of 18
Article
Human antibodies in Mexico and Brazil neutralizing
tick-borne flaviviruses
Graphical abstract
Highlights
d
Sera from Mexico and Brazil neutralize POWV and TBEV,
which are not known to circulate there
d
Monoclonal antibody P014 binds to POWV but not to
endemic flaviviruses
d
Monoclonal antibodies P002 and P003 neutralize POWV
lineage I
d
The P003 epitope is shared across 13 flaviviruses from tick
and mosquito serocomplexes
Authors
Toma
́
s Cervantes Rinco
́
n, Tania Kapoor,
Jennifer R. Keeffe, ..., Luca Varani,
Margaret R. MacDonald,
Davide F. Robbiani
Correspondence
macdonm@rockefeller.edu (M.R.M.),
drobbiani@irb.usi.ch (D.F.R.)
In brief
Cervantes Rinco
́
n et al. describe antibody
neutralizing activity against Powassan
virus from regions of the Americas where
this flavivirus transmitted by ticks is not
known to circulate. Two monoclonal
antibodies to the EDIII (P002 and P003)
are broadly cross-reactive against tick-
and mosquito-borne flaviviruses.
Cervantes Rinco
́
n et al., 2024, Cell Reports
43
, 114298
June 25, 2024
ª
2024 The Author(s). Published by Elsevier Inc.
https://doi.org/10.1016/j.celrep.2024.114298
ll
Article
Human antibodies in Mexico and Brazil
neutralizing tick-borne flaviviruses
Toma
́
s Cervantes Rinco
́
n,
1,18
Tania Kapoor,
2,18
Jennifer R. Keeffe,
3
Luca Simonelli,
1
Hans-Heinrich Hoffmann,
4
Marianna Agudelo,
2
Andrea Jurado,
4
Avery Peace,
4
Yu E. Lee,
3,17
Anna Gazumyan,
2
Francesca Guidetti,
2
Jasmine Cantergiani,
1
Benedetta Cena,
1
Filippo Bianchini,
1
Elia Tamagnini,
1
Simone G. Moro,
1
Pavel Svoboda,
5,6,7,8
Federico Costa,
9,10,11
Mitermayer G. Reis,
10,11,12
Albert I. Ko,
10,11
Brian A. Fallon,
13
Santiago Avila-Rios,
14
Gustavo Reyes-Te
́
ran,
14,15
Charles M. Rice,
4
Michel C. Nussenzweig,
2,16
Pamela J. Bjorkman,
3
Daniel Ruzek,
5,6,7
Luca Varani,
1
Margaret R. MacDonald,
4,19,
*
and Davide F. Robbiani
1,19,20,
*
1
Institute for Research in Biomedicine, Universita
`
della Svizzera italiana, 6500 Bellinzona, Switzerland
2
Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA
3
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
4
Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA
5
Veterinary Research Institute, Brno, Czech Republic
6
Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic
7
Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
8
Department of Pharmacology and Pharmacy, Faculty of Veterinary Medicine, University of Veterinary Sciences, Brno, Czech Republic
9
Institute of Collective Health, Federal University of Bahia, Salvador, BA 40025, Brazil
10
Gonc
̧alo Moniz Institute, Oswaldo Cruz Foundation, Ministry of Health, Salvador, BA 40296, Brazil
11
Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06511, USA
12
Faculty of Medicine of Bahia, Federal University of Bahia, Salvador 40025, Brazil
13
Department of Psychiatry, Columbia University, and New York State Psychiatric Institute, New York, NY 10027, USA
14
National Institute of Respiratory Diseases, Mexico City, CP 14080, Mexico
15
Coordination of the National Institutes of Health and High Specialty Hospitals, Ministry of Health, Mexico City, CP 14610, Mexico
16
Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA
17
Present address: Department of Biology, Stanford University, Stanford, CA 94305, USA
18
These authors contributed equally
19
Senior author
20
Lead contact
*Correspondence:
macdonm@rockefeller.edu
(M.R.M.),
drobbiani@irb.usi.ch
(D.F.R.)
https://doi.org/10.1016/j.celrep.2024.114298
SUMMARY
Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are spread by
mosquitoes and cause human disease and mortality in tropical areas. In contrast, Powassan virus
(POWV), which causes severe neurologic illness, is a flavivirus transmitted by ticks in temperate regions of
the Northern hemisphere. We find serologic neutralizing activity against POWV in individuals living in Mexico
and Brazil. Monoclonal antibodies P002 and P003, which were derived from a resident of Mexico (where
POWV is not reported), neutralize POWV lineage I by recognizing an epitope on the virus envelope domain
III (EDIII) that is shared with a broad range of tick- and mosquito-borne flaviviruses. Our findings raise the pos-
sibility that POWV, or a flavivirus closely related to it, infects humans in the tropics.
