key: cord-0740394-333mzfgr
authors: Zimmerman, O.; Altman Doss, A. M.; Kaplonek, P.; VanBlargan, L. A.; Liang, C.-Y.; Chen, R. E.; Monroy, J. M.; Wedner, H. J.; Kulczycki, A.; Mantia, T. L.; O'Shaughnessy, C. C.; Davis-Adams, H. G.; Bertera, H. L.; Adams, L. J.; Raju, S.; Zhao, F. R.; Rigell, C. J.; Biason Dy, T.; Kau, A. L.; Ren, Z.; Turner, J.; O'Halloran, J. A.; Presti, R.; Fremont, D. H.; Kendall, P. L.; Ellebedy, A. H.; Alter, G.; Diamond, M. S.
title: mRNA vaccine boosting enhances antibody responses against SARS-CoV-2 Omicron variant in patients with antibody deficiency syndromes
date: 2022-01-28
journal: nan
DOI: 10.1101/2022.01.26.22269848
sha: 7eb154719b88d1be62c19375c7c4ee6329a9f9df
doc_id: 740394
cord_uid: 333mzfgr

Patients with primary antibody deficiency syndromes (PAD) have poor humoral immune responses requiring immunoglobulin replacement therapy. We followed PAD patients after SARS-CoV-2 vaccination by evaluating their immunoglobulin replacement products and serum for anti-spike binding, Fc{gamma}R binding, and neutralizing activities. Immunoglobulin replacement products had low anti-spike and receptor binding domain (RBD) titers and neutralizing activity. In COVID-19-naive PAD patients, anti-spike and RBD titers increased after mRNA vaccination but decreased to pre-immunization levels by 90 days. Patients vaccinated after SARS-CoV-2 infection developed higher responses comparable to healthy donors. Most vaccinated PAD patients had serum neutralizing antibody titers above an estimated correlate of protection against ancestral SARS-CoV-2 and Delta virus but not against Omicron virus, although this was improved by boosting. Thus, currently used immunoglobulin replacement products likely have limited protective activity, and immunization and boosting of PAD patients with mRNA vaccines should confer at least short-term immunity against SARS-CoV-2 variants, including Omicron.

Common variable immune deficiency (CVID) and other primary antibody deficiency 51 syndromes (PAD) are associated with low immunoglobulin levels and impaired antibody 52 responses to pathogens and vaccines 1,2 . Patients with these immune disorders suffer from severe 53 and recurrent infections, autoimmunity, and are at increased risk for malignancies 3 . CVID has a 54 prevalence of 1 to 25,000 4-6 and is the most common primary immunodeficiency in patient 55 registries with more than 20% suffering from this condition. CVID is not a single disease but 56 rather a collection of hypogammaglobulinemia syndromes resulting from multiple genetic 57 defects 7-11 . Most patients with PAD require intravenous or subcutaneous immunoglobulin 58 replacement therapy that decreases their risk for infection [12] [13] [14] . There are more than 15 59 commercially available immunoglobulin products in the United States. The production of 60 immunoglobulin replacement products takes up to one year from sample donation to 61 distribution 15, 16 . Each vial contains immunoglobulins pooled from thousands of donors 16, 17 , and 62 each manufacturer has its own plasma donors. In patients with PAD, immunoglobulin 63 replacement is dosed every 1 to 4 weeks, depending on the route of administration. 64 SARS-CoV-2 is the causative agent of the global COVID-19 pandemic. From November 65 2019 until now, the virus has caused at least 5.5 million deaths. In the United States, the 66 emergency use authorization has been granted for two COVID-19 vaccines (mRNA-1273, 67 Moderna and Ad26.COV2.S, Johnson & Johnson/Janssen) and full approval has been given to 68 one mRNA vaccine (BNT162b2, Pfizer-BioNTech). Presently, there is limited data regarding the 69 effectiveness of mRNA or adenoviral vector vaccination against patients. Several studies showed variable seroconversion rates with detection of anti-spike, S1 or 71 RBD antibodies, in 20 to 90% of PAD patients following vaccination with BNT162b2, mRNA-72 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269848 doi: medRxiv preprint these differences did not reach statistical significance (P > 0.3). Moreover, 90 days after 130 boosting, serum titers against spike and RBD from PAD patients had decreased to levels 131 comparable to before boosting (Fig 1E-F) . 132

