key: cord-0989417-zp9lyy25
authors: Chen, Rita E.; Gorman, Matthew J.; Zhu, Daniel Y.; Carreño, Juan Manuel; Yuan, Dansu; VanBlargan, Laura A.; Burdess, Samantha; Lauffenburger, Douglas A.; Kim, Wooseob; Turner, Jackson S.; Droit, Lindsay; Handley, Scott A.; Chahin, Salim; Deepak, Parakkal; O’Halloran, Jane A.; Paley, Michael; Presti, Rachel M.; Wu, Gregory F.; Krammer, Florian; Alter, Galit; Ellebedy, Ali H.; Kim, Alfred H.J.; Diamond, Michael S.
title: Reduced antibody activity against SARS-CoV-2 B.1.617.2 Delta virus in serum of mRNA-vaccinated patients receiving Tumor Necrosis Factor-α inhibitors
date: 2021-11-18
journal: Med (N Y)
DOI: 10.1016/j.medj.2021.11.004
sha: aefccf4d42db46b59f22a9ced16167836030d27c
doc_id: 989417
cord_uid: zp9lyy25

Background Although vaccines effectively prevent COVID-19 in healthy individuals, they appear less immunogenic in individuals with chronic inflammatory diseases (CID) or receiving chronic immunosuppression therapy. Methods Here, we assessed a cohort of 77 CID patients treated as monotherapy with chronic immunosuppressive drugs for antibody responses in serum against historical and variant SARS-CoV-2 viruses after immunization with the BNT162b2 mRNA vaccine. Findings Longitudinal analysis showed the greatest reductions in neutralizing antibodies and Fc effector functions capacity in individuals treated with TNF-α inhibitors (TNFi), and this pattern appeared worse against B.1.617.2 Delta virus. Within five months of vaccination, serum neutralizing titers of all TNFi-treated patients tested fell below the presumed threshold correlate for antibody-mediated protection. However, TNFi-treated patients receiving a third mRNA vaccine dose boosted their serum neutralizing antibody titers by more than 16-fold. Conclusions Thus, vaccine boosting or administration of long-acting prophylaxis (e.g., monoclonal antibodies) likely will be required to prevent SARS-CoV-2 infection in this susceptible population. Funding. This study was supported by grants and contracts from NIH (R01 AI157155, R01AI151178, HHSN75N93019C00074, NIAID Centers of Excellence for Influenza Research and Response (CEIRR) contracts HHSN272201400008C and 75N93021C00014, and the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051).

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged and the global COVID-19 pandemic began. Since then, many antibody-based 48 therapeutics and vaccines have been developed 1,2 with some given Emergency Use 49 Authorization (EUA) or Food and Drug Administration approval (e.g., BNT162b2 mRNA 50 vaccine) in hopes of preventing infection and severe disease. While several of these 51 countermeasures show efficacy against historical (2019-early 2020) SARS-CoV-2 strains, the 52 emergence of variants of concern (VOC) has prompted questions as to whether they will retain 53 efficacy. 54 SARS-CoV-2 spike protein engages cell-surface receptor angiotensin-converting enzyme 55 2 (ACE2) for attachment and entry into human cells 3 . The S1 component of the spike protein 56 contains the N-terminal (NTD) and receptor binding (RBD) domains, the latter being the primary 57 target of neutralizing antibodies 4-7 . However, recent studies have shown that therapeutic 58 monoclonal and vaccine-elicited polyclonal antibodies have reduced neutralizing activity against 59 VOC, likely because these strains contain mutations within the RBD and the receptor binding 60 motif (RBM) [8] [9] [10] [11] [12] . This observation is concerning since serum neutralizing antibody titers are 61 believed to be an in vitro correlate of in vivo protection 13-15 . 62 Most studies on the immunogenicity and efficacy of SARS-CoV-2 vaccines have focused 63 on immunocompetent animals and humans. As SARS-CoV-2 has been documented to mutate 64 and evolve in immunocompromised hosts 16, 17 , it is important to understand whether vaccine-65 elicited responses are protective and durable in this population. Just recently, the Centers for 66 Disease Control and Prevention recommended an additional BNT162b2 mRNA dose for the 67 immunocompromised notwithstanding the relatively limited study of effects of 68 J o u r n a l P r e -p r o o f immunocompetent volunteers including TNFi (67%), antimetabolites (33%), antimalarial agents 143 (30%), NSAIDs (11%), and anti-IL-23 inhibitor (22%) ( Table 2) . 144 We separately grouped the individuals by disease and re-analyzed neutralization titers at 145 three months after second vaccination. Patient groups with certain diseases (e.g., ulcerative 146 colitis, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, and asthma) had 147 neutralization titers that were statistically similar to immunocompetent volunteers (Fig 2A and  148   S4 ). As a group, subjects with Crohn's disease ( (Fig 2A and S4) . 152

