Bone Marrow Suppression Secondary to the COVID-19 Booster Vaccine: A Case Report

TAuthors: oral Shastri 1Navkiran Randhawa 2Ragia Aly 3Masood Ghouse 3 PMID: 35210894PMCID: PMC8863340DOI: 10.2147/JBM.S350290 J Blood Med.  2022; 13: 69–74.Published online 2022 Feb 18. doi: 10.2147/JBM.S350290

Abstract

As of September 2021, SARS-CoV-2 booster shots became widely available in the US to ensure continued protection against the virus. A temporal relationship has been previously reported between the first or second dose of the COVID-19 vaccine and the development of thrombocytopenia. However, adverse events related to the third COVID-19 vaccine are still being reported and studied. We report a 74-year-old male who developed bone marrow suppression and pancytopenia recorded seven days after receiving the Pfizer SARS-CoV-2 vaccine. During his hospital stay, the patient’s hemoglobin, white blood cell, and platelet levels continued to trend downwards. However, all three levels showed improvement one week after discharge without robust intervention. Global vaccination is of utmost importance, as is understanding and documenting post-vaccination reactions including bone marrow suppression. Prompt evaluation and patient education are imperative to improve patient outcomes and combat hesitancy against vaccine administration.

Introduction

Since its emergence in December of 2019, the rapid spread of severe acute respiratory syndrome coronavirus (SARS-CoV-2) has quickly affected millions of lives across every continent.1 This highly transmittable and pathogenic viral infection has led to massive mitigation efforts and allocation of resources to prevent the spread of transmission and high mortality related to complications.2 The establishment of higher levels of community (herd) immunity and protection against SARS-CoV-2 via the widespread deployment of effective vaccines has become a global effort.3 In December of 2020, the FDA issued an Emergency use Authorization for the Pfizer-BioNTech and Moderna COVID-19 Vaccine as a two-dose series.4 In September 2021, booster vaccines became widely administered in the US due to waning immunity of the COVID-19 vaccines against variants of SARS-CoV-2 along with ensuring continued protection against the virus.5

Serious adverse events such as anaphylaxis, Guillain-Barre Syndrome, myocarditis, pericarditis, thrombocytopenia, and death have been previously reported following the first and/or second dose of vaccine.6 To our knowledge, no cases have been reported regarding bone marrow suppression related to the third COVID-19 vaccine. Adverse events reported between August 12-September 19, 2021 from the COVID-19 booster vaccine supported similar reactions to those after dose two.7 According to the Centers for Disease Control and Prevention (CDC), these initial findings indicate no unexpected patterns of adverse reactions after an additional dose of COVID-19 vaccination.7 However, adverse events related to the COVID-19 booster are still being reported and studied.6 This report presents a case of bone marrow suppression occurring after the third COVID-19 vaccine without a similar reaction after the first or second dose.Go to:

Case Report

A 74-year-old male with a history of polychondritis and hypothyroidism presented to the hospital after falling out of his chair and inability to ambulate. The patient was found to be mildly confused upon arrival to the emergency room, limiting our ability to obtain a full verbal history. Chart review revealed the patient had received his third Pfizer Covid vaccine shot seven days before admission followed by fatigue, decreased appetite, fever, and chills. The patient had received the second Pfizer Covid-19 shot nine months before the booster. No reactions to the previous two shots were noted.

Upon initial evaluation, vital signs were within normal limits and a physical exam revealed significant tenderness in the upper arm and no gross bleeding (Figure 1). Computed tomography (CT) imaging (Figure 2) was significant for enhancement of the left axillary lymph node. The patient’s initial complete blood count (CBC) was remarkable for a hemoglobin count of 9.9 g/dl and platelet count of 84 x 109/L; both values lower than his prior hemoglobin count of 13.7 g/dl and platelet count of 180 x 109/L from December of 2020. His mean corpuscular volume (MCV) was elevated at 101.3 femtolitres from his prior MCV value of 95.8 femtolitres in December of 2020. His white blood cell (WBC) count was recorded at 7.6 x 109/L.

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Figure 1

The patient’s upper arm showed erythema with no gross bleeding near the injection site

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Figure 2

The patient’s CT imaging of the thoracic region showed enhancement of the left axillary lymph node.

The hemoglobin, WBC, and platelet count further down trended from his baseline (Figures 3​5).5). Anemia labs including ferritin levels (554 ng/mL), vitamin B12 (253 pg/mL), total bilirubin (0.5 mg/dL), and reticulocyte count (0.8%) were nonsignificant during the patient’s hospital stay. The patient’s left shoulder presented with extensive bruising, erythema, papular rash, warmth, and tenderness on palpation during the hospitalization. An improvement in WBC and platelet levels was noted on day 4 of hospitalization.

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Figure 3

The patient’s hemoglobin count throughout his hospital course and 6 days after discharge.

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Figure 4

The patient’s WBC count throughout his hospital course and 6 days after discharge.

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Figure 5

The patient’s platelet count throughout his hospital course and 6 days after discharge.

Before discharge, the patient was fully alert and oriented and reported improvement in his symptoms. Examination of his lateral left arm showed decreased erythema and bruising with slight petechiae. The patient was discharged due to stabilization of labs and encouraged to take oral vitamin B12 supplements. During his outpatient follow-up six days after hospitalization, his hemoglobin increased to 10.5 g/dl, WBC count increased to 4.9 x 109/L, and platelets increased to 101 x 109/L.

Discussion

This paper presents a patient with pancytopenia recorded seven days after receiving the Pfizer booster vaccine. Interestingly, this patient did not report any reactions after the first or second dose of the Pfizer vaccine against SARS-CoV-2. Pancytopenia refers to a decrease in all peripheral bloodlines and is present when all three cell lines are below the normal reference range.8 The patient’s physical exam showed no signs of active bleeding along with his labs indicating no evidence of hemolysis. The patient’s hemoglobin, platelet, and white blood cell count presented below baseline followed by a decrease and slight improvement during his hospital stay. Six days after hospitalization, all three cell lines showed improvement. The temporal association with the booster vaccine and negative infectious disease workup raised suspicion for vaccine-induced bone marrow suppression. In addition, the patient’s reticulocyte count and lactate dehydrogenase value were consistent with hypoproliferation within the bone marrow.

Currently, there is a gap in knowledge of adverse events specific to the third vaccine against SARS-CoV-2 due to the recent initiation of administration and ongoing reporting of events.6 To our knowledge, bone marrow suppression after any dose of vaccine against SARS-CoV-2 has not been previously reported. However, a prior case of pancytopenia after the third vaccination with a recombinant hepatitis B vaccine has previously been reported.9 The patient’s bone marrow biopsy within this case displayed a paucity of late myeloid elements and CD8+ T cells.9 It was believed the patient’s CD8+T cells were causing excessive production of IFN-γ; a stimulant of negative regulators of hematopoiesis such as tumor necrosis factor and lymphotoxin.10 IFN-γ has also previously been reported to create immunological effects comprising an upregulation of histocompatibility gene transcription and alteration in class I and II antigen expression at the cell surface.11 It was predicted these changes resulted in an autoimmune reaction causing suppression of maturation of hematopoietic progenitor cells and pancytopenia.9 Via a similar mechanism, we believe that our patient’s pancytopenia was immune-mediated, potentially triggered by the vaccination.

Vaccines against SARS-CoV-2 (first or second dose) and the induction of Idiopathic Thrombocytopenic Purpura (ITP) have also been recently acknowledged in multiple cases.12 Our patient presented with low platelet levels and associated petechiae and purpura at the site of the vaccination. However, the patient’s presentation of low hemoglobin and white blood cells along with normal reticulocyte levels was more indicative of pancytopenia secondary to bone marrow suppression. In patients presenting with pancytopenia, the history and the physical exam should help assess the severity of the pancytopenia and comorbid illnesses that may complicate the disorder.13 In addition, suspicious medications and exposure to toxic agents should be ruled out.13 Initial screening laboratory evaluation should include the patient’s complete blood count, peripheral blood smear examination, reticulocyte count, complete metabolic panel, prothrombin time/partial thromboplastin time, and blood type and screen. Common interventions to alleviate bone marrow suppression and pancytopenia include treating the underlying cause and utilizing supplements to boost red blood cell production if indicated.