Immunoglobulin subclass and Fc-gamma receptor binding. Recent studies have 133 suggested that immunoglobulin subclass and antibody interactions with Fcγ receptors (FcγR) can 134 contribute to protective immunity against SARS-CoV-2 23,24 . Accordingly, we evaluated serum 135 from immunized PAD patients for their immunoglobulin subclasses (IgG1, IgG2, IgG3 and 136

IgG4, IgA, and IgM) that bind spike proteins and domains (S, S1, S2, and RBD) from historical 137 (Wuhan-1) and spike proteins from B.1.351 (Beta) and B.1.617.2 (Delta) SARS-CoV-2 strains 138 (Fig 2A-F; Fig S1) . Fourteen days after primary series immunization, COVID-19-naive PAD 139 had lower IgG2, IgG3, and IgM levels against spike and RBD proteins of all 3 tested virus 140 strains than vaccinated HD (Fig 2B-C and F) . COVID-19-naive PAD patients also had lower 141 IgG1 levels against Wuhan-1 SARS-CoV-2 spike and RBD than HD (Fig 2A) . In comparison, 142 vaccinated COVID-19-naive PAD patients had IgA titers against spike and RBD proteins that 143 were similar to HD (Fig 2E) . Vaccinated COVID-19-experienced PAD patients had lower IgG3 144 levels against Wuhan-1 spike, S1, and RBD (Fig 2C) , but similar levels of IgG1, IgG2, IgA, and 145

IgM against Wuhan-1 spike, S1, S2, and RBD and variant spike proteins compared to 146 immunized HD (Fig 2A-B and E-F) . Vaccinated, COVID-19-experienced PAD patients had 147 higher levels of IgG2, IgA, and IgM against Wuhan-1 spike and RBD than vaccinated, COVID-148 19-naive PAD patients. COVID-19-experienced PAD patients had higher IgG3, IgA and IgM 149 titers against S2 protein than COVID-19-naive PAD patients (Fig 2C, E-F) . The levels of IgG4 150 anti-spike or RBD protein in all groups were near the limit of detection (Fig 2D) . Although prior 151 infection with COVID-19 in PAD patients was associated with a better vaccine response, 152 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 COVID-19-naive and -experienced PAD patients had lower IgG3 responses than HD (Fig 2C  153 and S1). This result suggests that class switching to IgG3 is impaired in PAD patients following 154 infection or vaccination. 155

Given these results, we next evaluated anti-spike and anti-RBD antibody binding to 156 FcγRs (FcγR2A, FcγR2B, FcγR3A and FcγR3B) using a systems serology platform 25 . 157

Vaccinated, COVID-19-naive PAD patients had lower levels of FcγR binding to anti-spike and 158 RBD antibody than vaccinated, COVID-19-experienced PAD patients or HD (Fig 2G-J) . 159

FcγR2A, 3A and 3B binding was higher in serum from vaccinated, COVID-19-experienced PAD 160 patients than vaccinated, COVID-19-naive PAD patients for all viral antigens tested (Fig 2G, I-J  161 and S1). COVID-19-experienced PAD patient serum binding to FcγR2B was higher than 162 COVID-19-naive PAD patients for Wuhan-1 and B.1.617.2 spike proteins (Fig 2H) . FcγR2A, 163 FcγR2B, FcγR3A and FcγR3B binding was higher in HD than COVID-19-naive PAD patients 164 for most spike proteins (Fig 2G-J and S1 ). Serum anti-S2 responses from COVID-19-165 experienced PAD patients were higher than COVID-19 PAD patients for all tested FcγRs (Fig  166   2G -J). The higher levels of FcγR binding by anti-spike and anti-RBD antibodies in COVID-19-167 experienced compared to COVID-19-naive PAD patients after vaccination suggest that PAD 168 patients do not have an inherent defect in producing antibodies that mediate Fc effector 169

