RESEARCH ARTICLE

Differential Antibody Response to mRNA COVID-19 Vaccines in

Healthy Subjects
Sarah E. Wheeler,a Galina V. Shurin,a Mary Yost,a Adam Anderson,b Lisa Pinto,b Alan Wells,a Michael R. Shurina,c

a Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA

b Bio-Rad Laboratories, Inc., Benicia, California, USA
c Department of Immunology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA

ABSTRACT Knowledge about development and duration of virus-specific antibodies af-

ter COVID-19 vaccination is important for understanding how to limit the pandemic via
vaccination in different populations and societies. However, the clinical utility of postvac-
cination testing of antibody response and selection of targeted SARS-CoV-2 antigen(s)
has not been established. The results of such testing from clinical teams independent
from vaccine manufacturers are also limited. Here, we report the initial results of an
ongoing clinical study on evaluation of antibody response to four different SARS-CoV-2
antigens after first and second dose of Pfizer and Moderna mRNA vaccines and at later
time points. We revealed a peak of antibody induction after the vaccine boosting dose
with a gradual decline of antibody levels at later time. Anti-nucleocapsid antibody was Vaccines give you just the spike, NOT the nucleocapsid; this is why the
anti-nucleocapsid Ab can differentiate prior-infected from healthy
not induced by spike protein-encoding vaccines and this may continue to serve as a
marker of previous SARS-CoV-2 infection. No differences between the two vaccines in
terms of antibody response were revealed. Age and gender dependencies were deter-
mined to be minimal within the healthy adult (but not aged) population. Our results
suggest that postvaccination testing of antibody response is an important and feasible
tool for following people after vaccination and selecting individuals who might require a

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third dose of vaccine at an earlier time point or persons who may not need a second
dose due to previous SARS-CoV-2 infection.
IMPORTANCE Now that authorized vaccines for COVID-19 have been widely used, it
is important to understand how they induce antivirus antibodies, which antigens are
targeted, how long antibodies circulate, and how personal health conditions and
age may affect this humoral immunity. Here, we report induction and time course of
multiple anti-SARS-CoV-2 antibody responses in healthy individuals immunized with
Pfizer and Moderna mRNA vaccines. We also determined the age and gender de-
pendence of the antibody response and compared antibody levels to responses
seen in those who have recovered from COVID-19. Our results suggest the impor- Citation Wheeler SE, Shurin GV, Yost M,
tance of screening for antibody response to multiple antigens after vaccination in Anderson A, Pinto L, Wells A, Shurin MR. 2021.
order to reveal individuals who require early and late additional boosting and those Differential antibody response to mRNA
COVID-19 vaccines in healthy subjects.
who may not need second dose due to prior SARS-CoV-2 infection. Microbiol Spectr 9:e00341-21. https://doi.org/
10.1128/Spectrum.00341-21.
KEYWORDS antibody, COVID-19, SARS-CoV-2, vaccination, humoral immunity
Editor Miguel Angel Martinez, Fundacio
irsiCaixa
Copyright © 2021 Wheeler et al. This is an

S evere acute respiratory syndrome coronavirus 2, SARS-CoV-2, is a single-stranded

RNA virus which can be passed between humans and is the cause of the coronavi-
rus pandemic, which began in 2019. Infection with SARS-CoV-2 may result in coronavi-
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
rus disease 2019 (COVID-19), which commonly induces a robust and persistent Address correspondence to Michael R. Shurin,
shurinmr@upmc.edu.
immune response to SARS-CoV-2. This includes virus-specific antibodies, memory B cells,
Received 19 May 2021
and effector and memory CD81 T cells (1, 2). The U.S. Food and Drug Administration (FDA) Accepted 13 July 2021
issued an Emergency Use Authorization (EUA) for the first two SARS-CoV-2 vaccines in Published 4 August 2021
December 2020—the Pfizer-BioNTech and Moderna vaccines—for the prevention of

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Wheeler et al.

COVID-19 in the United States and the protection of persons who are at high risk for com-
plications (3, 4). Both vaccines use nucleoside-modified mRNA with a lipid nanoparticle-for-
mulation to encode the spike (S) protein of SARS-CoV-2.
It is considered critical that SARS-CoV-2 vaccines provoke a strong immune
response against the spike protein, particularly the receptor-binding domain (RBD) of
the spike protein which contains many neutralizing epitopes (5). Therefore, many vac-
cines utilize stabilizing mutations in the S glycoprotein to maintain the prefusion con-
formation and prevent shedding of the S1 subunit (6). Available information to date
seems to indicate that these mutated S proteins are more immunogenic than the wild-
type S protein. Both the mRNA-1273 vaccine (Moderna) and the BNT162b2 vaccine
(Pfizer) (7) encode the full-length viral S ectodomain with a transmembrane anchor
and two S-2P mutations which serve to stabilize the prefusion conformation (7, 8).
Initially, Pfizer clinically investigated the BNT162b1 vaccine candidate that incorpo-
rates modified mRNA to encode only the RBD portion of the S protein, thought to be
the key target of virus-neutralizing antibodies (9). The RBD antigen that resulted from
BNT162b1 had an additional T4 fibritin-derived fold in the trimerization domain which
served to increase immunogenicity (10). Later, Pfizer reported that the BNT162b2,
which encoded the full-length stabilized S glycoprotein, elicited dose-dependent
SARS-CoV-2 neutralizing antibody titers comparable or higher than those elicited by a
defined panel of convalescent SARS-CoV-2 serum samples (11). The full-length protein
vaccine was shown to have fewer side effects and was better tolerated than other vac-
cine candidates. Using the full-length glycoprotein vaccine, a randomized, placebo-
controlled trial was performed with over 40,000 participants. The trial found that two
doses of BNT162b2 demonstrated 95% protection against COVID-19 (7).
The Moderna mRNA-1273 vaccine encodes the S-2P antigen (15-1208; a mutant
recombinant version of the S glycoprotein with proline replacements at amino acids
986 and 987) (12, 13), a transmembrane anchor, and an S1-S2 cleavage site. The prefu-
sion conformation is stabilized by the consecutive proline substitutions which are
located in the S2 subunit at the top of the central helix (6, 14). Findings from the
30,000-participant phase III clinical trial demonstrated an efficacy of 94.1% after a me-
dian follow-up of 2 months in preventing symptomatic COVID-19 with laboratory con-

