The histologic and molecular correlates of COVID-19 vaccine-induced changes in the skin

A total of 22 patients who had developed an adverse cutaneous reaction to the Moderna or Pfizer vaccine underwent biopsies. Each patient was assessed light microscopically, and, in select biopsies, spike glycoprotein and cytokine assessment were also conducted. The patients developed self-limited cutaneous reactions often described clinically as urticarial or eczematous within 1 day to 4 weeks after receiving the first or second dose of the Pfizer or Moderna vaccine. Classic clinical and morphologic depictions of type IV cutaneous hypersensitivity with features of eczematous dermatitis, interface dermatitis, granulomatous inflammation, and/or lymphocytic vasculitic component were observed. Clinical and/or histologic features of perniosis, pityriasis rosea, pityriasis rubra pilaris, and guttate psoriasis were seen in select cases. In 2 cases the dominant picture was urticarial vasculitis, possibly reflective of an Arthus type III immune complex action. The biopsy specimens of normal skin post vaccine and of skin affected by the post-vaccine eruption showed rare deep microvessels positive for spike glycoprotein with no complement deposition contrasting with greater vascular deposition of spike protein and complement in skin biopsies from patients experiencing severe coronavirus disease 2019 (COVID-19). It is concluded that self-limited hypersensitivity reactions to the vaccine occur possibly owing to a substance found in the vaccine vehicle (eg, polyethylene glycol). An immune response that is directed against human-manufactured spike has to be considered because some of the reactions clinically and or histologically closely resemble mild COVID-19. Finally, vaccine-associated immune enhancement largely attributable to the adjuvant properties of the vaccine may unmask certain inflammatory milieus operational in psoriasis, atopic dermatitis, and subclinical hypersensitivity.


Introduction
More than 176 million cases of coronavirus disease 2019 , with more than 3.8 million deaths worldwide, have been reported. COVID-19 is a pandemic that has resulted in sweeping social changes but, at the same time, has drawn a unified front globally to bring its end to fruition. With the advent of a number of effective vaccines, worldwide herd immunity is predicted in the near future. Each COVID-19 vaccine has as its epicenter of functionality an immune response to the spike glycoprotein, the critical viral capsid protein that binds to angiotensin converting enzyme 2 (ACE2), obligatory for viral entry. [1][2][3] The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines do not prevent infection. They do however thwart serious forms of COVID-19 by eliciting an effective T-cell-mediated and humoral-(antibody-) mediated immunity response. The two most commonly administered vaccines in the United States are the Moderna vaccine, which is a nucleoside modified messenger RNA that encodes SARS-CoV-2 spike protein (mRNA-1273), and the Pfizer-BioNTech mRNA, which encodes the same protein (BNT162b2). 1 , 4 , 5 Also available is the Johnson & Johnson vaccine (Ad26.COV2.S), which is viral-vector based and uses a modified version of adenovirus 26 to encode the spike protein. The vaccines focus on the receptor binding protein of SARS-CoV-2, namely the spike glycoprotein, engineering our cells to produce this foreign protein and hence promoting an immune response that is effective in preventing significant disease complications from COVID-19. Paradoxically it is the spike glycoprotein that likely plays an important role in the pathogenesis of severe and critical COVID-19 and has been postulated as being at the crux of the vascular complications of  We have demonstrated in earlier studies that the SARS-CoV-2 spike glycoprotein attaches to endothelium via ACE2 and results in complement-mediated microvascular injury in the lung and in other microvascular fields where endothelia have high ACE2 + expression such as the skin and the brain. 6 The basis of the complement activation is that the spike glycoprotein has specific sugar moieties that are recognized by mannan-binding lectin, leading to the activation of MASP-2, ultimately resulting in the formation of C5b-9, which then damages the cell membranes of the endothelium. 7 Data have suggested that, in sites other than the lung and the nasopharynx, the spike glycoprotein engagement with endothelium is without intact virus, as revealed by the lack of viral particles on electron microscopy and the absence of any detectable viral RNA in situ . 7 Similarly, mice injected with large doses of the S1 subunit (but not S2 subunit) of the spike protein developed neurologic signs associated with central nervous system vascular injury; the spike protein was evident in their damaged central nervous system microvessels. 8 Thus, one could postulate that the spike glycoprotein that is synthesized by the myocytes after receiving the mRNA-based vaccine could disseminate to select ACE2 + microvessels. Based on the millions of people already vaccinated without incident, if this microvascular dissemination occurs, it appears to be at a level that is not clinically significant.
To date, no adverse microvascular or larger vessel thrombotic events that resemble severe and critical COVID- 19 have occurred with either the Moderna or the Pfizer vaccine. Catastrophic thrombotic complications involving the sagittal sinus and splenic veins with the adenovirus vector DNA vaccines manufactured by AstraZeneca and Johnson & Johnson have been reported. The exact basis is unknown, but a clinical parallel has been made with heparin-induced thrombocytopenia in which patients develop antibodies to heparin and platelet 4 complex . They have recently shown that human platelets express ACE2 and TMPRSS2, the surface serine protease for spike protein priming. They went on to demonstrate that SARS-CoV-2 spike protein directly enhanced platelet aggregation and clot retraction in vitro , and thereby spike protein resulted in thrombus formation in wildtype mice transfused with hACE2 transgenic platelets. Furthermore, recombinant human ACE2 protein and anti-spike monoclonal antibody-inhibited SARS-CoV-2 spike proteininduced platelet activation. 9 We encountered 22 patients who developed cutaneous reactions temporally associated with the vaccine administration. All the patients were biopsied, and the samples were sent to our laboratories for diagnostic evaluation. We studied the nature of the adverse immune response to the vaccine and assessed for any evidence of cutaneous viral spike protein localization and microvascular complement pathway activation and cytokine expression within the cutaneous microvessels in select cases. 10 Because a biopsy of normal skin can document systemic complement activation and the localization of spike glycoprotein in cutaneous microvessels in the setting of severe and critical COVID-19, 2 of the authors underwent a biopsy of unremarkable deltoid skin a few weeks after receiving the first dose of Pfizer vaccine (before receiving the second vaccination) and 1 of the authors underwent a biopsy after receiving the Johnson & Johnson vaccine. Our intent was to explore whether vaccine-derived spike glycoprotein could localize as pseudovirions to the cutaneous microvessels and, if so, whether it could have the same potential effect on endothelium that we observe in the cutaneous ACE2 + microvessels of patients with severe and critical COVID-19. 7 , 10 , 11