Serum neutralizing antibody responses. We evaluated the functional activity of 171 antibody preparations by performing focus reduction neutralization tests (FRNTs) with authentic 172 SARS-CoV-2 strains and variants (WA1/2020, B.1.617.2, and B.1.1.529). We first tested 173 neutralizing activity of the commercial immunoglobulin products (n = 17), serum from COVID-174 19-naive PAD patients after vaccination (n = 18), and serum from COVID-19-experienced 175 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 patients after vaccination (n = 9) (Fig 3A-B) . Fourteen of 17 different lots of immunoglobulin 176 product tested had no appreciable neutralizing activity against WA1/2020 or B.1.617.2 at 500 177 μg/ml (Fig 3A-B and S2) , which is approximately a 1/50 dilution of the mean IgG concentration 178 measured in our PAD patients on IgG replacement therapy (9.7 mg/ml) (Table S1 and S2). We 179 tested this concentration (rather than neat sample) since it corresponds to the serum dilution that 180 is the presumed cutoff for vaccine-mediated protection 26 . Two lots of Gammunex-C and one of 181 Hizentra had limited neutralizing activity against WA1/2020 strain at a 1/50 dilution, and only 182 one lot (Hizentra) had inhibitory activity against B.1.617.2 at this dilution (Fig 3A-B ; S1-S2, 183 Table S1 ). 184