Vaccines against SARS-CoV-2 undergo continuous safety monitoring; adverse events are very rare.14 However, vaccine hesitancy remains a barrier towards full population inoculation against SARS-CoV-2 and is influenced by misinformation regarding vaccine safety.15 One study using an anonymous online questionnaire found a person’s trust in the effectiveness of the vaccine was a major facilitative factor affecting willingness to vaccinate.16 The same study also found that 66.7% of unvaccinated participants thought the vaccine’s safety was not enough, making it the main reason for reluctance or hesitance to be vaccinated.16 Therefore, education of adverse events and available interventions post-vaccination is imperative to prevent the spread of misinformation and combat hesitancy towards vaccination.15

As of September 19, 2021, about 2.2 million people in the United States received a third vaccine against SARS-CoV-2.17 Among those who received the vaccine, 22,000 people reported the effects of the vaccine with no unexpected patterns of adverse reactions.17 Our patient demonstrates abnormal pancytopenia first recorded seven days after receiving the booster vaccine, possibly indicating a rare adverse event from the vaccination given the temporal relationship. While additional studies and observations are indicated to verify bone marrow suppression as an adverse reaction, this case report provides an opportunity for patient education and treatment planning before symptoms arise.

Conclusion

Our case report highlights pancytopenia secondary to bone marrow suppression following Pfizer vaccination against SARS-CoV-2. It is important to consider the possibility of bone marrow suppression following the third vaccine against SARS-CoV-2. Although additional studies are indicated to determine the risk factors and pathogenesis of vaccine-induced bone marrow suppression, prompt evaluation and initiation of interventions can improve patient outcomes.

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Severe aplastic anemia after COVID-19 mRNA vaccination: Causality or coincidence?

Authors: Shotaro Tabata 1Hiroki Hosoi 2Shogo Murata 1Satomi Takeda 1Toshiki Mushino 1Takashi Sonoki 1PMID: 34920343

PMCID: PMC8668346I: 10.1016/j.jaut.2021.102782 J Autoimmun. 2022 Jan; 126: 102782.Published online 2021 Dec 14. doi: 10.1016/j.jaut.2021.102782

Abstract

The development of various autoimmune diseases has been reported after COVID-19 infections or vaccinations. However, no method for assessing the relationships between vaccines and the development of autoimmune diseases has been established. Aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome. We report a case of severe AA that arose after the administration of a COVID-19 vaccine (the Pfizer-BioNTech mRNA vaccine), which was treated with allogeneic hematopoietic stem cell transplantation (HSCT). In this patient, antibodies against the SARS-CoV-2 spike protein were detected both before and after the HSCT. After the patient’s hematopoietic stem cells were replaced through HSCT, his AA improved despite the presence of anti-SARS-CoV-2 antibodies. In this case, antibodies derived from the COVID-19 vaccine may not have been directly involved in the development of AA. This case suggests that the measurement of vaccine antibody titers before and after allogeneic HSCT may provide clues to the pathogenesis of vaccine-related autoimmune diseases. Although causality was not proven in this case, further evaluations are warranted to assess the associations between vaccines and AA.

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of coronavirus disease 2019 (COVID-19). The introduction of SARS-CoV-2 vaccines has drastically reduced the transmission rate of the disease. Studies have confirmed the safety and efficacy of the available SARS-CoV-2 vaccines. However, rare cases of adverse immunological reactions to SARS-CoV-2 vaccines have been reported, including cases involving immune-mediated disease [[1][2][3]]. Although evaluating the associations between SARS-CoV-2 vaccines and the development of autoimmune diseases is important, no method for assessing such relationships has been established. Aplastic anemia (AA), a bone marrow failure syndrome, appears to be immune-mediated [4,5]. In addition to T lymphocytes and cytokines, autoantibodies are involved in the development of AA as immunological factors [4]. Here, we report a case of AA that developed after the administration of a SARS-CoV-2 vaccine and discuss the association between AA and vaccination.

2. Case description

A previously healthy 56-year-old male, who was not taking any medication, was referred to a clinic because of bleeding in the oral cavity after dental therapy. Laboratory tests showed that his white blood cell count (1.6 × 109/l) and platelet count (11 × 109/l) were decreased. Four days before his visit to the clinic, he had received a second dose of the Pfizer-BioNTech mRNA vaccine (three weeks after his first dose). He was admitted to our hospital due to progressive pancytopenia (Supplementary Table 1). He had no history of COVID-19 infection. The Elecsys® anti-SARS-CoV-2 immunoassay (Roche, Basel, Switzerland), which is used to detect anti-SARS-CoV-2 nucleocapsid protein antibodies, produced a negative result. Tests for immunoglobulin G against cytomegalovirus and Epstein-Barr virus produced positive results, but were not indicative of virus reactivation. Serological tests for hepatitis B, hepatitis C, and human immunodeficiency virus produced negative results. A bone marrow biopsy revealed a hypocellular marrow (Fig. 1 ). The patient was diagnosed with very severe AA [6]. Human leukocyte antigen (HLA) testing showed DRB1 04:05 04:05, which is not associated with a high frequency of AA. The administration of granulocyte-colony stimulating factor had no effect on his neutropenia. In spite of the administration of cyclosporine and eltrombopag, his pancytopenia progressed.

Fig. 1

Fig. 1

Histological findings of the bone marrow biopsy specimen at diagnosis. Panel A: Hematoxylin and eosin (H.E.) staining (x40) of the bone marrow after the administration of a SARS-CoV-2 vaccine showed a markedly hypocellular marrow. Panel B: H.E. staining (x400) showed the replacement of hematopoietic cells by fat and a few nucleated cells.

He underwent an allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA haploidentical related donor (Fig. 2 ). The donor had no history of COVID-19 infection and had not received a SARS-CoV-2 vaccine. The conditioning regimen consisted of 120 mg/m2 fludarabine, 100 mg/kg cyclophosphamide, 2.5 mg/kg anti-thymocyte globulin, and 2 Gy of total body irradiation. Tacrolimus and short-term methotrexate were used as a prophylaxis against graft-versus-host disease (GVHD). Post-transplant cyclophosphamide was not administered because the patient’s HLA-A, C, and DR were homologous, which would not increase the risk of GVHD. The transplanted cells collected from the donor’s bone marrow were transfused into the patient after the removal of red blood cells and plasma. Twenty-one days after the HSCT, neutrophil engraftment was achieved. Chimerism analysis performed on day 29 after the HSCT revealed complete chimerism in the peripheral blood. The patient developed acute GVHD (skin grade 1), which was ameliorated with a topical corticosteroid alone.

Fig. 2

Fig. 2

Evaluation of neutrophil count, The X-axis indicates the number of days after the 2nd dose of the COVID-19 vaccine was administered. The allogeneic BMT was conducted at 34 days after the 2nd dose of the COVID-19 vaccine was administered. The gray boxes indicate the titers of antibodies against the SARS-CoV-2 spike protein (log scale). BMT, bone marrow transplantation; COVID-19, coronavirus disease 2019; CyA, cyclosporine A; TAC, tacrolimus.

The titers of antibodies against SARS-CoV-2 were measured before and after the HSCT to examine the association between the SARS-CoV-2 vaccine the patient received and the development of AA. The measurement of anti-SARS-CoV-2 spike protein antibody titers was performed by SRL, Inc. (Tokyo, Japan) using the Elecsys® anti-SARS-CoV-2 S immunoassay (Roche, Basel, Switzerland). The titers of antibodies against SARS-CoV-2 before the conditioning regimen and 63 days after the HSCT were 540 and 34.9 U/mL (reference range, <0.8 U/mL), respectively (Fig. 2). These results suggest that the AA was ameliorated by the allogeneic HSCT even though anti-SARS-CoV-2 spike protein antibodies continued to be detected after the HSCT.Go to:

3. Discussion

We report a case in which AA developed after the administration of a SARS-CoV-2 vaccine. No association between new-onset AA and SARS-CoV-2 vaccines has been reported. The patient in this case underwent allogeneic HSCT. In this patient, antibodies against the SARS-CoV-2 spike protein were detected both before and after the HSCT. After the allogeneic HSCT, the patient’s AA was ameliorated despite the presence of antibodies against SARS-CoV-2. Our results did not reveal a direct association between antibodies derived from the SARS-CoV-2 vaccine and the development of AA. Further studies are needed to investigate the impairment of hematopoiesis induced by immune reactions after SARS-CoV-2 vaccine administration.

One of the most feared adverse reactions to vaccines is the development of autoimmune disease. To the best of our knowledge, only six cases of newly diagnosed acquired AA have been reported after vaccination [[7][8][9][10][11]] (Table 1 ). However, in general, AA is not recognized as a vaccine-related adverse event [12]. The mRNA vaccines against SARS-CoV-2 have a novel mechanism of action. Therefore, it is important to collect information about their adverse events. Various cases of autoimmune disease have been reported after SARS-CoV-2 vaccine administration, including autoimmune hepatitis, type 1 diabetes mellitus, immune thrombocytopenia, and acquired hemophilia [3,[13][14][15]]. Patients with AA after COVID-19 infection were also reported [16,17]. Further epidemiological evaluations of the incidence of AA after COVID-19 infection and SARS-CoV-2 vaccination are warranted.

