Vaccination boosts naturally enhanced neutralizing breadth to SARS-CoV-2 one year after infection

TL;DR: In this article, the authors report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines.

Abstract: Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies1,2. Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines3,4. In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naive individuals2,5-8. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern4,9. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.

Summary

Plasma SARS-CoV-2 Antibody Reactivity

• Antibody reactivity in plasma to the RBD and nucleoprotein (N) were measured by enzymelinked immunosorbent assay 3 .
• In contrast to anti-RBD antibody titers that were relatively stable, anti-N antibody titers decreased significantly between 6 and 12 months in this assay irrespective of vaccination (Fig. 1b and Extended Data Fig. 2l-n ).
• To determine the neutralizing activity against circulating variants of concern/interest, the authors performed neutralization assays on HIV-1 virus pseudotyped with the S protein of the following SARS-CoV-2 variants of concern/interest: B.1.1.7, B.1.351, B.1.526 and P.1 1, 14, 15 .
• These titers are an order of magnitude higher than the neutralizing titers the authors and others have reported against the wild-type SARS-CoV-2 at the peak of the initial response in infected individuals and in naïve individuals receiving both doses of mRNA vaccines (Fig. 1d and [2] [3] [4] [5] [6] [7] [8] ).

Memory B cells

• The authors limited their analysis to memory B cells that produce anti-RBD antibodies because they are the most numerous and potent 18, 19 .
• In the absence of vaccination, the number of RBD specific memory B cells present at 12 months was only 1.35-fold lower than the earlier timepoint (p= 0.027, Fig. 2a ).
• The number of variant RBD cross-reactive B cells was directly proportional to but 1.6 to 3.2-fold lower than wild-type RBD binding B cells (Fig. 2a ).
• The relative fraction of cells belonging to these clones varied from 7-54% of the repertoire with no significant difference between vaccinated and non-vaccinated groups.
• The overall clonal composition differed between 6 and 12 months in all individuals suggesting ongoing clonal evolution (Fig. 2b and Extended Data Fig 3d ).

Extended Data Fig 5b)

• There was no significant difference in mutation between conserved and newly arising clones at the 12-month time point in vaccinated individuals (Extended Data Fig 5c ).
• Moreover, phylogenetic analysis revealed that sequences found at 6 and 12 months were intermingled and similarly distant from their unmutated common ancestors (Extended Data Fig 6).
• The authors conclude that clonal re-expansion of memory cells in response to vaccination is not associated with additional accumulation of large numbers of somatic mutations as might be expected if the clones were re-entering and proliferating in germinal centers.

Neutralizing Activity of Monoclonal Antibodies

• Among the 174 antibodies tested, 173 bound to RBD indicating that the flow cytometry method used to identify B cells expressing anti-RBD antibodies was efficient (Supplementary Tables 4 and 5 ).
• Consistent with this observation there was an overall increase in the apparent avidity of plasma antibodies between 1.3 and 12 months 3,4 (p<0.0001, Extended data Fig. 7c ).
• All 174 RBD binding antibodies obtained from the 12-month time point were tested for neutralizing activity in a SARS-CoV-2 pseudotype neutralization assay.
• When compared to the earlier time points from the same individuals, the geometric mean half maximal inhibitory concentration (IC50) improved from 171 ng/mL (1.3 months) to 116 ng/mL (6 months) to 79 ng/mL (12 months), with no significant difference between vaccinated and non-vaccinated individuals (Fig. 3b and Extended data Fig. 7d , Supplementary Table 4 ).

Epitopes and Breadth of Neutralization

• The authors assayed 60 randomly selected antibodies with comparable neutralizing activity from the 1.3-and 12-month time points.
• The 60 antibodies were evenly distributed between the 2 time points and between neutralizers and non-neutralizers (Fig. 4 ).
• Antibody affinities for RBD were similar among neutralizers and non-neutralizers obtained at the same time point (Fig. 4b , Extended Data Fig. 8 ).
• Seven of the selected antibodies showed equivalent or decreased activity against wild-type SARS-CoV-2 after 12 months (Fig. 4g and Supplementary Table 8 ).

