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, 14 (2), e0008034
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Impact of Flavivirus Vaccine-Induced Immunity on Primary Zika Virus Antibody Response in Humans

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Impact of Flavivirus Vaccine-Induced Immunity on Primary Zika Virus Antibody Response in Humans

Stefan Malafa et al. PLoS Negl Trop Dis.

Abstract

Background: Zika virus has recently spread to South- and Central America, causing congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune due to prior infections with other flaviviruses (e.g. dengue virus) or flavivirus vaccinations. Since pre-existing cross-reactive immunity can potentially modulate antibody responses to Zika virus infection and may affect the outcome of disease, we analyzed fine-specificity as well as virus-neutralizing and infection-enhancing activities of antibodies induced by a primary Zika virus infection in flavivirus-naïve as well as yellow fever- and/or tick-borne encephalitis-vaccinated individuals.

Methodology: Antibodies in sera from convalescent Zika patients with and without vaccine-induced immunity were assessed by ELISA with respect to Zika virus-specificity and flavivirus cross-reactivity. Functional analyses included virus neutralization and infection-enhancement. The contribution of IgM and cross-reactive antibodies to these properties was determined by depletion experiments.

Principal findings: Pre-existing flavivirus immunity had a strong influence on the antibody response in primary Zika virus infections, resulting in higher titers of broadly flavivirus cross-reactive antibodies and slightly lower levels of Zika virus-specific IgM. Antibody-dependent enhancement (ADE) of Zika virus was mediated by sub-neutralizing concentrations of specific IgG but not by cross-reactive antibodies. This effect was potently counteracted by the presence of neutralizing IgM. Broadly cross-reactive antibodies were able to both neutralize and enhance infection of dengue virus but not Zika virus, indicating a different exposure of conserved sequence elements in the two viruses.