Table 1

Reported cases of newly diagnosed aplastic anemia after vaccinations.

Age (years)SexVaccineTime to symptom onsetTreatmentOutcomeReference
16FRecombinant hepatitis B3 weeks after 3rd doseCorticosteroidImprovedViallard et al. [7]
19FRecombinant hepatitis B10 days after 3rd doseCorticosteroidImprovedAshok Shenoy et al. [8]
25MHepatitis B7 days after 2nd doseAllogeneic HSCTN.A.Shah et al. [9]
19MAnthrax1 monthAllogeneic HSCTN.A.Shah et al. [9]
1.5FVaricella zoster3 weeksNoneImprovedAngelini et al. [10]
25MH1N1 influenza2 weeksAllogeneic HSCTImprovedDonnini et al. [11]
56MSARS-Cov-24 days after 2nd doseAllogeneic HSCTImprovedThis case

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HSCT, hematopoietic stem cell transplantation; N.A., not applicable; SARS-Cov-2, severe acute respiratory syndrome coronavirus 2.

Various cases of vaccine-related autoimmune disease have been reported. Most of these reports have linked vaccination to the development of autoimmune disease based on clinical observations of temporal associations. There is no established method for examining the relationships between vaccines and the development of autoimmune diseases. The pathogenetic mechanisms by which vaccines cause the development of autoimmune disease are still unclear. The major hypotheses relating to such immunological reactions involve epitope mimicry [18,19]. For example, it has been reported that vaccine-derived antibodies may exhibit structural similarities with autoantibodies [18,19]. There is significant evidence that AA is an immune-mediated condition, mainly based on the effectiveness of immunosuppressive therapy against AA. In addition to T cells and cytokines, autoantibodies are one of the factors that contribute to the pathogenesis of AA [4]. However, autoantibodies specific to AA and the role of autoantibodies for the pathogenesis of AA are unclear. The allogeneic HSCT replaces the recipient’s hematopoietic and associated immune systems with those of the donor. The measurement of vaccine antibody titers before and after allogeneic HSCT may provide a clue to the pathogenesis of vaccine-related autoimmune diseases. The clonal expansion of effector T cells was also reported to occur following vaccination [20]. To understand the link between COVID-19 vaccination and the development of AA, the following needs to be examined: the exploration of autoantibodies against stem cells, the role for molecular mimicry between mRNA vaccine encoded antigens and stem cells, and T-cell subset dynamics after vaccination.

In conclusion, the administered SARS-CoV-2 mRNA vaccine may have contributed to the pathogenesis of AA in this case. However, it is not clear whether antibodies derived from the SARS-CoV-2 vaccine directly contributed to the development of AA because the anti-SARS-CoV-2 antibodies remained after the patient’s pancytopenia had been ameliorated by the allogeneic HSCT. Further evaluations in large cohorts are warranted to elucidate the associations between AA and SARS-CoV-2 vaccines.Go to:

Authors’ contributions

Shotaro Tabata: Data curation, Investigation, Writing – original draft; Hiroki Hosoi: Conceptualization, Data curation, Investigation, Writing – original draft and Review & Editing; Shogo Murata: Investigation, Writing – review & editing; Satomi Takeda: Data curation, Writing – review & editing; Toshiki Mushino: Writing – review & editing; Takashi Sonoki: Writing – review & editing, Supervision.Go to:

Declaration of competing interest

There are no funding sources associated with the writing of this manuscript. Written consent for publication was obtained from the patient.Go to:

Acknowledgements

We thank the patients and clinical staff at Wakayama Medical University Hospital for their participation in this study. We also wish to thank Dr. Takashi Ozaki and Mr. Masaya Morimoto from Kinan Hospital for their helpful diagnostic support.Go to:

Footnotes

Appendix ASupplementary data to this article can be found online at https://doi.org/10.1016/j.jaut.2021.102782.Go to:

Appendix A. Supplementary data

The following is the Supplementary data to this article:Multimedia component 1:Click here to view.(12K, xlsx)Multimedia component 1Go to:

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Bone Marrow Suppression Secondary to the COVID-19 Booster Vaccine: A Case Report

Authors: Toral Shastri 1Navkiran Randhawa 2Ragia Aly 3Masood Ghouse 3

PMID: 35210894 PMCID: PMC8863340DOI: 10.2147/JBM.S350290

Abstract

As of September 2021, SARS-CoV-2 booster shots became widely available in the US to ensure continued protection against the virus. A temporal relationship has been previously reported between the first or second dose of the COVID-19 vaccine and the development of thrombocytopenia. However, adverse events related to the third COVID-19 vaccine are still being reported and studied. We report a 74-year-old male who developed bone marrow suppression and pancytopenia recorded seven days after receiving the Pfizer SARS-CoV-2 vaccine. During his hospital stay, the patient’s hemoglobin, white blood cell, and platelet levels continued to trend downwards. However, all three levels showed improvement one week after discharge without robust intervention. Global vaccination is of utmost importance, as is understanding and documenting post-vaccination reactions including bone marrow suppression. Prompt evaluation and patient education are imperative to improve patient outcomes and combat hesitancy against vaccine administration.

Introduction

Since its emergence in December of 2019, the rapid spread of severe acute respiratory syndrome coronavirus (SARS-CoV-2) has quickly affected millions of lives across every continent.1 This highly transmittable and pathogenic viral infection has led to massive mitigation efforts and allocation of resources to prevent the spread of transmission and high mortality related to complications.2 The establishment of higher levels of community (herd) immunity and protection against SARS-CoV-2 via the widespread deployment of effective vaccines has become a global effort.3 In December of 2020, the FDA issued an Emergency use Authorization for the Pfizer-BioNTech and Moderna COVID-19 Vaccine as a two-dose series.4 In September 2021, booster vaccines became widely administered in the US due to waning immunity of the COVID-19 vaccines against variants of SARS-CoV-2 along with ensuring continued protection against the virus.5

Serious adverse events such as anaphylaxis, Guillain-Barre Syndrome, myocarditis, pericarditis, thrombocytopenia, and death have been previously reported following the first and/or second dose of vaccine.6 To our knowledge, no cases have been reported regarding bone marrow suppression related to the third COVID-19 vaccine. Adverse events reported between August 12-September 19, 2021 from the COVID-19 booster vaccine supported similar reactions to those after dose two.7 According to the Centers for Disease Control and Prevention (CDC), these initial findings indicate no unexpected patterns of adverse reactions after an additional dose of COVID-19 vaccination.7 However, adverse events related to the COVID-19 booster are still being reported and studied.6 This report presents a case of bone marrow suppression occurring after the third COVID-19 vaccine without a similar reaction after the first or second dose.Go to:

Case Report

A 74-year-old male with a history of polychondritis and hypothyroidism presented to the hospital after falling out of his chair and inability to ambulate. The patient was found to be mildly confused upon arrival to the emergency room, limiting our ability to obtain a full verbal history. Chart review revealed the patient had received his third Pfizer Covid vaccine shot seven days before admission followed by fatigue, decreased appetite, fever, and chills. The patient had received the second Pfizer Covid-19 shot nine months before the booster. No reactions to the previous two shots were noted.

Upon initial evaluation, vital signs were within normal limits and a physical exam revealed significant tenderness in the upper arm and no gross bleeding (Figure 1). Computed tomography (CT) imaging (Figure 2) was significant for enhancement of the left axillary lymph node. The patient’s initial complete blood count (CBC) was remarkable for a hemoglobin count of 9.9 g/dl and platelet count of 84 x 109/L; both values lower than his prior hemoglobin count of 13.7 g/dl and platelet count of 180 x 109/L from December of 2020. His mean corpuscular volume (MCV) was elevated at 101.3 femtolitres from his prior MCV value of 95.8 femtolitres in December of 2020. His white blood cell (WBC) count was recorded at 7.6 x 109/L.

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Figure 1

The patient’s upper arm showed erythema with no gross bleeding near the injection site

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Figure 2

The patient’s CT imaging of the thoracic region showed enhancement of the left axillary lymph node.

The hemoglobin, WBC, and platelet count further down trended from his baseline (Figures 3​5).5). Anemia labs including ferritin levels (554 ng/mL), vitamin B12 (253 pg/mL), total bilirubin (0.5 mg/dL), and reticulocyte count (0.8%) were nonsignificant during the patient’s hospital stay. The patient’s left shoulder presented with extensive bruising, erythema, papular rash, warmth, and tenderness on palpation during the hospitalization. An improvement in WBC and platelet levels was noted on day 4 of hospitalization.