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firmation of infection. This trial excluded those previously infected with SARS-CoV-2
and was a randomized, double-blind, placebo-controlled trial (15).
The mRNA COVID-19 vaccine series is comprised of two doses administered intra-
muscularly: 30 m g, 0.3 ml each with 3 weeks apart for Pfizer-BioNTech and 100 m g,
0.5 ml each with 1 month apart for Moderna. Several reports documented a strong hu-
moral response to mRNA vaccines (16–18). These vaccines have shown efficacy against
the mutated strains of the virus (19–21). For instance, participants who were fully
immunized with the Moderna mRNA-1273 COVID-19 vaccine have sera that demon-
strates neutralizing activity against the SARS-CoV-2 variants: the Alpha variant (B.1.1.7),
the Beta variant (B.1.351-v1, B.1.351-v2, and B.1.351-v3), the Delta variant (B.1.617.2),
and the Gamma variant (P.1) (22). In addition, neutralization titers in vaccinated partici-
pants often exceed those of convalescent COVID-19 patients who were not hospital-
ized (23). Estimation of Pfizer-BioNTech BNT162b2 vaccine effectiveness against
mutated variants using a test-negative case-control study design revealed its effective-
ness against the B.1.1.7 and B.1.351 variants (23, 24). Pfizer-BioNTech vaccine is also
effective against Delta and Kappa variants of the coronavirus (25). Interestingly, SARS-
CoV-2 mRNA vaccination also induces cross-reactive antibodies to seasonal b -corona-
viruses (26).
Although the immune response against SARS-CoV-2 has been well characterized in
naturally infected people, development of immunity after administration of antiviral
vaccines, including mRNA vaccines, is not yet completely understood. For instance, si-
multaneous analysis and comparison of antibodies to four SARS-CoV-2 antigens in
healthy volunteers receiving two mRNA vaccines has not yet been conducted. The
availability of limited data about humoral response to vaccination may be in part due

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to the fact that the CDC does not recommend measuring postvaccination titers for
COVID-19: “Antibody testing is not currently recommended to assess for immunity to
SARS-CoV-2 following COVID-19 vaccination because the clinical utility of postvaccina-
tion testing has not been established” (27). However, to reduce the current pandemic
and limit the burden of COVID-19 worldwide by effectively vaccinating as many people
as possible, we must also select and validate appropriate assays and comprehensively
characterize antiviral immunity associated with COVID-19 vaccines.
Although a consistent proof of protective immunity after vaccination may only
come via reinfection challenge experiments or longitudinal studies of postvaccina-
tion individuals, studies analyzing antibody response to different SARS-CoV-2 anti-
gens after vaccination of healthy volunteers and recovered COVID-19 individuals
are important for establishing the “levels of protection” to be determined by FDA-
approved clinical laboratory assays. In addition, testing of vulnerable populations
for sufficient vaccine response will be critical in stemming the spread in these pop-
ulations and requires that we define a normal or sufficient vaccine response for
comparison. The goal of this study was to determine the level of antibodies to RBD,
S1, S2, and nucleocapsid (N) SARS-CoV-2 antigens in the sera of volunteers receiv-
ing Pfizer-BioNTech and Moderna vaccines by evaluating the feasibility of novel
BioPlex 2200 SARS-CoV-2 IgG panel. Our results demonstrate the dynamic of anti-
body production after the two-dose vaccine schedule and establish differences
between antibody generation induced by a vaccine and natural COVID-19 disease.
Although some of the immune mechanisms described here are not novel, our study
provides critical, confirming evidence that key antibody pathways are indeed
modulated under the COVID-19 vaccination procedure.

RESULTS
Dynamics of antibody response to vaccination. A common measure of the effec-
tiveness of a vaccine is the specific antibody response in serum samples. Common pat-
terns of antibody response to four different SARS-CoV-2 antigens after vaccination of
healthy individuals are shown in Fig. 1. All volunteers demonstrated no antibodies to
any tested antigens before vaccine administration. The first dose of a vaccine induced

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the production of specific antibodies within 2 weeks, although the levels varied from
negative to 580 (median, 71), 327 (median, 64), and 234 (median, 5) U/ml for RBD, S1,
and S2 antibodies, respectively. Interestingly, 13, 3, and 66% of participating volunteers
demonstrated negative (,10 U/ml) antibody responses to RBD, S1, and S2 antigens,
respectively, after the first dose vaccine administration. No anti-nucleocapsid antibod-
ies were detected during or after vaccination. The second vaccine dose caused a signif-
icant upregulation of tested antibodies of up to 100-fold in all volunteers, including
statistically significant increases in those who demonstrated negative or low response
to first vaccine dose (Fig. 1). The levels of antibodies reached 15,300 (median, 4,320),
5940 (median, 2,172), and 174 (median, 40) U/ml for RBD, S1, and S2, respectively
(Fig. 1). No nonresponders were detected after second vaccine dose administration for
anti-RBD and -S1 antibodies, and only one person demonstrated no response for anti-
S2 antibody (Fig. 1). Interestingly, the levels of anti-RBD and anti-S1 antibodies after secondary response
the second dose in all participants were higher than any levels seen after the first dose,
regardless of the responsiveness to the first dose; this suggests that even if scored as
negative, the initial vaccine dose did induce an immunologic response.
The next set of data includes anti-Spike antibody levels after a completed vacci-
nation on a monthly basis. Figure 2A summarizes these results for each antigen.
Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks with all pairwise
comparisons (Dunn’s method for unequal group sizes) revealed a statistically sig-
nificant difference in the levels of anti-RBD antibodies (nonlogarithmic values). For
instance, the “1st dose” group (median, 71.0) versus the “2nd dose” group (median,
4320.0) (P , 0.001) and the “45d” group (median, 2419.0) (P , 0.001) and the “75d”
group (median, 1514.0) (P , 0.001). A decrease in anti-RBD antibody after its peak

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Wheeler et al.