INTRODUCTION
Flaviviruses (the term flavivirus is used in the text to refer to mem-
bers of the genus
Orthoflavivirus
) include at least 30 human path-
ogens, which are transmitted by diverse arthropod vectors.
1
,
2
In
contrast to the many mosquito-borne flaviviruses that are
endemic to tropical areas, there is only one tick-borne flavivirus
known to cause human disease in the Americas: Powassan virus
(POWV), which is an emerging concern in the United States and
Canada.
3
Infection by POWV may cause encephalitis, which
can be lethal or lead to persisting neurological symptoms.
No vaccine or specific treatment exists for POWV. Although
there are two lineages of POWV, each with distinct enzootic cy-
cles, serological responses to both lineages are reported to be
indistinguishable.
3
Because flaviviruses are antigenically related, antibodies
induced by one flavivirus may bind to others. This complicates
serology-based diagnostics when multiple flaviviruses co-circu-
late in the same geographical region. Moreover, antibodies gener-
ated during primary infection by one flavivirus can protect against
disease upon secondary infection by the same or a distinct flavivi-
rusbutmayalsoenhancetheseverityofthediseaseinsomecases
due to antibody-dependent enhancement (ADE).
4–6
Tick- and
mosquito-borne flaviviruses are phylogenetically and antigenically
Cell Reports
43
, 114298, June 25, 2024
ª
2024 The Author(s). Published by Elsevier Inc.
1
This is an open access article under the CC BY license (
http://creativecommons.org/licenses/by/4.0/
).
ll
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divergent, leading to their classification in distinct serocomplexes:
POWV is a member of the tick-borne virus complex, which is
distinctfromthe complexes towhichdenguevirus (DENV), Zikavi-
rus (ZIKV), yellow fever virus (YFV), and West Nile virus (WNV)
belong.
7–10
We unexpectedly discovered human antibodies capable of
POWV neutralization from residents of tropical environments in
Mexico and Brazil that are hyperendemic for mosquito-borne fla-
viviruses but where the circulation of POWV or other known tick-
borne flaviviruses that infect humans has not been reported to
date. POWV-neutralizing, human monoclonal antibodies derived
from a resident of Mexico recognize an epitope on the virus en-
velope domain III (EDIII) that is broadly conserved across mos-
quito- and tick-transmitted flaviviruses.
RESULTS
POWV can be transmitted by the same species of tick (
Ixodes
scapularis
)thattransmits
Borrelia
species, including those that
cause Lyme disease.
3
Thus, to examine whether individuals from
the New York metropolitan area might have been exposed
to POWV, we examined by enzyme-linked immunosorbent assay
(ELISA) serum samples from suspected or confirmed Lyme
disease cases (
n
= 556).
11
As controls, we included samples
collected following a DENV outbreak in Salvador, Brazil, and after
a ZIKV outbreak in Santa Maria Mixtequilla, Oaxaca, Mexico (
n
=
49 and
n
=109,respectively;
Figure 1
;see
STAR Methods
). The
level of serum immunoglobulin G (IgG) reactivity to the EDIII of
POWV (lineage II) was significantly higher in the samples from
Mexico and Brazil compared to that of the New York cohort (
Fig-
ure 1
A). A similar result was obtained when measuring the IgG
reactivity to the EDIII of tick-borne encephalitis virus (TBEV), a
tick-borne flavivirus that causes human disease in Europe and
Asia (
Figure 1
B).
12
The samples from Mexico and Brazil were collected within a few
months of mosquito-borne flavivirus outbreaks, when antibody
cross-reactivity is high and specificity of binding is low. Thus, we
examined whether the sera binding to POWV and TBEV in ELISA
were also neutralizing using luciferase-encoding reporter virus
particles (RVPs) expressing the structural proteins of the corre-
sponding flaviviruses (see
STAR Methods
).
