Dengue plasmablast responses and their cross-reactivity to Zika virus: a single-cell analysis Open Access

Abstract

Antibodies are critical to viral clearance and protection in dengue virus (DENV) infections. While antibody titers to the infecting serotype are long-lived, cross-protective immunity wanes over time, leaving DENV-exposed individuals susceptible to secondary heterotypic infections. Studies have linked secondary dengue with more severe symptoms, implicating pre-existing immunity as a risk factor for increased disease severity. Previously, we showed that secondary dengue patients mount a massive DENV-specific plasmablast response early after fever onset. The appearance of plasmablasts in blood overlaps with a decrease in viremia but also with the onset of severe clinical symptoms. To better understand how plasmablasts contribute to dengue protection/immunopathology, we isolated plasmablasts from four secondary DENV2-infected patients, and generated monoclonal antibodies (mAbs) by in vitro expression cloning. By focusing on plasmablasts and not memory B cells, we avoided antigen-based cell screening for mAb synthesis, therefore performing unbiased analyses of patient B cell responses. We found that the plasmablasts were largely memory-derived, and that most mAbs were envelope-specific and neutralized multiple DENV serotypes. Although a majority of mAbs neutralized the infecting serotype, DENV2, several mAbs from two patients neutralized DENV1 more potently. This neutralization bias towards a heterologous serotype was reminiscent of original antigenic sin (OAS), and could potentially dampen protective immunity against the infecting virus. In view of the antigenic and structural similarities between DENV and Zika virus (ZIKV), we next examined whether the cross-reactive phenotype of dengue-induced antibodies could extend to ZIKV. Using the same mAbs characterized above, and acute and convalescent sera from additional dengue patients, we conclusively demonstrated immunological cross-reactivity between DENV and ZIKV. All dengue sera bound and neutralized ZIKV, and cross-reactive binding was broad at the mAb level. However, few dengue mAbs also neutralized ZIKV, thus displaying an OAS-like bias towards DENV. ZIKV-reactive dengue sera and mAbs also enhanced ZIKV infection in an FcγR-dependent manner. Overall, our findings suggest that pre-existing, cross-reactive dengue antibodies may modulate protective/pathological immune responses against future heterotypic DENV, or ZIKV infections. Our single-cell approach revealed the vast heterogeneity in the binding, neutralization and enhancement patterns of dengue antibodies, differences that might have remained concealed in serum-based analyses. These data are relevant to future vaccine efforts, given the high propensity of secondary DENV infections, and the overlapping distribution of ZIKV in DENV-endemic regions.

Table of Contents

Table of Contents

Abstract

Table of Contents

List of Figures and Tables

Chapter 1: Introduction ………………………………………………………………………………..……1

Part I: The human antibody response after dengue virus infection …………………………1

DENV classification and virion structure……………………………………………………………….2

DENV reservoir and tissue tropism……………………………………………………………………….5

Clinical presentation of disease…….………………………………………………………………………6

Human immune response to DENV……………………………………………………………………….8

Humoral responses: primary vs. secondary infection…………………………………………..10

Antigenic determinants of dengue antibodies ………………………………………….…………..12

Viral neutralization by antibody ………………………………………………………………………….14

Antibody dependent enhancement ……………………………………………………………………..16

Introduction to Chapter 2…………….……………………………………………………………………..17

Part II: Humoral cross-reactivity between Zika and dengue viruses ..…………….………21

Zika virus emergence and changing clinical features..……………………………….….………21

Viral transmission and tissue tropism..………………………………………………………………..23

Genetic and immunological relatedness between ZIKV and DENV………………………..24

Introduction to Chapter 3…………………………………………………………………………………...25

Figures………………………………………………………………………………………………………………27

References…………………………………………………………………………………………………………35

Chapter 2: B cell responses during secondary dengue infection are dominated by highly cross-reactive, memory-derived plasmablasts……..…………………………49

Abstract………………………………………………………………………………………………………..……50

Importance……………………………………………………………………………………………………..…52

Introduction………………………………………………………………………………………………………53

Materials and Methods………. ………………………………………………………………………………56

Results…………………….……….………………………………………………………………………………..61

Discussion………………………….………………………………………………………………………………67

Figures and figure legends…. ………………………………………………………………………………74

Tables….…………………………………………………………………………………………………………….82

References.….……………………………………………………………………………………………………..85

Chapter 3: Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus ……………………………………………………………………………...90

Abstract….………………………………………………………………………………………………………….91

Introduction…………………...….………………………………………………………………………………92

Significance Statement…. ……………………………………………………………………………………93

Materials and Methods…. ……………………………………………………………………………………96

Results….…………………………………………………………………………………………………………101

Discussion.………………………………………………………………………………………………………107

Figures and figure legends………………………………………………………………………………..113

Tables……………………………………………………………………………………………………………...121

References……………………………………………………………………………………………………….123

Chapter 4: Conclusions …………………………………………………………………………………..127

Part I……………………………………………………………………………………………………………….128

Part II……………………………………………………………………………………………………….…..….136

References……………………………………………………………………………………….………………143

Appendix………………………………………………………………………………………………………..146

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology Biology, Virology
Keyword	Zika virus Antibodies Dengue virus
Committee Chair / Thesis Advisor	Wrammert, Jens, Emory University
Committee Members	Lamb, Tracey, Emory University Suthar, Mehul S, Emory University Moore, Martin L, Emory University Jacob, Joshy, Emory University

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