登革热感染后NS1和抗NS1 IgG的动力学揭示了继发感染患者中可能早期形成免疫复合物的情况-动脉网

Kinetics of NS1 and anti-NS1 IgG following dengue infection reveals likely early formation of immune complexes in secondary infected patients

Nature 等信源发布 2025-02-25 11:24

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可切换为仅中文

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Abstract

摘要

Dengue virus (DENV) is a major health concern throughout the world infecting up to 390 million people globally each year. Infection with any one of the four DENV serotypes produces a spectrum of clinical illness ranging from a mild undifferentiated febrile disease through to severe dengue involving fever and haemorrhage.

登革病毒（DENV）是全球主要的健康问题，每年感染全球多达3.9亿人。感染四种DENV血清型中的任何一种都会产生一系列临床疾病，从轻微的非特异性发热疾病到伴有发烧和出血的严重登革热。

There is currently no antiviral treatment for dengue and only one licensed vaccine with limited distribution. This study characterises the kinetics of the serological dengue biomarker, NS1, and the appearance of anti-NS1 IgG, anti-E IgM and anti-E IgG responses in patients with primary and secondary infections.

目前尚无针对登革热的抗病毒治疗，且仅有一种获得许可但分发有限的疫苗。本研究描述了血清登革热生物标志物NS1的动力学特征，以及原发性和继发性感染患者中抗NS1 IgG、抗E IgM和抗E IgG反应的出现情况。

Blood samples were collected daily from a cohort of 52 Vietnamese patients during the acute phase of disease. NS1 was detected in 85% of patient samples from disease onset with detection decreasing throughout the acute phase of disease. Anti-NS1 IgG detected from the fourth day of illness and anti-E IgM and IgG from the third day of illness, were all observed to increase throughout the course of the disease.

在疾病急性期，每天从52名越南患者队列中采集血样。从发病开始，85%的患者样本中检测到NS1，随着疾病急性期进展，检出率逐渐下降。抗NS1 IgG从发病第四天开始检测到，抗E蛋白IgM和IgG从发病第三天开始检测到，并且在整个病程中均观察到它们的增加。

During secondary infection, NS1 levels rapidly decrease with the increasing levels of anti-NS1 IgG, suggesting the possibility of NS1 immune complex formation and a potential role in the pathogenesis of the severe forms of disease associated with secondary infections..

二次感染期间，随着抗NS1 IgG水平的升高，NS1水平迅速下降，这表明可能存在NS1免疫复合物的形成，并在与二次感染相关的严重疾病发病机制中起到潜在作用。

Introduction

介绍

Dengue virus (DENV) is a major public health concern throughout the tropical regions of the world, infecting up to 390 million people each year

登革热病毒（DENV）是全球热带地区主要的公共卫生问题，每年感染人数高达3.9亿。

. DENV is a single stranded RNA virus belonging to the genus

. DENV是一种属于黄病毒属的单链RNA病毒

Flavivirus

黄病毒属

of the family

家庭的

Flaviviridae

黄病毒科

and is spread primarily by mosquitoes to humans by a bite of the domestic mosquito

主要通过蚊子叮咬传播给人类，尤其是家蚊。

A. aegypti

埃及伊蚊

. Infection with any of the four DENV serotypes (DENV1–4) can produce a spectrum of clinical disease, ranging from a mild undifferentiated febrile illness through to the life-threatening forms of disease associated with secondary infection, characterized by increased vascular permeability, thrombocytopenia and hemorrhage.

感染任何一种四种DENV血清型（DENV1-4）均可产生一系列临床疾病，范围从轻微的非特异性发热病到与继发感染相关、以血管通透性增加、血小板减少和出血为特征的危及生命的疾病形式。

，

。

There is currently no antiviral therapy for dengue infection. Treatment is purely supportive, such as fluid replacement for patients. During the natural course of infection, an immune response is generated against viral antigens such as the DENV non-structural protein, NS1 which is often observed to be at elevated levels in patient plasma during the early stages of disease, particularly in secondary infection.

目前，登革热感染尚无抗病毒治疗方法。治疗纯粹是支持性的，例如为患者补充液体。在感染的自然过程中，会产生针对病毒抗原的免疫反应，例如登革病毒非结构蛋白NS1，这种蛋白在疾病早期，特别是在继发感染中，常被观察到在患者血浆中处于较高水平。

. As a result of these observations, NS1 has been proposed as a surrogate marker for viremia and a predictor of progression to severe disease in secondary infected patients

由于这些观察结果，NS1 已被提议作为病毒血症的替代标志物，以及继发感染患者进展为重症的预测因子。

. Furthermore, studies have identified NS1 as a viral toxin that contributes to an overactive innate immune response and endothelial cell dysfunction early in infection. The presence of NS1 in the serum of infected patients has been exploited as a useful early diagnostic biomarker

此外，研究已将NS1鉴定为一种病毒毒素，它在感染早期会导致先天免疫反应过度和内皮细胞功能障碍。感染者血清中的NS1已被用作一个有用的早期诊断生物标志物。

，

。

Primary dengue infection is characterized by rising levels of IgM appearing at day 3–5 and persisting for up to a period of months. Patients undergoing a secondary infection typically experience an anamnestic IgG antibody response with peak detection 6–9 days after the onset of symptoms. The DENV NS1 protein is secreted from infected cells during the natural course of infection with elevated levels of circulating secreted NS1 in patient plasma often observed during the early stages of disease.