During immune responses activated B cells interact with cognate T cells and begin dividing

• Whereas B cells expressing high affinity antibodies are favored to enter the long-lived plasma cell compartment, the memory compartment is more diverse and can develop directly from activated B cells or from a germinal center 17, [27] [28] [29] [30] [31] .
• Consistent with the longevity of bone marrow plasma cells, infection with SARS-CoV-2 leads to persistent serum anti-RBD antibodies, and corresponding neutralizing responses.
• Detailed characteristics of the symptomology and severity of the acute infection, symptom kinetics, and the immediate convalescent phase (7 weeks post-symptom onset until 6.2month visit) have been reported previously 4 .
• The study was performed in compliance with all relevant ethical regulations and the protocol (DRO-1006) for studies with human participants was approved by the Institutional Review Board of the Rockefeller University.

SARS-CoV-2 molecular tests

• Saliva was collected into guanidine thiocyanate buffer as described 42 .
• RNA was extracted using either a column-based (Qiagen QIAmp DSP Viral RNA Mini Kit, Cat#61904) or a magnetic beadbased method as described 43 .
• Reverse transcribed cDNA was amplified using primers and probes validated by the CDC or by Columbia University Personalized Medicine Genomics Laboratory respectively and approved by the FDA under the Emergency Use Authorization.
• Viral RNA was considered detected if Ct for two viral primers/probes were <40.

Blood samples processing and storage.

• Peripheral Blood Mononuclear Cells obtained from samples collected at Rockefeller University were purified as previously reported by gradient centrifugation and stored in liquid nitrogen in the presence of FCS and DMSO 3, 4 .
• Heparinized plasma and serum samples were aliquoted and stored at -20 ℃ or less.

ELISAs

• Monoclonal antibodies were tested at 10 μg/ml starting concentration and 10 additional fourfold serial dilutions.
• The average of its signal was used for normalization of all of the other values on the same plate with Excel software before calculating the area under the curve using Prism V9.1.
• For monoclonal antibodies, the EC50 was determined using four-parameter nonlinear regression (GraphPad Prism V9.1).

SARS-CoV-2 pseudotyped reporter virus

• A panel of plasmids expressing RBD-mutant SARS-CoV-2 spike proteins in the context of pSARS-CoV-2-S Δ19 has been described previously 2, 9, 26 The E484K and K417N/E484K/N501Y (KEN) substitution, as well as the deletions/substitutions corresponding to variants of concern were incorporated into a spike protein that also includes the R683G substitution, which disrupts the furin cleaveage site and increases particle infectivity.
• Neutralizing activity against mutant pseudoviruses were compared to a wildtype SARS-CoV-2 spike sequence (NC_045512), carrying R683G where appropriate.
• Briefly, 293T cells were transfected with pNL4-3DEnv-nanoluc and pSARS-CoV-2-SΔ19, particles were harvested 48 hpt, filtered and stored at -80°C.

Microneutralization assay with authentic SARS-CoV-2.

• The day prior to infection, Vero E6 cells were seeded at 1X10 4 cells/well into 96-well plates.
• After washing, cells were incubated for 1 hour at 37℃ with blocking solution of 5% goat serum in PBS (catalog no.
• A rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody (catalog no. GTX135357; GeneTex) was added to the cells at 1:1,000 dilution in blocking solution and incubated at 4 °C overnight.

Pseudotyped virus neutralization assay

• Fourfold serially diluted plasma from COVID-19-convalescent individuals or monoclonal antibodies were incubated with SARS-CoV-2 pseudotyped virus for 1 h at 37 °C.
• The mixture was subsequently incubated with 293TAce2 cells 3 (for comparisons of plasma or monoclonal antibodies from convalescent individuals) or HT1080Ace2 cl14 cells 13 (for analyses involving mutant/variant pseudovirus panels), as indicated, for 48h after which cells were washed with PBS and lysed with Luciferase Cell Culture Lysis 5× reagent .
• Nanoluc Luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System with the Glomax Navigator .
• The obtained relative luminescence units were normalized to those derived from cells infected with SARS-CoV-2 pseudotyped virus in the absence of plasma or monoclonal antibodies.
• The half-maximal neutralization titers for plasma (NT50) or halfmaximal and 90% inhibitory concentrations for monoclonal antibodies (IC50 and IC90) were determined using four-parameter nonlinear regression (least squares regression method without weighting; constraints: top=1, bottom=0) (GraphPad Prism).

