key: cord-0863575-3ato89qx
authors: Izmirly, Peter M.; Kim, Mimi Y.; Samanovic, Marie; Fernandez‐Ruiz, Ruth; Ohana, Sharon; Deonaraine, Kristina K.; Engel, Alexis J.; Masson, Mala; Xie, Xianhong; Cornelius, Amber R.; Herati, Ramin S.; Haberman, Rebecca H.; Scher, Jose U.; Guttmann, Allison; Blank, Rebecca B.; Plotz, Benjamin; Haj‐Ali, Mayce; Banbury, Brittany; Stream, Sara; Hasan, Ghadeer; Ho, Gary; Rackoff, Paula; Blazer, Ashira D.; Tseng, Chung‐E; Belmont, H. Michael; Saxena, Amit; Mulligan, Mark J.; Clancy, Robert M.; Buyon, Jill P.
title: Evaluation of Immune Response and Disease Status in SLE Patients Following SARS‐CoV‐2 Vaccination
date: 2021-08-04
journal: Arthritis Rheumatol
DOI: 10.1002/art.41937
sha: 716a178538b93aa13ddbf45f87402743987a79c1
doc_id: 863575
cord_uid: 3ato89qx

OBJECTIVE: To evaluate seroreactivity and disease flares after COVID‐19 vaccination in a multi‐ethnic/racial cohort of patients with systemic lupus erythematosus (SLE). METHODS: 90 SLE patients and 20 healthy controls receiving a complete COVID‐19 vaccine regimen were included. IgG seroreactivity to the SARS‐CoV‐2 spike receptor‐binding domain (RBD) and SARS‐CoV‐2 microneutralization were used to evaluate B cell responses; IFN‐γ production to assess T cell responses was measured by ELISpot. Disease activity was measured by the hybrid SLE disease activity index (SLEDAI) and flares were assigned by the SELENA/SLEDAI flare index. RESULTS: Overall, fully vaccinated SLE patients produced significantly lower IgG antibodies against SARS‐CoV‐2 spike RBD than controls. Twenty‐six SLE patients (28.8%) generated an IgG response below that of the lowest control (<100 units/ml). In logistic regression analyses, the use of any immunosuppressant or prednisone and a normal anti‐dsDNA level prior to vaccination associated with decreased vaccine responses. IgG seroreactivity to the SARS‐CoV‐2 Spike RBD strongly correlated with the SARS‐CoV‐2 microneutralization titers and antigen‐specific IFN‐γ production determined by ELISpot. In a subset of patients with poor antibody responses, IFN‐γ production was likewise diminished. Pre‐/post‐vaccination SLEDAI scores were similar. Only 11.4% of patients had a post‐vaccination flare; 1.3% were severe. CONCLUSION: In a multi‐ethnic/racial study of SLE patients 29% had a low response to the COVID‐19 vaccine which was associated with being on immunosuppression. Reassuringly, disease flares were rare. While minimal protective levels remain unknown, these data suggest protocol development is needed to assess efficacy of booster vaccination.

As scientific advances have been applied with unprecedented speed during the COVID-19 pandemic, physicians and their patients have pivoted from treatment of infection and passive immunization to full-scale preventative measures, particularly in high-risk individuals (1, 2) . Patients with systemic lupus erythematosus (SLE) comprise a unique population with regard to risk for infection and outcomes associated with SARS-CoV-2 given underlying demographics, associated organ damage and comorbidities. In addition, medications commonly used to treat SLE have been associated with an increased risk of death from COVID-19 (3) . Early data provided evidence that patients with SLE have a high risk for hospitalization from COVID-19, with factors including race/ethnicity, comorbidities such as cardiovascular disease and renal insufficiency, and higher body mass index identified as independent predictors of hospitalization (1, 4) . Further raising concern, infection was reported to associate with flares of disease (5) . In subsequent studies, patients with SLE and confirmed COVID-19

were demonstrated to generate and maintain serologic responses despite the use of a variety of immunosuppressants (6) . These data provided reassurance regarding the efficacy and durability of humoral immunity and protection against reinfection with

This article is protected by copyright. All rights reserved SARS-CoV-2, as well as potential insights into the efficacy of active immunization in SLE patients.