FIG 1 Individual antibody responses to two doses of mRNA vaccines. Blood specimens were collected from healthy individuals before and after the first
and second doses of the Pfizer or Moderna vaccines and then every month. IgG antibodies binding to RBD, S1, S2, and nucleocapsid SARS-CoV-2 structural
proteins were screened and differentiated by the BioPlex 2200 SARS-CoV-2 IgG multiplex panel as described in Materials and Methods. Each time point
represents individual values with the mean and SEM. 10 U/ml is the negative cutoff concentration.

in “2nd dose” group was not significant at day 45 (P = 0.056) but was significant on
day 75 (P , 0.001). At the same time, the difference between the “45d” and “75d”
groups was not statistically significant for anti-RBD antibody levels (P = 0.752).
Similar results were obtained for anti-S1 IgG. The “1st dose” group (median,
64.0) data were significantly lower than data obtained from all other groups: the

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“2nd dose” group (median, 2172.0), the “45d” group (median, 1053.5) and the
“75d” group (median, 821.0) (all P , 0.001). The decrease in anti-S1 antibody at
45 and 75 days after the second dose was significant (P = 0.013 and P , 0.001,
respectively), although no differences between the “45d” and “75d” groups were
seen (P = 1.0).
Changes in anti-S2 antibody levels were slightly different. Although a second dose
of a vaccine induced a significantly stronger anti-S2 IgG levels (median, 40.0 versus 5.0,
P , 0.001), the decrease on day 45 after a second shot (median, 20.0) did not reach sta-
tistical significance (P = 0.121), whereas it was significant on day 75 (median, 10.5,
P , 0.001). No differences between the “45d” and “75d” groups were detected
(P = 0.518).
To display the distribution of anti-SARS-CoV-2 antibody values and for visual
comparisons of distributions between different time points in vaccine groups, the
reverse cumulative distribution curves (RCDCs) are shown in Fig. 2B. In all groups
prevaccination, the RCD curves were similar for each of the tested antigens, indi-
cating a lack of baseline serological bias between study groups (data not shown).
Postvaccination, the proportion of subjects reaching higher antibody levels to
RBD, S1, and S2 proteins increased. For the RBD and S1 antigens, the RCD curves
for the second dose of a vaccine are above the curve for the first dose of a vaccine
at all antibody concentrations presented in a log scale. This suggests that the vac-
cine dose with the highest curve induced the greatest immune responses. For the
S2 antigen, two RCD curves coincide at the highest antibody levels, which demon-
strates comparable immune responses. While the postvaccination RCD curves
were similar for RBD and S1 antigens, for the S2 antigen there was a shift in the

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B1/2 - secondary response

FIG 2 Time-course of antibody response to vaccination with mRNA vaccines. Antibody responses to two doses of Pfizer and Moderna vaccination were
assessed for RBD, S1, S2, and nucleocapsid. (A) Summation of two-dose vaccine-induced antibody development to different SARS-CoV-2 antigens in healthy
volunteers. The x axis shows time points before and after two doses of vaccine administration and 45 and 75 days after a completed vaccination; the y axis
shows the logarithmic antibody concentrations shown as means 6 the SEM. Significant differences with corresponding P values for each antigen are
presented in the text. 10 U/ml is the negative cutoff concentration. (B) RCDCs of antibody response in healthy volunteers postvaccination for each of the
three antigens. The x axis represents the antibody concentration values in log scale; the y axis is the proportion of subjects having at least that antibody
level. The curve begins at 100% and then descends from left to right. When the RCD curves overlap, the two doses of vaccines induced comparable
immune responses. If one curve is above another one, it indicates the higher immune response.

“2nd dose” RCDC, demonstrating that a lower proportion of subjects achieved

antibody concentrations of .100 U/ml versus the “1st dose” group (Fig. 2B).
Together, these data demonstrate that none of volunteers had serologic signs of previous

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natural SARS-CoV-2 infection, all individuals responded to two-dose mRNA vaccination by a
significant production of antibodies specific to spike proteins encoded by mRNA vaccines,
and the peak of antibody response was seen 2 weeks after second dose of the vaccine.
Difference in antibody response to two vaccines. To compare the strength and du-
ration of antibody response to Pfizer and Moderna vaccines, we assessed levels of antibod-
ies to three antigens at different time points in individuals receiving Pfizer (51%; Table 1) or
Moderna (49%) vaccines. Analysis of results shown in Fig. 3 utilizing unpaired parametric t

TABLE 1 Demographic data of participated individuals

Characteristic Data
Total no. of participants 47

Age (yr)
Median 50.00
Mean 6 the SD 49.29 6 12.81
Range 19–70

No. (%) of subjects

Female 33 (70)
Pfizer 24 (51)
Female 16 (67)
Moderna 23 (49)
Female 16 (70)
Occupational risk 32 (68)
Healthcare workers 34 (72)

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Wheeler et al.