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Figure 3

The patient’s hemoglobin count throughout his hospital course and 6 days after discharge.

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Figure 4

The patient’s WBC count throughout his hospital course and 6 days after discharge.

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Figure 5

The patient’s platelet count throughout his hospital course and 6 days after discharge.

Before discharge, the patient was fully alert and oriented and reported improvement in his symptoms. Examination of his lateral left arm showed decreased erythema and bruising with slight petechiae. The patient was discharged due to stabilization of labs and encouraged to take oral vitamin B12 supplements. During his outpatient follow-up six days after hospitalization, his hemoglobin increased to 10.5 g/dl, WBC count increased to 4.9 x 109/L, and platelets increased to 101 x 109/L.

Discussion

This paper presents a patient with pancytopenia recorded seven days after receiving the Pfizer booster vaccine. Interestingly, this patient did not report any reactions after the first or second dose of the Pfizer vaccine against SARS-CoV-2. Pancytopenia refers to a decrease in all peripheral bloodlines and is present when all three cell lines are below the normal reference range.8 The patient’s physical exam showed no signs of active bleeding along with his labs indicating no evidence of hemolysis. The patient’s hemoglobin, platelet, and white blood cell count presented below baseline followed by a decrease and slight improvement during his hospital stay. Six days after hospitalization, all three cell lines showed improvement. The temporal association with the booster vaccine and negative infectious disease workup raised suspicion for vaccine-induced bone marrow suppression. In addition, the patient’s reticulocyte count and lactate dehydrogenase value were consistent with hypoproliferation within the bone marrow.

Currently, there is a gap in knowledge of adverse events specific to the third vaccine against SARS-CoV-2 due to the recent initiation of administration and ongoing reporting of events.6 To our knowledge, bone marrow suppression after any dose of vaccine against SARS-CoV-2 has not been previously reported. However, a prior case of pancytopenia after the third vaccination with a recombinant hepatitis B vaccine has previously been reported.9 The patient’s bone marrow biopsy within this case displayed a paucity of late myeloid elements and CD8+ T cells.9 It was believed the patient’s CD8+T cells were causing excessive production of IFN-γ; a stimulant of negative regulators of hematopoiesis such as tumor necrosis factor and lymphotoxin.10 IFN-γ has also previously been reported to create immunological effects comprising an upregulation of histocompatibility gene transcription and alteration in class I and II antigen expression at the cell surface.11 It was predicted these changes resulted in an autoimmune reaction causing suppression of maturation of hematopoietic progenitor cells and pancytopenia.9 Via a similar mechanism, we believe that our patient’s pancytopenia was immune-mediated, potentially triggered by the vaccination.

Vaccines against SARS-CoV-2 (first or second dose) and the induction of Idiopathic Thrombocytopenic Purpura (ITP) have also been recently acknowledged in multiple cases.12 Our patient presented with low platelet levels and associated petechiae and purpura at the site of the vaccination. However, the patient’s presentation of low hemoglobin and white blood cells along with normal reticulocyte levels was more indicative of pancytopenia secondary to bone marrow suppression. In patients presenting with pancytopenia, the history and the physical exam should help assess the severity of the pancytopenia and comorbid illnesses that may complicate the disorder.13 In addition, suspicious medications and exposure to toxic agents should be ruled out.13 Initial screening laboratory evaluation should include the patient’s complete blood count, peripheral blood smear examination, reticulocyte count, complete metabolic panel, prothrombin time/partial thromboplastin time, and blood type and screen. Common interventions to alleviate bone marrow suppression and pancytopenia include treating the underlying cause and utilizing supplements to boost red blood cell production if indicated.

Vaccines against SARS-CoV-2 undergo continuous safety monitoring; adverse events are very rare.14 However, vaccine hesitancy remains a barrier towards full population inoculation against SARS-CoV-2 and is influenced by misinformation regarding vaccine safety.15 One study using an anonymous online questionnaire found a person’s trust in the effectiveness of the vaccine was a major facilitative factor affecting willingness to vaccinate.16 The same study also found that 66.7% of unvaccinated participants thought the vaccine’s safety was not enough, making it the main reason for reluctance or hesitance to be vaccinated.16 Therefore, education of adverse events and available interventions post-vaccination is imperative to prevent the spread of misinformation and combat hesitancy towards vaccination.15

As of September 19, 2021, about 2.2 million people in the United States received a third vaccine against SARS-CoV-2.17 Among those who received the vaccine, 22,000 people reported the effects of the vaccine with no unexpected patterns of adverse reactions.17 Our patient demonstrates abnormal pancytopenia first recorded seven days after receiving the booster vaccine, possibly indicating a rare adverse event from the vaccination given the temporal relationship. While additional studies and observations are indicated to verify bone marrow suppression as an adverse reaction, this case report provides an opportunity for patient education and treatment planning before symptoms arise.

Conclusion

Our case report highlights pancytopenia secondary to bone marrow suppression following Pfizer vaccination against SARS-CoV-2. It is important to consider the possibility of bone marrow suppression following the third vaccine against SARS-CoV-2. Although additional studies are indicated to determine the risk factors and pathogenesis of vaccine-induced bone marrow suppression, prompt evaluation and initiation of interventions can improve patient outcomes

Consent for Publication

Institutional approval was not required to publish the case details. The publication of this study has been consented to by the patient.

Disclosure

The authors report no conflicts of interest in this work.

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SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans

Authors: Jackson S. TurnerWooseob KimElizaveta KalaidinaCharles W. GossAdriana M. RauseoAaron J. SchmitzLena HansenAlem HaileMichael K. KlebertIskra PusicJane A. O’HalloranRachel M. Presti & Ali H. Ellebedy 

Nature volume 595, pages421–425 (2021)

Abstract

Long-lived bone marrow plasma cells (BMPCs) are a persistent and essential source of protective antibodies1,2,3,4,5,6,7. Individuals who have recovered from COVID-19 have a substantially lower risk of reinfection with SARS-CoV-28,9,10. Nonetheless, it has been reported that levels of anti-SARS-CoV-2 serum antibodies decrease rapidly in the first few months after infection, raising concerns that long-lived BMPCs may not be generated and humoral immunity against SARS-CoV-2 may be short-lived11,12,13. Here we show that in convalescent individuals who had experienced mild SARS-CoV-2 infections (n = 77), levels of serum anti-SARS-CoV-2 spike protein (S) antibodies declined rapidly in the first 4 months after infection and then more gradually over the following 7 months, remaining detectable at least 11 months after infection. Anti-S antibody titres correlated with the frequency of S-specific plasma cells in bone marrow aspirates from 18 individuals who had recovered from COVID-19 at 7 to 8 months after infection. S-specific BMPCs were not detected in aspirates from 11 healthy individuals with no history of SARS-CoV-2 infection. We show that S-binding BMPCs are quiescent, which suggests that they are part of a stable compartment. Consistently, circulating resting memory B cells directed against SARS-CoV-2 S were detected in the convalescent individuals. Overall, our results indicate that mild infection with SARS-CoV-2 induces robust antigen-specific, long-lived humoral immune memory in humans.

Main

Reinfections by seasonal coronaviruses occur 6 to 12 months after the previous infection, indicating that protective immunity against these viruses may be short-lived14,15. Early reports documenting rapidly declining antibody titres in the first few months after infection in individuals who had recovered from COVID-19 suggested that protective immunity against SARS-CoV-2 might be similarly transient11,12,13. It was also suggested that infection with SARS-CoV-2 could fail to elicit a functional germinal centre response, which would interfere with the generation of long-lived plasma cells3,4,5,7,16. More recent reports analysing samples that were collected approximately 4 to 6 months after infection indicate that SARS-CoV-2 antibody titres decline more slowly than in the initial months after infection8,17,18,19,20,21. Durable serum antibody titres are maintained by long-lived plasma cells—non-replicating, antigen-specific plasma cells that are detected in the bone marrow long after the clearance of the antigen1,2,3,4,5,6,7. We sought to determine whether they were detectable in convalescent individuals approximately 7 months after SARS-CoV-2 infection.