13
In comparison to
the samples from New York, significant POWV RVP neutralization
was observed in a fraction of the samples from tropical regions,
particularly those originating from Mexico (
Figure 1
C). The neutral-
ization capacityofTBEVRVPsbythe samesamples was generally
lower than for POWV (
Figure 1
D). To confirm the results of the
screening, we measured the serum antibodies of MEX58, a study
participant from whom we were also able to obtain peripheral
blood mononuclear cells (PBMCs). The amount of IgG binding to
the POWV EDIII was comparable to a sample from an individual
vaccinated against TBEV, and the two samples also bound simi-
larly to the TBEV EDIII (
Figures 1
E and 1F). In contrast, MEX58 dis-
played selective neutralization of POWV RVPs (half-maximal
neutralization titer [NT
50
] = 4.9
3
10
3
)overTBEV(
Figures 1
Gand
1H). Conversely, the TBEV-vaccinated sample was mostly effec-
tive against TBEV RVPs (NT
50
=7.6
3
10
3
;
Figures 1
G and 1H).
The virus-neutralizing capacity of the serum from MEX58 was
confirmed with authentic POWV lineage I and lineage II (
Figure 1
I).
We conclude that individuals living in Mexico and Brazil have sera
that neutralize POWV and that although the antibodies of MEX58
recognize the EDIIIs of both POWV and TBEV flaviviruses, they
are more effective at neutralizing POWV.
To further characterize POWV antibodies, we used a previ-
ously established strategy to identify virus-binding B cells
from the PBMCs of MEX58 (
Figure 2
; see
STAR Methods
).
14
Single IgG-switched memory B cells binding to the fluorescently
labeled POWV EDIII were isolated by flow cytometry, and 31
antibodies were identified by sequencing (
Figures 2
A and
S1
;
Table S1
). Four monoclonal antibodies that were recombinantly
expressed bound to the POWV EDIII in ELISA: two representa-
tive antibodies from an expanded clone with IGHV1-46
paired with IGKV1-39 (P002 and P003) and two singlets (P014
and P020). With regard to cross-reactivity to endemic viruses,
P002, P003, and P020 cross-reacted with the EDIII of ZIKV
(and P002 weakly with DENV1 and DENV3), while P014 only
recognized POWV (
Figure 2
B). Thus, monoclonal antibodies
were obtained that exhibited narrow specificity to a tick-
borne flavivirus (POWV) not known to circulate in the region
(i.e., P014), while others were more broadly cross-reactive to
endemic viruses (i.e., P002, P003, P020).
Strikingly, in the ELISA, P002 and P003 recognized the EDIII
proteins of flaviviruses that can cause human disease and are
transmitted by
Ixodes
ticks (POWV, TBEV, Kyasanur forest dis-
ease virus, Omsk hemorrhagic fever virus, Louping ill virus, Lan-
gat virus), as well as
Aedes
(ZIKV, YFV) and
Culex
(WNV, Japa-
nese encephalitis virus, Murray Valley encephalitis virus, Saint
Louis encephalitis virus, Usutu virus) mosquitoes (
Figures 3
A
and 3B). Thus, P002 and P003 bind to a broad range of flavivirus
envelopes. To assess the neutralizing potential of the antibodies,
we performed plaque reduction neutralization tests. P002 and
P003 neutralized POWV lineage I with a half-maximal inhibitory
concentration of 6.8
m
g/mL. Weak neutralization was detected
for WNV, but not for POWV lineage II, ZIKV, or TBEV, even
upon incubation at 40

C to increase virus breathing to expose
cryptic epitopes (
Figures 3
C and
S2
). Thus, although P002 and
P003 recognize the EDIIIs of all 5 viruses, they selectively
neutralize POWV lineage I and, to a lesser extent, WNV.
To map the P002/P003 epitope, we performed sandwich
ELISAs and structural analysis (
Figure 4
). P002, P003, and previ-
ously characterized anti-ZIKV antibodies that recognize the EDIII
lateral ridge (Z004, Z021, and Z039) and a non-lateral ridge
epitope (Z015)
14
,
15
were immobilized on an ELISA surface, and
the ZIKV EDIII antigen was captured by the bound antibodies.
