels of PF4/heparin polyanionic antibodies as reported from the Oslo University Hospital [26]. The authors concluded that the condition, also named “vaccine​-induced immune thrombotic thrombocytopenia (VITT),” was a new and rare but potentially fatal thrombotic event manifested in otherwise healthy young adults.

Allergic reactions to vaccine components have been hypothesized by some research groups as the triggering factor [34-36]. Kounis et al. [36] hypothesized an allergic inflammatory reaction triggering some cardiovascular manifestations resembling the typical Kounis syndrome. Questions, however, on why venous thrombosis primarily occurs with adenoviral vector vaccines and in the female gender most remain open. A common pathway connects these two conditions: these vaccines contain polysorbate excipient, a mixture of esters and etherates synthesized by oleic acid, ethylene oxide, sorbitan, and isosorbide largely added to creams and cosmetics used by women and capable of inducing hypersensitivity reactions and haemolysis.

Altogether, the cascade of GAG heparins, polysorbate, and sulphates could also explain the “non heparin” HIT-like thrombus formation in predisposed subjects [6, 8, 31, 33-36].

Many features were in common between HIT and VIPIT/VITT. HIT is a fascinating and still partially known immune syndrome that is linked to a harmful prothrombotic state and may lead to life-threatening complications as well [37]. The trigger is when PF4 is released from platelet α-granules during these cells' activation and, due to its high positive charge, binds to many negatively charged polyanions, heparin included. PF4 and heparin form large complexes that are highly immunogenic. The resulting immunoglobulin G antibodies are the cause of HIT, a prothrombotic adverse drug effect. In HIT, multimolecular complexes composed of PF4, heparin, and anti-PF4/heparin IgG cross-link platelet Fcγ IIa receptors, triggering platelet activation, microparticle formation, and thrombin generation with about half of the affected patients developing thrombosis. A minority of patients may develop symptoms and signs of HIT without any exposition to heparin, especially after orthopaedic surgery [38].

Restrictions to the use of the vaccine in Europe failed to attract much attention in the United States, as the vaccine had not been authorized by the FDA at the time. Millions of Americans who had received the vaccine suddenly became aware of the concerns following a temporary suspension by USA public-health officials on the use of the Johnson & Johnson vaccine due to the finding of a few cases of a similar, rare blood clotting syndrome at unusual locations [27, 29].

The world is currently undertaking a vaccination program, the likes of which have never been witnessed, accompanied by an unprecedented inquiry into ultra-rare severe adverse events. After careful consideration, the EMA concluded that the unusual clotting events should indeed be considered a side effect of the AstraZeneca vaccine. Since then, approximately 220 cases of dangerous blood abnormalities had been identified. To date, a mere handful of cases have been documented among vaccinated American people: 1 case per 100,000 vaccine recipients for the ChAdOx1 nCoV-19 vaccine [AstraZeneca] and 1 per 1,000,000 for the Ad26.COV2.S vaccine [Johnson & Johnson/Janssen], mainly in women. The cases have been characterized by immune thrombotic thrombocytopenia mediated by platelet-activating antibodies to PF4 [39].

5. DISCUSSION

Crucially, the biological relationship between vaccine administration and immune-mediated blood clotting disorders should be thoroughly investigated. This is the first challenge faced by the global immunization program undertaken to protect against the coronavirus pandemic. The new frontiers may potentially be exposed to the need to manage the onset of a series of severe side effects and to provide a response to unanswered questions. No major safety warnings, apart from rare cases of anaphylaxis, were reported in clinical trials conducted on tens of thousands of adults, and the risk of severe adverse effects continues to remain remarkably low following the vaccination of more than 400 million people worldwide [18].

Thrombosis of the large veins manifested in an atypical location (mainly cerebral, but also digestive associated with thrombocytopenia or coagulation disorders) have raised particular concern amongst the scientific community and heavily influenced public opinion The cause of such a strange location is still unknown, at the best of our knowledge [40].

COVID-19 infection is known to carry an important risk of blood clotting disorders itself. It would, therefore, be possible to speculate that the spike protein of COVID-19 may have maintained some of its thrombogenic properties, although to a significantly lesser extent than the whole virus. In this respect, Marschalek and Coll., from Goethe-University of Frankfurt, experimentally showed that potential splice events lead to the production of Spike protein variants thathave lost the membrane anchor. These variants may trigger severe side effects when binding to ACE2-receptor expressed on the surface of blood vessels endothelial cells. This mechanism has been then named the “Vaccine-Induced COVID-19 Mimicry” syndrome (VIC19M syndrome) [41].

Adjuvant vector vaccines may carry a further inherent menace. Adenovirus is a highly evolutionary pathogen extensively distributed throughout the animal kingdom, capable of poisoning a series of hosts ranging from mice to humans. The human adenovirus (HAdv) is remarkably efficient at infecting and replicating in human cells, thus rendering Adv-based vectors a convenient platform for the development of new therapeutic options. Regrettably, early studies have indicated HAdv as a potent activator of the innate immune response, at times giving rise to a manifold acute inflammatory response [34].

Mice studies have shown that rapid activation of the innate immune system produced by the intravenous administration of large quantities of adenovirus-based vectors may result in hepatic uptake and subsequent cytokine storm, activation of a coagulation cascade likely to lead to thrombocytopenia, disseminated intravascular coagulation and multi-visceral damage. Approximately 50 billion virus particles are needed to achieve a similarly toxic effect in a 20g mouse (2500 billion virus/kg) [34].

