key: cord-0933947-egfri75f
authors: Mantus, Grace; Nyhoff, Lindsay E.; Edara, Venkata-Viswanadh; Zarnitsyna, Veronika I.; Ciric, Caroline R.; Flowers, Maria W.; Norwood, Carson; Ellis, Madison; Hussaini, Laila; Manning, Kelly E.; Stephens, Kathy; Anderson, Evan J.; Ahmed, Rafi; Suthar, Mehul S.; Wrammert, Jens
title: Pre-existing SARS-CoV-2 immunity influences potency, breadth, and durability of the humoral response to SARS-CoV-2 vaccination.
date: 2022-03-29
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2022.100603
sha: 0335df6cc9738c2b577ad7e40e376179d41a9a20
doc_id: 933947
cord_uid: egfri75f

The ongoing SARS-CoV-2 pandemic highlights the importance of determining the breadth and durability of humoral immunity to SARS-CoV-2 mRNA vaccination. Herein, we characterize the humoral response in 27 naïve and 40 recovered vaccinees. SARS-CoV-2-specific antibody and MBC responses are durable up to six months, although antibody half lives are shorter for naïve recipients. The magnitude of the humoral responses to vaccination strongly correlates with responses to initial SARS-CoV-2 infection. Neutralization titers are lower against SARS-CoV-2 variants in both recovered and naïve vaccinees, with titers more reduced in naïve recipients. While RBD is the main neutralizing target of circulating antibodies, Moderna-vaccinated naïves show a lesser reliance on RBD, with >25% neutralization remaining after depletion of RBD-binding antibodies. Overall, we observe that vaccination induces higher peak titers and improves durability in recovered as compared to naïve vaccinees. These findings have broad implications for current vaccine strategies deployed against the SARS-CoV-2 pandemic.

SARS-CoV-2 is an ongoing public health crisis with over 450 million infections and 6 million deaths attributed 45 to the virus worldwide two years after its emergence. 1 In numerous study cohorts, overwhelming evidence has 46 illustrated the importance of antibodies targeting the trimeric spike (S) protein on the viral surface, especially the receptor binding domain (RBD), in controlling SARS-CoV-2 infections. 2,3,4,5,6 RBD-specific antibodies in specific MBCs up to 6 months post-vaccination, reaching comparable numbers as recovered individuals at this 126 timepoint ( Figure 1E ).These data show that mRNA vaccination induces robust RBD-specific MBC formation in 127 both recovered and naïve vaccinees, underlines that one dose may be sufficient for recovered individuals, and 128 shows no significant difference between the two available mRNA-based vaccines.

The RBD-specific MBC response to vaccination is dominated by IgG in both naïve and recovered individuals.

To further characterize the RBD-specific MBC response, we separated the MBC compartment by expression of 133 IgM, IgG, and IgA. We did not find significant RBD-specific IgM+ MBC, even at early timepoints. Though IgM+ 

Although not observed in Moderna recipients, the recovered Pfizer group exhibited a significant increase in 143 IgA+ RBD-specific MBC over baseline after the first dose (p=0.047) and retained a small but significant 144 increase six months after vaccination (p=0.005). Naïve vaccinees also generated slight but significant IgA+ 145 RBD-specific MBCs six months following vaccination in both Moderna (p=0.044) and Pfizer (0.015) cohorts 146 ( Figure 1G ).

RBD-specific MBC exhibit sustained activation, as measured by expression of CD71.

We have recently reported that RBD-specific MBC upregulate the activation marker CD71 during acute 151 To determine the duration of activation, we assessed CD71 expression in RBD-binding and non- 

We conducted serological analyses using multiplexed antigen panels, containing SARS-CoV-2 S antigens were observed in antibodies against spike derived from either HKU1 or OC43, suggesting that vaccination has 210 minimal effect on these pre-existing antibody titers (Supplemental Figure 2A ). As expected, titers against N 211 were unaffected throughout vaccination; recovered individuals displayed a higher baseline titer due to their 212 previous exposure to SARS-CoV-2 (Supplemental Figure 2B ).