原发性登革热感染的特征是IgM水平在第3至5天升高，并持续数月。经历继发性感染的患者通常会出现回忆性IgG抗体反应，在症状出现后的6至9天达到峰值。在感染的自然过程中，DENV NS1蛋白从受感染细胞中分泌出来，在疾病早期阶段常观察到患者血浆中循环分泌的NS1水平升高。

，

。

The kinetics of these dengue biomarkers have been intensively researched, and many studies have investigated various combinations of biomarkers in either primary, secondary infections or both. Several key studies by Papa et al. and Koraka et al. characterized NS1 and anti-NS1

这些登革热生物标志物的动力学已被深入研究，许多研究探讨了在原发感染、继发感染或两者中各种生物标志物的组合。Papa等人和Koraka等人的一些关键研究表征了NS1和抗NS1。

，

, while the study by Hu et al. determined the kinetics of NS1, anti-dengue E IgM and IgG

，而胡等人进行的研究则确定了NS1、抗登革热E蛋白IgM和IgG的动力学特性。

. While these studies provide solid evidence for the kinetics of the appearance of these biomarkers, the combined story has to be inferred by outbreaks from different geographic locations, outbreaks, dengue serotypes and times using various methods. However, no single prospective study has been performed to characterize the kinetics of NS1, anti-NS1, anti-E IgM, and anti-E IgG in a single patient cohort.

虽然这些研究为这些生物标志物出现的动力学提供了坚实的证据，但综合的情况必须通过不同地理位置、疫情、登革病毒血清型和时间使用各种方法来推断。然而，尚未进行任何单一的前瞻性研究来表征NS1、抗NS1、抗E IgM和抗E IgG在单个患者群体中的动力学特征。

In the study presented in this manuscript, we investigated the kinetics of these biomarkers in a single prospective study of primary and secondary infected patients from a single dengue outbreak occurring in Vietnam where dengue virus types 1 and 2 were circulating. Here we characterise the kinetics of serological dengue biomarkers during the acute phase of disease.

在本手稿中介绍的研究中，我们调查了这些生物标志物的动力学，研究对象为越南单一登革热爆发期间的一次前瞻性研究中的原发和继发感染患者，当时登革病毒1型和2型正在流行。在此，我们描述了疾病急性期血清登革热生物标志物的动力学特征。

Levels of free NS1 circulating during the course of infection in patient sera were determined by quantitative capture ELISA. Titres were determined for anti-NS1 antibodies circulating in sera by indirect ELISA. Anti-E IgM and IgG levels were assessed by capture IgM and IgG ELISA, respectively.

通过定量捕获ELISA测定患者血清中感染过程中循环的游离NS1水平。通过间接ELISA测定血清中循环的抗NS1抗体的滴度。分别通过捕获IgM和IgG ELISA评估抗E IgM和IgG水平。

。

Results

结果

Analysis of NS1 protein and anti-NS1 IgG kinetics in infected patients

感染患者中NS1蛋白和抗NS1 IgG动力学分析

In primary patients, NS1 could be detected in blood from the onset of illness through to 9 days post infection, with peak NS1 levels occurring between day 1–4. In secondary patients, NS1 could be detected from day 1 to 8 post onset of illness. However, lower NS1 levels were observed later in infection when compared to primary patients with NS1 peaking on days 3 and 4.

在初次感染的患者中，从发病开始到感染后9天内都可以在血液中检测到NS1，峰值出现在第1至4天。在二次感染的患者中，NS1从发病第1天到第8天可以被检测到。然而，与初次感染的患者相比，在感染后期观察到NS1水平较低，其峰值出现在第3和第4天。

NS1 levels ranged from undetectable to 7.8 µg/ml. Patients with DENV-1 had higher levels of NS1 during primary infection, than those patients with primary DENV-2 infection whose NS1 levels were consistently low (Fig. .

NS1水平范围从检测不到到7.8微克/毫升。感染DENV-1的患者在初次感染时的NS1水平高于初次感染DENV-2的患者，后者的NS1水平一直较低（图。

a).