Materials and methods
The skin biopsy specimens of 22 patients who developed cutaneous eruptions after receiving the COVID vaccine in which the clinical diagnosis was one of a vaccine triggered hypersensitivity reaction were studied. Deltoid skin biopsies from 3 people who died of COVID-19 and 5 pre-COVID-19 skin biopsy specimens were also included.  The biopsy shows an interface dermatitis mediated by lymphocytes with concomitant papillary dermal edema and a subjacent necrotizing lymphocytic and granulomatous vasculitis with evidence of vascular compromise as characterized by red cell extravasation (hematoxylin and eosin [H and E], 200 ×). Higher power magnification demonstrates the extent and nature of the vascular injury pattern. It is one that involves the capillaries and venules largely confined to the superficial corium whereby inflammatory cells course through the vessel wall, and it is associated with fibrin along with red cell extravasation. (B) The infiltrate is predominated by lymphocytes and histiocytes defining a hybrid lymphocytic and granulomatous vasculitis, but there is also a smattering of neutrophils (H and E, 400 ×). (C, D) A microvessel is visible in the deep dermis, demonstrating spike and interleukin 6. The microvessels in the papillary dermis, where the inflammation is has no spike glycoprotein. Using a previously published protocol 6 , 7 blinded to the clinical information, we tested tissues for the viral spike protein (a cocktail that can detect the S1, S2, and receptorbinding domain subunits), the SARS-CoV-2 membrane and envelope proteins, interleukin (IL)-6, caspase 3, ACE2, tumor necrosis factor α (TNF α), C3d, C4d, MASP-2, and C5b-9. Coexpression analysis was used with the Nuance system (Nuance, Burlington, MA). 6 , 7 We examined deltoid skin biopsy specimens from 3 physician authors of this report who did not have COVID-19 infection and who had received the mRNA-based COVID-19 vaccine at days 10 and 14, and at 10 days with the Johnson & Johnson vaccine, respectively, post-vaccination. To evaluate for evidence of systemic complement pathway activation, a fourth deltoid biopsy specimen from the normal skin of a 21-year-old woman was tested to evaluate for evidence of systemic complement pathway activation. She developed myocardial insufficiency temporally associated with the administration of the Moderna vaccine. The study is covered under the institutional review board protocol 20-02021524.