FIG 3 Comparative analysis of antibody response to two mRNA vaccines. Antibody responses to two doses of Pfizer and Moderna vaccination of healthy
volunteers were assessed by the BioPlex 2200 SARS-CoV-2 IgG multiplex panel. Results represent individual values (dots) and means 6 the SEM (bars) for
IgG antibodies recognizing RBD, S1, and S2 SARS-CoV-2 antigens. P values were determined using an unpaired t test. 10 U/ml (dotted line) is the negative
cutoff concentration.

test with Welch’s correction (after evaluation for normal distribution by Kolmogorov-
Smirnov test) revealed no statistically significant differences in the potency of immune
response to RBD protein after first and second doses of either mRNA vaccine. No statisti-
cally significant differences in the duration of anti-RBD responses to Pfizer and Moderna
vaccines were also detected (Fig. 3). Antibody response to S1 protein showed a signifi- Dawn pointed this out
cantly higher response to first dose of Moderna (96.23 6 18.13 U/ml) than Pfizer
(51.61 6 9.54) vaccine, while no statistically significant differences were detected at all fol-
lowing time points. Finally, no differences between two vaccines in antibody response to
S2 protein after vaccination were determined (Fig. 3). Overall, we did not reveal differences
between Pfizer and Moderna in terms of the antibody response to spike protein after two-
dose vaccination in healthy volunteers.
Relative analysis (age and gender). We next evaluated whether the antibody
response to mRNA vaccination was similar in males and females. Comparative results
are shown in Fig. 4A. An unpaired t test analysis revealed no statistically significant dif-

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ferences between vaccinated males and females in anti-RBD and anti-S1 antibody lev-
els after the first and second doses and at the later time points, that is, 45 and 75 days
after the second shot. However, a statistically significant difference between males
(28.91 6 4.19 U/ml) and females (55.93 6 8.11 U/ml) in anti-S2 response after the sec-
ond dose was demonstrated (Fig. 4A). Although at 45 days after the second injection
the levels of anti-S2 antibodies were higher in females (41.31 6 11.44 U/ml) than in
males (19.60 6 4.64 U/ml), these differences did not reach statistical significance. Thirty
days later, this difference was minimal: ;19 U/ml versus ;13 U/ml. Thus, we did not
observe gender-associated differences in a long-term response to mRNA vaccination.
Next, we sought to determine whether specific antibody responses to mRNA adminis-
tration may be age dependent within adults aged 19 to 70. Figure 4B shows antibody
responses versus age separated based on the median age (51 years old). Pearson correla-
tion analysis utilizing all age groups revealed a statistically significant relationship between
the age and level of anti-RBD IgG after first dose of vaccine administration (Fig. 4B).
However, no age dependency of the antibody response was seen after the second dose,
suggesting that immune boosting was significant in all age categories. No correlations
between the anti-RBD antibody levels and age were detected at later time points of blood
collection (45 and 75 days after second dose administration). Similarly, induction of anti-S1
IgG after first vaccine dose was age dependent (R = 0.410, P = 0.005), whereas this correla-
tion disappeared after second dose (R = 0.234, P = 0.152) and was not detected at later
time points (R , 0.02, P . 0.9). No significant correlations between age and anti-S2
response were determined (data not shown). Thus, induction of anti-RBD and anti-S2 anti-
bodies by vaccination was initially inversely correlated with volunteers’ age, while the later
response and its duration were age independent.

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Age difference did exist in the first dose but disappeared in the
second dose based on p-values

FIG 4 Gender and age differences in antibody responses to mRNA vaccination. Antibody responses to two doses of Pfizer and Moderna vaccinations of
healthy volunteers were assessed by the BioPlex 2200 SARS-CoV-2 IgG multiplex assay. (A) Results represent individual values (dots) and means 6 the SEM
of IgG antibodies recognizing RBD, S1, and S2 SARS-CoV-2 antigens in males and females 2 weeks after the first and second doses and 45 and 75 days
after the second shot. The y axis represents antibody levels in U/ml in logarithmic scale. P values were calculated using an unpaired t test. (B) Pearson
correlation analysis of antibody responses versus age of healthy volunteers. Two age cohorts separated based on the median age (50) are shown for clarity.
Induction of anti-RBD antibody after first (left panel) and second (right panel) doses of a vaccine are shown in the y axis. Regression lines and correlation
coefficients with the corresponding P values are shown. CIs (95%) and prediction intervals (95%) are also indicated.

Comparative examples (COVID-19, vaccination interval, and vaccination after a

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natural disease). For comparative presentation of anti-SARS-CoV-2 serum antibody
levels determined by a multiplexed assay, we determined the antibody response in
randomly selected samples obtained from COVID-19-confirmed and COVID-19-recov-
ered patients who donated their blood for convalescent plasma evaluation. A compre-
hensive analysis of antibody levels in different cohorts of COVID-19 patients have been
recently reported by our lab (N. Cook et al., unpublished data). Figure 5A demonstrates
a range of anti-SARS-CoV-2 antibodies in individuals recovered (3 to 6 months) after
mild COVID-19 incidence. The levels of tested antibodies were similar to the levels of
antibodies seen in individuals receiving a first dose of COVID-19 mRNA vaccines, with
the only difference being the absence of anti-nucleocapsid antibody in vaccinated
healthy volunteers (Fig. 6). As an additional confirmation of positive anti-SARS-CoV-2
immunity in COVID-19-recovered patients, we utilized the EuroImmun assay detecting
anti-S IgG antibodies in the same blood specimens (Fig. 5B). Together, these results
suggest that anti-N antibody may serve as a marker of previous COVID-19 disease and
that two-dose mRNA vaccination induces a significantly higher antibody response than
that seen in people recovered after mild or medium SARS-CoV-2 infection.
A few examples from participating volunteers open additional opportunities for fur-
ther studies. Figure 6 demonstrates individual results for comparison: a representative
antibody response to a two-dose vaccination in a volunteer in a linear (Fig. 6B) and a
logarithmic (Fig. 6A) scale. The peak of antibody response to RBD antigen reached
;3,000 U/ml. The level of anti-SARS-CoV-2 antibodies in an individual recovered after
mild COVID-19 was within 50 to 150 U/ml during ;8 months after a disease, with a
slight but consistent declining of anti-RBD and anti-S1 antibodies and a marked reduc-
tion of anti-N antibody (Fig. 6): the results are shown in a linear (Fig. 6D) and a

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Wheeler et al.

