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Diagnostic Laboratory Immunology

Antibody Responses to Zika Virus Infections in Environments of Flavivirus Endemicity

Sarah L. Keasey, Christine L. Pugh, Stig M. R. Jensen, Jessica L. Smith, Robert D. Hontz, Anna P. Durbin, Dawn M. Dudley, David H. O'Connor, Robert G. Ulrich
Herman F. Staats, Editor
Sarah L. Keasey
aDepartment of Biology, University of Maryland—Baltimore County, Baltimore, Maryland, USA
bMolecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA
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Christine L. Pugh
bMolecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA
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Stig M. R. Jensen
bMolecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA
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Jessica L. Smith
bMolecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA
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Robert D. Hontz
cNaval Medical Research Center, Silver Spring, Maryland, USA, and U.S. Naval Medical Research Unit No. 6 (NAMRU-6), Lima, Peru
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Anna P. Durbin
dCenter for Immunization Research, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
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Dawn M. Dudley
eDepartment of Pathology and Laboratory Medicine, University of Wisconsin—Madison, Madison, Wisconsin, USA
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David H. O'Connor
eDepartment of Pathology and Laboratory Medicine, University of Wisconsin—Madison, Madison, Wisconsin, USA
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Robert G. Ulrich
bMolecular and Translational Sciences Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA
fVirology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA
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Herman F. Staats
Duke University Medical Center
Roles: Editor
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DOI: 10.1128/CVI.00036-17
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  • FIG 1
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    FIG 1

    Phylogenetic relationships and recognition of microarrayed antigens by virus-specific antibody standards. (A) The phylogenies of flaviviruses examined in this study were inferred from an alignment of amino acid sequences from envelope (E) proteins. (B) Microarrays of E, nonstructural protein 1 (NS1), and premembrane (pM) proteins probed with mouse polyclonal antibodies generated against each virus shown (centered labels above each row of bar graphs). Antibody binding data are shown as log10-transformed mean fluorescence intensities (±standard errors of the means [SEM] [error bars]), and the arrows indicate the virus-specific antigens. Heterologous antigens that exhibit increased recognition compared to the virus-specific antigen are labeled with an asterisk (P < 0.05, one-way ANOVA with Tukey's range test). Virus abbreviations: YFV, yellow fever virus; SLEV, St. Louis encephalitis virus; DENV, dengue virus; DENV1, dengue virus serotype 1; POWV, Powassan virus; TBEV-E, tick-borne encephalitis virus, Eastern strain; TBEV-EUR, tick-borne encephalitis virus, European strain; MVEV, Murray Valley encephalitis virus; WNV, West Nile virus; ZIKV, Zika virus; ZIKV-AFR, ZIKV from Africa; ZIKV-AS, ZIKV from Asia; JEV, Japanese encephalitis virus; ROCV, Rocio virus.

  • FIG 2
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    FIG 2

    Specificity and kinetics of the humoral immune response to ZIKV. (A and B) ZIKV-challenged nonhuman primate (NHP) IgG recognition of ZIKV particles harvested early (48 h) or late (144 h) postinfection of HEK293T cells (A) and ZIKV proteins (envelope [E], nonstructural protein 1 [NS1], and premembrane protein [pM]) (B) from five Asian (AS) and six African (AFR) lineages (Table 1). ZIKV-specific antibody responses are denoted by scatter plots with center horizontal lines representing the mean binding of serum antibodies from NHPs challenged with either an AFR (n = 3) (circles) or AS (n = 3) (squares) lineage ZIKV at 0 to 2 days postinfection (dpi) (open symbols) and 21 to 28 dpi (filled symbols). Error bars indicate SEM. Statistically significant differences between mean antibody binding of all ZIKV-challenged NHPs to ZIKV antigens at 0 to 2 dpi and 21 to 28 dpi were calculated using a one-tailed Student's t test (*, P < 7.5e−5; ns, not significant), while no significant differences were observed between mean antibody binding of ZIKV-AS- and ZIKV-AFR-challenged groups to AS and AFR ZIKV antigens at 21 to 28 dpi (two-tailed Student's t test). (C) IgM and IgG binding profiles to ZIKV particles (harvest at 144 h) and ZIKV E protein are compared to viral load (Zika Open-Research Portal [https://zika.labkey.com ]) from preinfection (day 0) to 28 dpi for ZIKV-challenged NHPs (n = 9). Second-order (IgM), third-order (IgG), and fourth-order (viral load) polynomial curves were fitted to the data, with fitted lines and shading under the curve consistent with data point colors.