Post-COVID-19 vaccine cutaneous eruptions in patients (22 cases)
Skin biopsies were encountered in our routine diagnostic dermatopathology practices to evaluate generalized skin eruptions that had developed after patients had received the COVID-19-associated vaccines ( Table 1 ). The patient population was represented by 10 women, and 12 men ranging in age from 23 to 96 years, with a median age of 53 years. In all cases, the onset of the eruption was temporally associated with the administration of the vaccine and was characterized by a generalized papulovesicular, eczematous dermatitis, and/or urticarial eruption in most cases ( Figures 1 A, 2 A, 3 A, 4 A and B, 5 A, 6 A, 7 A, 8 , 9 A, 10 ). One patient developed Grover disease 1 week after receiving the Moderna vaccine. Another patient's symptoms were consistent with guttate psoriasis, presenting with red scaly macules all over the body for 2 weeks after the second dose of the Pfizer vaccine ( Figure 8 A). A vasculitic presentation was noted in four patients, including two patients who had acral lesions resembling perniosis and two patients who had urticarial vasculitis. After receiving the Moderna vaccine, 11 patients developed symptoms. After receiving the Pfizer vaccine, seven patients developed symptoms. In four patients the vaccine administered was not known. The reactions occurred after the first dose in five patients and after the second dose in nine patients. In eight patients it was not known whether the eruption occurred after the first or second dose. The reactions developed 1 day to 4 weeks after receiving the vaccine. In 17 pa-  tients a more precise timing between vaccine administration and eruption development was known. In eight patients the eruption developed within 1 week after receiving either the first or second dose of the vaccine, including five patients where the eruption developed within 48 hours after receiving the vaccine. In nine patients the reaction was more delayed, developing 8 days, 9 days, 10 days, 12 days, 2 weeks, 3 weeks, and 4 weeks after receiving the vaccine. Some degree of arthralgias and fever were common. Joint swelling was observed in two patients including one patient who had a folliculocentric vesiculopustular eruption ( Figure 9 A) and another who had thrombocytopenia and hemolysis. One patient had peripheral blood eosinophilia.

Light microscopy
The dominant histologic patterns included eczematous dermatitis (10 cases . 12 In addition, in 1 case there were pustules noted clinically although there was no histologic documentation of a pustular diathesis. The clinical impression was acute generalized exanthematous pustulosis. Another case presented a photo-distributed papular eruption on an erythematous base. In most cases there were other overlapping morphologic reaction patterns defining a hybrid dermatitis, including cases showing combined eczematous, interface, vasculitic, and interstitial granulomatous features. The most common pattern was concurrent interface and eczematous dermatitis identified in six cases. Tissue eosinophilia was common. In all cases the clinical impression was congruous with the histologic findings, and the eruptions resolved either spontaneously or with topical or systemic steroid therapy except the case in which the eruption had persisted for 4.5 months. The patient was then given a trial of ustekinumab. One case was compatible with guttate psoriasis, another T-cell-mediated process in which it has been established that an exogenous antigenic trigger is frequently implicated albeit typically in the context of streptococcal antigen ( Figure 8 B). In another case an unusual picture of interface dermatitis with concomitant features of pityriasis rubra pilaris was observed. The type I interferon signature was upregulated in four out of five cases tested including 1 case of perniosis.

Immunohistochemical assessment for spike glycoprotein, complement deposition, and endothelial cytokine expression (IL-6, caspase 3, and TNF α)
The immunohistochemical stain to assess for spike glycoprotein was conducted on 12 of the cases. The basis for doing the stain was to document evidence of human synthesis and establish the ability of spike glycoprotein to dock to ACE2 + vessels as a pseudovirion, a hypothesis proffered as the basis of systemic complement activation in the setting of severe and critical COVID-19. We were able to document spike glycoprotein in the cutaneous microvasculature in 10 cases tested. In particular, there were rare deep-seated vessels in the reticular dermis and subcutaneous fat that showed fo- cal detection of spike glycoprotein in endothelium reflecting the preferential expression of ACE2 in the deeper microvessels. There were typically only one or at most a few positive staining vessels (fewer than 5) ( Figures 11 C and D, 12 D). A similar distribution was observed for IL-6, caspase 3, and/or TNF α, but with no significant microvascular complement deposition ( Figure 10 C). The overall amount of microvascular spike glycoprotein and ACE2 expression was much less than that observed in the setting of thrombotic retiform purpura of severe and critical COVID-19.