FIG 5 Serum anti-SARS-CoV-2 antibody levels in COVID-19 recovered individuals. Antibody levels
were assessed by using the BioPlex 2200 SARS-CoV-2 IgG multiplex panel (A) and the EuroImmun IgG
ELISA (B) in 59 COVID-19-recovered persons 3 to 8 months after disease. Antibody levels are
expressed in U/ml in logarithmic scale (A) and index values (B), as described in Materials and
Methods. 10 U/ml (dotted line) is the negative cutoff concentration for panel A. A, index value of 1
(dotted line) is the negative cutoff concentration for panel B. Results from a representative cohort of
patients are shown.

logarithmic (Fig. 6C) scale. This suggests that vaccine-induced antibodies are seen at
higher levels compared to mild natural disease-induced antibodies at least during a
few months after vaccination. Interestingly, vaccination of a person several months af-
ter mild confirmed COVID-19 recovery resulted in a dramatic induction of anti-RBD,
-S1, and -S2, but not anti-N, antibodies up to 40,000 U/ml, which is almost 1,000-fold
higher than in healthy vaccinated volunteers (Fig. 6E and F). Importantly, although the
second vaccine dose resulted in the highest boosting of antibody production, even the
first dose stimulated a dramatic antibody response in a COVID-19-recovered person,
which was significantly higher than the average response in previously COVID-19-free

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volunteers. Together, these results of the multiplexed analysis of anti-SARS-CoV2 anti-
bodies support the idea that both anti-N and anti-S2 antibodies may be helpful in dif-
ferentiation of individuals who have had a previous infection for at least several
months earlier and were or were not vaccinated.
Figure 7 shows prolonged serum antibody levels in a healthy volunteer after the
first dose of the Moderna vaccine for up to 70 days prior to administration of the sec-
ond dose of the vaccine. As expected, a relatively low antibody response, determined
by the BioPlex SARS-CoV-2 IgG assay, was seen after first injection (Fig. 7B) to both RBD
and S1 antigens, as shown in the linear (lower panel) and logarithmic (upper panel)
scales. Antibody levels slowly and gradually decreased for 70 days, while a boosting
vaccination induced 30- to 40-fold increase in antibody levels, peaking 1 week after
injection. The following decline in serum antibodies was also anticipated. These results
were verified by determining antibodies utilizing Siemens SARS-CoV-2 total assay
(Fig. 7A). The results, shown linear (lower panel) and logarithmic (upper panel) scales,
confirmed a persistent low level of specific antibodies after the first dose and a signifi-
cant boosting induced by the second dose of a vaccine. Together, these data demon-
strate that waiting longer between the two doses could achieve the same effect in
healthy individuals.
In summary, our results demonstrate clinical usefulness of multiplexed detection of
anti-SARS-CoV-2 antibodies in vaccinated healthy volunteers. Specifically, we revealed
a compatible efficacy of the Pfizer-BioNTech and Moderna vaccines in the induction of
various anti-SARS-CoV-2 antibodies, a similar immune response to vaccination in males
and females, an initial dose-limited age dependence of antibody production, and a
marked monthly decline in antibody levels after vaccination.

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Antibody Response to Vaccination
Artificial active natural active natural active + vaccine
Below: healthy vaccine covid (no vaccine) covid and then vaccinated
redundancy since you have anti N -
trade off!

vaccinated people retained higher than if you got it natural

if you got infected and vaccinated made you much better; getting the vaccine helps
even if you got covid

FIG 6 Vignettes to compare healthy to previously infected volunteers. Examples of anti-SARS-CoV-2 antibody levels in a vaccinated healthy volunteer (A
and B), a mild COVID-19 recovered person (C and D), and a previously SARS-CoV-2-infected individual receiving a two-dose mRNA vaccine (E and F) are
shown. Antibody levels were assessed by using a BioPlex 2200 SARS-CoV-2 IgG multiplex panel. Results are expressed as antibody levels in U/ml (y axis) in
the linear (B, D, and F) and logarithmic (A, C, and E) scales for comparison. The x axis indicates time points before, 2 weeks after the first and second
vaccine doses, and 45, 75, and 105 days after the second shot (A, B, E, and F) or days after natural disease (C and D). 10 U/ml (dotted line) is the negative
cutoff concentration.

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DISCUSSION
Currently, there are three basic types of tests to determine whether an individual
has been infected with SARS-CoV-2: viral RNA detection, viral antigen detection, and
detection of antibodies to the virus. Viral tests are used to assess acute infection,
whereas antibody tests provide evidence of prior infection. Although the FDA has not
authorized the use of antibody tests for the diagnosis of acute infection, they are the
key tests for assessing immune response to a COVID-19 vaccination, distinguishing
immune response to a vaccine or natural disease and identification of prior asymptom-
atic infections.
There are four major structural proteins in SARS-CoV-2: spike, envelope, membrane,
and nucleocapsid encoded by the S, E, M, and N genes, as well as nonstructural pro-
teins (13, 28). The S protein contains the S1 and S2 subunits which mediate receptor
binding and membrane fusion, respectively. Viral entry is achieved through virus-host
cell membrane fusion, which requires a conformational change in the S protein. The
RBD in S1 binds to angiotensin-converting enzyme 2 (ACE2) on the host cell, which ini-
tiates the necessary conformational change in the S2 subunit that allows for mem-
brane fusion (28). Both antibody and T cell responses are detectable to all major viral
antigens during or after COVID-19 (29, 30). Similar immune responses can be seen after
COVID-19 vaccination. Although more than 200 vaccine candidates are in develop-
ment, of which more than 60 are in clinical development, as of May 2021 the WHO has
determined that the following vaccines against COVID-19 have met the necessary crite-
ria for safety and efficacy: vaccines made by SinoPharm, AstraZeneca/Oxford, Johnson
and Johnson, Moderna, and Pfizer-BioNTech. Some national regulators have also

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Wheeler et al.