As expected, serum of COVID-19-naive PAD patients had no neutralizing activity 185 against WA1/2020 prior to vaccination, whereas COVID-19-experienced patients did (Fig  186   3A ). Fourteen days after immunization, 15 of 18 (83%) COVID-19-naive PAD patients had 187 levels of serum neutralizing antibodies against WA1/2020 that are considered protective 26 (titer > 188 50). By 90 days, neutralizing activity had waned, with 12 of 17 (71%) PAD patients in this group 189 still having titers above the presumed protective threshold. Following boosting, neutralizing 190 titers increased in 11 of 12 (92%) COVID-19-naive PAD to levels greater than 50 against 191 WA1/2020 (Fig 3A) . In the cohort of COVID-19-experienced PAD patients, serum neutralizing 192 activity against WA1/2020 exceeded the protective cutoff at all tested time points in all subjects 193 ( Fig 3A) . Indeed, 14 and 90 days post-primary immunization series, COVID-19-experienced 194 PAD patients had serum neutralizing titers against WA1/2020 that were 20 and 8-fold higher 195 than COVID-19-naive PAD patients, respectively (Fig 3A) . 196 We repeated FRNTs with PAD patient serum and the B.1.617.2 Delta strain, which can 197 evade neutralizing antibodies due to amino acid substitutions in the RBD 27 . Although pre-198 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. (Fig 3B) . Fourteen and 90 days post-vaccination 14 of 18 (78%) and 11 of 17 (65%) 201 COVID-19-naive patients, respectively had serum neutralizing titers against B.1.617.2 that were 202 above 50. Following boosting, 10 of 12 (83%) COVID-19-naive PAD patients had neutralizing 203 titers above 50 (Fig 3B) . We next analyzed PAD patients (n = 17) who received an mRNA vaccine booster (Fig  210   3C ). We included in this analysis two patients who initially received an Ad26.COV2.S vaccine 211 (Table 1 and Fig S3) . At 14 days and 90 post-primary vaccination, 14 of 17 (82%) and 11 of 16 212 (69%) patients had serum neutralization titers above 50 against WA1/2020. At 14 days post-213 boosting, 16 of 17 (94%) patients had neutralizing titers against WA1/2020 that exceeded 50 214 (Fig 3C) , and the highest titers (GMT: 786) showed a 7-fold increase over levels at 90 days post-215 primary series (Fig 3C) . Ninety days post-boosting, 10 of 11 (91%) patients had neutralization 216 titers above 50 (Fig 3C) . Similar findings were observed against B.1.617.2, with 12 of 17 (71%), 217 10 of 16 (63%), 15 of 17 (88%) and 8 of 11 (73%) PAD patients having neutralizing titers 218 greater than 50, at 14 and 90 days post-primary immunization series or at 14 and 90 days post-219 boosting, respectively (Fig 3D) . Similar to WA1/2020, the highest neutralization titers against 220 B.1.617.2 were detected 14 days after boosting (Fig 3D) . 221 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269848 doi: medRxiv preprint The B.1.1.529 Omicron variant has >30 substitutions, deletions, and insertions in its 222 spike protein, which jeopardizes the efficacy of vaccines designed against historical SARS-CoV-223 2 strains 28-30 . Accordingly, we evaluated serum neutralizing activity against B.1.1.529 in our 224 patient cohort (Fig 3E-F) . Fourteen days after completing the primary vaccination series, only 8 225 of 27 (30%) PAD patients had serum levels of neutralizing antibody above 50 against B.1.1.529 226 (Fig 3E) , and only 1 of 8 patients in this group was COVID-19-naive. Fourteen days following 227 boosting, 12 of 17 (71%) PAD patients had neutralizing titers against B.1.1.529 that exceeded 50 228 ( Fig 3F) . Ninety days post-boosting, 5 of 11 (45%) PAD patients had serum neutralization titers 229 against B.1.1.529 that exceeded 50 (Fig 3G) . The mean neutralization titer against B.1.1.529 was 230 lower than against WA1/2020 and B.1.617.2 at 14 days after primary immunization (~10-fold, P 231 < 0.0001), 14 days post-boosting (~4 to 8-fold, P < 0.05), and 90 days post-boosting (~6-fold, P 232 < 0.05), which is consistent with recent studies in immunized healthy cohorts 28-30 . 233 234 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. In patients with no prior history of COVID-19 infection, the immune response to two 251 doses of mRNA vaccine was lower in magnitude and less durable than in HD or PAD patients 252 with a history of infection. Of note, the increase in serum neutralizing titers following boosting 253 was higher than the increase in anti-spike and anti-RBD titers (4.5-fold compared to 2-fold). This 254 observation highlights the utility of performing antibody neutralization assays in addition to 255 spike or RBD binding assays for assessing the quality of humoral immune responses. Although 256 further studies that sample and sequence B cells in blood from PAD patients are needed, we 257 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269848 doi: medRxiv preprint speculate that B cells from at least some PAD patients undergo antibody maturation after 258 infection, vaccination, and boosting. 259

One limitation of our study is the heterogeneity in the PAD patient cohort, which 260 included those with CVID, hypogammaglobulinemia, or specific antibody deficiency. Although 261 this was a limitation of the cohort available for study, we did not observe substantive differences 262 between the patient subgroups. Instead, the most significant differences in antibody response to 263 mRNA vaccines were between patients that had or lacked a prior history of SARS-CoV-2 264 infection. 265

Many of our PAD patients who historically had poor immune responses to bacterial and 266 other protein antigens (e.g., Streptococcus pneumoniae polysaccharides, tetanus toxoid, and 267 diphtheria toxin) as part of their initial immune workup (Table S3) Ad26.COV2.S adenoviral-vectored vaccine (Fig S3) . Because PAD is a heterogeneous clinical 273 entity, with many of the genetic defects unknown 7-11 , certain classes of adjuvants or antigens 274 may overcome specific deficiencies and promote B cell responses, albeit at lower levels than 275 healthy counterparts. Our data suggest that the mRNA platform may have utility for vaccination 276 of PAD patients. That said, their less durable response, lower level of anti-spike and anti-RBD 277

IgG3, and lower levels of complement-fixing and FcγR-engaging antibodies, suggest that more 278 frequent boosters may be required to establish and maintain protective immunity. 279