Post-vaccine normal deltoid biopsies in patients without symptoms
Serial sections of each of the two normal post-vaccine skin biopsy specimens demonstrated a lymphocytic vascular reaction localized to a single vessel in the subcutaneous fat in one biopsy specimen. In both biopsies there was expression of ACE2 in endothelium in deeper dermal and subcutaneous microvessels. The deltoid skin biopsy specimens from patients who died of COVID-19 showed significant microvascular endothelial cell localization of spike glycoprotein, whereas the pre-COVID-19 skin samples were negative ( Figure 15 ). In the two normal biopsy specimens taken after the patients had received the Pfizer vaccine, occasional endothelial cells in the microvessels of the deep dermis and subcutis were positive for spike glycoprotein (fewer than 5 microvessels with some degree of positivity in   the endothelial lining cells). Unlike the deltoid skin biopsy specimens from the patients with severe COVID-19 that also contained the envelope and membrane viral proteins within the endothelium, these additional viral capsid proteins were absent in the biopsy specimens taken after the patients had received the Pfizer vaccine (data not presented) ( Figure 16 ). Complement studies to assess for C3d, C4d, and C5b-9 deposition were conducted on both cases, and only rare vessels showed any evidence of complement deposition and hence did not support a diagnosis of systemic complement activation. Nuance software (Nuance) coexpression analysis showed coexpression of spike glycoprotein with caspase 3, TNF α, and IL-6 ( Figure 16 ). An additional deltoid biopsy specimen was procured from another physician author who received the Johnson & Johnson vaccine. Spike glycoprotein was not identified Figure 17 .

Post-vaccine normal deltoid biopsy in a patient with post-vaccine acute myocardial insufficiency
A deltoid biopsy specimen was procured from a 21-year-old patient who went to the emergency room on March 12, 2021, with chest pain radiating into her left arm 2 days after she received her second Moderna vaccination. The skin biopsy specimen exhibited a minimal perivascular lymphocytic infiltrate. The C5b-9 studies showed roughly 8 positive staining vessels including capillaries, venules, and a single arteriole, and hence did not meet criteria for evidence of systemic complement activation. A single deep dermal vessel showed spike glycoprotein, TNF α, and caspase 3 positivity. .

Discussion
We have presented a series of adverse cutaneous responses temporally associated with the administration of the first or second dose of the Pfizer or Moderna COVID-19 vaccines. Patients from all age ranges could develop the vaccine reaction with either the Pfizer or Moderna. The eruptions were typically generalized and had an urticarial or eczematous appearance. In our series, reactions were more commonly observed with the Moderna vaccine rather than with the Pfizer vaccine. In addition, the adverse reactions could develop either after the first dose or the second dose, although it was almost twice as common after the second dose compared with the first dose. The reaction could develop as quickly as 48 hours after the vaccine was administered or could be delayed for as long as 4 weeks after the vaccine was administrated, with almost half of the cases developing within a week of administration of the vaccine.

Histologic patterns resembling hypersensitivity
The more common histologic patterns were eczematous dermatitis, interface dermatitis, interstitial granulomatous dermatitis, and lymphocytic vasculitis including two cases of perniosis. One of the hallmarks of the vaccine reactions that we encountered was a hybrid inflammatory pattern. A case could show a mixed pattern best exemplified by cases of interface and eczematous dermatitis with an accompanying mild lymphocytic vasculitis. These specific histologic patterns are commonly reflective of underlying type IV hypersensitivity. One case associated with a striking pattern of interface dermatitis had concomitant pityriasis rubra pilaris-like changes. This patient has had a persistent severe generalized skin eruption for at least 4 months refractory to prednisone and has begun ustekinumab therapy. One biopsy specimen showed a distinctive folliculocentric immune   reaction that falls under the category of sterile neutrophilic folliculitis with folliculocentric vascular injury. 13 Pathogenetically, this type of sterile neutrophilic follicular reaction has been hypothesized to represent a TH1 dominant type IV immune response in which the cytokine milieu is conducive to a neutrophilic influx into the skin. 13 , 14 A minor subset of cases demonstrated an urticarial vasculitis, a morphologic subset of leukocytoclastic vasculitis characteristically triggered by immune complex deposition but other proinflammatory pathways can be implicated. One might consider a scenario in which antibodies bound to an undefined foreign protein introduced by the vaccine could be deposited in microvessels as an immune complex and trigger the classic complement pathway to result in a neutrophil-rich inflammatory reaction.