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FIG 7 Anti-SARS-CoV-2 antibody levels in a vaccinated healthy volunteer with a delayed boosting vaccination. Antibody levels were assessed by using the
Siemens SARS-CoV-2 total assay (A) and the BioPlex 2200 SARS-CoV-2 IgG multiplex panel (B). The results are shown with linear (lower panels) and
logarithmic (upper panels) scales (y axis) in index values (A) and U/ml (B). The x axis indicates the days after the first dose of a vaccine administration. An
index unit of 1 (dotted line) is the negative cutoff concentration for panel A. 10 U/ml (dotted line) is the negative cutoff concentration for panel B.

assessed other COVID-19 vaccine products for use in their countries. All major first-gen-
eration COVID-19 vaccines approved in the United States utilize spike protein coding
for immune response targeting and thus induce RBD, S1, and S2 antibody production.
Interestingly, the Pfizer phase I/II COVID-19 vaccine trial with BNT162b1, an mRNA that
encodes the RBD of the spike protein, showed that a detectable level of RBD-binding
IgG was seen before the second dose, but the peak was 1 week after the booster (31).
For Moderna, there were detectable antibodies by the day of second dose with the
peak on day 15 after second injection (14). The follow-up data for the Moderna mRNA-
1273 vaccine provide immunogenicity data set 90 days after the second vaccination,
showing high levels of RBD binding and neutralizing antibodies that declined slightly
over time, as expected (32). Similarly, blood from six adults receiving the mRNA-based
SARS-CoV-2 vaccines was analyzed for the immune responses to S protein and RBD by
ELISA (16). This study found that immune responses were highest 1 week after the sec-
ond dose of vaccine with a subsequent decline in antibody titers. Neutralizing anti-
body titers displayed a comparable trend for all vaccinees (16). These results were con-
firmed by retrospective analysis of healthy donors’ serologic response to immunization
with the Pfizer BNT162b2 vaccine. All participants demonstrated no S protein (RBD)
antibodies before vaccination and were all positive for S protein antibodies by 2 weeks
after the first vaccine dose. The serum levels of S protein antibodies peaked at 4 to

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Antibody Response to Vaccination

5 weeks following the initial vaccine dose (33). Though correlates of protection against
SARS-CoV-2 infection are not yet widely proven, our results and those of others signify
that even with a slight and expected decrease in the level of circulating binding and
neutralizing antibodies, mRNA vaccines have the ability to deliver lasting humoral
immunity.
Utilizing a new multiplexed antibody detection approach here, we demonstrated
induction of diverse anti-spike antibodies after both the first and the second doses of
spike protein encoding Moderna and Pfizer vaccines, including a strong response to
RBD, which is the major target of neutralizing antibodies in convalescent patients (34).
However, some isolated neutralizing antibodies may exert their function without inter-
fering with RBD-ACE2 recognition or even without binding to RBD (35–37). Indeed,
while RBD-binding antibodies are commonly referred to as “neutralizing” antibodies,
there is not a full correlation between the level of RBD-binding antibodies and the real
neutralizing potential of plasma or serum. For example, it was reported that the con-
cordance between seven different anti-SARS-CoV-2 immunoassays and virus neutrali-
zation tests varied widely (38). Although these observations may suggest that neutrali-
zation potency, as opposed to antibody to epitope specificity, is responsible for the
putative protection of anti-SARS-CoV-2 antibodies (39), these results also advocate that
detection and characterization of immune response to multiple viral protein domains
and antigens is well justified.
In our study, the anti-RBD response was the strongest, followed by the anti-S1 response,
whereas the anti-S2 response was the lowest in all tested individuals. While RBD and S1
antibodies are commonly associated with their ability to neutralize a virus, the clinical sig-
nificance of antibodies recognizing the S2 subunit is less clear. Recently, Voss et al.
reported that more than 80% of the anti-spike IgG repertoire bound to epitopes outside
the RBD, with about 40% of the circulating antibodies targeting the S2 subunit (40).
Protective activity of the neutralizing anti-S2 antibody has been also reported (35, 39, 41).
Thus, our data demonstrate that tested vaccines, in addition to the anti-RBD and anti-S1
responses, are also eliciting low but significant antibodies targeting the S2 subunit, which
are likely providing another layer of protection against the virus. Interestingly, recent char-
acterization of binding and neutralizing antibodies isolated from SARS-CoV-2-infected sub-