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10. 1101 /2022 Our study also showed that many of the immunoglobulin replacement products currently 280 used have low levels of inhibitory anti-SARS-CoV-2 antibodies. This may be due to the long 281 lead time required for collection from donors, purification, and testing. Neutralization assays 282 performed by one manufacturer and by us showed low inhibitory activity against Wuhan-1 and 283 less activity against SARS-CoV-2 variants 38 . The three products we identified with some activity 284 against Wuhan-1 and B.1.617.2 had titers that likely would not confer protection against 285 B.1.1.529 given the more extensive antibody evasion by this strain 31, 32, 39, 40 . Indeed, neutralizing 286 titers were below the presumed protective cutoff in the 4 COVID-19-naive PAD patients who 287 donated pre-vaccination blood samples, even though all had received immunoglobulin 288 replacement every 3 to 4 weeks before study enrollment. It is unclear when commercially 289 available products will have sufficient levels of specific and neutralizing anti-SARS-CoV-2 290 antibodies to protect PAD patients. Further binding and neutralization studies are warranted 291 once anti-SARS-CoV-2 antibodies become more widespread in plasma pools. While many PAD 292 patients might be eligible for long-acting combination monoclonal antibody prophylaxis (e.g., 293

Evusheld [AZD7442]) against COVID-19, recent studies showed substantial (~33-fold) losses in 294 potency against B.1.1.529 Omicron virus. Thus, for now, immunization of PAD patients with 295 mRNA vaccines that includes a booster may be the most effective way to induce a protective 296 antibody response against SARS-CoV-2 and its variants. 297 298 299 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. (n = 20, blue circles), COVID-19-naive (n = 18, red circles) and COVID-19-experienced (n = 9, 364 green circles) PAD patients 14 days following the completion of mRNA vaccine series. Two-365 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269848 doi: medRxiv preprint way ANOVA with Tukey post-test; mean; Only significant differences are shown: *, P < 0.05; 366 **, P<0.01; ***, P< 0.001; ****, P < 0.0001). 367 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Dunn's post-test; Only significant differences are shown: *, P < 0.05; **, P<0.01; ***, P< 0.001; 389 ****, P < 0.0001). CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269848 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted January 28, 2022. ;

Patients and samples. The study was approved by the Institutional Review Board of 423 Washington University School of Medicine (Approval # 202104138). Patients were identified by 424 a medical record search for PAD, and their records were reviewed to confirm their diagnosis and 425 verify they met the inclusion criteria. COVID-19 vaccination status was reviewed, and subjects 426 were contacted if they were within the vaccination window or not yet immunized. Laboratory 427 values ( Table S2- . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 469 charts were reviewed, and 160 subjects were contacted. A total of 30 adults (27 444 females, 3 males) with PAD met eligibility requirements and agreed to enroll in the study (see 445   Table 1 ); we note a sex-bias in the enrollees from our PAD cohort, which is not typical for the 446 disease itself. Ages ranged from 20 to 82, with an average age of 48.4 years old. Twenty PAD 447 patients had CVID, six had specific antibody deficiency, and four had hypogammaglobulinemia. 448

Twenty-seven of these subjects had received immunoglobulin replacement therapy before and 449 during the study period from 9 different products. Nineteen subjects received the BNT162b2 pre-vaccination blood sample was collected up to 14 days before receiving vaccine. For subjects 457 who received a two-dose series of mRNA vaccines, the first post-vaccination blood collection 458 occurred 7 -28 days after the second dose. For subjects receiving the Ad26.COV2.S single-dose 459 vaccine, the first post-vaccination blood sample was collected 21-35 days after immunization. 460