Post-vaccine reactions resembling a dermatosis with an underlying genetic predisposition
One patient developed eruptive psoriasis, expanding the clinical and morphologic spectrum of type IV T-cell immune responses.
Based on certain COVID-19 vaccine trials cutaneous reactions are becoming increasingly recognized and while psoriasis was not originally described in some of the initial trials, the influenza vaccine has been recognized as a trigger for guttate psoriasis. 15 -18 Molecular mimicry between streptococcal antigens and keratins in the epidermis of patients with eruptive psoriasis underlies the association between streptococcal pharyngitis and guttate psoriasis. There is structural homology between spike glycoprotein and M6 protein implicated in guttate psoriasis although not specifically the spike glycoprotein of SARS-CoV-2. Vaccine-associated immune enhancement, however, may play a role in unmasking psoriasis in a genetically predisposed patient. Two patients also showed changes of Grover disease histologically although mechanistically its basis is unclear.

Post-vaccine cutaneous reactions literature review
Other authors have described cutaneous eruptions in patients who have received the Pfizer or Moderna COVID-19 vaccine, both in the context of generalized eruptions as well as in erythema at the vaccination site. Farinazzo et al. described the first registered cases of cutaneous adverse reactions in North-East Italy after patients had received the Comirnaty-BioNTech/Pfizer mRNA COVID-19 vaccine in Trieste. 19 They reported one or more cutaneous adverse effects in 0.22% of all vaccinated individuals and 16.54% of communicated adverse effects in all vaccinated individuals. The reactions were divided into those at the vaccine site and those that were discontiguous and more generalized. The reactions included urticaria, malar erythema, a hand eruption, pityriasis rosea, and a reaction that resembled a fixed drug eruption. Reactions were seen in patient who had received the first dose or the second dose of the vaccine in the absence of a reaction to the first dose and could appear as quickly as 60 hours from the time of the initial vaccination. In trying to establish the trigger, it was suggested that polyethyleneglycol 200, which is a known cause of immediate type I hypersensitivity as well as delayed reactions, could be responsible. Other terms have been used to describe the polymers such as macrogol, oxyethylene polymer, and laureth. In a second review that examined more than 400 adverse cutaneous reactions collected from a US-based database, more than 80% of cases were seen in association with the Moderna vaccine. 17 Less than 20% of the reactions were associated with the Pfizer vaccine. 17 There was a predominance of the adverse vaccine reaction in women. The adverse reaction could occur after the patients received the first or second dose of the vaccine, and if it occurred after the first dose there was a 40% likelihood that it would occur again after the second dose. There were two basic time frames: an immediate one that occurred at 1 to 3 days and a delayed one developing 6 to 7 days after the vaccine. The reactions included swelling at the vaccine site, morbilliform eruptions, pityriasis rosea-like, and chilblains/perniosis. We have seen cases of post-vaccination erythema at the site of the vaccine including one case with concomitant lymphangitis but with no biopsy to confirm the nature of the inflammatory response. Authors have suggested certain agents used in the vaccine vehicle as the putative trigger such as polyethylene glycol. 17

Role of a product in the vaccine vehicle as an allergen
When one considers vaccines in general and not specifically in regard to the SARS-CoV-2 mRNA vaccines, the common constituents found in diverse vaccines have elicited a variety of systemic allergic contact dermatitis reactions. The most common culprits are antibiotics (eg, neomycin) used as cer-tain key preservatives such as formaldehyde, propylene glycol, and sorbic acid. In each patient that develops an adverse reaction, one could consider patch testing to determine which component elicits the immune response.
The cutaneous reactions associated with the COVID-19 vaccine appear to be self-limited. The histology suggests that two common limbs of hypersensitivity observed with other exogenous antigens could be the basis, although unproven. The dominant pattern is one that would be consistent with a systemic eczematoid hypersensitivity reaction. In our series and consistent with other reported studies, the most common pattern is one resembling type IV hypersensitivity characterized by an eczematous dermatitis and or a concomitant cytotoxic interface dermatitis. One might suggest that the antigen could be a substance in the vehicle used to administer the vaccine, although a T-cell and/or humoral reaction to the myocyte-manufactured spike glycoprotein emerges as a putative antigenic trigger, especially given its localization to the cutaneous microvessels. An antigen unrelated to the vaccine, but in which an immune response to the antigen becoming unmasked owing to vaccine-associated immune enhancement, is possible.
All COVID-19-vaccine reactions were observed in patients after they received the Moderna vaccine or the Pfizer vaccine. The Johnson & Johnson vaccine is a viral vector vaccine using a replication-incompetent recombinant adenovirus vector that expresses the SARS-COV-2 spike protein in a stabilized conformation. The stabilized version includes two mutations in which amino acids are replaced with prolines. In addition, the vaccine has inactive ingredients like citric acid monohydrate but does not include polyethylene glycol. The combination of these factors may potentially define the basis for why the Johnson & Johnson vaccine has not been associated with these hypersensitivity reactions. [18][19][20]