Biphasic decay of anti-S antibody titres

Blood samples were collected approximately 1 month after the onset of symptoms from 77 individuals who were convalescing from COVID-19 (49% female, 51% male, median age 49 years), the majority of whom had experienced mild illness (7.8% hospitalized, Extended Data Tables 12). Follow-up blood samples were collected three times at approximately three-month intervals. Twelve convalescent participants received either the BNT162b2 (Pfizer) or the mRNA-1273 (Moderna) SARS-CoV-2 vaccine between the last two time points; these post-vaccination samples were not included in our analyses. In addition, bone marrow aspirates were collected from 18 of the convalescent individuals at 7 to 8 months after infection and from 11 healthy volunteers with no history of SARS-CoV-2 infection or vaccination. Follow-up bone marrow aspirates were collected from 5 of the 18 convalescent individuals and from 1 additional convalescent donor approximately 11 months after infection (Fig. 1a, Extended Data Tables 34). We first performed a longitudinal analysis of circulating anti-SARS-CoV-2 serum antibodies. Whereas anti-SARS-CoV-2 spike protein (S) IgG antibodies were undetectable in blood from control individuals, 74 out of the 77 convalescent individuals had detectable serum titres approximately 1 month after the onset of symptoms. Between 1 and 4 months after symptom onset, overall anti-S IgG titres decreased from a mean loge-transformed half-maximal dilution of 6.3 to 5.7 (mean difference 0.59 ± 0.06, P < 0.001). However, in the interval between 4 and 11 months after symptom onset, the rate of decline slowed, and mean titres decreased from 5.7 to 5.3 (mean difference 0.44 ± 0.10, P < 0.001; Fig. 1a). In contrast to the anti-S antibody titres, IgG titres against the 2019–2020 inactivated seasonal influenza virus vaccine were detected in all control individuals and individuals who were convalescing from COVID-19, and declined much more gradually, if at all over the course of the study, with mean titres decreasing from 8.0 to 7.9 (mean difference 0.16 ± 0.06, P = 0.042) and 7.9 to 7.8 (mean difference 0.02 ± 0.08, P = 0.997) across the 1-to-4-month and 4-to-11-month intervals after symptom onset, respectively (Fig. 1b).

figure 1
Fig. 1: SARS-CoV-2 infection elicits durable serum anti-S antibody titres.

Induction of S-binding long-lived BMPCs

The relatively rapid early decline in the levels of anti-S IgG, followed by a slower decrease, is consistent with a transition from serum antibodies being secreted by short-lived plasmablasts to secretion by a smaller but more persistent population of long-lived plasma cells generated later in the immune response. The majority of this latter population resides in the bone marrow1,2,3,4,5,6. To investigate whether individuals who had recovered from COVID-19 developed a virus-specific long-lived BMPC compartment, we examined bone marrow aspirates obtained approximately 7 and 11 months after infection for anti-SARS-CoV-2 S-specific BMPCs. We magnetically enriched BMPCs from the aspirates and then quantified the frequencies of those secreting IgG and IgA directed against the 2019–2020 influenza virus vaccine, the tetanus–diphtheria vaccine and SARS-CoV-2 S by enzyme-linked immunosorbent spot assay (ELISpot) (Fig. 2a). Frequencies of influenza- and tetanus–diphtheria-vaccine-specific BMPCs were comparable between control individuals and convalescent individuals. IgG- and IgA-secreting S-specific BMPCs were detected in 15 and 9 of the 19 convalescent individuals, respectively, but not in any of the 11 control individuals (Fig. 2b). Notably, none of the control individuals or convalescent individuals had detectable S-specific antibody-secreting cells in the blood at the time of bone marrow sampling, indicating that the detected BMPCs represent bone-marrow-resident cells and not contamination from circulating plasmablasts. Frequencies of anti-S IgG BMPCs were stable among the 5 convalescent individuals who were sampled a second time approximately 4 months later, and frequencies of anti-S IgA BMPCs were stable in 4 of these 5 individuals but had decreased to below the limit of detection in one individual (Fig. 2c). Consistent with their stable BMPC frequencies, anti-S IgG titres in the 5 convalescent individuals remained consistent between 7 and 11 months after symptom onset. IgG titres measured against the receptor-binding domain (RBD) of the S protein—a primary target of neutralizing antibodies—were detected in 4 of the 5 convalescent individuals and were also stable between 7 and 11 months after symptom onset (Fig. 2d). Frequencies of anti-S IgG BMPCs showed a modest but significant correlation with circulating anti-S IgG titres at 7–8 months after the onset of symptoms in convalescent individuals, consistent with the long-term maintenance of antibody levels by these cells (r = 0.48, P = 0.046). In accordance with previous reports22,23,24, frequencies of influenza-vaccine-specific IgG BMPCs and antibody titres exhibited a strong and significant correlation (r = 0.67, P < 0.001; Fig. 2e). Nine of the aspirates from control individuals and 12 of the 18 aspirates that were collected 7 months after symptom onset from convalescent individuals yielded a sufficient number of BMPCs for additional analysis by flow cytometry. We stained these samples intracellularly with fluorescently labelled S and influenza virus haemagglutinin (HA) probes to identify and characterize antigen-specific BMPCs. As controls, we also intracellularly stained peripheral blood mononuclear cells (PBMCs) from healthy volunteers one week after vaccination against SARS-CoV-2 or seasonal influenza virus (Fig. 3a, Extended Data Fig. 1a–c). Consistent with the ELISpot data, low frequencies of S-binding BMPCs were detected in 10 of the 12 samples from convalescent individuals, but not in any of the 9 control samples (Fig. 3b). Although both recently generated circulating plasmablasts and S- and HA-binding BMPCs expressed BLIMP-1, the BMPCs were differentiated by their lack of expression of Ki-67—indicating a quiescent state—as well as by higher levels of CD38 (Fig. 3c).

figure 2
Fig. 2: SARS-CoV-2 infection elicits S-binding long-lived BMPCs.
figure 3
Fig. 3: SARS-CoV-2 S-binding BMPCs are quiescent and distinct from circulating plasmablasts.

Robust S-binding memory B cell response

Memory B cells form the second arm of humoral immune memory. After re-exposure to an antigen, memory B cells rapidly expand and differentiate into antibody-secreting plasmablasts. We examined the frequency of SARS-CoV-2-specific circulating memory B cells in individuals who were convalescing from COVID-19 and in healthy control individuals. We stained PBMCs with fluorescently labelled S probes and determined the frequency of S-binding memory B cells among isotype-switched IgDloCD20+ memory B cells by flow cytometry. For comparison, we co-stained the cells with fluorescently labelled influenza virus HA probes (Fig. 4a, Extended Data Fig. 1d). S-binding memory B cells were identified in convalescent individuals in the first sample that was collected approximately one month after the onset of symptoms, with comparable frequencies to influenza HA-binding memory B cells (Fig. 4b). S-binding memory B cells were maintained for at least 7 months after symptom onset and were present at significantly higher frequencies relative to healthy controls—comparable to the frequencies of influenza HA-binding memory B cells that were identified in both groups (Fig. 4c).

figure 4
Fig. 4: SARS-CoV-2 infection elicits a robust memory B cell response.

Discussion

This study sought to determine whether infection with SARS-CoV-2 induces antigen-specific long-lived BMPCs in humans. We detected SARS-CoV-2 S-specific BMPCs in bone marrow aspirates from 15 out of 19 convalescent individuals, and in none from the 11 control participants. The frequencies of anti-S IgG BMPCs modestly correlated with serum IgG titres at 7–8 months after infection. Phenotypic analysis by flow cytometry showed that S-binding BMPCs were quiescent, and their frequencies were largely consistent in 5 paired aspirates collected at 7 and 11 months after symptom onset. Notably, we detected no S-binding cells among plasmablasts in blood samples collected at the same time as the bone marrow aspirates by ELISpot or flow cytometry in any of the convalescent or control samples. Together, these data indicate that mild SARS-CoV-2 infection induces a long-lived BMPC response. In addition, we showed that S-binding memory B cells in the blood of individuals who had recovered from COVID-19 were present at similar frequencies to those directed against influenza virus HA. Overall, our results are consistent with SARS-CoV-2 infection eliciting a canonical T-cell-dependent B cell response, in which an early transient burst of extrafollicular plasmablasts generates a wave of serum antibodies that decline relatively quickly. This is followed by more stably maintained levels of serum antibodies that are supported by long-lived BMPCs.

Although this overall trend captures the serum antibody dynamics of the majority of participants, we observed that in three participants, anti-S serum antibody titres increased between 4 and 7 months after the onset of symptoms, after having initially declined between 1 and 4 months. This could be stochastic noise, could represent increased net binding affinity as early plasmablast-derived antibodies are replaced by those from affinity-matured BMPCs, or could represent increases in antibody concentration from re-encounter with the virus (although none of the participants in our cohort tested positive a second time). Although anti-S IgG titres in the convalescent cohort were relatively stable in the interval between 4 and 11 months after symptom onset, they did measurably decrease, in contrast to anti-influenza virus vaccine titres. It is possible that this decline reflects a final waning of early plasmablast-derived antibodies. It is also possible that the lack of decline in influenza titres was due to boosting through exposure to influenza antigens. Our data suggest that SARS-CoV-2 infection induces a germinal centre response in humans because long-lived BMPCs are thought to be predominantly germinal-centre-derived7. This is consistent with a recent study that reported increased levels of somatic hypermutation in memory B cells that target the RBD of SARS-CoV-2 S in convalescent individuals at 6 months compared to 1 month after infection20.