  • FIG 3
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    FIG 3

    Differentiation of nonhuman primates challenged with ZIKV or DENV by specific IgG binding to E antigens. (A) Binding of convalescent-phase serum antibodies from nonhuman primates (NHPs) challenged with either an Asian (H/PF) (n = 3) (red) or African (MR-766) (n = 3) (royal blue) lineage ZIKV, or DENV (n = 4 each for the DENV1 [black], DENV2 [green], DENV3 [orange] groups; n = 3 for the DENV4 group [magenta]) to whole viruses (144 h) and E proteins. Values shown are antibody binding signals relative to the virus used for challenge (±SEM). (B) Principal-component analyses of relative IgG binding to E proteins and viruses (144 h) by NHP antibodies. Individual data points and virus-specific clusters are colored according to the challenge virus as in panel A. PC1, principal component 1.

  • FIG 4
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    FIG 4

    Antibody specificity of primary and secondary flavivirus infections. Relative binding (±SEM) of convalescent-phase serum antibodies from nonhuman primate (NHP) and human flavivirus infections to 15 flavivirus E proteins is shown. (A) Sera from primary infections are indicated by color as follows: gray, DENV-challenged NHPs (individual data for each NHP group are overlaid in a scatter plot; n = 4 each for the DENV1 [black], DENV2 [green], and DENV3 [orange] groups and n = 3 for the DENV4 group [magenta]); green, human (Hu) rDEN2Δ30 (n = 8) (primary infection); red, pooled African and Asian lineage ZIKV NHPs (n = 6); white, YFV-vaccinated NHPs (n = 3). (B) Sera from confirmed human flaviviral infections with unknown infection histories are indicated by color as follows: gray, DENV (individual data are overlaid in a scatter plot; the colors correspond to the most recent DENV infection); green, DENV2 (n = 5); orange, DENV3 (n = 2); red, ZIKV (n = 4); white, YFV vaccination (n = 13); cyan, WNV (n = 20). (C) Predicted infection histories of human secondary DENV (gray in panel B) and primary ZIKV (red in panel B) infections, based on a supervised SVM classifier. Individual human sera are shown at the bottom (Z for ZIKV, D2 for DENV2, and D3 for DENV3; virus followed by serum identification [ID] number), with probability values for each viral class (left) gradient colored from low to high (white to royal blue) (right). Predicted infection histories are designated by colored bars above serum ID (DENV1 [black], DENV4 [magenta], no prediction [no bar]).

  • FIG 5
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    FIG 5

    Quantitative comparisons of antibodies directed to the infecting virus versus all other flaviviruses. Antibody recognition of microarrayed E proteins displayed as mean fluorescence intensity (±SEM). Antibodies from primary flavivirus infections of NHPs (ZIKV, DENV1 to DENV4, and YFV) and humans (rDEN2Δ30) exhibited significantly decreased recognition of heterologous E antigens compared to virus-specific E (dark gray) (P < 0.05 by one-way ANOVA with Tukey's range test). DENV E proteins are separated from all other flavivirus E proteins (including YFV, SLEV, POWV, TBEV, MVEV, WNV, JEV, and ROCV) to show DENV antibody cross-reactivity between serotypes.

  • FIG 6
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    FIG 6

    Overlap in rising and waning antibody responses to independent infections. The primary infection of a flavivirus-naive individual with dengue virus occurs at day 0 (solid black arrowhead). Levels of virus-specific antibody (gray bars and shading) begin to increase shortly after the acute phase of infection, peak after convalescence, and subside thereafter. A second infection with Zika virus (solid red arrowhead) is followed by an increase in virus-specific antibody (red bars and shading), resulting in detection of a mixture of anti-dengue virus and anti-Zika virus antibodies that will vary with time from infections. The ratio of dengue virus-to-Zika virus antibodies, as shown, will be further increased if the secondary infection results in a less potent activation of serological immune responses.