Mechanism of systemic contact dermatitis could be implicated in the post-vaccine reactions
Prior studies on systemic contact dermatitis 21 , 22 have suggested that cross reactivity between the systemically administered antigen and a topical agent to which the patient has been sensitized could underlie the pathophysiology of the systemic eczematoid reaction. Antigenically primed memory T cells along with Langerhans cells are involved, 21 with recruitment of memory T cells to sites on the skin where the topical agent was previously applied. Discontiguous sites could become inflamed analogous to the cutaneous interface dermatitis reaction occurring at cutaneous sites distant from the primary dermatosis. The allergens, rather than being topical, are travelling through the circulation by varying routes such as ingestion, intravenous administration or intramuscular injection such as through a vaccine. The clinical presentation is somewhat diverse-among the cutaneous manifestations are an eruption at the prior site of allergic contact dermatitis, an eruption at a site of a prior patch test, vesicular hand dermatitis, and pruritic papules on the elbows and knees, erythroderma, and small vessel vasculitis like lesions. After metabolism of the causative systemic antigen, such as a drug in the skin where it functions as a hapten-carrier complex, the antigen is processed by antigen-presenting cells and leads to clonal expansion of T cells in the local lymph node, which can then migrate to the skin and elicit a type IV immune response. There is a preferential sequestration of memory T cells expressing cutaneous lymphocyte antigen into the skin, leading to a relative decrease in memory T cells in the peripheral blood. Another mechanism is the so-called p-1 concept whereby the drugs are bound directly to a T-cell receptor. There is no direct presentation with the major histocompatibility complex and there is no prior metabolism. Regardless of the exact mechanisms, one can explain at least some of the vaccine-associated cutaneous reactions through the mechanisms that have been advanced for systemic contact dermatitis because the well-known cutaneous sensitizer-polyethylene glycol-is found in both Pfizer and Moderna COVID-19 vaccine preparations. 21 Spike glycoprotein as the potential stimulus to the post-vaccine reaction Given that some of the reactions resemble classic cutaneous manifestations encountered in mild COVID-19, such as pityriasis rosea (ie, those cases showing a hybrid interface and eczematous dermatitis with a concomitant low-grade lymphocytic vascular injury), interstitial granulomatous inflammation, small vessel vasculitis, and chilblains/perniosis, one has to consider that an additional potential candidate for this vaccine reaction could be the novel protein manufactured by the genetically altered myocytes.
Another question is whether the localization of the hypersensitivity in any way relates to the in situ cutaneous localization of spike glycoprotein because of the relatively high level of ACE2 expression in the cutaneous microvessels relative to other organ sites. Indeed, the immunohistochemical stain to assess for spike glycoprotein showed consistent reproducible findings in all samples tested. The basis for doing the stain was to document evidence of human synthesis and to establish the ability of spike glycoprotein to dock to ACE2 + vessels as a pseudovirion, a hypothesis offered as the basis of systemic complement activation in the setting of severe and critical COVID-19. We were able to show spike glycoprotein in the cutaneous microvasculature in all cases tested. In particular, there were occasional deep-seated vessels in the deep reticular dermis and fat that showed focal expression of spike glycoprotein in endothelium, which is not surprising as it reflects the preferential expression of ACE2 in deeper dermal and subcutaneous microvessels. There were only rare positive-staining vessels, however. A similar distribution was observed for IL-6, caspase 3, and/or TNF α, but with no significant microvascular complement deposition observed. The overall amount of spike glycoprotein in the microvessels was much less than what we observed in the setting of thrombotic retiform purpura of severe and critical COVID-19. Even though the spike is produced for the lifetime of the muscle cell (ie, 10 to 16 years), the neutralizing antibody in response to the manufactured spike would likely prevent spike glycoprotein localization to distant ACE2 positive microvascular beds after humoral immunity is achieved; however, during that nascent period during which complete adaptive immunity has not been reached to neutralize spike glycoprotein binding, an immune response to circulating pseudovirions is very possible ( Figure 18 ).