The binding to the antibody-EDIII complex by His-tagged anti-
body fragment (Fab) of anti-ZIKV or P002 and P003 antibodies
was then evaluated. P002 and P003 recognize similar epitopes
on the ZIKV EDIII that do not overlap with those of Z004, Z021,
Z039, or Z015 (
Figure 4
A). Therefore, P002 and P003 recognize
the same or similar EDIII epitopes as each other, which does not
include the lateral ridge.
Since attempts to crystallize Fab-EDIII complexes were unsuc-
cessful, to uncover the epitope recognized by P002 and P003, we
performed nuclear magnetic resonance (NMR) epitope mapping
(see
STAR Methods
and Simonelli et al.,
17–19
Bardelli et al.,
20
Wang et al.,
21
and Frontzek et al.
22
) on P003 Fab binding to the
ZIKV EDIII. The ZIKV EDIII was used instead of the POWV EDIII
2
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because NMR assignment for the former was available from previ-
ous work.
21
Briefly, the addition of P003 results in changes to the
NMR signal of antigen residues due to the direct contact with the
antibody or due to allosteric effects. The NMR-derived epitope
and cross-competition information was used to validatecomputa-
tional models yielding three-dimensional structures of the P003/
ZIKV EDIII complex. The model in best agreement with the exper-
imentaldatawasfurthersubjectedtofullyatomisticcomputational
moleculardynamicssimulations
21
toincreaseitsprecisionandac-
curacy (
Figures S3
A and S3B). The computational simulations
place P003 and its epitope away from the lateral ridge
(
Figures 4
B and 4C). To further validate the model, we generated
mutant ZIKV EDIII proteins with amino acid changes at surface-
exposed residues within or near the epitope determined by
the NMR data. In agreement with the model, P003 binding to
ZIKV EDIII proteins mutated at positions A361, T366, M374, and
M375, at the core of the epitope, is greatly reduced, whereas pe-
ripheral mutations have a lesser impact (
Figures 4
C, 4D, and
S3
C). These results are also consistent with those from the sand-
wichELISA(
Figure4
A)andthe biolayerinterferometryassays(
Fig-
ure S3
D) and with the crystal structures of the complexes formed
between the EDIII and the Z021- or Z004-related antibody Z006
Figure 1. Serum neutralizing activity against POWV in Mexico and Brazil
(A and B) Screening for serum IgG antibodies binding to the EDIII of POWV (lineage II, A) and TBEV (European subtype, B) in ELISA. Each dot represents the
IgG
reactivity of an individual participant at 1:500 serum dilution (single well). Samples with optical density above 0.6 (dotted lines) were selected f
or the neutralization
screening in (C) and (D).
(C and D) Screening of select sera (
n
= 66) for neutralization of RVPs corresponding to POWV (lineage II, C) and TBEV (European subtype, D). Shown is the rank-
ordered luciferase activity relative to ‘‘no-serum’’ control (lower values correspond to higher neutralization). Each bar represents an individu
al participant sample
analyzed at 1:1,200 dilution; average reading of duplicate wells.
(E and F) ELISA IgG binding to POWV (E) and TBEV (F) EDIIIs by serial serum dilutions. Representative of two experiments.
(G and H) Neutralization of POWV (G) and TBEV (H) RVPs by serial serum dilutions. Luciferase (Luc) activity relative to no-serum control. Mean
±
SD of triplicates.
Representative of two experiments.
(I) Neutralization of authentic POWV lineage I and lineage II by MEX58 serum using a microscopy-based neutralization assay. One experiment, mean
±
SD of
triplicates.
In (A)–(D): black, dark gray, and light gray represent samples from New York, Brazil, and Mexico, respectively. In red is MEX58 and in blue a sample from
a TBEV-
vaccinated individual. The
p
values in (A)–(D) were determined with the Mann-Whitney test.
Cell Reports
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(
Figure 4
E; PDB: 6DFI and 5VIG
14
,
23
). Additional validation of the
model was provided by evaluating POWV lineage I EDIII mutant
proteins (
Figures 4
F, 4G, and
S3
E).