Biotinylation of viral protein for use in flow cytometry

• Purified and Avi-tagged SARS-CoV-2 RBD or SARS-CoV-2 RBD KEN mutant (K417N, E484K, N501Y) was biotinylated using the Biotin-Protein Ligase-BIRA kit according to manufacturer's instructions as described before 3 .
• Ovalbumin (Sigma, A5503-1G) was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit according to the manufacturer's instructions (Thermo Scientific).

Flow cytometry and single cell sorting

• Briefly, peripheral blood mononuclear cells were enriched for B cells by negative selection using a pan-B-cell isolation kit according to the manufacturer's instructions (Miltenyi Biotec, 130-101-638).
• B cells were sorted into individual wells of 96-well plates containing 4 μl of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units/ml RNasin Ribonuclease Inhibitors (Promega, N2615) per well using a FACS Aria III and FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis.
• The sorted cells were frozen on dry ice, and then stored at −80 °C or immediately used for subsequent RNA reverse transcription.

Antibody sequencing, cloning and expression

• In brief, RNA from single cells was reverse-transcribed (SuperScript III Reverse Transcriptase, Invitrogen, 18080-044) and the cDNA stored at −20 °C or used for subsequent amplification of the variable IGH, IGL and IGK genes by nested PCR and Sanger sequencing.
• Amplicons from the first PCR reaction were used as templates for sequence-and ligation-independent cloning into antibody expression vectors.
• Recombinant monoclonal antibodies were produced and purified as previously described 3 .

Biolayer interferometry

• Epitope-binding assays were performed with protein A biosensor (ForteBio 18-5010), following the manufacturer's protocol 'classical sandwich assay'.
• Sensors immersed 10 min with Ab1 at 30 μg/ml, also known as (2) Capture first antibody.
• IgGs binding were corrected by subtracting the signal obtained from traces performed with IgGs in the absence of WT RBD.
• 200sec immersion in buffer, also known as (3) baseline.
• Curve fitting was performed using a fast 1:1 binding model and the Data analysis software .

Computational analyses of antibody sequences

• Antibody sequences were trimmed based on quality and annotated using Igblastn v.1.14.
• Annotation was performed systematically using Change-O toolkit v.0.4.540 48 .
• All scripts and the data used to process antibody sequences are publicly available on GitHub (https://github.com/stratust/igpipeline).
• Based on the 91 distinct V genes that make up the 6902 analyzed sequences from Ig repertoire of the 10 participants present in this study, the authors selected the IgH and IgL sequences from the database that are partially coded by the same V genes and counted them according to the constant region.

Figures

Figures 287

Full text

Naturally enhanced neutralizing breadth to SARS-CoV-2 after one year
1
2
3
Zijun Wang
1,*
, Frauke Muecksch
2,*
, Dennis Schaefer-Babajew
1,*
, Shlomo Finkin
1,*
, Charlotte
4
Viant
1,*
, Christian Gaebler
1,*
, Hans- Heinrich Hoffmann
3
, Christopher O. Barnes
4
, Melissa
5
Cipolla
1
, Victor Ramos
1
, Thiago Y. Oliveira
1
, Alice Cho
1
, Fabian Schmidt
2
, Justin da Silva
2
, Eva
6
Bednarski
2
, Lauren Aguado
3
, Jim Yee
5
, Mridushi Daga
1
, Martina Turroja
1
, Katrina G. Millard
1
,
7
Mila Jankovic
1
, Anna Gazumyan
1, 6
, Zhen Zhao
5
, Charles M. Rice
3
, Paul D. Bieniasz
2, 6
, Marina
8
Caskey
1
, Theodora Hatziioannou
2
, Michel C. Nussenzweig
1, 6
9
10
11
12
1
Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA
13
2
Laboratory of Retrovirology, The Rockefeller University, New York, NY 10065, USA
14
3
Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY,
15
10065, USA.
16
4
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena,
17
CA, USA.
18
5
Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY,
19
10065, USA
20
6
Howard Hughes Medical Institute
21
22
*equal contribution
23
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted June 2, 2021. ; https://doi.org/10.1101/2021.05.07.443175doi: bioRxiv preprint