Since the Phase III clinical studies of all three vaccines excluded patients treated with immunosuppressants or immune-modifying drugs within six months of enrollment, data on SLE is virtually absent (7) (8) (9) . Furthermore, given the potential of disease flares following immunization, it is not surprising that a recent study reported hesitancy for vaccination in patients with rheumatic diseases including SLE (10) . Accordingly, the current study was initiated to address these critical gaps and examine the efficacy of these promising COVID-19 vaccines in patients with SLE. This was accomplished by evaluation of a multi-ethnic/racial cohort of SLE patients with assessment of serologic responses and compared to healthy controls. The assays included antibodies to the spike protein receptor-binding domain (RBD), virus-neutralizing antibodies, and antigenspecific T cell production of interferon IFN-γ, both prior to and post-vaccination. Factors associated with the level of responsiveness were sought. In addition, SLE disease activity pre and post vaccination was measured as well as the rate of flare post vaccination. 

This article is protected by copyright. All rights reserved following: 1) the American College of Rheumatology (ACR) revised classification criteria (11) ; 2) the Systemic Lupus International Collaborating Clinics (SLICC) classification criteria (12) ; 3) the European League Against Rheumatism (EULAR)/ACR classification criteria (13) . All NYU lupus cohort patients and controls signed an informed consent, which is available in English, Spanish and Mandarin. All adult patients with SLE planning on receiving any of the available COVID-19 vaccines were eligible for inclusion. Exclusion criteria included unwillingness to provide blood after the second dose of the vaccine, incomplete vaccination schedule, and speaking a language other than English, Spanish, or Mandarin. Healthy controls were ≥18 years of age, had no known rheumatologic diseases and were on no immunosuppressive medications. The study protocol and the NYU Lupus Cohort and recruitment of controls were approved by the NYU and Bellevue Hospital Institutional Review Boards.

Patients were recruited using convenience sampling with inclusion and exclusion criteria as stated above. For most patients, blood samples were available pre-and post-vaccination. Pre-vaccination disease activity measures and laboratory data were available as part of the NYU Lupus Cohort but were limited to patients seen within four months of their first vaccine dose. Post-vaccination follow-ups were scheduled approximately two weeks post-second dose of the mRNA vaccines or post-one dose of the J&J vaccine to collect post-vaccination blood samples and assess for any change in SLE activity. Disease activity was measured by the hybrid SELENA/SLEDAI (urine protein/creatinine ratios > 0.5 were always counted) and flares assessed by the SELENA/SLEDAI flare index (14) (15) (16) . In addition, use and dosages of

This article is protected by copyright. All rights reserved immunosuppressive medications were recorded at each visit, including, among others, glucocorticoids, hydroxychloroquine (HCQ), azathioprine, mycophenolate mofetil (MMF), methotrexate, belimumab, and tacrolimus. Any cyclophosphamide, obinutuzumab, or rituximab administered within six months of the patient's visit was also recorded. Given our patients were largely enrolled before the American College of Rheumatology updated their guidelines to hold MMF used to treat more severe manifestations such as nephritis, that medication was not held. We did advise patients to hold methotrexate and adjusted other medications as recommended per their guidelines (17) .

Ninety-six well plates were coated with 1 µg/ml recombinant SARS-CoV-2 spike RBD (R&D Systems, #BT10500), diluted in PBS and incubated overnight at 4°C. Plates were blocked with 0.1% gelatin in PBS. Plasma (spun 10,000 RPM for one minute) were diluted 1:200-1:100,000 and added to the plate for 1 hour at room temperature.

Samples were run in triplicate. With each run, two positive controls were included in the 96-well plate: plasma from control non-SLE participants post-vaccination, with high and low IgG titers, each diluted 1:500 to ensure that measurements were captured across the assay range. Detection relies on an enzyme-labeled secondary antibody, alkalinephosphatase conjugated rabbit anti-human IgG (γ-chain specific) (Sigma, St Louis, MO) diluted 1:2,000. After developing with the addition of phosphatase substrate, the optical density (OD) was measured at 405 nm, and the reaction was evaluated when the low positive control reached an OD of 1. The OD measured for a tested sample was multiplied by the dilution factor which gave an OD in the range of 0.3-0.8.

This article is protected by copyright. All rights reserved

Viral neutralization activity of plasma was measured in an immunofluorescencebased microneutralization assay by detecting the neutralization of infectious virus in 

This article is protected by copyright. All rights reserved 

Categorical variables were summarized by computing counts and proportions of patients (%). Continuous variables are expressed as mean (SD), median with interquartile range (IQR), or range, as appropriate. Two group comparisons were performed using the Chi-square or Fisher's exact tests for categorical variables and the two-sample T-test or Mann-Whitney U tests for continuous variables. The Spearman rank correlation coefficient was computed for the association between the ELISA and microneutralization assays. An exploratory logistic regression analysis was also conducted to identify potential independent predictors of low post-vaccine ELISA antibody response (≤100 units/ml, the lowest value seen in controls). Variable selection

This article is protected by copyright. All rights reserved in the final model was based on both statistical significance (p<0.10 given limited sample size and power of the study) as well as clinical considerations. All statistical analyses were performed using SAS version 9.4.