To explain the breadth of P003 reactivity, we aligned the refer-
encesequences of POWV and other flaviviruses at the EDIII region
(
Figure S4
A). Mapping of the P003 contact residues revealed
strong epitope conservation overall (
Figures 5
A–5C). Consistent
with the ELISA results (
Figure 2
B), DENV1–4 were among the
most dissimilar flaviviruses in this analysis. In particular, EDIII res-
idue E329
ZIKV
is negatively charged in POWV and all other viruses
recognized by P003, whereas it loses its negative charge (Q) or
gains a positive charge (R, K) in DENV1 or DENV2, DENV3, and
DENV4, respectively (
Figures 5
A–5C). The analysis was extended
to all flavivirus envelope sequences deposited in the NCBI Virus
database(see
STARMethods
),demonstratinghighsequencesim-
ilarity in correspondence of the P003 epitope (
Figures S4
Band
S4C). Finally, K-to-E substitution at position 321 (K321E
DENV3
,
equivalent to E329
ZIKV
) rescued the binding of P002 and P003 to
the DENV3 EDIII, pointing to a central role of this residue for
P002/P003 binding to all flaviviruses that were assayed except
DENV1–4 (
Figure 5
C). Therefore, P003 recognizes an epitope on
the EDIII that is broadly shared among flaviviruses belonging to
several serocomplexes.
DISCUSSION
Symptomatic POWV infection or Powassan disease has been re-
ported in Canada and the northeast of the United States, where
POWV is present at variable frequencies in ticks (

3.5%).
24–26
While searching for antibodies against POWV, we first evaluated
samples of individuals from the New York metropolitan area with
suspected or confirmed Lyme disease because POWV is trans-
mitted by the same
Ixodes
ticks that also transmit
Borrelia
spe-
cies. To our surprise, the New York sera displayed significantly
lower POWV-binding and -neutralizing activity compared to con-
trol sera from tropical regions of Mexico and Brazil. Central and
South America are hyperendemic for flaviviruses transmitted by
mosquitoes, but to our knowledge, there are no accounts of tick-
borne flaviviruses that infect humans in those regions. Thus, the
discovery of POWV-neutralizing sera and the isolation of POWV-
neutralizing monoclonal antibodies from tropical regions of the
Americas were unexpected.
There are atleast two possibilities to explain the development of
thesePOWVantibodies.Thefirstoneiscross-reactivity.Residents
of tropical regions are recurrently exposed to various DENV sero-
types and ZIKV and are at risk of WNV and YFV infection; they also
receive the YFV vaccine in some regions. Sequential exposure
to different combinations of these mosquito-borne flaviviruses
could, in theory, produce antibodies that cross-react with, and
even neutralize, phylogenetically more distant viruses of the
same genus. This hypothesis clashes with the common view that
serum neutralizing activity is flavivirus specific, which is why sero-
logicassaysthatmeasurevirusneutralizationareconsideredmore
specific for diagnostic purposes.
27
Nonetheless, monoclonal anti-
bodies that broadly cross-react with multiple flaviviruses have
beendescribed,eventhoughtheymorecommonlytargetthevirus
EDII region, which is more conserved at the sequence level, and
notthe EDIIIthatisrecognizedbytheP002and P003antibodies.
28
Reports of monoclonal antibodies that bind to both tick-borne
and mosquito-borne flaviviruses, however, are scarce, which is
Figure 2. Identification of human monoclonal antibodies binding to POWV EDIII from a resident of Mexico
(A) POWV-EDIII binding antibodies from the memory B cells of MEX58. Top, flow cytometry plot identifying human B cells binding to POWV EDIII proteins
corresponding to lineage I and II (gate). The frequency of antigen-binding B cells is shown. Bottom, pie chart showing the distribution of the 31 antib
odies.
Antibodies that share the same immunoglobulin heavy- and light-chain genes are represented by the colored slices, and singlets are in white.
(B) Monoclonal antibody binding to the EDIIIs of the indicated flaviviruses by ELISA. Z039 is a ZIKV antibody that broadly binds the EDIIIs of all four DE
NV
serotypes and was tested alongside as positive control.
14
Average of two independent experiments.
4
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consistent with the increased antigenic distance between flavivi-
ruses belonging to distinct serocomplexes.
29
,
30
We find that
P002 and P003 cross-react with a broad range of flaviviruses
from at least three serocomplexes, including ZIKV (
Aedes
sero-
complex) and WNV (
Culex
serocomplex) but not DENV1–4, which
is the most common flavivirus infection in Mexico and Brazil.
Remarkably, they fail to effectively neutralize WNV or ZIKV but
are effective against POWV instead, a tick-borne flavivirus that
the immune system of the individual from which P002 and P003
were derived has presumably not even encountered.
