Letters
RESEARCH LETTER Live-virus focus reduction neutralization tests (FRNTs)
Neutralizing Antibodies Against SARS-CoV-2
Variants After Infection and Vaccination
Serum neutralizing antibodies rapidly appear after SARS-
CoV-2 infection1 and vaccination2 and are maintained for sev-
eral months.3,4 The emergence of SARS-CoV-2 variants has
raised concerns about the
breadth of neutralizing-
Related article page 1898
antibody responses. We com-
pared the neutralizing-anti-
Supplemental content body response to 4 variants in
infected and vaccinated indi-
viduals to determine how mutations within the spike protein
are associated with virus neutralization.
Methods | Serum samples were obtained from 3 groups of
individuals. At Emory University, hospitalized adults with
SARS-CoV-2 infection (polymerase chain reaction con-
firmed) were enrolled 5 to 19 days after symptom onset
(July 2020). Infected convalescent individuals (polymerase
chain reaction or antigen test confirmed) were enrolled 32
to 94 days after symptom onset (March to August 2020).
Deidentified serum samples drawn 14 days after the second
dose (100-μg cohort) from individuals in the mRNA-1273
phase 1 clinical trial2 were obtained from the National Insti-
tutes of Health. See the eAppendix in the Supplement for
participant details. Institutional review board approval was
obtained from Emory University and Advarra; all partici-
pants provided written informed consent.
Four variants were examined, chosen to represent the
original SARS-CoV-2 strain and emerging variants with
mutations in the spike protein. The first variant, nCoV/
USA_WA1/2020 (A.1 lineage), closely resembled the original
Wuhan strain and the spike used in the mRNA-1273 vac-
cine, and was propagated from an infectious SARS-CoV-2
clone. The second variant, EHC-083E (B.1 lineage), contain-
ing a D614G mutation within the spike, was the predomi-
nant circulating strain at the time of the study and was
isolated from a residual nasopharyngeal swab from a patient
in Atlanta, Georgia, in March 2020 (SARS-CoV-2/human/
USA/GA-EHC-083E/2020). The third variant, B.1.1.7 (SARS-
CoV-2/human/USA/CA_CDC_5574/2020), was originally
identified in the UK and of concern because of increased
transmissibility. It contained several spike mutations and
was isolated from a residual nasopharyngeal swab from a
patient in San Diego, California, in December 2020. The
fourth variant, N501Y SARS-CoV-2 virus, containing a muta-
tion in the critical receptor binding domain of the spike that
is present across multiple emerging variants, including the
B.1.1.7 variant in this study, was generated from an infec-
tious clone as previously described.5 This virus is not found
in nature.
1896 JAMA May 11, 2021 Volume 325, Number 18 (Reprinted)
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were performed as previously described.6 See the eAppendix
in the Supplement for details on the laboratory methods.
FRNT50 titers, which represent the reciprocal dilution of se-
rum that neutralizes 50% of the input virus, were interpo-
lated with a 4-parameter nonlinear regression, and geomet-
ric mean titers (GMTs) were calculated with 95% CI in GraphPad
Prism version 8.4.3. Kruskal-Wallis test was used to compare
FRNT50 GMTs between the variants, followed by Dunn’s mul-
tiple comparison post hoc test. We determined P < .05 (2 sided)
to define statistical significance.
Results | Twenty acutely infected COVID-19 patients pro-
vided serum samples (mean age, 56.6 years; 50% men).
The FRNT 50 GMT for the A.1 variant was 186 (95% CI,
90-383); for B.1, 110 (95% CI, 57-209); for B.1.1.7, 116 (95%
CI, 62-215); and for N501Y, 141 (95% CI, 74-269). Comparison
of the FRNT50 GMT of the variants was not statistically sig-
nificant (Figure).
Twenty convalescent individuals provided serum samples
(mean age, 45 years; 55% men). The FRNT50 GMT for the A.1
variant was 168 (95% CI, 113-249); for B.1, 91 (95% CI, 60-138);
for B.1.1.7, 145 (95% CI, 96-220); and for N501Y, 145 (95% CI,
76-172). Comparison of the FRNT50 GMT of the variants was
not statistically significant.
Serum samples were available for 14 mRNA-1273 vacci-
nated individuals2 (age range, 18-55 years; 43% men). The
FRNT 50 GMT for the A.1 variant was 1709 (95% CI, 1412-
2069); for B.1, 804 (95% CI, 632-1023); for B.1.1.7, 965 (95% CI,
695-1341); and for N501Y, 994 (95% CI, 777-1272). Compari-
sons of the FRNT50 GMT of B.1, B.1.1.7, and the N501Y variant
were not statistically significant. The FRNT50 GMTs for the B.1
(P < .001), B.1.1.7 (P = .02), and N501Y (P = .02) variants were
statistically significantly lower than that for the A.1 variant.