Since TNFi is an important therapeutic target in Crohn's disease, we analyzed this group by 153 treatment class. Compared to 36% (27 of 75) of all CID patients at 3 months after second 154 vaccination that had a neutralization titer of less than 1/50 against B.1.617.2, 39% (12 of 31) of 155

Crohn's disease patients fell below this threshold (Fig S4) . However, 67% (6 of 9) of Crohn's 156 disease patients receiving TNFi had neutralization titers against B.1.617.2 below the 1/50 cut-off 157 compared to 27% (6 of 22) of those on other treatment regimens (Fig S4) . Multivariable 158 regression analysis indicated that the lower neutralization titers against B.1.617.2 were 159 associated with TNFi treatment and not Crohn's disease (Table S3) . 160

Recent studies have shown that the Fc effector functions of antibodies can contribute to 161 protection against SARS-CoV-2 infection and disease [27] [28] [29] [30] [31] [32] . To address whether our immunized 162 CID patients had distinct Fc effector function profiles, we analyzed their SARS-CoV-2 specific 163 antibodies in sera for IgG subclass distribution and C1q and Fc receptor (FcR) binding. We 164 focused our analyses on groups with at least n = 5: anti-integrin inhibitors, antimetabolites, 165

TNFi, antimalarial agents, and immunocompetent volunteers, and measured responses against 166

Wuhan-1 D614G, B.1.617.2, and B.1.351 spike proteins (Fig 3 and S5 ). There were no 167 differences in levels of IgG1, IgG3, and IgM against SARS-CoV-2 spike proteins, and C1q or 168 FcR (FcR2A, FcR2B, or FcR3A) binding (Fig 3 and S5) . However, compared to 169 immunocompetent volunteers, patients treated with TNFi had decreased anti-SARS-CoV-2 IgG, 170

IgG2, and IgG4 levels against Wuhan-1 D614G (Fig 3A) , B.1.617.2 (Fig 3B) , and B.1.351 (Fig  171   S5 ) whereas all other groups had no significant difference. We also assessed for antibody-172 mediated innate immune effector functions. While all groups showed similar antibody-dependent 173 neutrophil phagocytosis (ADNP) (Fig 3C-D and S5 ), patients receiving TNFi had decreased 174 antibody-dependent cellular phagocytosis (ADCP) against B.1.617.2 (Fig 3D) and B.1.351 (Fig  175   S4 ). For unexplained reasons, patients receiving anti-integrin inhibitors had enhanced ADCP 176 against Wuhan-1 D614G (Fig 3C) and antibody-dependent complement fixation (ADCD) 177 against B.1.617.2 (Fig 3D) and B.1.351 (Fig S5) . When we combined the data, only patients 178 receiving TNFi showed substantive decreases in antibody effector functions at three months after 179 second vaccination (Fig 3E) . 180

As serum antibody titers generated by the Pfizer BNT162b2 vaccine wane over time 33,34 , 181 we also assessed neutralizing activity at five months after immunization in CID patients (n = 43); 182 for comparison, we used the later, six-month time point from our immunocompetent cohort (n 183 (Fig S2) . Sera from both 187 patient and immunocompetent subject groups also were less efficient at neutralizing Wash-188 J o u r n a l P r e -p r o o f B.1.351 and B.1.617.2 than WA1/2020 D614G (Fig 4) . While there were limited numbers of 189 patients in each drug treatment group at the time 5-month point, decreased inhibitory activity 190 against all three viruses tested was seen in serum from patients receiving TNFi (Fig S2B) . 191