To our knowledge, the current study provides the first direct evidence for the induction of antigen-specific BMPCs after a viral infection in humans. However, we do acknowledge several limitations. Although we detected anti-S IgG antibodies in serum at least 7 months after infection in all 19 of the convalescent donors from whom we obtained bone marrow aspirates, we failed to detect S-specific BMPCs in 4 donors. Serum anti-S antibody titres in those four donors were low, suggesting that S-specific BMPCs may potentially be present at very low frequencies that are below the limit of detection of the assay. Another limitation is that we do not know the fraction of the S-binding BMPCs detected in our study that encodes neutralizing antibodies. SARS-CoV-2 S protein is the main target of neutralizing antibodies17,25,26,27,28,29,30 and a correlation between serum anti-S IgG binding and neutralization titres has been documented17,31. Further studies will be required to determine the epitopes that are targeted by BMPCs and memory B cells, as well as their clonal relatedness. Finally, although our data document a robust induction of long-lived BMPCs after infection with SARS-CoV-2, it is critical to note that our convalescent individuals mostly experienced mild infections. Our data are consistent with a report showing that individuals who recovered rapidly from symptomatic SARS-CoV-2 infection generated a robust humoral immune response32. It is possible that more-severe SARS-CoV-2 infections could lead to a different outcome with respect to long-lived BMPC frequencies, owing to dysregulated humoral immune responses. This, however, has not been the case in survivors of the 2014 Ebola virus outbreak in West Africa, in whom severe viral infection induced long-lasting antigen-specific serum IgG antibodies33.

Long-lived BMPCs provide the host with a persistent source of preformed protective antibodies and are therefore needed to maintain durable immune protection. However, the longevity of serum anti-S IgG antibodies is not the only determinant of how durable immune-mediated protection will be. Isotype-switched memory B cells can rapidly differentiate into antibody-secreting cells after re-exposure to a pathogen, offering a second line of defence34. Encouragingly, the frequency of S-binding circulating memory B cells at 7 months after infection was similar to that of B cells directed against contemporary influenza HA antigens. Overall, our data provide strong evidence that SARS-CoV-2 infection in humans robustly establishes the two arms of humoral immune memory: long-lived BMPCs and memory B cells. These findings provide an immunogenicity benchmark for SARS-CoV-2 vaccines and a foundation for assessing the durability of primary humoral immune responses that are induced in humans after viral infections.

Methods

Data reporting

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded during outcome assessment.

Sample collection, preparation and storage

All studies were approved by the Institutional Review Board of Washington University in St Louis. Written consent was obtained from all participants. Seventy-seven participants who had recovered from SARS-CoV-2 infection and eleven control individuals without a history of SARS-CoV-2 infection were enrolled (Extended Data Tables 14). Blood samples were collected in EDTA tubes and PBMCs were enriched by density gradient centrifugation over Ficoll 1077 (GE) or Lymphopure (BioLegend). The remaining red blood cells were lysed with ammonium chloride lysis buffer, and cells were immediately used or cryopreserved in 10% dimethyl sulfoxide in fetal bovine serum (FBS). Bone marrow aspirates of approximately 30 ml were collected in EDTA tubes from the iliac crest of 18 individuals who had recovered from COVID-19 and the control individuals. Bone marrow mononuclear cells were enriched by density gradient centrifugation over Ficoll 1077, and the remaining red blood cells were lysed with ammonium chloride buffer (Lonza) and washed with phosphate-buffered saline (PBS) supplemented with 2% FBS and 2 mM EDTA. Bone marrow plasma cells were enriched from bone marrow mononuclear cells using the CD138 Positive Selection Kit II (Stemcell) and immediately used for ELISpot or cryopreserved in 10% dimethyl sulfoxide in FBS.

Antigens

Recombinant soluble spike protein (S) and its receptor-binding domain (RBD) derived from SARS-CoV-2 were expressed as previously described35. In brief, mammalian cell codon-optimized nucleotide sequences coding for the soluble version of S (GenBank: MN908947.3, amino acids (aa) 1–1,213) including a C-terminal thrombin cleavage site, T4 foldon trimerization domain and hexahistidine tag cloned into the mammalian expression vector pCAGGS. The S protein sequence was modified to remove the polybasic cleavage site (RRAR to A) and two stabilizing mutations were introduced (K986P and V987P, wild-type numbering). The RBD, along with the signal peptide (aa 1–14) plus a hexahistidine tag were cloned into the mammalian expression vector pCAGGS. Recombinant proteins were produced in Expi293F cells (Thermo Fisher Scientific) by transfection with purified DNA using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Supernatants from transfected cells were collected 3 (for S) or 4 (for RBD) days after transfection, and recombinant proteins were purified using Ni-NTA agarose (Thermo Fisher Scientific), then buffer-exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore). For flow cytometry staining, recombinant S was labelled with Alexa Fluor 647- or DyLight 488-NHS ester (Thermo Fisher Scientific); excess Alexa Fluor 647 and DyLight 488 were removed using 7-kDa and 40-kDa Zeba desalting columns, respectively (Pierce). Recombinant HA from A/Michigan/45/2015 (aa 18–529, Immune Technology) was labelled with DyLight 405-NHS ester (Thermo Fisher Scientific); excess DyLight 405 was removed using 7-kDa Zeba desalting columns. Recombinant HA from A/Brisbane/02/2018 (aa 18–529) and B/Colorado/06/2017 (aa 18–546) (both Immune Technology) were biotinylated using the EZ-Link Micro NHS-PEG4-Biotinylation Kit (Thermo Fisher Scientific); excess biotin was removed using 7-kDa Zeba desalting columns.

ELISpot

Plates were coated with Flucelvax Quadrivalent 2019/2020 seasonal influenza virus vaccine (Sequiris), tetanus–diphtheria vaccine (Grifols), recombinant S or anti-human Ig. Direct ex vivo ELISpot was performed to determine the number of total, vaccine-binding or recombinant S-binding IgG- and IgA-secreting cells present in BMPC and PBMC samples using IgG/IgA double-colour ELISpot Kits (Cellular Technology) according to the manufacturer’s instructions. ELISpot plates were analysed using an ELISpot counter (Cellular Technology).

ELISA

Assays were performed in 96-well plates (MaxiSorp, Thermo Fisher Scientific) coated with 100 μl of Flucelvax 2019/2020 or recombinant S in PBS, and plates were incubated at 4 °C overnight. Plates were then blocked with 10% FBS and 0.05% Tween-20 in PBS. Serum or plasma were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween-20 in PBS. Goat anti-human IgG–HRP (Jackson ImmunoResearch, 1:2,500) was diluted in blocking buffer before adding to wells and incubating for 60 min at room temperature. Plates were washed 3 times with 0.05% Tween-20 in PBS, and then washed 3 times with PBS before the addition of o-phenylenediamine dihydrochloride peroxidase substrate (Sigma-Aldrich). Reactions were stopped by the addition of 1 M HCl. Optical density measurements were taken at 490 nm. The half-maximal binding dilution for each serum or plasma sample was calculated using nonlinear regression (GraphPad Prism v.8). The limit of detection was defined as 1:30.

Statistics

Spearman’s correlation coefficients were estimated to assess the relationship between 7-month anti-S and anti-influenza virus vaccine IgG titres and the frequencies of BMPCs secreting IgG specific for S and for influenza virus vaccine, respectively. Means and pairwise differences of antibody titres at each time point were estimated using a linear mixed model analysis with a first-order autoregressive covariance structure. Time since symptom onset was treated as a categorical fixed effect for the 4 different sample time points spaced approximately 3 months apart. P values were adjusted for multiple comparisons using Tukey’s method. All analyses were conducted using SAS v.9.4 (SAS Institute) and Prism v.8.4 (GraphPad), and P values of less than 0.05 were considered significant.