Discussion | This study found neutralizing activity of
infection- and vaccine-elicited antibodies against 4 SARS-
CoV-2 variants, including B.1, B.1.1.7, and N501Y. Because
neutralization studies measure the ability of antibodies to
block virus infection, these results suggest that infection-
and vaccine-induced immunity may be retained against the
B.1.1.7 variant. As additional variants emerge, neutralizing-
antibody responses after infection and vaccination should
be monitored.
Limitations include the small sample size, possible selec-
tion bias, lack of clinical outcomes, and how neutralization ti-
ters correlate with protection.
Venkata Viswanadh Edara, PhD
William H. Hudson, PhD
Xuping Xie, PhD
Rafi Ahmed, PhD
Mehul S. Suthar, PhD
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Figure. Neutralizing Antibody Responses Against SARS-CoV-2 Variants
A Infected patients (acute) (n = 20) B Convalescent individuals (n = 20)
104 104
103 103
FRNT50 titer
FRNT50 titer
102 102
101 101
A.1 B.1 B.1.1.7 N501Y
SARS-CoV-2 variants
A, Data from 20 patients with acute COVID-19 infection (5-19 days after
symptom onset). B, Data from 20 convalescent COVID-19 individuals (32-94
days after symptom onset). C, Data from 14 healthy individuals (aged 18-55
years) who received the Moderna (mRNA-1273) vaccine, 100-μg dose, on day 14
(post–second dose). The geometric mean titers (GMTs) with 95% CI are shown
for samples against the A.1, B.1, B.1.1.7, and N501Y variants. The horizontal
dashed lines indicate the limit of detection (FRNT50 GMT = 20). Statistical
significance was determined with the Kruskal-Wallis test to compare GMTs
Author Affiliations: Emory University Department of Pediatrics, Atlanta,
Georgia (Edara, Suthar); Emory Vaccine Center, Atlanta, Georgia (Hudson,
Ahmed); University of Texas Medical Branch, Galveston (Xie).
Corresponding Author: Mehul S. Suthar, PhD, Yerkes National Primate
Research Center, Emory University, 954 Gatewood Rd, Room 2022, Atlanta, GA
30329-4208 (msuthar@emory.edu).
Accepted for Publication: March 8, 2021.
Published Online: March 19, 2021. doi:10.1001/jama.2021.4388
Author Contributions: Dr Suthar had full access to all of the data in the study
and takes responsibility for the integrity of the data and the accuracy of the data
analysis.
Concept and design: Edara, Ahmed, Suthar.
Acquisition, analysis, or interpretation of data: Edara, Hudson, Xie, Suthar.
Drafting of the manuscript: Edara, Suthar.
Critical revision of the manuscript for important intellectual content: All authors.
Statistical analysis: Edara, Hudson, Suthar.
Obtained funding: Suthar.
Administrative, technical, or material support: Xie, Suthar.
Supervision: Edara, Ahmed, Suthar.
Data visualization: Hudson.
Conflict of Interest Disclosures: None reported.
Funding/Support: This work was supported in part by grants NIH P51
OD011132, 3U19AI057266-17S1 CCHI Immune Memory Supplement,
U19AI090023, R01AI127799, R01AI148378, K99AI153736, and UM1AI148684
to Emory University; R00AG049092 and R24AI120942 to the University of
Texas Medical Branch from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health; the Oliver S. and Jennie R. Donaldson
Charitable Trust; the Emory Executive Vice President for Health Affairs Synergy
Fund award; the Pediatric Research Alliance Center for Childhood Infections and
Vaccines and Children’s Healthcare of Atlanta; COVID-Catalyst-I3 Funds from the
Woodruff Health Sciences Center and Emory School of Medicine; Woodruff
Health Sciences Center 2020 COVID-19 CURE Award; and the Vital Projects/
Proteus funds.
Role of the Funder/Sponsor: The funders had no role in the design and
conduct of the study; collection, management, analysis, and interpretation of
the data; preparation, review, or approval of the manuscript; and decision to
submit the manuscript for publication.
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Letters
C Vaccinated individuals (n = 14)
104
103
FRNT50 titer
102
101
A.1 B.1 B.1.1.7 N501Y A.1 B.1 B.1.1.7 N501Y
SARS-CoV-2 variants SARS-CoV-2 variants
between the variants, followed by the Dunn’s multiple comparison post hoc
test. For A (acutely infected patients) and B (convalescent individuals), no
comparisons were statistically significant. For C (vaccinated individuals),
significant differences were found for variant A.1 vs B.1 (P < .001), variant A.1 vs
B.1.1.7 (P = .02), and variant A.1 vs N501Y (P = .02). FRNT50 indicates live-virus
focus reduction neutralization tests with the reciprocal dilution of serum that
neutralizes 50% of the input virus.