A total of 90 patients with SLE and 20 controls were included in this study. Table   1 shows the demographics of the patients and controls in addition to SLE-specific information. Cases and controls were relatively well matched; however, controls were more likely to be male (p<0.01). Whereas controls only received the Pfizer/BioNTech and Moderna vaccines, SLE patients received all three vaccines currently available in the US, inclusive of J&J. In addition, 12% of the SLE patients had a history of prior COVID-19 compared to 10% of controls. Forty-four percent of patients had a history of lupus nephritis (LN), 10% had secondary antiphospholipid syndrome (APLS), and 5.6% had a kidney transplant. The majority of patients were on HCQ (79%), and 29% were on systemic glucocorticoids (mean dose of 7 mg of prednisone). Forty-two percent were on at least one immunosuppressant, with MMF (21%) being the most common, followed by belimumab (11%). In addition, 17% were on more than one immunosuppressant. Figure   1 shows the number of SLE patients included in each subsequent analysis. Table 2 . In unadjusted analyses, low responders were more likely to be on prednisone, MMF or mycophenolic acid, a combination of prednisone and at least one immunosuppressant, or two or more immunosuppressants, whereas high responders were more likely to be on only antimalarials or no medication. In addition, low responders were more likely to have received the J&J vaccine, although sample sizes were limited, and to have had a normal anti-dsDNA level prior to vaccination, Further details of the non-responders are provided in Table 3 .

Sixteen SLE patients (of whom four were pre-and post-vaccine) and two controls (both pre-and post-vaccine) were further evaluated addressing T cell reactivity which was operationally reported by the release of IFN-γ in response to challenge of PBMCs in the absence and presence of the full length S1 protein (COVID-19 antigen), as described in the Methods. Individuals were chosen to represent a range of responses to the SARS-CoV-2 spike protein and SARS-CoV-2 microneutralization assay but with a particular focus on the patients with low seroreactivity on both assays. As shown in Supplemental Figure 1 , there was a correlation of the post-vaccine ELISpot number and ELISA evaluations, (R=0.57; p=0.0135). In a subset of patients with poor antibody responses, IFN-γ production was likewise diminished (Supplemental Figure 1) .

This article is protected by copyright. All rights reserved Table 1 ). Overall, there was no meaningful difference in SLEDAI score between pre-and post-vaccine visits (3.2 vs 2.9). There were no changes in the percentages of patients with abnormal anti-dsDNA antibodies and/or abnormal complement levels; likewise, the levels of C3 and C4 were similar pre-and postvaccination (Supplemental Table 1 ). Nine of the 79 patients (11.4%) had a postvaccination flare, with all but one considered to be mild/moderate (two in new organ systems: arthritis with no treatment, and pericarditis treated with naproxen). The severe flare was characterized by arthritis and treated with methotrexate; the patient had discontinued HCQ due to maculopathy several years prior to vaccination. Further details of the flares are provided in Table 4 .

To our knowledge, this is the first reported study focused on patients with SLE who received full regimens of a COVID-19 vaccine, overall IgG antibody responses against the RBD of SARS-CoV-2 spike protein were significantly decreased compared to controls, with 28.8% of patients generating responses falling below the lowest level observed in the healthy controls. Being on any immunosuppressive agent other than antimalarials and having a normal anti-dsDNA prior to vaccination were identified as independent predictors for poor response to the COVID-19 vaccine. Seroreactivity to the SARS-CoV-2 spike RBD strongly correlated with the functional SARS-CoV-2

This article is protected by copyright. All rights reserved microneutralization assay and with the ELISpot assay. Overall, there was no change in SLEDAI pre-and post-vaccination, with 11.4% of patients having a flare, 1.3% being severe, supporting the relative safety of the vaccination in SLE patients.