Post-COVID-19 vaccine reaction is largely a cutaneous confined reaction but not every case
The lack of systemic and/or multiorgan parenchymal dysfunction implies that the skin is selectively targeted in these adverse cutaneous reactions. In many cases, the cutaneous reaction is not part of a multiorgan adverse hypersensitivity response triggered by the vaccine but rather that inflammation appears to be limited to the skin in most cases.
A few outliers developed in which the reaction was characterized by more severe joint swelling and a folliculocentric vasculitic process in a 27-year-old woman and a 23-yearold man who developed fever, hemolysis, thrombocytopenia, and cutaneous lymphocytic and granulomatous vasculitis. Cases of hemolysis occurred after the COVID-19 vaccine was administered to patients with antecedent histories of hemolysis attributable to paroxysmal nocturnal hemoglobinuria where it has been postulated that the inflammatory response associated with the vaccine is a trigger to complement pathway activation as opposed to the direct effect of spike glycoprotein on causing red cell hemolysis. 23 In this particular case, although the skin eruption was typical for a COVID-19-vaccine reaction given the combination of interface dermatitis along with lymphocytic and granulomatous vasculitis, there was also evidence of systemic complement pathway activation. The patient was not known to have any prior disease associated with hemolysis such as atypical hemolytic uremic syndrome. Another 21-year-old patient developed myocardial dysfunction, and imaging studies suggested myocarditis. The deltoid skin biopsy did not disclose any evidence of excessive type I interferon signaling nor was there evidence of systemic complement pathway activation unlike the pattern of excessive complement vascular deposition we see in the normal deltoid skin biopsies of patients with severe COVID-19, including a previously reported patient who developed significant myocardial disease likely representing a small vessel vasculitic variant of myocarditis in the setting of severe COVID-19. 7 In addition, the amount of spike glycoprotein localized to the microvessels and the endothelial based cytokine response was similar to the two healthy adults who underwent deltoid biopsy after receiving the vaccine. There have been 226 cases of myocarditis or pericarditis in people aged 30 years and younger who have received an mRNA COVID-19 vaccine, occurring more commonly after the second dose and with a higher incidence occurring in male individuals. Symptoms include chest pain, elevated cardiac enzymes, ST-or T-wave changes, dyspnea, and abnormal echocardiography/imaging. The patients typically make a full recovery. 24 The myocardium is rich in ACE2 positive microvessels. We have already demonstrated that human synthesized spike glycoprotein localizes to ACE2 positive vessels of the deeper dermis and fat. We would expect a similarly low level of localization to other organs in which the microvessels express ACE2 as does the heart. As T-cell and B-cell responses are invariably elicited to the human spike glycoprotein, some degree of inflammation could be occurring in the heart, reflective of the localization of the antigenic target.

Role of the vaccine as an adjuvant in unmasking an adaptive immune response in a predisposed host
All vaccines have adjuvants that are added to enhance the adaptive and innate immune response. For example, the bacille Calmette-Guerin enhances the tumoricidal capacity of the autoreactive T cells targeting bladder cancer. The adjuvant plays a role in activating molecules involved in antigen presentation and other pro-inflammatory cytokines and therefore kick-start the immune system. The adjuvants in mRNA vaccines are lipid or polymer-based nanoparticles that protect and stabilize the fragile mRNA and improve its uptake by our immune cells. The mRNA nucleic acid itself is an inherent immunostimulatory molecule owing to its recognition by a variety of innate immune receptors localized at the cell surface, endosome, and cytoplasm. It is logical that our innate immune system would be hardwired to recognize foreign nucleic acid as a threat. 25 Because of the high levels of proinflammatory cytokines associated with the adaptive TH1 or TH2 immune response, a microenvironment conducive to the influx of inflammatory cells associated with either TH1 or TH2 immune polarization could be operational in some cases. If there is a genetic tendency for a psoriatic diathesis or atopic dermatitis, the vaccine could trigger the inflammatory cascade that could eventuate in a particular dermatosis such as dermatitis. Conversely, a subclinical hypersensitivity reaction could become unmasked. 26 , 27 Owing to the lack of data on safety of novel mRNA COVID-19 vaccines, concern regarding its impact on patients suffering from inflammatory diseases has been raised. In general, vaccination is an uncommon factor triggering psoriasis flares. The association of vaccination with the new development or exacerbation of this skin disease has been reported. The mechanisms responsible for psoriasis exacerbation after vaccination are yet to be understood. It is possible that, similar to influenza vaccines, this mechanism may be caused by both dysregulation of immune system due to viral components and vaccine adjuvants. 16 , 28