Since the study was non-interventional, patients were informed if they mounted an immune 461 response to the vaccine, but the decision to receive a booster was made between the patient and 462 their physician. Subjects who opted for boosting provided a blood sample up to 14 days prior to 463 receiving the booster dose, unless the subject previously provided a sample within 2 weeks as 464 part of the optional post-vaccine assessments. Subjects returned for an additional sample 7-28 465 days after receiving the booster (range 7-27 days, median 17 days, mean 17 days. One patient 466 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269848 doi: medRxiv preprint had her post-booster sample drawn at day 35), with a second post-booster visit and sample 467 collection at 90 ±14 days. Immunoglobulin replacement product vials that were used in PAD 468 patients were collected at each study visit and/or post-infusion at the Washington University 469

Allergy and Immunology Division infusion centers. 470

Healthy donor controls. Immunocompetent healthy donor volunteer blood samples were 471 obtained as previously described 41 . 472 SARS-CoV-2 spike and RBD protein expression. Genes encoding SARS-CoV-2 spike 473 protein (residues 1-1213, GenBank: MN908947.3) and RBD (residues 319-514) were cloned into 474 a pCAGGS mammalian expression vector with a C-terminal hexahistidine tag. The spike protein 475 was stabilized in a prefusion form using six proline substitutions (F817P, A892P, A899P, 476 A942P, K986P, V987P) 42 , and expression was optimized with a disrupted S1/S2 furin cleavage 477 site and a C-terminal foldon trimerization motif (YIPEAPRDGQAYVRKDGEWVLLSTFL) 43 . 478

Expi293F cells were transiently transfected, and proteins were purified by cobalt-affinity 479 chromatography (G-Biosciences) as previously described 44, 45 . 480 ELISA. Maxisorp ELISA (Thermo Fisher) plates were coated with SARS-CoV-2 481 Whuan-1 spike or RBD (2 µg/ml) overnight in sodium bicarbonate buffer, pH 9.3. Plates were 482 washed four times with PBS and 0.05% Tween-20 and blocked with 4% BSA in PBS for 1 h at 483 25ºC. Plates were then incubated with 50 μL of patient and healthy donor serially-diluted 484 samples (starting at 1/50) in 2% BSA PBS, for 2 hours at 25ºC on a shaker. Immunoglobulin 485 replacement products were diluted to 10 mg/ml (average patient IgG level) and then treated as 486 described above. Plates were washed with PBS and 0.05% Tween-20 and incubated with 487 horseradish peroxide (HRP)-conjugated goat anti-human IgG (H + L) (1:2000 dilution, Jackson 488

ImmunoResearch) for 1 h at room temperature. After washing, plates were developed with 100 489 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269848 doi: medRxiv preprint μL of 3,3'-5,5' tetramethylbenzidine substrate (Thermo Fisher) for 120 sec and fixed with 50 μL 490 of 2N H 2 SO 4 . Plates were read at 450 nM using a microplate reader (Synergy H1; BioTek). 491

Luminex profiling. Serum samples were analyzed by a customized Luminex assay to 492 quantify the levels of antigen-specific antibody subclasses and FcγR binding profiles, as 493 previously described 46, 47 . Briefly, SARS-CoV-2 antigens were coupled to magnetic Luminex 494 beads (Luminex Corp) by carbodiimide-NHS ester-coupling (Thermo Fisher). Antigen-coupled 495 microspheres were washed and incubated with plasma samples at an appropriate sample dilution 496

(1:100 for antibody isotyping and 1:1000 for all low-affinity FcγRs) overnight in 384-well plates 497 (Greiner Bio-One). Unbound antibodies were washed away, and antigen-bound antibodies were 498 detected by using a PE-coupled detection antibody for each subclass and isotype (IgG1, IgG2, 499

IgG3, IgA1, and IgM; Southern Biotech), and FcγRs were fluorescently labeled with PE before 500 addition to immune complexes (FcγR2a, FcγR2b, FcγR3a, FcγR2b; Duke Protein Production 501 facility). After one hour of incubation, plates were washed, and flow cytometry was performed 502 with an iQue (Intellicyt), and analysis was performed on IntelliCyt ForeCyt (v8.1). PE median 503 fluorescent intensity (MFI) is reported as a readout for antigen-specific antibody titers. 504