Tables

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  • TABLE 1

    Flavivirus strains used for production of whole viruses and recombinant proteins

    VirusaIDbIsolateCountryYrHostGenBank accession no.
    ZIKV
        Asian lineage H/PF/2013cFrench Polynesia2013Human KJ776791
    1SV0127/14Thailand2014Human KU681081
    2CPC-0740dPhilippines2012Human KU681082
    3VABC59USA (Puerto Rico)2015Human KU501215
    4SPH2015Brazil2015Human KU321639
    5YAPdMicronesia2007Human EU545988
        African lineage1MR-766c,dUganda1947 Macaca mulatta KU955594
    2IBH30656Nigeria1968Human HQ234500
    3DAKAR41525Senegal1984 Aedes africanus KU955591
    4DAKAR 41662Senegal1984 A. africanus KU955592
    5ARB7701Central Africa1976 A. africanus KF268950
    6ArD_41519Senegal1984 A. africanus HQ234501
    DENV1 HAWAIIUSA1944Human KM204119
    DENV2 NGCNew Guinea1944Human KM204118
    DENV3 H87Philippines1956Human M93130
    DENV4 H241Philippines1956Human AY947539
    WNV NY99USA1999Owl NC_009942
    YFV 17-D-204USA1985Vaccine JX503529
    JEV SA14-14-2South Korea2006Vaccine JN604986
    SLEV PARTONUSA1933Human EF158070
    MVEV 1-51Australia1952Human NC_000943
    ROCV SPH34675Brazil1975Human AY632542
    POWV LBCanada1958Human NC_003687
    TBEV-E SOFJIN-HORussia1937Human AB062064
    TBEV-EUR NEUDOERFLAustria1971 Ixodes ricinus NC_001672
    • ↵a Virus abbreviations: ZIKV, Zika virus; DENV, dengue virus; WNV, West Nile virus; YFV, yellow fever virus; JEV, Japanese encephalitis virus; SLEV, St. Louis encephalitis virus; MVEV, Murray Valley encephalitis virus; ROCV, Rocio virus; POWV, Powassan virus; TBEV-E, tick-borne encephalitis virus, Eastern strain; TBEV-EUR, tick-borne encephalitis virus, European strain.

    • ↵b The identification (ID) number corresponds to the ZIKV strain labels shown in Fig. 2A and B.

    • ↵c Used for challenge of M. mulatta.

    • ↵d Representative antigens are shown in Fig. 2C to 4 and Fig. S2 to S4 (virus for CPC-0740; E protein for YAP).

Additional Files

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    • Supplemental file 1 -

      Fig. S1. Flavivirus disease sera. Fig. S2. The kinetics and magnitude of humoral immune responses to ZIKV by nonhuman primates are not affected by challenge dose. Fig. S3. Antibody-E protein binding patterns are distinct for ZIKV- or DENV-infected humans. Fig. S4. Statistical analysis of antibody specificity of primary and secondary flavivirus infections. Fig. S5. Human and nonhuman primate sera excluded from analysis.

      PDF, 1.0M

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Antibody Responses to Zika Virus Infections in Environments of Flavivirus Endemicity
Sarah L. Keasey, Christine L. Pugh, Stig M. R. Jensen, Jessica L. Smith, Robert D. Hontz, Anna P. Durbin, Dawn M. Dudley, David H. O'Connor, Robert G. Ulrich
Clinical and Vaccine Immunology Apr 2017, 24 (4) e00036-17; DOI: 10.1128/CVI.00036-17

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Antibody Responses to Zika Virus Infections in Environments of Flavivirus Endemicity
Sarah L. Keasey, Christine L. Pugh, Stig M. R. Jensen, Jessica L. Smith, Robert D. Hontz, Anna P. Durbin, Dawn M. Dudley, David H. O'Connor, Robert G. Ulrich
Clinical and Vaccine Immunology Apr 2017, 24 (4) e00036-17; DOI: 10.1128/CVI.00036-17
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    • ABSTRACT
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
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    • REFERENCES
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KEYWORDS

Antibodies, Viral
Antibody Formation
Cross Reactions
Endemic Diseases
Flaviviridae Infections
Zika
cross-reactivity
flavivirus
humoral immunity
protein microarray

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