The finding of anti-dsDNA antibodies positively correlating with higher responses to COVID-19 vaccination was initially unexpected, especially given that this finding persisted even after controlling for medication use. Moreover, disease activity per se was not associated with more effective seroreactivity. It could be hypothesized that the presence of anti-dsDNA antibodies is a proxy of elevated type I interferon activity in these patients. Indeed, studies have shown that high IFN-α activity in patients with SLE associates with the presence of disease-specific autoantibodies, such as anti-dsDNA (18). These autoantibodies can form immune complexes, further stimulating type I IFN production (19) . Beside their potent antiviral properties, type I IFNs induce the maturation and activation of myeloid dendritic cells, and promote B cell survival and differentiation into antibody-producing cells (20, 21) . These considerations support the hypothesis that higher responders to the COVID-19 vaccines could be those with higher baseline type I IFN activity, due to its potential to enhance antibody responses to foreign antigens. Thus, patients with anti-dsDNA antibodies, despite being on immunosuppression, may be more likely to develop a strong humoral response to the COVID-19 vaccines. Alternatively, these analyses did not account for patient adherence with medication, or the possibility that elevated dsDNA reflects inefficacy of immunosuppression, which might account for these findings. These potential insights merit further investigation.

This article is protected by copyright. All rights reserved (27) . A subsequent analysis of 89 patients that included 10 patients with SLE showed that rituximab was associated with impaired serologic response to the

This article is protected by copyright. All rights reserved SARS-CoV-2 vaccine (28) . Haberman et al. (29) demonstrated that methotrexate adversely affected both the humoral and cellular immune responses to COVID-19 mRNA vaccines in patients with immune-mediated inflammatory diseases. A large study from Furer et al., including 101 patients with SLE, showed that older age and treatment with glucocorticoids, rituximab, MMF, and abatacept associated with reduced immunogenicity as measured by serum IgG antibody levels against SARS-CoV-2 spike S1/S2 proteins 2-6 weeks after vaccination (30) . Our study showed that being on any non-antimalarial immunosuppression independently associated with decreased response to COVID-19 vaccines in patients with SLE.

In addition to concerns regarding inefficient immune responses to COVID vaccination, it may be the case that vaccination induces increased autoantibody production and disease activity. As speculated by Tang et al, delivery of mRNA encoding S protein via the vaccine, likely degraded by normal cellular processes, could interact with a number of cytoplasmic RNA-binding proteins involved in the posttranscriptional regulation of inflammation and result in worsening SLE (5) . Similarly, RNA vaccines may trigger Toll-like receptors, generating further production of type I IFN, already well-recognized to be elevated in most SLE patients (19) . It has been reported that influenza vaccines triggered transient increase in several autoantibody specificities in 72 SLE patients, with a flare rate of 19.4% within six weeks postvaccination; 10 (13.9%) were mild/moderate and 4 (5.6%) were severe (31) . In a study evaluating SLE flares after immunization against poliomyelitis, only 4 (5%) of 73 patients flared (32) . In aggregate, despite apprehensions, the data presented herein did not support significantly increased anti-dsDNA autoantibody production or flares post-

This article is protected by copyright. All rights reserved vaccination. These results are consistent with a recent study which showed the majority of vaccinated SLE patients had no change or decrease in disease activity after COVID-19 vaccination as measured by the SLEDAI (30) .

Our study has several limitations. Similar to other studies evaluating potential surrogate markers for vaccine efficacy, it is premature to assign a threshold level of protection based on either the IgG response to the anti-RBD of SARS-CoV-2 spike protein or the microneutralization assay given the number of controls. There was vaccine hesitancy amongst patients in the NYU Lupus Cohort, in large part because of concern regarding the potential effect on lupus activity, and thus the patients in this study may not be fully representative of the patients seen in our cohort. While known prior COVID-19 was accounted for in all patients, it remains possible that asymptomatic or mild infection occurred between pre-vaccine blood draw and vaccination, which could This study has several strengths. In contrast to previous reports, the focus was limited to patients with SLE and, to our knowledge, is the first to assess the COVID-19

vaccines' effects on lupus-specific disease activity with availability of a validated

This article is protected by copyright. All rights reserved disease index pre-and post-vaccination in the majority of patients. Flares were rare, with only 1.3% being severe. These data are reassuring and support that vaccines do not exacerbate disease activity, a finding which should alleviate vaccine hesitancy. Our study assessed two surrogate markers for B cell reactivity and a surrogate for T-cell mediated responses. Although the latter was limited to fewer patients, it was particularly applied to evaluate those with lower humoral responses and reinforced the concern about vaccine efficacy in a subset of these individuals.