Human-manufactured spike glycoprotein does not result in systemic complement pathway activation and vascular injury
Despite microvascular localization of spike glycoprotein, significant microvascular sequelae do not appear to occur, reflecting the low burden of manufactured spike glycoprotein in the systemic circulation and the progressive neutralization of human synthesized spike protein by antibodies produced by the host. The data show successful production of the spike glycoprotein post-mRNA vaccination. During that nascent period before the adaptive immune response to neutralize the spike glycoprotein, it is not surprising that circulating spike protein localizes to ACE2 positive endothelium situated in the deeper skin vessels. The human-derived spike glycoprotein that was endocytosed through the endothelium resulted in an endothelial cell response, given the focal expression of caspase 3, IL-6, and TNF α; however, it was not associated with activation of the complement pathway-the critical pathway that is triggered in severe and critical COVID-19-and contributes significantly to the microvascular and procoagulant complications that characterize severe and critical COVID-19. The basis for the lack of complement activation is unclear but the amount of the protein localized to the receptor binding site might be a factor, as could be a difference in the glycosylation pattern of the wild-type spike protein versus the protein synthesized by vaccinated myocytes. In the young woman who developed myocardial insufficiency after receiving the Moderna COVID-19 vaccine, there were no thrombotic changes and no evidence of complement pathway activation. In addition, the amount of spike glycoprotein deposition was minimal and consistent with the extent of deposition observed in the other cases. A summary of the pathogenetic events that underlie the localization of human-manufactured spike glycoprotein to distant microvascular beds is highlighted in Figure 18 .
Not surprisingly, based on our identification of spike glycoprotein of presumptive human myocyte origin in cutaneous microvessels in vaccinated patients, the spike protein product of the mRNA vaccine is detectable in peripheral blood. A prospective study of 13 Boston healthcare workers who had received the Moderna mRNA-1273 vaccine showed the S1 spike subunit in plasma beginning on day 1, peaking at day 525. Although the S1 subunit became undetectable by day 14 as antibody levels rose, intact spike protein persisted much longer in a few of the healthcare workers. There is evidence that the mRNA vaccines themselves may distribute widely through the body. In one manufacturer's report to Japanese regulators, the biodistribution of lipid Fig. 18 The nucleoside modified mRNA severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine is effective at producing spike protein in human myocytes with subsequent cutaneous microvascular localization. 1. The mRNA to encode spike protein is delivered via a lipid nanoparticle envelope. 2. Myocytes synthesize spike protein, which is then released into the circulation. 3. Spike travels as a pseudovirion into the bloodstream. 4. Angiotensin converting enzyme 2 (ACE2) on endothelium of deeper dermal/subcutaneous vessels (red chromagen highlighting ACE2 positive vessels, blue arrow ). 5. Spike binds to the ACE2-positive microvessels of the skin (red chromagen highlighting spike in endothelium, blue arrow ). In the initial phase of vaccine administration spike protein can escape into the blood stream and localize at low levels to ACE2 positive vessels. nanoparticles in injected rats showed that up to 75% of the inoculum escaped the injection site and was found circulating in the blood and pooled in the spleen, liver, bone marrow, adrenal glands, ovaries, and other tissues. 29 , 30

Conclusions
In summation, the data demonstrate that the basis of most post-COVID-19-vaccine skin eruptions suggest a type IV hypersensitivity reaction and, less commonly, immunecomplex-mediated hypersensitivity. The exact antigenic trigger is not yet established, but the possibilities include an exogenous agent found within the vaccine vehicle, namely polyethylene glycol versus the foreign spike protein manufactured by human myocytes functioning as a systemic antigen capable of eliciting classic forms of hypersensitivity, especially type IV but also antibody-mediated reactions resulting in immune complex deposition. An unrelated antigen or an underlying genetic predisposition for an inflammatory dermatosis like psoriasis, which becomes unmasked owing to immune-enhancing properties of the vaccine, has to be considered as well. The exceptionally small amount of spike glycoprotein that disseminates to ACE2 + microvascular never results in a disease process that resembles severe or critical COVID-19 and hence, not surprisingly, complement pathway activation is not observed, and thrombotic vascular complications, although rarely reported after administration of the COVID-19 vaccine, have not been proven to be caused by the vaccine.