Analternative possibility isthe circulation inMexicoand Brazilof
POWV or of a flavivirus that is antigenically closely related to it and
that infects humans. A public health conference report from 1962
mentions POWV seroreactivity in the Mexican state of Sonora,
which is a non-tropical area in the north.
31
Some tick-borne flavivi-
ruses, such as Kyasanur forest disease virus, which causes hem-
orrhagic fever in India, are known to infect humans in tropical
areas, but they are not known to be present in the Americas.
32
Nevertheless, ticks, including the
Ixodes
species that are vectors
for POWV in temperate regions, are present in the tropics.
33
Such a flavivirus could easily remain undiscovered if it rarely or
never causes human disease or if the infection causes symptoms
that are indistinguishable from a common circulating virus such as
DENVthatisoftendiagnosedsolelybasedonclinicalpresentation.
The existence of such a virus could have significant implica-
tions. On one hand, circulation of yet another flavivirus in tropical
Americas would further complicate diagnostics. On the other, it
could potentially induce antibodies that alter the disease of sub-
sequent flavivirus infections due to antibody cross-reactivity and
the possibility of disease enhancement (ADE). Further studies
are required to determine the origin of POWV-neutralizing anti-
bodies in individuals in Brazil and Mexico.
Limitations of the study
One limitation of the study is that the infection history of the study
participants as well as their demographic information (age, sex/
gender, ethnicity) were not available or obtained, and thus the in-
fluence (or association) of these factors on the results of the study
is not reported. Furthermore, the samples were collected within a
few months of outbreaks by mosquito-borne flaviviruses (DENV in
Brazil and ZIKV in Mexico): serum antibodies elicited by one flavi-
virus can bind to others (especially shortly after an outbreak),
possibly leading to false positive results in the ELISA. However,
the cross-reactivity of binding is not expected to significantly
influence the ability of the antibodies to neutralize flaviviruses
belonging to distinct serocomplexes.
29
,
34–36
The study cannot
resolve whether the antibodies neutralizing POWV were induced
by a tick-borne flavivirus or by sequential exposure to different
combinations of mosquito-borne flaviviruses but prompts further
investigations that include the analysis of ticks in these regions
of the Americas.
STAR
+
METHODS
Detailed methods are provided in the online version of this paper and include
the following:
d
KEY RESOURCES TABLE
d
RESOURCE AVAILABILITY
B
Lead contact
B
Materials availability
Figure 3. P002 and P003 human monoclonal antibodies are broadly cross-reactive to flaviviruses and neutralize POWV
(A and B) ELISA binding of recombinant human monoclonal IgG antibodies P002 and P003 to the EDIIIs of flaviviruses transmitted by ticks or
Aedes
mosquitoes
(A) and
Culex
mosquitoes (B). Representative of two independent experiments.
(C) Neutralization of POWV lineage I by P002 and P003 antibodies. Neutralization was determined with the plaque reduction neutralization test assay
and values
normalized to no-antibody control. Two experiments (mean
±
SD).
In (A): KFDV, Kyasanur forest disease virus; OHFV, Omsk hemorrhagic fever virus; LIV, Louping ill virus; LGTV, Langat virus. In (B): JEV, Japanese enc
ephalitis
virus; MVEV, Murray Valley encephalitis virus; SLEV, Saint Louis encephalitis virus; USUV, Usutu virus.
Cell Reports
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B
Data and code availability
d
EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
B
Viruses
B
Cell lines
B
Human samples
d
METHOD DETAILS
B
Reagents
B
ELISA assays
B
Sandwich ELISA
B
Neutralization of luciferase-encoding RVPs
Figure 4. P003 recognizes an epitope away from the EDIII lateral ridge
(A) Sandwich ELISA shows that P002 and P003 recognize an EDIII epitope distinct from previously reported ZIKV antibodies. His-tagged Fabs were asses
sed for
binding to the ZIKV EDIII immobilized by a specific IgG. Binding of the Fab suggests a non-overlapping epitope with the IgG; no binding suggests a simila
ror
overlapping epitope with the IgG. Mean
±
SD of quadruplicates.
(B) Model of the P003-ZIKV EDIII complex from NMR-validated computational simulations. EDIII residues affected by antibody binding are in green on t
he surface
of EDIII (PDB: 5OMZ). P003 is the orange cartoon, with the light chain in lighter tones. See also
Figures S3
A and S3B.