IgM titers against RBD and S were significantly lower than IgG and IgA titers, rapidly declined, and 214 returned to baseline by 1-month post-vaccination in both recovered and naïve groups (Supplemental Figure   215 2C). Anti-NTD, -RBD and -S IgG titers increased rapidly in recovered subjects following the first dose,

increasing significantly compared to naïve subjects ( Figure 4A ). Following dose 2, IgG titers in naïve 217 individuals were comparable to their recovered counterparts (Supplemental Table 7 ). IgG titers against NTD,

RBD, and S fell more rapidly in naïve versus recovered groups, shown by significantly higher titers in 219 recovered subjects one, three, and six months after vaccination ( Figure 4A , Supplemental Table 7 ). IgA titers 220 followed a similar pattern: recovered groups peaked following one dose while naïve groups required two. IgA 

J o u r n a l P r e -p r o o f Samples were run against SARS-CoV-2 (WA1\2020). All vaccinated individuals had detectable neutralizing 237 titers against SARS-CoV-2 at 1-month post-vaccination ( Figure 4C ). Recovered individuals had significantly 238 higher titers than naïve individuals with no difference in titers between vaccine brand ( Figure 4C , Supplemental 239 Figure 3B ). Neutralizing titers from both recovered and naïve individuals were significantly higher than titers 240 from samples collected 1-2 months after initial infection with SARS-CoV-2 ( Figure 4C ). Additionally, we were 241 able to compare neutralization titers between recovered (n=37) and naïve (n=25) vaccinees at 6 months after 242 vaccination ( Figure 4C ). This data has recently been published in a study of Omicron neutralization in infected 243 and vaccinated individuals and is shown here for comparative reasons only. 17 We observed that recovered 244 individuals continued to have significantly higher neutralization that naïve individuals at this timepoint but note 245 that the majority of individuals in both groups retain neutralizing titers against wild-type virus even 6 months 246 from initial vaccination ( Figure 4C ).

We also assessed the binding and neutralizing response to SARS-CoV-2 variants, specifically beta 

( Figure 5B ). RBD + MBCs and IgG titers did not correlate in naïve vaccinees (p=0.73) ( Figure 5B ).

We also sought to determine if levels of binding titers, neutralizing titers, and antigen-specific MBCs In a previous study, we determined that the majority of the circulating neutralizing activity in acutely infected 292 COVID-19 patients was driven by RBD-specific antibodies. 4 Here, we sought to determine whether the 293 circulating response to vaccination had a similar reliance on RBD-binding antibodies. We depleted RBD-294 specific antibodies, as previously described 4 , from plasma in a subset of recovered (n=23) and naïve (n=12) 295 individuals at 1-month post-vaccination and assessed subsequent binding and neutralization activity. We Figure 4B ). In addition, we also assayed binding titers towards SARS-

CoV-2 S1 NTD, another epitope on the spike protein shown to elicit neutralizing antibodies 28,29 , and we again 314 observed no significant correlation between IgG titers and percent reduction of neutralization ( Figure 6E ). The strength, breadth, and durability of the immune response to SARS-CoV-2 following vaccination is a 338 topic of great importance as variants continue to emerge, and regulatory and governmental agencies across 339 the world debate the benefits of booster shots for the general public. Additionally, the comparison of infection-340 versus vaccine-generated immunity is of public interest. Our study is uniquely suited to assess these factors 341 due to the inclusion of a convalescent cohort followed for up to a year before vaccination as well as a naïve 

In addition to the humoral durability to wild-type SARS-CoV-2, we sought to determine whether the 

To further assess differences in repertoire breadth between naïve and recovered individuals, we Moderna is more efficacious in preventing hospitalization when compared to Pfizer, especially many months 451 out from vaccination. 11 Further investigation into the specific differences in repertoire generated by these two 452 vaccines is necessary.

SARS-CoV-2 continues to be a critical worldwide public health threat. Vaccination, especially with the 454 highly efficacious mRNA vaccines, remains the best possible strategy for combatting the continuing pandemic.

The comparison of antibody-binding, B cell memory, and neutralizing activity in recovered and naïve 

Because this study continues to collect participant samples, including following booster administration, we will 466 be able to further assess and evaluate our predictions on this important immune response.