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jects revealed that the anti-S2 antibody was the only one that was unaffected by mutations
found in the recently emerged South African variant (39). The authors of that study con-
cluded that these antibodies can serve as blueprints for the development of immunogens
to elicit protective neutralizing antibody responses against multiple coronaviruses. Our
findings suggest that these vaccines elicit polyclonal responses against multiple epitopes,
which may offer enhance protection against variants.
In our study, no differences between Pfizer-induced and Moderna-induced antibody
responses were detected (Fig. 3). This agrees with other work in a study of a cohort of
20 volunteers who received either the Moderna or the Pfizer/BioNTech vaccines. Wang
et al. demonstrated high levels of IgM and IgG anti-SARS-CoV-2 spike protein and RBD
binding titers 8 weeks after the second vaccine injection (18).
We also did not detect significant differences in immune response between males
and females but demonstrated that the initial response to different antigens was age
dependent. However, this correlation was observed only during the relatively weak
response to the first dose and disappeared after boosting of the immune reaction with
a second dose (Fig. 4). These results have been recently confirmed (23). The results of
another study, analyzing the antibody titer 7 days after the second dose of Pfizer
BNT162b2 vaccine in health care workers, revealed that females, lean people, and
young people have an increased capacity to mount humoral immune responses com-
pared to male, overweight, and older populations (42). Although these data confirm
the age dependence of vaccine-induced humoral immune response detected in our
study, we did not see a significant gender-dependent responses in our cohort of
healthy volunteers. Importantly, our study includes only IgG antibody evaluation.
Recent data revealed that in response to BNT162b2, volunteers developed only

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Wheeler et al.

moderate levels of anti-S1 IgA and IgM antibodies after both the first and the second
doses of the vaccine, in contrast to the strong IgG response (23). Similarly, the
BNT162b2 vaccine compared to natural infection induces low anti-S and anti-RBD IgM
and IgA responses but does induce a strong IgG response (43). One possible explana-
tion for the relatively low IgM and IgA responses to the vaccine are the lipid compo-
nents of the vaccine formulation, which are relatively uncharacterized with respect to
their effect on the human immune system. Some early work indicates that the lipid
components may increase Th1-polarized CD41 T cell responses, thus creating early IgG
class-switching that could produce the observed high IgG and low IgA and IgM
responses (43). However, Wang et al. reported high levels of IgM and IgG anti-S and
anti-RBD binding titers in volunteers 8 weeks after the second vaccine injection (18).
Therefore, additional studies of different cohorts of vaccinated people are needed in
order to draw conclusions regarding the significance of polarized antibody responses
after COVID-19 vaccination.
Our data demonstrating a strong antibody response in an individual who experi-
ence a natural disease 9 months earlier (Fig. 6C) are in agreement with the results dem-
onstrating that the antibody response to the first vaccine dose in individuals with pre-
existing immunity is equal to or even exceeds the titers found in naive individuals after
the second dose (17, 44). These authors studied more than 100 vaccine recipients,
;40% of whom were seropositive for SARS-CoV-2 at the time of vaccination. They
found that median antibody titers among seropositive vaccinees after the first dose
were more than 10 times higher than titers among seronegative vaccinees after the
second dose. The fact that after a single dose of COVID-19 vaccine, people with a prior
COVID-19 infection had antibody levels similar to those of people without prior infec-
tion after two vaccine doses suggests the importance of testing anti-SARS-CoV-2 anti-
bodies prior to vaccination in order to prevent immune overboosting and limit poten-
tial adverse effects, as well as to minimize the unnecessary utilization of vaccines in
countries with restricted vaccine availability.
In conclusion, our results suggest that postvaccination testing of multiple antibody
responses is a vital and practicable instrument for following vaccinated people for
selecting individuals who need additional boosting because of low responsiveness or

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might require a third dose of vaccine at an earlier time point or persons who may do
not need second dose due to previous SARS-CoV-2 infection. For instance, measure-
ment of SARS-CoV-2 IgG production in patients with hematological malignancy who
received two mRNA vaccine doses revealed that 46% of these patients did not produce
antibodies and were therefore vaccine nonresponders. Patients with B-cell CLL were at
a particularly high risk, with only 23% having a detectable antibody response even
though almost 70% of the B-cell CLL patients were not concurrently receiving cancer
therapy (45). Alternatively, if a cohort of presumably healthy individuals demonstrates
an unexpectedly low response to vaccination, concerns can be raised regarding a par-
ticular batch of a vaccine. Our and other studies characterizing the clinically testable
response to COVID-19 vaccination pave the way toward developing effective tools to
combat the pandemic.

MATERIALS AND METHODS

Study design and participants. The goal of this prospective panel study was to examine healthy
individuals receiving the Pfizer/BioNTech and Moderna COVID-19 vaccines to characterize the serum lev-
els of antibodies recognizing four virus antigens before and at different time points after the first and
second doses of vaccines. Eligible participants were healthy individuals aged 19 or older. A total of 47
volunteers who received two injections of either vaccine 3 or 4 weeks apart (Pfizer/BioNTech and
Moderna, respectively) participated in the study (Table 1). The key exclusion criteria included an axillary
temperature of more than 37°C, a history of allergy to any vaccine components, anemia and treatment
for anemia or iron deficiency, and immunosuppression whether from disease or treatment. Written
informed consent was obtained from each participant before enrollment. As a control (natural disease),
remnant samples from patients who had SARS-CoV-2 antibody ordered as part of their clinical care or
for convalescent plasma donation were utilized. Written approval of the study protocol and informed
consent form were obtained from the relevant Independent Ethics Committee/Institutional Review
Board (IRB) of the University of Pittsburgh (studies 20040072 and 20120157). The study was conducted