Address correspondence to: Paul D. Bieniasz, pbieniasz@rockefeller.edu; Marina Caskey,
24
mcaskey@rockefeller.edu; Theodora Hatziioannou, thatziio@rockefeller.edu; or Michel C.
25
Nussenzweig, nussen@rockefeller.edu.
26
27
28
Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic
29
caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains
30
difficult to control despite the availability of several excellent vaccines. Progress in
31
controlling the pandemic is slowed by the emergence of variants that appear to be more
32
transmissible and more resistant to antibodies
1,2
. Here we report on a cohort of 63 COVID-
33
19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of
34
whom also received mRNA vaccines
3,4
. In the absence of vaccination antibody reactivity to
35
the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number
36
of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination
37
increases all components of the humoral response, and as expected, results in serum
38
neutralizing activities against variants of concern that are comparable to or greater than
39
neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naïve
40
individuals
2,5-8
. The mechanism underlying these broad-based responses involves ongoing
41
antibody somatic mutation, memory B cell clonal turnover, and development of
42
monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations,
43
including those found in variants of concern
4,9
. In addition, B cell clones expressing broad
44
and potent antibodies are selectively retained in the repertoire over time and expand
45
dramatically after vaccination. The data suggest that immunity in convalescent individuals
46
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted June 2, 2021. ; https://doi.org/10.1101/2021.05.07.443175doi: bioRxiv preprint

will be very long lasting and that convalescent individuals who receive available mRNA
47
vaccines will produce antibodies and memory B cells that should be protective against
48
circulating SARS-CoV-2 variants.
49
50
We initially characterized immune responses to SARS-CoV-2 in a cohort of convalescent
51
individuals 1.3 and 6.2 months after infection
3,4
. Between February 8 and March 26, 2021, 63
52
participants between the ages of 26 and 73 years old (median 47 years) returned for a 12-month
53
follow-up visit. Among those, 26 (41%) had received at least one dose of either the Moderna
54
(mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines, on average 40 days (range 2-82 days)
55
before their study visit and 311 days (range 272-373 days) after onset of acute illness
56
(Supplementary Table 1). Participants were was almost evenly split between sexes (43% female)
57
and of the individuals that returned for a 12-month follow-up, only 10% had been hospitalized
58
and the remainder had experienced relatively mild initial infections. Only 14% of the individuals
59
reported persistent long-term symptoms after 12 months, reduced from 44% at the 6-month time
60
point
4
. Symptom persistence was not associated with the duration and severity of acute disease
61
or with vaccination status (Extended Data Fig. 1 a-c). All participants tested negative for active
62
infection at the 12-month time point as measured by a saliva-based PCR assay
4
. The
63
demographics and clinical characteristics of the participants are shown in Supplementary Tables
64
1 and 2.
65
66
Plasma SARS-CoV-2 Antibody Reactivity
67
Antibody reactivity in plasma to the RBD and nucleoprotein (N) were measured by enzyme-
68
linked immunosorbent assay (ELISA)
3
. We limited our analysis to RBD because plasma anti-
69
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted June 2, 2021. ; https://doi.org/10.1101/2021.05.07.443175doi: bioRxiv preprint