(C) Structure of ZIKV EDIII (white) with selected amino acids colored according to the half-maximal effective concentration (EC
50
) value of P003 binding to the
EDIII with the amino acid change shown in (D). Contours indicate the P003 epitope.
(D) Binding of P003 and P002 to ZIKV EDIII mutated at selected epitope residues assessed by ELISA. Summary of EC
50
binding values. Z015 is a ZIKV antibody
binding to a distinct epitope. See also (C) and
Figure S3
C.
(E) P003 (orange) binds to a different EDIII region than Z021 (blue) or Z006 (pink).
(F and G) Same as in (C) and (D) for validation of the model using POWV (lineage I) EDIII wild-type and mutant proteins. T036 is a TBEV antibody that also bi
nds
POWV
16
and recognizes a distinct epitope. See also
Figure S3
E.
6
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B
Plaque reduction neutralization test (PRNT)
B
Microscopy-based neutralization assay
B
Isolation of virus-specific B cells
B
Antibody sequencing, cloning and production
B
Biolayer interferometry
B
NMR spectroscopy
B
Antibody modeling, docking and MD simulations
B
EDIII sequence analysis
d
QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at
https://doi.org/10.1016/j.
celrep.2024.114298
.
ACKNOWLEDGMENTS
We thank all study participants as well as the clinical and research teams at
Columbia University (New York), The Oswaldo Cruz Foundation (Brazil), and
the National Institute of Respiratory Diseases (Mexico). We thank Ted Pierson
(NIH) for providing the plasmids pZIKV-HPF-CprME and pWNVII-Rep-REN-IB,
Pei Yong Shi for providing pFLWNV, and Aaron Brault (CDC) for providing the
DTV 2-plasmid infectious clone system prior to publication. We thank Andrea
Celoria for technical assistance, William Schneider for assistance in viral RNA
sequencing, and Kristie Gordon for fluorescence-activated cell sorting. We
thank Alison Ashbrook for POWV stock genome copy determinations and Tyler
Lewy for providing the WNV NY99 virus stock. The graphical abstract was pre-
pared with BioRender. This work was supported in part by the Swiss Vaccine
Research Institute (SVRI) and by a Czech-Swiss collaborative project funded
jointly by the Czech Science Foundation (no. GF21-05445L to D.R.) and the
Figure 5. The P003 epitope on the EDIII is highly conserved across flaviviruses except for DENV
(A) Alignment of the P003 contact residues with the sequences of the EDIII proteins used in ELISA (
Figures 2
and
3
). Highlighted in green are the identical residues
and in yellow those that are similar by side-chain functionality to the corresponding residues in POWV. The arrow points to the P003 epitope at positio
n 329 (ZIKV
numbering). Related to
Figure S4
A.
(B) Coldmaps representing on the ZIKV EDIII the frequency of identical or similar residues at the P003 epitope (dashed contour) across the flaviviruse
s recognized
by P003 and indicated in (A).
(C) Rescue of P002 and P003 binding by the K321E substitution in DENV3 (equivalent to E329
ZIKV
). Average of two independent ELISA experiments.
Cell Reports
43
, 114298, June 25, 2024
7
Article
ll
OPEN ACCESS
Swiss National Science Foundation (no. 310030L_196866 to D.F.R.) and NIH
grants U01 AI151698 (United World Antiviral Research Network [UWARN] to
M.C.N. and D.F.R.); P01 AI138938 and U19 AI111825 (to C.M.R., M.C.N.,
P.J.B., D.F.R.); R01 AI052473, R01 TW009504, and R01 AI174105 (to A.I.K.);
and R21 AI142010 (to M.R.M.). The study was also possible thanks to the
IRB-Rockefeller University Stavros Niarchos Foundation Partnership for
Global Infectious Disease Research supported by the Fondazione Leonardo
and was supported in part by a pilot project award (to M.R.M) through grant
no. UL1TR001866 from the National Center for Advancing Translational Sci-
ences (NCATS, NIH Clinical and Translational Science Award [CTSA] pro-
gram), and the Mexican government (Programa Presupuestal P016; Anexo
13 del Decreto del Presupuesto de Egresos de la Federacio
́
n). M.C.N. is a Ho-
ward Hughes Medical Institute (HHMI) Investigator. This article is subject to
HHMI’s open access to publications policy. HHMI lab heads have previously
granted a non-exclusive CC BY 4.0 license to the public and a sublicensable
license to HHMI in their research articles. Pursuant to those licenses, the
author-accepted manuscript of this article can be made freely available under
a CC BY 4.0 license immediately upon publication.