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Antibody Response to Vaccination

in compliance with Good Clinical Practice and all applicable laws and guidelines consistent with ethical
principles of the Declaration of Helsinki.
Blood samples were collected 6 to 72 h prior to the initial vaccine administration, 2 weeks (14 to
16 days) after the first dose of either vaccine, 2 weeks after the second dose, and then monthly at day 45
(42 to 46 days), day 75 (73 to 78 days), 105 (102 to 108 days), and 135 (132 to 138) after the completed
vaccination.
Detection of virus-specific antibodies. Blood samples were collected into BD serum gel separator
tubes and centrifuged after complete clotting at room temperature (1,200  g, 10 min). All specimens
were deidentified and aliquoted for assessment. Serum samples were stored at 230°C for 1 to 3 months
before analysis.
SARS-CoV-2 antibody assays were performed in CLIA certified high-complexity clinical laboratories at
the (University of Pittsburgh Medical Center). For screening and differentiation of the antibody response
to COVID-19 vaccines, we used the BioPlex 2200 CoV-2 IgG Panel (Bio-Rad Laboratories, Inc., Hercules,
CA). The BioPlex 2200 SARS-CoV-2 IgG panel is a multiplex assay for the qualitative (IgG screen) and
semiquantitative (U/ml) detection of IgG class antibodies against the RBD, S1, S2, and nucleocapsid (N)
proteins of the SARS-CoV-2 virus in human serum and plasma. The analytical measuring range was 1 to
100 U/ml, with onboard dilutions of 1:8, 1:16, and 1:32. Samples that displayed any antibody levels
higher than 3,200 U/ml were manually diluted 1:5 or 1:10 using the Bio-Rad dilution solution. The SARS-
CoV-2 IgG calibrator set included five levels of each antibody specificity, with a 4PL calibration curve fit;
for the SARS-CoV-2 IgG quality control, two levels were utilized. Performance studies showed an overall
specificity of 99.8% and an overall sensitivity of 96.3%, according to the manufacturer’s instructions for
use. Evaluation and validation of this assay in our lab confirmed these characteristics (Cook et al., unpub-
lished). Results that are ,10 U/ml are considered negative, and positivity is $10 U/ml.
For the expansion and verification of serum antibody results, we also used an ADVIA Centaur SARS-
CoV-2 total (COV2T) chemiluminescent immunoassay (Siemens USA, Malvern, PA) intended for qualita-
tive detection of total antibodies, including IgG and IgM, to SARS-CoV-2. The COV2T assay on ADVIA
Centaur XP system is a fully automated one-step antigen sandwich immunoassay using acridinium ester
chemiluminescent technology, in which antigens are bridged by antibodies present in the patient sam-
ple. The solid phase contains a preformed complex of streptavidin-coated microparticles and biotinyl-
ated SARS-CoV-2 recombinant S1-RBD antigen. A direct relationship exists between the level of SARS-
CoV-2 antibodies present in the patient sample and the amount of relative light units (RLUs) detected
by the system. A result of reactive or nonreactive is determined according to the index value established
with the high and low calibrators. Within the measuring interval 0.05 to 10.00 index, results were
reported as nonreactive (,1.0 index) or reactive ($1.0 index). Manual dilution of serum samples (1:10 to
1:1,000) was utilized if required. This assay was evaluated in the lab and the results were published (46).
For additional confirmation of antibody results, we used an anti-SARS-CoV-2 ELISA (EuroImmun, NJ)
that provides semiquantitative determination of human IgG antibodies targeting spike protein, which
has been evaluated and verified in our lab previously (46, 47). The assay was run according to the manu-
facturer provided protocol using an iMark microplate absorbance reader (Bio-Rad Laboratories). The ab-

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sorbance of sample wells was measured immediately at 450 and 630 nm, with output reports generated
with the optical density (OD) at 630 nm subtracted from the OD at 450 nm. Data were then analyzed as
recommended by the manufacturer as a ratio based on the sample OD divided by the averaged OD of
the calibrators. This ratio was interpreted as follows: ,0.8, negative; $0.8 to ,1.0, borderline; and $1.1,
positive. Antibody level results are expressed as the “index value.”
Statistical analysis. First, data were compared using t tests on nontransformed and log-transformed
values. Evolution of antibody levels over time was assessed using paired t tests. For a single comparison
of two groups, a Student t test was used after evaluation of normality. If the data distribution was not
normal (Kolmogorov-Smirnov normality test), a Mann-Whitney rank sum test was performed. To com-
pare multiple groups, ANOVA was applied. We used ANOVA to compare log-transformed and nontrans-
formed antibody levels. When comparison of all groups showed a significant difference, we performed
pairwise comparisons. Correlation between tested parameters was done using Pearson correlation.
Hypothesis testing was two-sided, and we considered P values of ,0.05 to be significant.
To analyze variability and central tendencies and summarize immune response profiles after vaccina-
tion, reverse cumulative distribution curves (RCDCs) were generated. These graphic techniques were
used by Salk to display distribution of anti-polio antibodies (48). The method was further developed by
Reed et al. to analyze antibody response in vaccine studies (49). RCDCs are step functions based on the
order statistics of the data. The curves begin with a value of 1.0 or 100% at an antibody titer of virtually
zero and fall to a value of zero above the largest titer value in steps of 1/n. If there are ties, then the step
size is equal to the number of tied values times 1/n (50). We calculated antibody levels and correspond-
ing 95% confidence intervals (CIs) on the basis of standard normal distribution of the log-transformed
antibody concentrations.
We used SigmaPlot (version 14; Systat Software, Inc., San Jose, CA) and Prism (version 9; GraphPad,
San Diego, CA) for all analyses and data presentation. The data are presented as means 6 the standard
errors of the mean (SEM) or medians with a 95% confidence interval (CI), as stated in the corresponding
figure legends or in the text.

ACKNOWLEDGMENTS
We thank Mary Yost, Vincent Maskivish, and Sharon Palmosina for help with blood
collection and preparation of serum samples, and we thank Bio-Rad Laboratories for

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Wheeler et al.

providing key reagents and supporting antibody detection. We also thank all
volunteers participating in the study.
This research received no specific grant from any funding agency in the public,
commercial or not-for-profit sectors.
We confirm that all relevant ethical guidelines have been followed, and all necessary
IRB and/or ethics committee approvals have been obtained. Bio-Rad Laboratories had
no role in study design, data collection, data analysis, data interpretation, or writing of
the report. All authors had full access to all the data in the study and had final
responsibility for the decision to submit for publication.

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