Flow cytometry

Staining for flow cytometry analysis was performed using cryo-preserved magnetically enriched BMPCs and cryo-preserved PBMCs. For BMPC staining, cells were stained for 30 min on ice with CD45-A532 (HI30, Thermo Fisher Scientific, 1:50), CD38-BB700 (HIT2, BD Horizon, 1:500), CD19-PE (HIB19, 1:200), CXCR5-PE-Dazzle 594 (J252D4, 1:50), CD71-PE-Cy7 (CY1G4, 1:400), CD20-APC-Fire750 (2H7, 1:400), CD3-APC-Fire810 (SK7, 1:50) and Zombie Aqua (all BioLegend) diluted in Brilliant Stain buffer (BD Horizon). Cells were washed twice with 2% FBS and 2 mM EDTA in PBS (P2), fixed for 1 h using the True Nuclear permeabilization kit (BioLegend), washed twice with perm/wash buffer, stained for 1h with DyLight 405-conjugated recombinant HA from A/Michigan/45/2015, DyLight 488- and Alexa 647-conjugated S, Ki-67-BV711 (Ki-67, 1:200, BioLegend) and BLIMP-1-A700 (646702, 1:50, R&D), washed twice with perm/wash buffer, and resuspended in P2. For memory B cell staining, PBMCs were stained for 30 min on ice with biotinylated recombinant HAs diluted in P2, washed twice, then stained for 30 min on ice with Alexa 647-conjugated S, IgA-FITC (M24A, Millipore, 1:500), IgG-BV480 (goat polyclonal, Jackson ImmunoResearch, 1:100), IgD-SB702 (IA6-2, Thermo Fisher Scientific, 1:50), CD38-BB700 (HIT2, BD Horizon, 1:500), CD20-Pacific Blue (2H7, 1:400), CD4-BV570 (OKT4, 1:50), CD24-BV605 (ML5, 1:100), streptavidin-BV650, CD19-BV750 (HIB19, 1:100), CD71-PE (CY1G4, 1:400), CXCR5-PE-Dazzle 594 (J252D4, 1:50), CD27-PE-Cy7 (O323, 1:200), IgM-APC-Fire750 (MHM-88, 1:100), CD3-APC-Fire810 (SK7, 1:50) and Zombie NIR (all BioLegend) diluted in Brilliant Stain buffer (BD Horizon), and washed twice with P2. Cells were acquired on an Aurora using SpectroFlo v.2.2 (Cytek). Flow cytometry data were analysed using FlowJo v.10 (Treestar). In each experiment, PBMCs were included from convalescent individuals and control individuals.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

Relevant data are available from the corresponding author upon reasonable request.

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Trained Innate Immunity, Epigenetics, and Covid-19

Authors: Alberto Mantovani, M.D., and Mihai G. Netea, M.D.

Innate immunity is mediated by different cell types and cell-associated or fluid-phase pattern-recognition molecules and plays a key role in tissue repair and resistance against pathogens.1 Exposure to selected vaccines, such as bacille Calmette–Guérin (BCG) or microbial components, can increase the baseline tone of innate immunity and trigger pathogen-agnostic antimicrobial resistance (known as trained innate immunity). Such training is directly relevant to resistance against infectious diseases, including Covid-19. A recent study by de Laval et al.2 pinpoints a driver of durable innate immune memory conferred by myeloid cells (monocytes, macrophages, and neutrophils).

Myeloid cells are central players in innate immunity: they produce effector molecules and contribute to the activation, orientation, and regulation of adaptive immune responses. Diversity and plasticity are fundamental properties of myeloid cells, particularly macrophages. To some extent, these properties are imprinted through ontogenetic origin (embryonal vs. adult bone marrow), but they are also influenced by environmental cues in the tissue. Moreover, in response to microbial molecules, metabolic products, or cytokines, macrophages increase effector function (“activation”), are primed for short-term responses (“priming”), or become unresponsive (“tolerance”). Microbial components can also cause long-term imprinting (“training”) of innate immunity and myeloid-cell function (Figure 1).3 (This type of imprinting is distinct from genomic imprinting whereby methyl groups are added to DNA in or near specific genes.)

For More Information: https://www.nejm.org/doi/10.1056/NEJMcibr2011679

Multi-layered transcriptomic analyses reveal an immunological overlap between COVID-19 and hemophagocytic lymphohistiocytosis associated with disease severity

Authors: Lena F. Schimkea,5, Alexandre H.C. Marquesa, Gabriela Crispim Baiocchia, Caroline Aliane de Souza Pradob Dennyson Leandro M. Fonsecab , Paula Paccielli Freirea , Desirée Rodrigues Plaçab , Igor Salerno Filgueirasa ,Ranieri Coelho Salgadoa, Gabriel Jansen-Marquesc, Antonio Edson Rocha liveirab
, Jean PierreSchatzmann Perona, José Alexandre Marzagão Barbutoa,d, Niels Olsen Saraiva Camaraa
, Vera Lúcia Garcia Calicha , Hans D. Ochse, Antonio Condino-Netoa, Katherine A. Overmyerf,g, Joshua J. Coonh,i, JosephBalnisj,k, Ariel Jaitovichj,k, Jonas Schulte-Schreppingl, Thomas Ulasm, Joachim L. Schultzel,m, Helder I.Nakayab, Igor Jurisican,o,p, Otavio Cabral-Marquesa,b,q

ABSTRACT
Clinical and hyperinflammatory overlap between COVID-19 and hemophagocytic lymphohistiocytosis (HLH) has been reported. However, the underlying mechanisms are unclear. Here we show that COVID-19 and HLH have an overlap of signaling pathways and gene signatures commonly dysregulated, which were defined by investigating the transcriptomes of
1253 subjects (controls, COVID-19, and HLH patients) using microarray, bulk RNA-sequencing (RNAseq), and single-cell RNAseq (scRNAseq). COVID-19 and HLH share pathways involved in cytokine and chemokine signaling as well as neutrophil-mediated immune responses that associate with COVID-19 severity. These genes are dysregulated at protein level across several
COVID-19 studies and form an interconnected network with differentially expressed plasma proteins which converge to neutrophil hyperactivation in COVID-19 patients admitted to the intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils.


Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention.

INTRODUCTION
More than one year of Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome Coronavirus (SARS-CoV)-2, more than 197 million cases and 4,2 million deaths have been reported worldwide (July 30th 2021, WHO COVID-19 Dashboard). The clinical presentation ranges from asymptomatic to severe disease manifesting as pneumonia, acute respiratory distress syndrome (ARDS), and a life-threatening hyperinflammatory syndrome associated with excessive cytokine release (hypercytokinaemia)1–3 . Risk factors for severe manifestation and higher mortality include old age as well as hypertension, obesity, and diabetes4. Currently, COVID-19 continues to spread, new variants of SARS-CoV-2 have been reported and the number of infections resulting in death of young individuals with no comorbidities has increased the mortality rates among the young population 5,6. In addition, some novel SARS-CoV-2 variants of concern appear to escape neutralization by vaccine-induced humoral immunity7 . Thus, the need for a better understanding of the immunopathologic mechanisms associated with severe SARS-CoV-2 infection.


Patients with severe COVID-19 have systemically dysregulated innate and adaptive immune responses, which are reflected in elevated plasma levels of numerous cytokines and chemokines including granulocyte colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), interleukin (IL)-6, IL-6R, IL18, CC chemokine ligand 2 (CCL2) and CXC chemokine ligand 10
(CXCL10)8–10 , and hyperactivation of lymphoid and myeloid cells11. Notably, the hyperinflammation in COVID-19 shares similarities with cytokine storm syndromes such as those triggered by sepsis, autoinflammatory disorders, metabolic conditions and malignancies12–14 ,often resembling a hematopathologic condition called hemophagocytic lymphohistiocytosis
(HLH)15. HLH is a life-threatening progressive systemic hyperinflammatory disorder characterized by multi-organ involvement, fever flares, hepatosplenomegaly, and cytopenia due to hemophagocytic activity in the bone marrow15–17 or within peripheral lymphoid organs such as pulmonary lymph nodes and spleen. HLH is marked by aberrant activation of B and T lymphocytes and monocytes/macrophages, coagulopathy, hypotension, and ARDS. Recently, neutrophil hyperactivation has been shown to also play a critical role in HLH development18,19. This is in agreement with the observation that the HLH-like phenotype observed in severe COVID-19 patients is due to an innate neutrophilic hyperinflammatory response associated with available under aCC-BY-NC-ND 4.0 International license. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

It is made bioRxiv preprint doi: ttps://doi.org/10.1101/2021.07.30.454529; this version posted August 1, 2021. The copyright holder for this preprint
virus-induced hypercytokinaemia which is dominant in patients with an unfavorable clinical course17 . Thus, HLH has been proposed as an underlying etiologic factor of severe COVID191,3,20. HLH usually develops during the acute phase of COVID-191,20–27 . However, a case of HLH that occurred two weeks after recovery from COVID-19 has recently been reported as the cause
of death during post-acute COVID-19 syndrome28
.
The familial form of HLH (fHLH) is caused by inborn errors of immunity (IEI) in different genes encoding proteins involved in granule-dependent cytotoxic activity of leukocytes such as AP3B1, LYST, PRF1, RAB27A, STXBP2, STX11, UNC13D29–31. In contrast, the secondary form (sHLH) usually manifests in adults following a viral infection (e.g., adenovirus, EBV, enterovirus, hepatitis viruses, parvovirus B19, and HIV)32,33, or in association with autoimmune /rheumatologic, malignant, or metabolic conditions that lead to defects in T/NK cell functions and excessive inflammation16,31. fHLH and sHLH affect both children and adults, however, the clinical and genetic distinction of HLH forms is not clear since immunocompetent children can develop sHLH 34,35, while adult patients with sHLH may also have germline mutations in HLH genes36. Of note, germline variants in UNC13D and AP3B1 have also been
identified in some COVID-19 patients with HLH phenotype37, thus, indicating that both HLH forms may be associated with COVID-19.


