Antibodies to Envelope Glycoprotein of Dengue Virus during the Natural Course of Infection Are Predominantly Cross-Reactive and Recognize Epitopes Containing Highly Conserved Residues at the Fusion Loop of Domain II

ABSTRACT The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.

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Demonstration of marmosets (Callithrix jacchus) as a non-human primate model for secondary dengue virus infection: high levels of viraemia and serotype cross-reactive antibody responses consistent with secondary infection of humans

Journal of General Virology ◽

10.1099/vir.0.060384-0 ◽

2014 ◽

Vol 95 (3) ◽

pp. 591-600 ◽

Cited By ~ 18

Author(s):

Meng Ling Moi ◽

Tomohiko Takasaki ◽

Tsutomu Omatsu ◽

Shinichiro Nakamura ◽

Yuko Katakai ◽

...

Keyword(s):

Dengue Virus ◽

Primary Infection ◽

Secondary Infection ◽

Denv Infection ◽

Callithrix Jacchus ◽

Antibody Responses ◽

Infection Model ◽

Primate Model ◽

Bhk Cells ◽

Denv Serotypes

There are four dengue virus (DENV) serotypes. Primary infection with one does not confer protective immunity against the others. We have reported previously that the marmoset (Callithrix jacchus) is a useful primary DENV infection model. It has been reported that secondary DENV infection with a heterotypic serotype induces viraemia kinetics and antibody responses that differ from those in primary infection. Thus, it is important to determine the utility of the marmoset as a model for secondary DENV infection. Marmosets were infected with heterologous DENV by secondary inoculation, and viraemia kinetics and antibody responses were analysed. The marmosets consistently developed high levels of viraemia after the secondary inoculation with heterologous DENV serotypes. IgM responses were lower compared with primary inoculation responses, whilst IgG responses were rapid and high. Neutralizing activities, which possessed serotype cross-reactive activities, were detected as early as 4 days after inoculation. In addition, infectious viraemia titres were higher when assayed with Fcγ receptor-expressing baby hamster kidney (BHK) cells than when assayed with conventional BHK cells, suggesting the presence of infectious virus–antibody immune complexes. After secondary infection with heterotypic DENV, the marmosets demonstrated viraemia kinetics, IgM and IgG responses, and high levels of serotype cross-reactive neutralizing antibody responses, all of which were consistent with secondary DENV infection in humans. The results indicate the marmoset as a useful animal for studying secondary, as well as primary, DENV infection.

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A Critical Role for Perivascular Cells in Amplifying Vascular Leakage Induced by Dengue Virus Nonstructural Protein 1

mSphere ◽

10.1128/msphere.00258-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Yin P. Cheung ◽

Valeria Mastrullo ◽

Davide Maselli ◽

Teemapron Butsabong ◽

Paolo Madeddu ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Dengue Virus ◽

Nonstructural Protein ◽

Critical Role ◽

Vascular Leakage ◽

World Health ◽

Severe Dengue ◽

Perivascular Cells ◽

Nonstructural Protein 1

ABSTRACT Dengue is the most prevalent arthropod-borne viral disease affecting humans, with severe dengue typified by potentially fatal microvascular leakage and hypovolemic shock. Blood vessels of the microvasculature are composed of a tubular structure of endothelial cells ensheathed by perivascular cells (pericytes). Pericytes support endothelial cell barrier formation and maintenance through paracrine and contact-mediated signaling and are critical to microvascular integrity. Pericyte dysfunction has been linked to vascular leakage in noncommunicable pathologies such as diabetic retinopathy but has never been linked to infection-related vascular leakage. Dengue vascular leakage has been shown to result in part from the direct action of the secreted dengue virus (DENV) nonstructural protein NS1 on endothelial cells. Using primary human vascular cells, we show here that NS1 also causes pericyte dysfunction and that NS1-induced endothelial hyperpermeability is more pronounced in the presence of pericytes. Notably, NS1 specifically disrupted the ability of pericytes to support endothelial cell function in a three-dimensional (3D) microvascular assay, with no effect on pericyte viability or physiology. These effects are mediated at least in part through contact-independent paracrine signals involved in endothelial barrier maintenance by pericytes. We therefore identify a role for pericytes in amplifying NS1-induced microvascular hyperpermeability in severe dengue and thus show that pericytes can play a critical role in the etiology of an infectious vascular leakage syndrome. These findings open new avenues of research for the development of drugs and diagnostic assays for combating infection-induced vascular leakage, such as severe dengue. IMPORTANCE The World Health Organization considers dengue one of the top 10 global public health problems. There is no specific antiviral therapy to treat dengue virus and no way of predicting which patients will develop potentially fatal severe dengue, typified by vascular leakage and circulatory shock. We show here that perivascular cells (pericytes) amplify the vascular leakage-inducing effects of the dengue viral protein NS1 through contact-independent signaling to endothelial cells. While pericytes are known to contribute to noncommunicable vascular leakage, this is the first time these cells have been implicated in the vascular effects of an infectious disease. Our findings could pave the way for new therapies and diagnostics to combat dengue and potentially other infectious vascular leakage syndromes.