Moreover, at this time point, all other therapy groups generally had less serum neutralizing 192 activity against Wash-B.1.351 and B.1.617.2 compared to WA1/2020 D614G (Fig 4 and S6) . In this study, we evaluated the functional antibody responses after immunization with the 212 Pfizer BNT162b2 mRNA vaccine against historical and emerging SARS-CoV-2 strains in a 213 cohort of adult CID patients with a range of diagnoses and treatment interventions. These results 214 were compared to a separate cohort of similarly vaccinated immunocompetent adults 24 and 215 showed consistently lower serum antibody neutralizing titers in most CID patients after two 216 doses, with a substantial fraction falling below an estimated 1/50 cutoff against B.1.617.2 (Delta) 217 that has been proposed as a correlate of protection 26 . Subgroup analysis suggested that 218 individuals on TNFi had lower inhibitory titers than other therapeutic groups. Thus, these 219 patients might be at greatest risk for breakthrough infections, especially with VOC. Similarly, in 220 studies that evaluated Fc effector function of serum antibodies, those receiving TNFi showed 221 greater decreases in antibody effector functions, providing a second possible mechanism for risk 222 of vaccine failure in these populations. 223

Our results contrast with a recent report on 84 psoriasis patients and 17 healthy controls 224 immunized with the Pfizer BNT162b2 mRNA vaccine 37 . In that study, neutralizing activity 225 against SARS-CoV-2 Wuhan-1 was lower in patients receiving methotrexate than TNFi. The 226 variation in results could be explained by the following differences in study design and analysis: 227 (a) we had only one psoriatic arthritis patient in our CID cohort; (b) we grouped methotrexate 228 with other antimetabolites due to small numbers; (c) differences in the specific TNFi used; and 229 (d) the neutralization assays used were not the same. In comparison, our data suggesting that 230

TNFi blunts the humoral response to vaccines is consistent with a meta-analysis of 25 231 observational studies with 5,360 patients who received Pfizer BNT162b2, Moderna mRNA-1273 232 or other platforms 38 . 233

Our findings corroborate studies that report an association between patients with CID, 234 including those on TNFi, and reduced antibody responses after vaccination 18,39-41 . Poor 235 seroconversion in these patients has been described after vaccination against hepatitis A 42 , 236 hepatitis B 43,44 , and influenza [45] [46] [47] which also may confer protection; and (6) The small sample size precluded assessment of 282 comorbidities, age, sex, and race as independent variables on humoral responses. We note there 283 was some sex-skewing between the immunocompetent (64% male) and CID (68% female) 284 cohorts. 285 (14) 4 (57) 3 (43) Anti-integrin 9 0 (0) 1 (11) 0 (0) 3 0 (0) 0 (0) 1 (33) NSAIDs 9 1 (11) 1 (11) 1 (11) 5 1 (20) 2 (40) 2 (40) Anti-IL-23 9 1 (11) 2 (22) 2 (22) 3 0 (0) 1 (33) 1 (33)

Lead Contact. Further information and requests for resources and reagents should be 470 directed to the Lead Contact, Michael S. Diamond (diamond@wusm.wustl.edu). 471 Materials Availability. All requests for resources and reagents should be directed to the 472 Lead Contact author. This includes mice and viruses. All reagents will be made available on 473 request after completion of a Materials Transfer Agreement. 474

Data and code availability. 475 (a) Data. All serological results described in this study are available within the body of 476 the paper. All data (including raw data used to generate neutralizing and binding 477 curves) reported in this paper will be shared by the lead contact upon request 478 Ollmann Saphire (La Jolla Institute for Immunology). 532

Luminex profiling. Serum samples were analyzed by customized Luminex assay to 533 quantify the relative concentration of antigen-specific antibody isotypes, subclasses, and Fcγ-534 receptor (FcγR) binding profiles, as previously described 77,78 . Briefly, SARS-CoV-2 antigens 535 were used to profile specific humoral immune responses. Antigens were coupled to magnetic 536 Luminex beads (Luminex Corp) by carbodiimide-NHS ester-coupling (Thermo Fisher). Antigen-537 coupled microspheres were washed and incubated with plasma or serum samples at an 538 appropriate sample dilution (1:5000 for IgG1 and all low affinity FcγR, and 1:200 for all other 539 readouts) for 2 h at 37°C in 384-well plates (Greiner Bio-One). Unbound antibodies were 540 washed away, and antigen-bound antibodies were detected by using a PE-coupled detection 541 antibody for each subclass and isotype (IgG1, IgG3, IgA1, and IgM; Southern Biotech), and 542

FcγR were fluorescently labeled with PE before addition to immune complexes (FcγR2a, 543

