Dengue virus immune protection is specific to the serotype encountered and is thought to persist throughout one’s lifetime. Many serotype cross-reactive memory B cells isolated from humans with previous dengue infection are specific for the nonstructural and the prM structural viral proteins, and they can enhance infection in vitro. However, plasmablasts circulating in enormous numbers during acute secondary infection have not been studied. In this study, we analyzed single plasmablasts from two patients by sorting the cells for Ig sequence analysis and for recombinant expression of Abs. In contrast to memory B cells, most plasmablast-derived Abs bound to the structural E protein of dengue, and protection experiments in mice revealed that virus serotypes encountered during past infections were neutralized more efficiently than were the serotypes of the current infection. Together with genetic analyses, we show evidence that plasmablasts in dengue patients are a polyclonal pool of activated E protein–specific memory B cells and that their specificity is not representative of the serum Abs secreted by long-lived plasma cells in the memory phase. These results contribute to the understanding of the phenomenon of original antigenic sin in dengue.

A therapy for dengue is still elusive. We describe the neutralizing and protective capacity of a dengue serotype-cross-reactive antibody isolated from the plasmablasts of a patient. Antibody SIgN-3C neutralized all four dengue virus serotypes at nano to picomolar concentrations and significantly decreased viremia of all serotypes in adult mice when given 2 days after infection. Moreover, mice were protected from pathology and death from a lethal dengue virus-2 infection. To avoid potential Fc-mediated uptake of immune complexes and ensuing enhanced infection, we introduced a LALA mutation in the Fc part. SIgN-3C-LALA was as efficient as the non-modified antibody in neutralizing dengue virus and in protecting mice while antibody-dependent enhancement was completely abrogated. The epitope of the antibody includes conserved amino acids in all three domains of the glycoprotein, which can explain its cross-reactivity. SIgN-3C-LALA neutralizes dengue virus both pre and post-attachment to host cells. These attributes likely contribute to the remarkable protective capacity of SIgN-3C.

Dengue is endemic in tropical countries worldwide and the four dengue virus serotypes often co-circulate. Infection with one serotype results in high titers of cross-reactive antibodies produced by plasmablasts, protecting temporarily against all serotypes, but impairing protective immunity in subsequent infections. To understand the development of these plasmablasts, we analyzed virus-specific B cell properties in patients during acute disease and at convalescence. Plasmablasts were unrelated to classical memory cells expanding in the blood during early recovery. We propose that only a small subset of memory B cells is activated as plasmablasts during repeat infection and that plasmablast responses are not representative of the memory B cell repertoire after dengue infection.

BackgroundThe human leukocyte antigen alleles have been implicated as probable genetic markers in predicting the susceptibility and/or protection to severe manifestations of dengue virus (DENV) infection. In this present study, we aimed to investigate for the first time, the genotype variants of HLA Class 1(-A and -B) of DENV infected patients against healthy individuals in Malaysia.Methodology/Principal FindingsThis study was carried out with 92 dengue disease patients and 95 healthy controls from three different ethnic groups (Malay, Chinese and Indian) in Malaysia. All patients with clinical and laboratory confirmation of DENV infection were typed for the HLA-A and B loci, using polymerase chain reaction-sequence specific primer techniques. In our total population, a significant increase for HLA-B*53 (P = 0.042, Pc = 1.008) allele and a significant decrease for A*03 (P = 0.015, Pc = 0.18, OR = 5.23, 95% CI = 1.19–23.02) and B*18 (P = 0.017, Pc = 0.408) alleles were noted in DHF patients as compared to healthy donors. We also observed that in the Malay DHF patients, allele B*13 (P = 0.049, Pc = 1.176, OR = 0.18, 95% CI = 0.03–0.90) was present at a significantly higher frequency in this population while allele HLA-B*18 (P = 0.024, Pc = 0.576) was seen to be negatively associated with DHF.Conclusions/SignificanceThese are the first findings on genetic polymorphisms in our population and we conclude that: (1) In our total population, HLA-B*53 probably involve in disease susceptibility, while the HLA-A*03 and HLA-B*18 may confer protection from progression to severe disease; (2) In the Malay population, HLA-B*13 and B*18 are probably associated in disease susceptibility and protection, respectively. These results could furnish as a valuable predictive tool to identify ethnically different individuals at risk and/or protection from severe forms of DENV infection and would provide valuable informations for the design of future dengue vaccine.

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas in the world. Attempts to develop effective vaccines have been hampered by the lack of understanding of the pathogenesis of the disease and the absence of suitable experimental models for dengue viral infection. The magnitude of T-cell responses has been reported to correlate with dengue disease severity. Sixty Malaysian adults with dengue viral infections were investigated for their dengue virus-specific T-cell responses to 32 peptides antigens from the structural and nonstructural regions from a dengue virus isolate. Seventeen different peptides from the C, E, NS2B, NS3, NS4A, NS4B, and NS5 regions were found to evoke significant responses in a gamma interferon enzyme-linked immunospot (ELISPOT) assay of samples from 13 selected patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). NS3 and predominantly NS3 422-431 were found to be important T-cell targets. The highest peaks of T-cell responses observed were in responses to NS3 422-431 and NS5 563-571 in DHF patients. We also found almost a sevenfold increase in T-cell response in three DHF patients compared to three DF patient responses to peptide NS3 422-431 . A large number of patients' T cells also responded to the NS2B 97-106 region. The ELISPOT analyses also revealed high frequencies of T cells that recognize both serotype-specific and cross-reactive dengue virus antigens in patients with DHF.

Abstract. Plasma leakage in severe dengue has been postulated to be associated with skewed cytokine immune responses. In this study, the association of cytokines with vascular permeability in dengue patients was investigated. Human serum samples collected from 48 persons (13 with dengue fever, 29 with dengue hemorrhagic fever, and 6 healthy) were subjected to cytokines analysis by using Luminex Multiplex Technology. Selected serum samples from patients with dengue hemorrhagic fever sera and recombinant human cytokines were then tested for roles on inducing vascular permeability by treatment of human umbilical vein endothelial cells. Confocal immunofluorescence staining indicated morphologic alteration of human umbilical vein endothelial cells treated with serum samples from patients with dengue hemorrhagic fever compared with serum samples from healthy persons. The findings suggest that cytokines produced during dengue hemorrhagic infections could induce alterations in the vascular endothelium, which may play a fundamental role in the pathophysiology of dengue.

Dengue virus has four serotypes and is endemic globally in tropical countries. Neither a specific treatment nor an approved vaccine is available, and correlates of protection are not established. The standard neutralization assay cannot differentiate between serotype-specific and serotype cross-reactive antibodies in patients early after infection, leading to an overestimation of the long-term serotype-specific protection of an antibody response. It is known that the cross-reactive response in patients is temporary but few studies have assessed kinetics and potential changes in serum antibody specificity over time. To better define the specificity of polyclonal antibodies during disease and after recovery, longitudinal samples from patients with primary or secondary DENV-2 infection were collected over a period of 1 year. We found that serotype cross-reactive antibodies peaked 3 weeks after infection and subsided within 1 year. Since secondary patients rapidly produced antibodies specific for the virus envelope (E) protein, an E-specific ELISA was superior compared to a virus particle-specific ELISA to identify patients with secondary infections. Dengue infection triggered a massive activation and mobilization of both naïve and memory B cells possibly from lymphoid organs into the blood, providing an explanation for the surge of circulating plasmablasts and the increase in cross-reactive E protein-specific antibodies.

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