Conclusions: Our data point to an important role of flavivirus-specific IgM during the transient early stages of infection, by contributing substantially to neutralization and by counteracting ADE. In addition, our results highlight structural differences between strains of Zika and dengue viruses that are used for analyzing infection-enhancement by cross-reactive antibodies. These findings underscore the possible impact of specific antibody patterns on flavivirus disease and vaccination efficacy.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Structure of Zika virus and antigenic relationships of flaviviruses.
(A) Zika virus particle (PDB: 6CO8, [22]). (B) Distance relationships between flavivirus E proteins based on amino acid sequence differences. (C) Ribbon diagram of the Zika virus E dimer (side view) and (D) surface representation of the Zika virus E dimer (top view). Color code: domain I (DI), red; domain II (DII), yellow; domain III (DIII), blue; stem and transmembrane domains, gray; fusion loop (FL), green. Figures were prepared with Pymol (Schrödinger LLC, www.pymol.org) and Chimera (http://www.rbvi.ucsf.edu/chimera, [23]). Sequences of E were obtained from the ViPR data bank (https://www.viprbrc.org). GenBank accession numbers are as follows: U27495 for tick-borne encephalitis virus, AY640589 for yellow fever virus, DQ211652for West Nile virus, D90194 for Japanese encephalitis virus, KJ776791 for Zika virus, AF226687 for dengue 1 virus, DQ863638 for dengue 3 virus, M29095 for dengue 2 virus, GQ398256 for dengue 4 virus, and NC_003675 for Rio Bravo virus.
Fig 2
Fig 2. Zika virus-specific and broadly cross-reactive antibody titers of four serum pools (colored columns) and the individual serum samples of these pools (black circles).
The mean values of individual sera are shown as red open circles. (A) Zika virus IgM ELISA units, (B) Rio Bravo virus IgM ELISA absorbance values (cross-reactive antibodies), (C) Zika virus IgG ELISA titers, (D) Rio Bravo virus IgG ELISA titers (cross-reactive antibodies), (E) relative Zika virus IgG avidities (%), (F) Zika virus NT50 titers. The cut-off is indicated by a dotted line. Mean values were calculated from at least three independent experiments except panel B (two independent experiments). Error bars represent the standard error of the mean (SEM; A, C-F) or the range (B). The statistics shown in the figure refer to the pool data. Statistical comparisons of pool data and mean values of individual sera are given in S1 Table. (A, C, D, F): One-way ANOVA followed by Dunnett´s test, (E): General linear model and Wald’s test. Significant differences between the naïve pool and the pre-immune pools are indicated; *, p < 0.05; **, p < 0.01; ***, p < 0.001. ZIKV, Zika virus; RBV, Rio Bravo virus; naïve, flavivirus naïve serum pool; YF+, yellow fever pre-vaccinated serum pool; TBE+, tick-borne encephalitis pre-vaccinated serum pool; YF+TBE+: yellow fever and tick-borne encephalitis pre-vaccinated serum pool.
Fig 3
Fig 3. Zika virus neutralization NT50 titers by IgM after IgG depletion from serum pools.
Colored columns: mock depleted pools containing IgM and IgG; empty columns: IgM only after IgG-depletion. NT titers are shown as mean values +/- SEM from three independent experiments. Numbers in the empty columns represent the means of percent IgM-mediated neutralizing activity (IgM NT) derived from three independent experiments. The dotted line indicates the cut-off of the assay. Asterisks indicate significant differences between the NT50 titers of mock and IgG-depleted pools (2-way ANOVA with linear contrasts); *, p < 0.05; **, p < 0.01. ZIKV, Zika virus; naïve, flavivirus naïve serum pool; YF+, yellow fever pre-vaccinated serum pool; TBE+, tick-borne encephalitis pre-vaccinated serum pool; YF+TBE+: yellow fever and tick-borne encephalitis pre-vaccinated serum pool; mock dep, mock-depleted serum pool; IgG dep, IgG-depleted serum pool.
Fig 4
Fig 4. Effect of IgM depletion from serum pools on ADE of Zika virus infection.
Serum pools were depleted with IgM agarose beads as described in Methods. Fcγ receptor-positive K562 cells were infected with Zika virus in the presence of serially diluted (A) flavivirus-naïve pool, (B) YF pre-vaccinated pool, (C) TBE pre-vaccinated pool, and (D) YF and TBE pre-vaccinated pool. Colored symbols: mock depletion, empty symbols: IgM depletion. Data are shown as mean values +/- range from two independent experiments. Dotted line indicates no enhancement of infection. Comparison of fold-enhancement of Zika virus infection in the presence of depleted and mock-depleted pools was performed by applying a GLM as described in Methods. p-values are indicated in the graphs. ZIKV, Zika virus; naïve, flavivirus naïve serum pool; YF+, yellow fever pre-vaccinated serum pool; TBE+, tick-borne encephalitis pre-vaccinated serum pool; YF+TBE+: yellow fever and tick-borne encephalitis pre-vaccinated serum pool; mock dep, mock-depleted serum pool; IgM dep, IgM-depleted serum pool.
Fig 5
Fig 5. Effect of IgM and IgG depletion on ADE of Zika virus infection.
The four serum pools were depleted from IgM or IgG as described in Methods. Fcγ receptor-positive K562 cells were infected with Zika virus in the presence of a 1:1000 dilution of each pool, either non-depleted (non dep), IgM depleted (IgM dep), IgG depleted (IgG dep) or a reconstituted sample, i.e. a mixture of the IgM and IgG-depleted pools (mixt). Data are shown as mean values +/- SEM from three independent experiments. Dotted line indicates no enhancement of infection. Statistical comparisons (with the original and reconstituted samples) were performed by applying a GLM with linear contrasts and a Bonferroni-Holm adjustment and are indicated at the top of the figure. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Naïve, flavivirus naïve serum pool; YF+, yellow fever pre-vaccinated serum pool; TBE+, tick-borne encephalitis pre-vaccinated serum pool; YF+TBE+: yellow fever and tick-borne encephalitis pre-vaccinated serum pool.