The research reported in this publication was supported in part with Federal funds from the National Institute of 

CD14 (61D3; eBioscience), CD16 (CB16; 636 eBioscience), CD19 (SJ25C1; BD Biosciences), CD20 (2H7; BD Biosciences), CD27 (O323; BioLegend or 637 MT271

RBD was conjugated as previously described. 4 After staining, PBMCs were washed 640 and then fixed for 15 min using 2% paraformaldehyde

Data were acquired on 641 a BD FACSymphony A5 and analyzed using FlowJo 10.8.0 (BD Biosciences)

Antibody Binding Assay 644 Binding analyses were performed on plasma and serum samples using one or more of the following 645 multiplexed antigen panels: V-PLEX COVID-19 Coronavirus Panel 1 (K15362/64U)

V-PLEX SARS-CoV-2 Panel 11 (K15455U), and/or V-PLEX SARS-CoV-2 Panel 13 647 (K15463U). Briefly, plates were blocked with 150 L/well of PBS + 5% BSA for 30 minutes shaking at 700 rpm

50 L /well of sample diluted at 1:20,000 was added to the plate 649 in duplicate and incubated for 2 hours shaking at 700 rpm. After washing, 50 L /well of SULFO-TAG 650 secondary (Anti-Human IgM, IgG, or IgA as appropriate) was incubated for 1 hour shaking at 700 rpm. After a 651 final wash, 150 L/well of MSD GOLD Read Buffer was added, and plates were read immediately on the 652 MESO QuickPlex SQ 120

FRNT assays were performed as previously described. 39 Briefly, samples were diluted at 3-fold in 8 serial 657 dilutions using DMEM (VWR, #45000-304) in duplicates with an initial dilution of 1:10 in a total

Serially diluted samples were incubated with an equal volume of WA1/2020 or B.1.351 or B.1.617

37 o C for 1 hour in a round-bottomed 96-well culture plate. The antibody-660 virus mixture was then added to VeroE6-TMPRSS2 cells and incubated at 37 o C for 1 hour. Post-incubation, the 661 antibody-virus mixture was removed and 100 µl of pre-warmed 0.85% methylcellulose (Sigma-Aldrich, #M0512-662 250G) overlay was added to each well. Plates were incubated at 37 o C for 16 hours

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Convergent antibody responses to SARS-CoV-2 in 724 convalescent individuals

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The Advisory Committee on Immunization

Comparative Effectiveness of Moderna

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Anti-SARS-CoV-2 receptor binding domain antibody evolution 747 after mRNA vaccination

mRNA vaccines induce durable immune memory 750 to SARS-CoV-2 and variants of concern

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mRNA-1273 and BNT162b2 mRNA vaccines have reduced 756 neutralizing activity against the SARS-CoV-2 omicron variant

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Med 2, 100354

Robust memory responses against influenza vaccination in pemphigus patients previously 763 treated with rituximab

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CoV-2 naive and recovered individuals following mRNA vaccination

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Defining antigen-specific plasmablast and 805 memory B cell subsets in human blood after viral infection or vaccination

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methylcellulose overlay was removed, and cells were washed three times with PBS. Cells were then fixed with 664 2% paraformaldehyde in PBS for 30 minutes. Following fixation, plates were washed twice with PBS and 100 µl 665 of permeabilization buffer, was added to the fixed cells for 20 minutes. Cells were incubated with an anti-SARS-

CoV spike primary antibody directly conjugated with alexaflour-647 (CR3022-AF647) for up to 4 hours at room 667 temperature. Cells were washed three times in PBS and foci were visualized and imaged on an ELISPOT reader 668 (CTL).

Depletion of RBD-specific antibodies from plasma was conducted as previously described. 4 Briefly, plasma 672 samples were diluted 1:10 with superparamagnetic beads coupled to RBD according to the manufacturer's 673 protocol. Samples were incubated with rotation at RT for 1 hour after which the diluted plasma was separated 674 from beads and transferred to tubes containing the same amount of RBD-coupled beads separated from 675 storage buffer. Samples were incubated again rotating at RT for 1 hour, and the diluted plasma was separated 676 from beads and transferred to fresh tubes for analysis. Removal of RBD-binding antibodies was confirmed 677 through binding analysis (as described previously), and neutralization assays were performed as described 678 previously using an initial dilution of 1:50 in 100 µL. • SARS-CoV-2 specific MBC remain activated and increase over time in naïve subjects.• Antibody response to vaccination is broader and more durable in recovered vs naïve.• Naïve vaccinees have higher proportion of non-RBD-specific neutralizing antibodies.