AUTHOR CONTRIBUTIONS
T.C.R., T.K., J.R.K., L.S., H.-H.H., M.A., A.J., A.P., Y.E.L., A.G., F.G., J.C.,
B.C., F.B., E.T., and P.S. produced reagents, performed experiments, and
analyzed the data; L.S. and S.G.M. performed computational structural and
sequence analyses; M.G.R., F.C., A.I.K., B.A.F., S.A.-R., and G.R.-T. provided
human samples; C.M.R., M.C.N., P.J.B., D.R., and L.V. supervised portions of
the work; J.R.K., M.R.M., and D.F.R. conceived the study, coordinated and su-
pervised the execution, analyzed the data, and wrote the manuscript, with
contributions by the other authors.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: November 9, 2023
Revised: April 11, 2024
Accepted: May 14, 2024
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STAR
+
METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
Anti-IgG (human) Sheep Polyclonal
Antibody HRP
Cytiva
Cat. No: NA933; RRID:AB_772208
Human recombinant antibody 10-1074
Mouquet et al.
37
N/A
Human recombinant antibody Z004, Z015,
Z021, Z039
Robbiani et al.
14
N/A
Human recombinant antibody T036
Agudelo et al.
16
N/A
Human recombinant antibody hr2.023
Bianchini et al.
38
N/A
PE-Cy
TM
7 Mouse anti-human CD20
BD Biosciences
Cat. No: 560735; RRID:AB_1727450
APC Mouse anti-human IgG
BD Biosciences
Cat. No: 562025; RRID:AB_10892809
THE
TM
His Tag HRP-conjugated
Genscript
Cat. No: A00612; RRID:AB_915573
Bacterial and virus strains
E.coli
BL21(DE3)
New England Biolabs
Cat No: 2527I
POWV (lineage I); Byers strain
CDC
N/A
POWV (lineage II); Spooner strain
CDC
N/A
TBEV; Hypr strain
Collection of Arboviruses, Institute of
Parasitology, Biology Center of the
Czech Academy of Sciences, Cask
Budejovice, Czech Republic
N/A
WNV; NY99 strain
Shi et al.
39
ZIKV; PRVABC59 strain
CDC
N/A
Chemicals, peptides, and recombinant proteins
Albumin (BSA) Fraction V (pH 7.0)
PanReac AppliChem
Cat. No : A1391
Avicel
FMC BioPolymer
Type RC-581; CAS#9004-34-6
Carboxymethylcellulose
Sigma-Aldrich
Cat. No : C4888
Dimethylsulfoxide (DMSO)
Sigma-Aldrich
Cat. No: 41640
DMEM, high glucose, GlutaMAX
TM
Themo Fisher Scientific
Cat. No: 61965-026
Dulbecco’s Phosphate Buffered Saline
(PBS)
Sigma-Aldrich
Cat. No: D8537
EDTA solution pH 8.0 (0.5M)
PanReac AppliChem
Cat. No: A3145
Fetal Bovine Serum (FBS)
Gibco
Cat. No: A5256701
Formaldehyde
Fisher
Cat. No: F79-1
L-glutamine
Sigma-Aldrich
Cat. No: G7513
Leibovitz (L-15)
Biosera
Cat. No: LM-L1050
Medium 199
Gibco
Cat. No: 11150059
MEM w/ Earles Salts w/ Glutamine
Biowest
Cat. No: L0415
Methylcellulose
Fisher
Cat. No: M352-500
Naphthalene black solution
Sigma-Aldrich
Cat. No: 195243
Octet 1M Ethanolamine
Sartorius
Cat. No: 18-1071
Opti-MEM
Gibco
Cat. No: 31985062
Pierce
TM
TMB Substrate Kit
Themo Fisher Scientific
Cat. No: 34021
Streptavidin APC Conjugate
Thermo Fisher Scientific
Cat. No: 17-4317-82
Streptavidin PE Conjugate
Thermo Fisher Scientific
Cat. No: 12-4317-87
Sulfuric Acid
Sigma-Aldrich
Cat. No: 258105
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Cell Reports
43
, 114298, June 25, 2024
11
Article
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