FcγR3a; Duke Protein Production facility). After one hour of incubation, plates were washed, 544 and flow cytometry was performed with an iQue (Intellicyt) and analyzed using IntelliCyt 545 ForeCyt (v8.1). PE median fluorescent intensity (MFI) is reported as a readout for antigen-546 specific antibody titers. 547

Antibody-dependent complement deposition (ADCD) Antibody-dependent 548 complement deposition (ADCD) was conducted as previously described 79 . Briefly, SARS-CoV-2 549 antigens were coupled to magnetic Luminex beads (Luminex Corp) by carbodiimide-NHS ester-550 coupling (Thermo Fisher). Coupled beads were incubated for 2 h at 37°C with serum samples 551

(1:10 dilution) to form immune complexes and then washed to remove unbound 552 immunoglobulins. To measure antibody-dependent deposition of C3, lyophilized guinea pig 553 complement (Cedarlane) was diluted in gelatin veronal buffer with calcium and magnesium 554 (GBV++) (Boston BioProducts) and added to immune complexes. Subsequently, C3 was 555 detected with an anti-C3 fluorescein-conjugated goat IgG fraction detection antibody (Mpbio). 556

Flow cytometry was performed 5 Laser LSR Fortessa Flow Cytometer and analyzed using 557 FlowJo V10.7.1. ADCD was reported as the median of C3 deposition. 558

Antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent neutrophil 560 phagocytosis (ADNP) were conducted according to the previously described protocols 80,81 . In 561 detail, SARS-CoV-2 antigens were biotinylated using EDC (Thermo Fisher) and Sulfo-NHS-562 LCLC biotin (Thermo Fisher) and coupled to yellow-green (505/515) fluorescent 563 Neutravidinconjugated beads (Thermo Fisher), respectively. To form immune complexes, 564 antigen-coupled beads were incubated for 2 h at 37°C with 1:100 diluted serum samples and then 565 washed to remove unbound immunoglobulins. For ADCP, the immune complexes were 566 incubated for 16-18 hours with THP-1 cells (1.25×10 5 THP-1 cells/mL) and for ADNP for 1 567 hour with RBC-lyzed whole blood. Following the incubation, cells were fixed with 4% PFA. For 568 ADNP, RBC-lyzed whole blood was washed, stained for CD66b + (Biolegend) to identify 569 neutrophils, and then fixed in 4% PFA. Flow cytometry was performed to identify the percentage 570 of cells that had phagocytosed beads as well as the number of beads that had been phagocytosis 571 (phagocytosis score = % positive cells × Median Fluorescent Intensity of positive cells/10000). 572

Flow cytometry was performed with 5 Laser LSR Fortessa Flow Cytometer and analyzed using 573 The data used to generate Fig 3e was graphed and analyzed using Python version 3.8.5 and the 581 'plotly' package 82 . For each feature, data was first standardized by computing the Z-score, 582 scaling values to zero mean and unit variance. The median resulting values are represented on 583 each polar plot. All other data were graphed and analyzed in GraphPad Prism v8.4.3. Tobit linear 584 regression was performed using Stata/MP 13.1, and the effects were refined to account for left-585 censoring of data below the limit of detection (LoD). 586 J o u r n a l P r e -p r o o f

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Context and significance: In most individuals, mRNA vaccines effectively prevent severe disease following SARS-CoV-2 infection. However, the protective immunity induced by mRNA vaccines is diminished in immunocompromised patients, and the impact of variant strains is unexplored. Here, we evaluated serum antibody responses in patients with chronic inflammatory diseases after immunization with the Pfizer BNT162b2 mRNA vaccine. The lowest neutralizing antibody titers were observed in individuals treated with TNF- inhibitors, and this pattern appeared worse against Delta virus, with the antibody levels falling below the presumed threshold correlate of protection. Administration of a third vaccine dose boosted serum neutralizing titers substantially. Our data suggest vaccine boosting likely is needed to prevent SARS-CoV-2 infection in some immunocompromised patient populations

assess serum antibodies from BNT162b2 mRNA vaccinated patients with chronic inflammatory diseases receiving single immunosuppressive drug therapies. Patients receiving TNF- inhibitors (TNFi) had reduced antibody neutralizing and Fc effector function activity against B.1.351 and B.1.617.2 variants. A third vaccine dose markedly boosted neutralizing titers in TNFi recipients

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