Here, we characterized the signaling pathways and gene signatures commonly dysregulated in both COVID-19 and HLH patients by investigating the transcriptomes of 1253 subjects (controls, COVID-19, and HLH patients) assessed by microarray, bulk RNA-sequencing (RNAseq), and single-cell RNAseq (scRNAseq) (Table 1). We found shared gene signatures and cellular signaling pathways involved in cytokine and chemokine signaling as well as neutrophilmediated immune responses that associate with COVID-19 severity.

For More Information: https://www.biorxiv.org/content/10.1101/2021.07.30.454529v1.full.pdf

Good news: Mild COVID-19 induces lasting antibody protection

People who have had mild illness develop antibodyproducing cells that can last lifetime

Authors: by Tamara Bhandari•May 24, 2021

Months after recovering from mild cases of COVID-19, people still have immune cells in their body pumping out antibodies against the virus that causes COVID-19, according to a study from researchers at Washington University School of Medicine in St. Louis. Such cells could persist for a lifetime, churning out antibodies all the while.

The findings, published May 24 in the journal Nature, suggest that mild cases of COVID-19 leave those infected with lasting antibody protection and that repeated bouts of illness are likely to be uncommon.

“Last fall, there were reports that antibodies wane quickly after infection with the virus that causes COVID-19, and mainstream media interpreted that to mean that immunity was not long-lived,” said senior author Ali Ellebedy, PhD, an associate professor of pathology & immunology, of medicine and of molecular microbiology. “But that’s a misinterpretation of the data. It’s normal for antibody levels to go down after acute infection, but they don’t go down to zero; they plateau. Here, we found antibody-producing cells in people 11 months after first symptoms. These cells will live and produce antibodies for the rest of people’s lives. That’s strong evidence for long-lasting immunity.”

For More Information: https://medicine.wustl.edu/news/good-news-mild-covid-19-induces-lasting-antibody-protection/

COVID-19 Science Update released: June 4, 2021 Edition 92

Authors: From the Office of the Chief Medical Officer, CDC COVID-19 Response, and the CDC Library, Atlanta GA. Intended for use by public health professionals responding to the COVID-19 pandemic.

PEER-REVIEWED

Safety, immunogenicity, and efficacy of the BNT162b2 COVID-19 vaccine in adolescents.external icon Frenck et al. NEJM (May 27, 2021).

Key findings:

  • Vaccine efficacy was 100% (95% CI 75.3%-100%) in 12- to 15-year-olds.
    • There were no cases in the vaccinated group compared with 16 cases among the placebo group, 7 or more days after dose 2.
  • Compared with baseline, geometric mean neutralizing antibody titers were 118.3-fold higher 1 month after dose 2.
  • Vaccine reactions were mainly transient, mild to moderate, and similar to a comparator group of 16–25-year-olds.
    • Injection-site pain was reported by 79% to 86%, fatigue was reported by 60% to 66%, and headache was reported by 55% to 65% of participants (Figure).

Methods: A randomized, placebo-controlled, observer-blinded trial of Pfizer/BioNTech BNT162b2 in 2,260 adolescents 12–15 years old (1,129 received placebo). Efficacy of the vaccine was assessed based on confirmed SARS-CoV-2 infection with onset 7 or more days after dose 2. Reactogenicity events (assessed for 7 days after each dose) and unsolicited adverse events compared with 16–25 age group (n = 3,610). SARS-CoV-2 serum neutralization assays were performed. LimitationsRacial and ethnic diversity of participants 12-15 years does not reflect the general US population; short (1 month) post-vaccination safety evaluation.

Implications: Vaccination of adolescents with BNT162b2 was safe and effective. Vaccinating adolescents will broaden community protection, and it will likely facilitate reintegration into society and resumption of in-person learning.

Figure:Graphs showing systemic events with 7 days after dose 1 or dose 2 of vaccine or placeboresize iconView Larger

Note: Adapted from Frenck et al. Systemic events reported within 7 days after receiving dose 1 (top) or dose 2 (bottom) of vaccine or placebo. 1 participant in the 12-to-15-year-old group had a fever with a temperature >40°C after dose 1. From the New England Journal of Medicine, Frenck et al., Safety, immunogenicity, and efficacy of the BNT162b2 COVID-19 vaccine in adolescents. May 27, 2021, online ahead of print. Copyright © 2021 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.

Occurrence of severe COVID-19 in vaccinated transplant patientsexternal icon. Caillard et al. Kidney International. (May 21, 2021).

Key findings:

  • 55 solid organ transplant recipients developed COVID-19 after receiving 2 doses of mRNA vaccine.
    • Symptoms began a median of 22 days after the second vaccine dose (Figure).
    • 15 cases required hospitalization; of these, 6 were admitted to an intensive care unit, and 3 died.
  • Of 25 patients with post-vaccination serology, 24 were antibody negative; 1 was antibody positive but had low titers.

For More Information: https://www.cdc.gov/library/covid19/06042021_covidupdate.html

A long-term perspective on immunity to COVID

Determining the duration of protective immunity to infection by SARS-CoV-2 is crucial for understanding and predicting the course of the COVID-19 pandemic. Clinical studies now indicate that immunity will be long-lasting.

Authors: Andreas Radbruch & Hyun-Dong Chang

Generating immunity against the SARS-CoV-2 coronavirus is of the utmost importance for bringing the COVID-19 pandemic under control, protecting vulnerable individuals from severe disease and limiting viral spread. Our immune systems protect against SARS-CoV-2 either through a sophisticated reaction to infection or in response to vaccination. A key question is, how long does this immunity last? Writing in NatureTurner et al.1 and Wang et al.2 characterize human immune responses to SARS-CoV-2 infection over the course of a year.

There is ongoing discussion about which aspects of the immune response to SARS-CoV-2 provide hallmarks of immunity (in other words, correlates of immunological protection). However, there is probably a consensus that the two main pillars of an antiviral response are immune cells called cytotoxic T cells, which can selectively eliminate infected cells, and neutralizing antibodies, a type of antibody that prevents a virus from infecting cells, and that is secreted by immune cells called plasma cells. A third pillar of an effective immune response would be the generation of T helper cells, which are specific for the virus and coordinate the immune reaction. Crucially, these latter cells are required for generating immunological memory — in particular, for orchestrating the emergence of long-lived plasma cells3, which continue to secrete antiviral antibodies even when the virus has gone.

Immunological memory is not a long-lasting version of the immediate immune reaction to a particular virus; rather, it is a distinct aspect of the immune system. In the memory phase of an immune response, B and T cells that are specific for a virus are maintained in a state of dormancy, but are poised to spring into action if they encounter the virus again or a vaccine that represents it. These memory B and T cells arise from cells activated in the initial immune reaction. The cells undergo changes to their chromosomal DNA, termed epigenetic modifications, that enable them to react rapidly to subsequent signs of infection and drive responses geared to eliminating the disease-causing agent4. B cells have a dual role in immunity: they produce antibodies that can recognize viral proteins, and they can present parts of these proteins to specific T cells or develop into plasma cells that secrete antibodies in large quantities. About 25 years ago5, it became evident that plasma cells can become memory cells themselves, and can secrete antibodies for long-lasting protection. Memory plasma cells can be maintained for decades, if not a lifetime, in the bone marrow6.

The presence in the bone marrow of long-lived, antibody-secreting memory plasma cells is probably the best available predictor of long-lasting immunity. For SARS-CoV-2, most studies so far have analyzed the acute phase of the immune response, which spans a few months after infection, and have monitored T cells, B cells and secreted antibodies7. It has remained unclear whether the response generates long-lived memory plasma cells that secrete antibodies against SARS-CoV-2.

For More Information: https://www.nature.com/articles/d41586-021-01557-z