Disclaimer: The findings and conclusions in this report are those of the authors
and do not necessarily represent the official position of the Centers for Disease
Control and Prevention.
Additional Contributions: We acknowledge the following individuals for
providing reagents, discussion, and editing of the manuscript: Emory University
School of Medicine, Atlanta, Georgia: Katharine Floyd, BS, Lilin Lai, MD,
Meredith Gardner, PhD, Anne Piantadosi, MD, Jesse J. Waggoner, MD, Ahmed
Babiker, MBBS, David S. Stephens, MD, Evan J. Anderson, MD, Srilatha
Edupuganti, MD, MPH, Nadine Rouphael, MD, MSc, Grace Mantus, MS, Lindsay
Nyhoff, PhD, Jens Wrammert, PhD, Max W. Adelman, MD, MSc, Rebecca
Fineman, BS, Shivan Patel, MD, Rebecca Byram, ME, Dumingu Nipuni Gomes,
MPH, Garett Michael, BS, BA, Hayatu Abdullahi, MD, Erin M. Scherer, PhD, Nour
Beydoun, MD, Bernadine Panganiban, BS, Nina McNair, BS, Kieffer Hellmeister,
BA, Jamila Pitts, BS, Joy Winters, MS, Jennifer Kleinhenz, BS, Jacob Usher, BS,
and James O’Keefe, MD; UC San Diego School of Medicine, California: Louise C.
Laurent, MD, PhD, Peter De Hoff, PhD, Holly Valentine, BA, MPH, Rob Knight,
PhD, Phoebe Seaver, BA, MPH, Gene W. Yeo, PhD, MBA, Shashank Sathe, BTech,
MS, and Aaron Carlin, MD, PhD; The Scripps Research Institute, La Jolla,
California: Kristian G. Andersen, PhD, Mark Zeller, PhD, Karthik Gangavarapu,
BS, Catie Anderson, BA, and Alaa Abdel Latif, BA, MPhil, BS; University of Texas
Medical Branch, Galveston: Kumari Lokugamage, PhD, Vineet Menachery, PhD,
Pei-Yong Shi, PhD; and Centers for Disease Control and Prevention, Atlanta,
Georgia: Natalie Thornburg, PhD, Azaibi Tamin, PhD, Jennifer L. Harcourt, PhD,
Maureen Diaz, PhD, Suxiang Tong, PhD, Ying Tao, PhD, Jing Zhang, PhD, Phili
Wong, MS, Shilpli Jain, PhD, and Jennifer Folster, PhD. No one received financial
compensation for his or her contributions. We thank the mRNA-1273 phase 1
study team and the Division of Microbiology and Infectious Diseases for
providing clinical samples.
1. Suthar MS, Zimmerman MG, Kauffman RC, et al. Rapid generation of
neutralizing antibody responses in COVID-19 patients. Cell Rep Med. 2020;1(3):
100040. doi:10.1016/j.xcrm.2020.100040
2. Jackson LA, Anderson EJ, Rouphael NG, et al; mRNA-1273 Study Group. An
mRNA vaccine against SARS-CoV-2. N Engl J Med. 2020;383(20):1920-1931.
doi:10.1056/NEJMoa2022483
3. Dan JM, Mateus J, Kato Y, et al. Immunological memory to SARS-CoV-2
assessed for up to 8 months after infection. Science. 2021;371(6529):eabf4063.
doi:10.1126/science.abf4063
(Reprinted) JAMA May 11, 2021 Volume 325, Number 18 1897
 Letters
4. Widge AT, Rouphael NG, Jackson LA, et al; mRNA-1273 Study Group.
Durability of responses after SARS-CoV-2 mRNA-1273 vaccination. N Engl J Med.
2021;384(1):80-82. doi:10.1056/NEJMc2032195
5. Liu Y, Liu J, Xia H, et al. Neutralizing activity of BNT162b2-elicited serum:
preliminary report. N Engl J Med. Published online February 17, 2021. doi:10.
1056/NEJMc2102017
6. Vanderheiden A, Edara VV, Floyd K, et al. Development of a rapid focus
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Spike Antibody Levels of Nursing Home Residents
With or Without Prior COVID-19 3 Weeks After
a Single BNT162b2 Vaccine Dose
Recent studies have suggested that, to reach immunity,
immunocompetent SARS-CoV-2 seropositive adults may
only require 1 dose rather than 2 doses of a messenger RNA
vaccine 1,2 ; however, these
studies did not include older
Related article page 1896
adults. Older adults living in
nursing homes are at higher
risk for severe COVID-19, and the immune response to the vac-
cine may differ from that of younger, healthier adults.