RBD antibodies are strongly correlated with neutralizing activity
3,10-12
. Convalescent
70
participants who had not been vaccinated maintained most of their anti-RBD IgM (103%), IgG
71
(82%), and IgA (72%) titers between 6 and 12 months (Fig. 1a and Extended Data Fig. 2a-k).
72
Consistent with previous reports
5-8
, vaccination increased the anti-RBD plasma antibody levels,
73
with IgG titers increasing by nearly 30-fold compared to unvaccinated individuals (Fig. 1a right).
74
The 2 individuals who did not show an increase had been vaccinated only 2 days before sample
75
collection. In contrast to anti-RBD antibody titers that were relatively stable, anti-N antibody
76
titers decreased significantly between 6 and 12 months in this assay irrespective of vaccination
77
(Fig. 1b and Extended Data Fig. 2l-n).
78
79
Plasma neutralizing activity in 63 participants was measured using an HIV-1 pseudotyped with
80
the SARS-CoV-2 spike protein
3,4,13
(Fig. 1c-d and Extended Data Fig. 2o). Twelve-months after
81
infection, the geometric mean half-maximal neutralizing titer (NT
50
) for the 37 individuals that
82
had not been vaccinated was 75, which was not significantly different from the same individuals
83
at 6.2 months (Fig. 1c). In contrast, the vaccinated individuals showed a geometric mean NT
50
of
84
3,684, which was nearly 50-fold greater than unvaccinated individuals and slightly better
85
compared to the 30-fold increase in anti-RBD IgG antibodies (Fig. 1a, c, and d). Neutralizing
86
activity was directly correlated with IgG anti-RBD (Extended Data Fig.2p) but not with anti-N
87
titers (Extended Data Fig.2r). We conclude that neutralizing titers remain relatively unchanged
88
between 6 to 12 months after SARS-CoV-2 infection, and that vaccination further boosts this
89
activity by nearly 50-fold.
90
91
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted June 2, 2021. ; https://doi.org/10.1101/2021.05.07.443175doi: bioRxiv preprint

To determine the neutralizing activity against circulating variants of concern/interest, we
92
performed neutralization assays on HIV-1 virus pseudotyped with the S protein of the following
93
SARS-CoV-2 variants of concern/interest: B.1.1.7, B.1.351, B.1.526 and P.1
1,14,15
. Twelve-
94
months after infection neutralizing activity against the variants was generally lower than against
95
wild-type SARS-CoV-2 virus in the same assay with the greatest loss of activity against B.1.351
96
(Fig. 1e). After vaccination the geometric mean NT
50
rose to 11,493, 48,341, 22,109 and 26,553
97
against B.1.351, B.1.1.7, B.1.526 and P.1, respectively. These titers are an order of magnitude
98
higher than the neutralizing titers we and others have reported against the wild-type SARS-CoV-
99
2 at the peak of the initial response in infected individuals and in naïve individuals receiving both
100
doses of mRNA vaccines (Fig. 1d and
2-8
). Similar results were also obtained using authentic
101
SARS-CoV-2 WA1/2020 and B.1.351 (Extended Data Fig.2s).
102
103
Memory B cells
104
The memory B cell compartment serves as an immune reservoir that contains a diverse collection
105
of antibodies
16,17
. Although antibodies to the N-terminal domain and other parts of S can also be
106
neutralizing, we limited our analysis to memory B cells that produce anti-RBD antibodies
107
because they are the most numerous and potent
18,19
. To enumerate RBD-specific memory B
108
cells, we performed flow cytometry using a biotin-labeled RBD
3
(Fig. 2a and Extended Data Fig.
109
3a and b). In the absence of vaccination, the number of RBD specific memory B cells present at
110
12 months was only 1.35-fold lower than the earlier timepoint (p= 0.027, Fig. 2a). In contrast
111
and consistent with previous reports
5,8,20
, convalescent individuals that received mRNA vaccines
112
showed an average 8.6-fold increase in the number of circulating RBD specific memory B cells
113
(Fig. 2a). B cells expressing antibodies that bound to both wild-type and K417N/E484K/N501Y
114
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted June 2, 2021. ; https://doi.org/10.1101/2021.05.07.443175doi: bioRxiv preprint