In summary, in a multi-racial/ethnic study of SLE patients receiving a complete COVID-19 vaccine regimen, nearly 30% had a low response. Having a normal anti-dsDNA and taking any immunosuppressive medication other than antimalarials independently associated with a decreased vaccine response. While minimal protective antibody levels remain unknown, these results, supported by other studies, raise concerns for our lupus patients, many of whom rely on medications to maintain low disease activity. Accordingly, the next phase of scientific inquiry and advance should focus on protocols addressing additional vaccination. Reassuringly, disease flares are infrequent, which should encourage patients to consider vaccination. This article is protected by copyright. All rights reserved 

Leveraging the United States Epicenter to Provide Insights on COVID-19 in Patients with Systemic Lupus Erythematosus. Arthritis Rheumatol. 2020. 2. Fernandez-Ruiz R, Paredes JL, Niewold TB. COVID-19 in patients with systemic lupus erythematosus: lessons learned from the inflammatory disease

Factors associated with COVID-19-related death in people with rheumatic diseases: results from the COVID-19 Global Rheumatology Alliance physician-reported registry

Characteristics associated with hospitalisation for COVID-19 in people with rheumatic disease: data from the COVID-19 Global Rheumatology Alliance physician-reported registry

SARS-CoV-2 vaccines in patients with SLE

Evaluation of SARS-CoV-2 IgG antibody reactivity in patients with systemic lupus erythematosus: analysis of a multi-racial and multi-ethnic cohort

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine

Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-19

Perspective of patients with autoimmune diseases on COVID-19 vaccination

Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus

Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus

Systemic lupus erythematosus disease activity index

The effect of combined estrogen and progesterone hormone replacement therapy on disease activity in systemic lupus erythematosus: a randomized trial

Development and initial validation of the systemic lupus erythematosus disease activity index 2000 responder index 50

American College of Rheumatology Guidance for COVID-19 Vaccination in Patients With Rheumatic and Musculoskeletal Diseases: Version 2. Arthritis Rheumatol. 2021. 18. Niewold TB

Type I interferon in the pathogenesis of systemic lupus erythematosus

A pathogenic IFNα, BLyS and IL-17 axis in Systemic Lupus Erythematosus patients

Type I interferon-mediated autoimmune diseases: pathogenesis, diagnosis and targeted therapy

Immunogenicity of a Single Dose of SARS-CoV-2 Messenger RNA Vaccine in Solid Organ Transplant Recipients

Antibody Response to 2-Dose SARS-CoV-2 mRNA Vaccine Series in Solid Organ Transplant Recipients

High antibody response to two-dose SARS-CoV-2 messenger RNA vaccination in patients with rheumatic and musculoskeletal diseases

Immunogenicity and safety of anti-SARS-CoV-2 mRNA vaccines in patients with chronic inflammatory conditions and immunosuppressive therapy in a monocentric cohort

Cell Depleting Agents Substantially Impair Immunogenicity of mRNA Vaccines to SARS-CoV-2. medRxiv

but not other antirheumatic therapies, is associated with impaired serological response to SARS-CoV-2 vaccination in patients with rheumatic diseases

Methotrexate hampers immunogenicity to BNT162b2 mRNA COVID-19 vaccine in immune-mediated inflammatory disease

Immunogenicity and safety of the BNT162b2 mRNA COVID-19 vaccine in adult patients with autoimmune inflammatory rheumatic diseases and in the general population: a multicentre study

Influenza vaccination responses in human systemic lupus erythematosus: impact of clinical and demographic features

Poliovaccines and the course of systemic lupus erythematosus--a retrospective study of 73 patients

This article is protected by copyright. All rights reserved Only antimalarials (HCQ+CHLOR)** among those taking any meds

*Continuous variables summarized as mean (SD)

MMF= mycophenolate mofetil, MYF= Mycophenolic acid

Laboratory measures and SLEDAI based on N=66 patients with pre-vaccine data available within 4 months of vaccine

The authors would like to thank the patients who participated in the study. They would also like to acknowledge Ranit Shriky and Rebecca Cohen for their assistance with regulatory matters and Benjamin Wainwright for his contributions to the manuscript.

This article is protected by copyright. All rights reserved

This article is protected by copyright. All rights reserved

This article is protected by copyright. All rights reserved

This article is protected by copyright. All rights reserved

This article is protected by copyright. All rights reserved 

This article is protected by copyright. All rights reserved Supplemental Figure 1 . IFN-γ secretion from peripheral blood mononuclear cells (PBMCs) in response to SARS-CoV-2 spike Protein S1. PBMCs obtained from 16 SLE patients and two controls were plated in enzyme-linked immunospot (ELISpot) plates and stimulated by Spike Protein S1. The numbers of interferon-γ (IFN-γ)-producing cells were then detected by ELISpot. PBMCs were shown to release IFN-γ in response to antigen (Panel A). There is a correlation between spike-RBD IgG in sera with IFN-γ