In comparison, the AstraZeneca vaccine contains 50 billion adenoviruses (0.7 billion viruses/kg based on a subject weighing 70 kg, a ratio 35,000 times lower than that administered to mice). It is feasible, therefore, to suppose that the vaccine is not overtly responsible for the reaction produced but more likely acts as an initial trigger for this rare mechanism [35].

Adenovirus capture by heparan sulphate chains on the surface of endothelial cells and the direct action of complement may be involved in the harmful side effects of the vaccine. The complement activation may also be linked with an autoimmune humoral reaction due to antigenic interplay between adenoviral epitopes and host epitopes, such as in type 2 HIT, where antibodies identify the heparin-PF4 complex as foreign. These antibodies activate platelets and cause specific thrombocytopenia. This may also be related to antiphospholipid antibody, which is responsible for autoimmune syndrome and has already been shown for SARS-CoV-2. The delay in the production of these autoantibodies may explain the onset of Adenovirus response within 4-14 days after injection, such as in type 2 HIT [34, 35].

The difference between the numbers of vaccinated individuals in Europe and the United Kingdom may provide additional clues, particularly in view of the apparent higher prevalence of disease in patients under the age of 55. In the UK, the majority of adults over the age of 50 have received the AstraZeneca vaccine, whereas, in Europe, the same vaccine was administered largely to adults below the age of 50 prior to the pharmacovigilance process set up in March 2021. Based on this observation, the vaccine response intensity may be relevant in the manifestation of the observed rare forms of thrombocytopenic thrombosis [40].

Excipients may play a role in triggering an extra intrinsic risk. The use of combined oral contraception in women under the age of 50 (4 out of 12 cases reported) and/or smoking may be further prognosticators for this rare side effect. In the same way as HIT, antibodies directed against a complex of heparin and PF4 are induced, in turn activating the CD32 receptor present on platelets, thus triggering the coagulation cascade, which leads to thrombosis even in patients who had never received heparin treatment [35].

Also, the presence in this kind of vaccine of polysorbate excipient, largely added to creams and cosmetics, mainly used by women, may be capable of inducing hypersensitivity cross reactions and haemolysis, as hypothesized by Kounis [36].

6. FUTURE PERSPECTIVE

So far, the vaccines approved in the United States and Europe seem to be capable of coping with the most concerning SARS-CoV-2 mutations. However, the fast-multiplying variants led investigators to wonder if a vaccine mixing doses of Pfizer and AstraZeneca could increase protection against these variants. A Strong Vaccine Cocktail trial is on the way in the United Kingdom to test the immune response in patients who receive a dose of the Pfizer mRNA vaccine and then a dose of AstraZeneca adenovirus viral vector vaccine, or vice versa, 4 and 12 weeks apart [42].

As a general rule, a stronger antibody response increases the body's chances to deal with more resistant variants. The strategy has been used in animal studies for years, and it is well known that this promotes a much better immune response than when immunizing with the same vaccine twice. This has also been shown in early human studies HIV trials over 25 years, though CDC warns against mixing.

How much protection does prior SARS-CoV-2 infection or vaccination provide against future disease is still an open question. Apparently, especially in the case of mild infection, the number of detectable antibodies against SARS-CoV-2 declines with time, thus raising concerns that humoral immunity against the virus may be short-time. However, in bone marrow aspirates of patients 7 to 8 months after infection, the presence of quiescent bone marrow plasma cells, which are a persistent source of protective antibodies, was demonstrated, along with a consequent long-time and possibly life-long immunity. However, viral variants potentially may make less strong some of the protection they offer [43].

CONCLUSION

Management of the COVID-19 pandemic is an onerous task for public health administrations worldwide, with vaccination against SARS-CoV-2 providing critical protection.

COVID-19 disease is known to be associated with venous thrombosis caused by the induced presence of hypercoagulable blood in approx. 1 in 5 patients. The risk of venous clots (thrombosis) manifested in the lungs as pulmonary embolism, in the legs as deep vein thrombosis, or, less often, in the brain as cerebral venous sinus thrombosis is highest in those with severe COVID-19 disease treated in an intensive care unit [44, 45].

A pathogenic PF4-dependent syndrome, unrelated to the use of heparin therapy, may be manifested following the administration of viral vector vaccines. A thorough risk/benefit profile should be evaluated for VITT, which, although rare, represents a newly devastating phenomenon manifested in otherwise healthy young adults.

A better understanding of the specific biological mechanism implicated in the above- described clinical syndrome is mandatory. Clinical and laboratory awareness of this novel syndrome allows for its rapid identification as an appropriate therapeutic approach and prevents the onset of VIPIT/VITT/VIC19M syndrome. Current evidence, however, indicates the risk for CSVT [46] to be 10 times higher in COVID-19 infected patients compared with those receiving the COVID-19 vaccines.

The combination of stimulated autoantibodies against PF4, impurities of the vaccine itself, and potential splice events with a production of Spike protein variants is a strong pro-inflammatory and pro-thrombotic environment that leads to severe clinical events approximately in 1 out of 110.000 persons. Very few cases of VIPIT have been described with mRNA vaccines as well, but they seem to be anecdotal [47].

We are still far from winning the war against COVID-19 direct and indirect harmful consequences [48].

CONSENT FOR PUBLICATION

Not applicable

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.