We compared IgG antibody levels after a single dose of
BNT162b2 (Pfizer-BioNTech) vaccine in nursing home resi-
dents with or without prior COVID-19.
Methods | Between March and June 2020, we studied resi-
dents from nursing homes in Montpellier, France, facing a
COVID-19 outbreak. 3 As soon as a resident developed
COVID-19, the testing recommendations from the European
Geriatric Medicine Society were followed4 in that all resi-
dents were repeatedly tested using reverse transcriptase–
polymerase chain reaction (RT-PCR) on nasopharyngeal
swabs until no new cases were diagnosed. Participants
provided written informed consent and the study was
approved by the Montpellier University hospital institu-
tional review board.
Six weeks after the end of the outbreak, all residents
underwent blood testing for levels of IgG antibody against
the SARS-CoV-2 nucleocapsid (N) protein. 3 All residents
from 6 nursing homes were offered a first vaccine dose
in January 2021. Three weeks later, all residents underwent
blood testing to quantitatively assess IgG antibody levels
Table. Demographic Characteristics and Seroconversion Level of 96 Residents by COVID-19 Status
During Past 7 to 10 Months
Prior COVID-19
Yes (n = 36)a
Age, mean (SD), y 89.06 (6.69)
Sex
Female 29 (80.5)
Male 7 (19.5)
SARS-CoV-2 IgG level, No. (%)
N protein >0.8 signal 26 (72.2)
to cutoff ratio
S protein >50 AU/mL 36 (100)
S-protein IgG antibody, ≥40 000 (22 801-≥40 000)
median (IQR) [range], AU/mL [588-≥40 000]
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against the SARS-CoV-2 spike (S) protein and N protein.
Levels of IgG antibody against the SARS-CoV-2 receptor-
binding domain were quantified using the SARS-CoV-2
IgG II Quant assay (Abbott Diagnostics). The results were
expressed as arbitrary units (AU) per milliliter (positive
threshold: 50 AU/mL; upper limit: 40 000 AU/mL). The IgG
antibodies against the SARS-CoV-2 N protein were detected
using the SARS-CoV-2 IgG assay (Abbott Diagnostics). The
results were expressed as the signal to cutoff ratio (Abbott
Alinity; Abbott Diagnostics) (positive threshold: 0.8 signal
to cutoff ratio).
In residents with or without a prior history of COVID-19,
we compared IgG antibody levels against SARS-CoV-2 pro-
teins S and N by using 2-sided Wilcoxon Mann-Whitney tests.
The statistical significance threshold was set at 5%. Analyses
were performed using SAS Enterprise Guide version 7.3 (SAS
Institute Inc).
Results | Of the 102 residents, 60 had no prior SARS-CoV-2
infection (COVID-19), 36 had a positive RT-PCR result and
were seropositive for SARS-CoV-2 N-protein IgG in June
2020, and 6 had a positive RT-PCR result or were seroposi-
tive for SARS-CoV-2 N-protein IgG. Of the 36 residents who
had a positive RT-PCR result and were seropositive for
SARS-CoV-2 N-protein IgG in June 2020, 26 remained sero-
positive in January-February 2021 (72.2%).
All 36 residents with prior COVID-19 were seropositive for
S-protein IgG after 1 vaccine dose vs 29 of 60 residents (49.2%)
without prior COVID-19. Among residents with prior COVID-
19, the median level of S-protein IgG was 40 000 AU/mL or
greater (interquartile range [IQR], 22 801-≥40 000 AU/mL) vs
48.0 AU/mL (IQR, 14.0-278.0 AU/mL) in those without prior
COVID-19 (P < .001; Table).
One resident with a positive RT-PCR result in April 2020
tested seronegative for N-protein IgG in June 2020 and
January 2021; the resident had a robust S-protein IgG level
(≥40 000 AU/mL). Five residents were found to be seroposi-
tive for N-protein IgG in June 2020 while having repeated
negative RT-PCR results in April 2020. All 5 of these resi-
dents had high levels of S-protein IgG antibody (median,
≥40 000 AU/mL; IQR, ≥40 000-≥40 000 AU/mL). Among
the 6 residents with a positive RT-PCR result or who were
No (n = 60)b P value
83.91 (8.38) .002
42 (70.0) Abbreviations: AU, arbitrary units;
.25 IQR, interquartile range.
18 (30.0)
a
Positive reverse transcriptase–
polymerase chain reaction (RT-PCR)
0 <.001 result for COVID-19 and seropositive
for N-protein IgG.
29 (49.2) <.001 b
Negative RT-PCR result for
48.0 (14.0-278.0) <.001 COVID-19 and seronegative for
[1-1426] N-protein IgG.
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