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Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Viruses ◽

10.3390/v13071393 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1393

Author(s):

Thanyaporn Dechtawewat ◽

Sittiruk Roytrakul ◽

Yodying Yingchutrakul ◽

Sawanya Charoenlappanit ◽

Bunpote Siridechadilok ◽

...

Keyword(s):

Dengue Virus ◽

Nonstructural Protein ◽

Antiviral Drug ◽

Denv Infection ◽

Site Directed Mutagenesis ◽

Phosphorylation Sites ◽

Post Translational Modification ◽

Multifunctional Protein ◽

Nonstructural Protein 1 ◽

Underlying Mechanisms

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

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Establishment and characterization of dengue virus type 2 nonstructural protein 1 specific T cell lines

Comparative Immunology Microbiology and Infectious Diseases ◽

10.1016/j.cimid.2010.01.002 ◽

2010 ◽

Vol 33 (6) ◽

pp. e75-e80 ◽

Cited By ~ 1

Author(s):

Yang Xiao-meng ◽

Jiang Li-fang ◽

Tang Yun-xia ◽

Yin Yue ◽

Liu Wen-quan ◽

...

Keyword(s):

T Cell ◽

Dengue Virus ◽

Cell Lines ◽

Virus Type ◽

Nonstructural Protein ◽

Nonstructural Protein 1 ◽

T Cell Lines ◽

Dengue Virus Type 2

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Relative contribution of nonstructural protein 1 in dengue pathogenesis

Journal of Experimental Medicine ◽

10.1084/jem.20191548 ◽

2020 ◽

Vol 217 (9) ◽

Cited By ~ 2

Author(s):

Pei Xuan Lee ◽

Donald Heng Rong Ting ◽

Clement Peng Hee Boey ◽

Eunice Tze Xin Tan ◽

Janice Zuo Hui Chia ◽

...

Keyword(s):

Nonstructural Protein ◽

Critical Role ◽

Health Concern ◽

Relative Contribution ◽

Nonstructural Protein 1 ◽

Structural Region ◽

Strain Dependent ◽

Highly Virulent

Dengue is a major public health concern in the tropical and subtropical world, with no effective treatment. The controversial live attenuated virus vaccine Dengvaxia has boosted the pursuit of subunit vaccine approaches, and nonstructural protein 1 (NS1) has recently emerged as a promising candidate. However, we found that NS1 immunization or passive transfer of NS1 antibodies failed to confer protection in symptomatic dengue mouse models using two non–mouse-adapted DENV2 strains that are highly virulent. Exogenous administration of purified NS1 also failed to worsen in vivo vascular leakage in sublethally infected mice. Neither method of NS1 immune neutralization changed the disease outcome of a chimeric strain expressing a vascular leak-potent NS1. Instead, virus chimerization involving the prME structural region indicated that these proteins play a critical role in driving in vivo fitness and virulence of the virus, through induction of key proinflammatory cytokines. This work highlights that the pathogenic role of NS1 is DENV strain dependent, which warrants reevaluation of NS1 as a universal dengue vaccine candidate.

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