Focus reduction neutralization test. Serial dilutions of immunoglobulins products or 505 sera were incubated with 10 2 focus-forming units (FFU) of different strains of SARS-CoV-2 for 506 1 h at 37°C. Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-507 well plates and incubated at 37°C for 1 h. Subsequently, cells were overlaid with 1% (w/v) 508 methylcellulose in MEM supplemented with 2% FBS. Plates were harvested 30 h later by 509 removing overlays and fixed with 4% PFA in PBS for 20 min at room temperature. Plates were 510 washed and sequentially incubated with an oligoclonal pool of SARS2-2, SARS2-11, SARS2-16, 511 SARS2-31, SARS2-38, SARS2-57, and SARS2-71 48,49 anti-spike antibodies and HRP-512 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269848 doi: medRxiv preprint conjugated goat anti-mouse IgG (Sigma) in PBS supplemented with 0.1% saponin and 0.1% 513 BSA. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate 514 (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies). 515

Quantification and statistical analysis. Statistical significance was assigned using 516

Prism Version 9 (GraphPad) when P < 0.05. All data reported in this paper will be shared upon request. Any additional information 523 required to reanalyze the data reported in this paper is available from the lead contact upon 524 request. 525 526 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10.1101/2022.01.26.22269848 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 28, 2022. ; https://doi.org/10. 1101 /2022 

International Consensus Document (ICON): 528 Common Variable Immunodeficiency Disorders

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Distinct Early Serological Signatures Track with 584 SARS-CoV-2 Survival

Systems serology for evaluation of HIV vaccine 586 trials

Neutralizing antibody levels are highly 588 predictive of immune protection from symptomatic SARS-CoV-2 infection

SARS-CoV-2 Variants and Vaccines. N Engl 591

Omicron extensively but incompletely escapes 593

Pfizer BNT162b2 neutralization

Neutralization of SARS-CoV-2 Omicron variant by sera 595 from BNT162b2 or Coronavac vaccine recipients

Effectiveness of BNT162b2 597 Vaccine against Omicron Variant in South Africa

An infectious SARS-CoV-2 B

Omicron virus escapes neutralization by therapeutic monoclonal antibodies

Striking Antibody Evasion Manifested by the Omicron 602 Variant of SARS-CoV-2

The antigenic anatomy of SARS-CoV-2 604 receptor binding domain

COVID-19 Vaccines for Moderately or Severely Immunocompromised People

Lipid nanoparticles for mRNA delivery

Lipid nanoparticles enhance the efficacy of 611 mRNA and protein subunit vaccines by inducing robust T follicular helper cell and 612 humoral responses

The mRNA-LNP 614 platform's lipid nanoparticle component used in preclinical vaccine studies is highly 615 inflammatory. iScience

Anti-SARS-CoV-2 antibodies in healthy donor plasma 617 pools and IVIG products-an update

Omicron escapes the majority of existing SARS-CoV-2 619 neutralizing antibodies

Omicron-B.1.1.529 leads to widespread escape 621 from neutralizing antibody responses

SARS-CoV-2 mRNA vaccines induce 623 persistent human germinal centre responses

Structure-based design of prefusion-625 stabilized SARS-CoV-2 spikes

Structure of bacteriophage T4 627 fibritin: a segmented coiled coil and the role of the C-terminal domain

A Potently Neutralizing Antibody Protects Mice 630 against SARS-CoV-2 Infection

A SARS-CoV-2 Infection Model in Mice 632

Multiplexed Fc array for evaluation of 634 antigen-specific antibody effector profiles

High-throughput, multiplexed IgG subclassing of 636 antigen-specific antibodies from clinical samples

Identification of SARS-CoV-2 spike mutations 639 that attenuate monoclonal and serum antibody neutralization

A potently neutralizing SARS-CoV-2 antibody 642 inhibits variants of concern by utilizing unique binding residues in a highly conserved 643 epitope