key: cord-0265921-l5ueoviq
authors: Yamanaka, Atsushi; Imad, Hisham Ahmed; Phumratanaprapin, Weerapong; Phadungsombat, Juthamas; Konishi, Eiji; Shioda, Tatsuo
title: Antibody-dependent enhancement representing in vitro infective progeny virus titer correlates with the viremia level in dengue patients
date: 2020-11-23
journal: bioRxiv
DOI: 10.1101/2020.11.20.392357
sha: 713f8a462a9087e203e9ea490344b101c3a428d5
doc_id: 265921
cord_uid: l5ueoviq

Dengue virus (DENV) distributes throughout tropical and subtropical countries and causes dengue fever (DF) and dengue hemorrhagic fever in humans. Some DF patients suddenly develop severe symptoms after the defervescent period. Although the pathogenic mechanism of the severe symptoms has not been fully elucidated, the viremia level in the early phase has been shown to correlate with the disease severity. One of the hypotheses is that a phenomenon called antibody-dependent enhancement (ADE) of infection leads to a high level of viremia. To examine the plausibility of this hypothesis, we examined the relationship between in vitro ADE activity and in vivo viral load quantity in six patients with dengue diseases. An autologous DENV strain was isolated from each of the six patients. Blood samples were then collected at multiple time points between the acute and defervescent phases, and the balance between neutralizing and enhancing activities against the autologous and prototype viruses was examined. As the antibody levels against DENV were rapidly increased, ADE activity was decreased over time or partially maintained against some viruses at low serum dilution. In addition, positive correlations were observed between ADE activity representing in vitro progeny virus production and viremia levels in patient plasma samples. Therefore, the measurement of ADE activity in dengue-seropositive samples may help to predict the impact of viral load in the subsequent DENV infection. IMPORTANCE It has not been fully elucidated how the phenomenon of antibody-dependent enhancement (ADE) affects the pathogenesis of severe dengue diseases, although high viremia levels have been epidemiologically demonstrated to be associated with the disease severity. Here, we show that ADE in the acute-phase patient sera exhibited significantly different activities against autologous and lab strains than ADE in the defervescent-phase sera. Further, the enhancement of progeny virus production activity, which is one of the factors to evaluate ADE in vitro, was significantly correlated with the levels of viral load in the patient blood circulation. This suggests that measurement of the in vitro enhancing progeny virus titers might be used to predict the impact of in vivo DENV viremia level. Our present findings could contribute to a method to forecast disease severity for seropositive populations who would be at risk of developing severe disease in the event of heterotypic DENV infection.

ABSTRACT (222 words) 30 Dengue virus (DENV) distributes throughout tropical and subtropical countries and 31 causes dengue fever (DF) and dengue hemorrhagic fever in humans. Some DF patients 32 suddenly develop severe symptoms after the defervescent period. Although the 33 pathogenic mechanism of the severe symptoms has not been fully elucidated, the 34 viremia level in the early phase has been shown to correlate with the disease severity. 35 One of the hypotheses is that a phenomenon called antibody-dependent enhancement suggesting that the relative capacity for neutralization might be easily affected by the 106 balance between NAbs and EAbs.

In the present study, we evaluated the balance between neutralizing and enhancing 108 activities in sera collected from dengue patients at multiple time points between the 109 acute and defervescent phases. The six autologous viruses isolated from the respective 110 patients were used as assay antigens, allowing us to examine the balance antibody assay 111 with autologous combinations between patient sera and virus antigens. We also 112 measured the number of viral RNA copies in plasma samples collected at multiple time 113 points, and revealed a correlation between the in vitro ADE activity and in vivo viral 114 load quantity. In conclusion, the present NAb/EAb-balance assay might be used to predict the 264 impact of the viremia level when a human is subsequently infected with DENV. 

Blood samples 275 The present study was conducted using the serum and plasma collected from The NAb/EAb-balance assay was conducted using semi-adherent K562 cells as 

When the number of infected cells was higher than the mean + three SD, it was defined 316 as enhancing activity. In contrast, when the infected cell number was lower than the 317 mean − three SD, it was defined as neutralizing activity.

In addition, enhancing activity was also evaluated by titrating the progeny virus Immunochemical staining was performed essentially as described previously (34). 

The authors declare that they have no competing interests. The NAb/EAb-balance assay was conducted with a combination of 2-fold serial serum dilutions (starting from 1:10) in patient #49, and each DENV strain (the autologous, Mochizuki, NGC, H87 or H241 strain). Culture supernatants were harvested at 24 h after the mixture of serum, virus and K562 cells. The infective titers (FFU/ml) were determined on Vero cells. The blue, green, yellow and red triangles, corresponding to Fig.  1A , indicate the hours after hospitalization. The abscissa indicates the serum dilutions and the ordinate shows the progeny virus titers (both expressed as log10). Dotted lines indicate the mean progeny virus titers calculated from four negative (no serum) controls. (B) Decreasing trend of ADE at low serum dilution with time progression. Fold enhancement was calculated from the infective progeny virus titers obtained in Fig. 3A (specifying data at 1:10 serum dilution), and was expressed in log10 as the increase in the progeny virus titer relative to the negative control. The fold enhancement was plotted as the ordinate against time (h) as the abscissa. 

World Health Organization. 2020. Dengue and severe dengue

Refining the global spatial limits of dengue 373 virus transmission by evidence-based consensus

The global distribution and burden 378 of dengue

Dengue 382 infection