Fig 6
Fig 6. Effect of depletion of cross-reactive antibodies from serum pools with Rio Bravo virus E on Zika virus antibody titers.
(A) Zika virus IgG ELISA titers, (B) relative avidity of Zika virus-specific IgG, and (C) Zika virus NT50 titers. Colored columns: mock depletion, empty columns: RB depletion. Percentages in the empty columns of (A) and (C) indicate residual ELISA reactivity and neutralizing activity after depletion, respectively. Data are shown as mean values +/- SEM from three independent experiments. The dotted line indicates the cut-off of the assay. Significant differences between mock depletion and RB depletion are indicated by asterisks (2-way ANOVA with linear contrasts); *, p < 0.05; **, p < 0.01; ***, p < 0.001. ZIKV, Zika virus; mock dep, IgM and mock-depleted serum pool; RB dep, IgM and Rio Bravo virus E-depleted serum pool.
Fig 7
Fig 7. Effect of depletion of cross-reactive antibodies from serum pools with Rio Bravo virus E on ADE of Zika virus infection.
Fcγ receptor-positive K562 cells were infected with Zika virus in the presence of serially diluted (A) flavivirus-naïve pool, (B) YF pre-vaccinated pool, (C) TBE pre-vaccinated pool, and (D) YF and TBE pre-vaccinated pool. IgG-containing serum pools were further depleted with RB virus E protein as described in Methods. Colored symbols: mock depletion, empty symbols: RB depletion. Data are shown as mean values +/- SEM from three independent experiments. Dotted line indicates no enhancement of infection. Comparison of fold-enhancement of Zika virus infection in the presence of depleted and mock-depleted pools was performed by applying a general linear model as described in Methods. p-values are indicated in the graphs. ZIKV, Zika virus; mock dep, IgM and mock-depleted serum pool; RB dep, IgM and Rio Bravo virus E-depleted serum pool.
Fig 8
Fig 8. Effect of depletion of cross-reactive antibodies from serum pools with Rio Bravo virus E on TBE and dengue virus neutralization.
(A) TBE virus NT. (B) Dengue virus NT. IgG-containing serum pools were further depleted with RB virus E protein as described in Methods. Colored columns: mock depletion, empty columns: RB depletion. The dotted line indicates the cut-off (c.o.) of the assay. Mean NT50 titers were calculated from two (A) or three (B) independent experiments. Error bars represent the range (A) or standard error of the mean (B). Significant differences between mock depletion and RB depletion are indicated by asterisks (2-way ANOVA with linear contrasts); ***, p < 0.001. TBEV, tick-borne encephalitis virus; DENV1, dengue virus serotype 1; mock dep, IgM and mock-depleted serum pool; RB dep, IgM and Rio Bravo virus E-depleted serum pool.
Fig 9
Fig 9. Effect of depletion of cross-reactive antibodies from serum pools with Rio Bravo E on ADE of dengue virus infection.
Fcγ receptor-positive K562 cells were infected with dengue virus in the presence of serially diluted (A) flavivirus-naïve pool, (B) YF pre-vaccinated pool, (C) TBE pre-vaccinated pool, and (D) YF and TBE pre-vaccinated pool. IgG-containing serum pools were further depleted with RB E protein as described in Methods. Colored symbols: mock depletion, empty symbols: RB depletion. Data are shown as mean values +/- SEM from three independent experiments. Dotted line indicates no enhancement of infection. Comparison of fold-enhancement of dengue virus infection in the presence of depleted and mock-depleted pools was performed by applying a general linear model as described in Methods. p-values are indicated in the graphs. DENV2, dengue virus serotype 2; mock dep, IgM and mock-depleted serum pool; RB dep, IgM and Rio Bravo virus E-depleted serum pool.
Fig 10
Fig 10. Quantification of Zika virus-specific IgM and IgG antibodies and broadly cross-reactive IgG antibodies using single serum samples from Zika cases.
(A) Zika virus IgM ELISA units, (B) Zika virus IgG ELISA titers, and (C) Rio Bravo virus IgG ELISA titers of individual samples are plotted against days after disease onset. Data points represent means from two to three (A) or three (B, C) independent experiments. Flavivirus pre-immune patients are color-coded in blue, flavivirus naïve patients in red. Dotted lines indicate the cut-off of each assay. The significance of differences between the extents of naïve and pre-immune antibody responses were calculated based on the maxima reached, applying a general linear model as described in Methods. p-values are indicated in the graphs. ZIKV, Zika virus; RBV, Rio Bravo virus.
Fig 11
Fig 11. Zika and dengue virus neutralization tests using single serum samples from Zika cases.
(A) Zika virus and (B) dengue virus NT50 titers from individual serum samples were plotted against days after disease onset. Data points represent mean NT50 titers from at least three independent experiments. Color code as in Fig 10. Dotted lines indicate the cut-off of each assay. The significance of differences between the extents of naïve and pre-immune antibody responses were calculated based on the maxima reached, applying a general linear model as described in Methods. p-values are indicated in the graphs. ZIKV, Zika virus; DENV1, dengue virus serotype 1.

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Grant support

The study has been funded by the Austrian Science Fund FWF (https://www.fwf.ac.at; grant P27501-B21 to FXH; grant P29928-B30 to KS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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