Fig. 1

图1

Daily patient samples tested for NS1 and anti-NS1 levels. (

每日检测患者的NS1和抗NS1水平的样本。

) Primary patient NS1 levels and (

) 初级患者 NS1 水平和 (

) Secondary patient NS1 levels during the acute phase of illness. (

）急性病期次级患者NS1水平。（

) Primary patient anti-NS1 IgG responses. (

) 初级患者抗NS1 IgG反应。(

) Secondary patient anti-NS1tested by indirect ELISA and plotted as 50% titres. Anti-NS1 IgG titre plotted against NS1 levels in (

通过间接ELISA检测的次级患者抗NS1，并绘制成50%滴度。抗NS1 IgG滴度相对于NS1水平绘制。

) primary and (

) 主要和 (

) secondary patients. Blue circles: DENV-1 DF (Dengue Fever) patient, Red circles: DENV-2 DF patient, Blue squares: DENV-1 DHF (Dengue Haemorrhagic Fever) patient, Red squares: DENV-2 DHF patient,

) 继发性患者。蓝色圆圈：DENV-1 DF（登革热）患者，红色圆圈：DENV-2 DF患者，蓝色方块：DENV-1 DHF（登革出血热）患者，红色方块：DENV-2 DHF患者，

LoD

层次细节 (Level of Detail)

limit of detection.

检测限。

Full size image

全尺寸图像

NS1 was successfully detected throughout the acute phase of the disease with detection above 80% for the first four days of illness in primary patients. However, detection was lower in secondary infections, ranging from 50 to 63% during the first 4 days of infection. NS1 was detected in 100% of primary DENV-1 patients during the first 3 days of illness and above 80% through the rest of the acute period.

在疾病的急性期，NS1在整个过程中成功检出，在初发患者发病的前四天检测率超过80%。然而，在继发感染中检出率较低，感染前4天的检出率为50%至63%。在初发DENV-1患者中，NS1在发病前三天的检出率达到100%，并在急性期其余时间保持在80%以上。

However, the results for primary DENV-2 patients revealed lower detection rates ranging from 50 to 67% during the first 4 days of illness. During DENV-1 secondary infection, NS1 was detected from day 1 for only one patient, with detection decreasing through to day 8 where no NS1 was detected. DENV-2 secondary infection patients had no detectable NS1 on day 1 (.

然而，原发性DENV-2患者的结果显示，在发病的前4天内，检出率较低，范围为50%至67%。在DENV-1继发感染期间，仅有一名患者从第1天开始检测到NS1，随后检出率逐渐下降，直到第8天未检测到NS1。DENV-2继发感染患者在第1天没有检测到NS1。

= 1), with detection rising to 83% on day 2 and peaking at 85% detection on day 3 before steadily decreasing throughout infection until NS1 was undetectable by day 8 (Fig.

= 1），第二天检测率上升至83%，第三天达到85%的峰值，随后在整个感染过程中逐渐下降，直至第8天NS1无法检测到（图。

b).

Primary DENV patients had poor early anti-NS1 IgG responses, as expected for an initial antigen exposure with NS1 specific IgG only detected in one patient on day 4 during acute illness and at 8% detection in the follow-up samples (Fig.

原发性DENV患者早期抗NS1 IgG反应较弱，这是由于初次抗原暴露所致，正如预期，在急性病程第4天仅一名患者检测到NS1特异性IgG，随访样本中的检出率为8%（图。

c). In contrast, and as expected of an anamnestic response, secondary patients showed a rapid anti-NS1 IgG response. Anti-NS1 IgG was detected as early as 4 days post onset of illness (Fig.

c). 相比之下，符合回忆反应的预期，继发性感染患者表现出快速的抗NS1 IgG反应。抗NS1 IgG在发病后最早4天就被检测到（图。

d). Anti-NS1 IgG titers and detection increased during infection, with 88% of secondary patients having detectable anti-NS1 IgG levels in the follow-up samples. Primary patients had high levels of free circulating NS1 while having undetectable anti-NS1 IgG titers, whereas secondary infected patients had decreasing levels of NS1 at the same time that anti-NS1 IgG levels were rising (Fig. .

d). 感染期间抗NS1 IgG滴度和检测率增加，88%的继发感染患者在随访样本中可检测到抗NS1 IgG水平。初次感染患者有高水平的游离循环NS1，而抗NS1 IgG滴度无法检测到，而继发感染患者在抗NS1 IgG水平上升的同时，NS1水平下降（图. 。

e). The appearance of anti-NS1 IgG was inversely correlated (Pearsons two tailed

e). 抗NS1 IgG的出现呈负相关（皮尔逊双尾

test, 95% CI) with NS1 levels in secondary patients, suggesting the formation of NS1/IgG immune complexes or IgG-mediated NS1 clearance from circulation (Fig.

测试，95% CI）与继发感染患者的NS1水平相关，表明NS1/IgG免疫复合物的形成或IgG介导的NS1从循环中清除（图。

f).

Appearance of anti-E IgM and IgG in patient serum samples

患者血清样本中抗E IgM和IgG的出现

The virion surface E protein is a major target of an infected patient’s immune response. The presence of anti-E IgM and IgG was determined using IgM and IgG specific capture ELISAs (Fig.

病毒颗粒表面的E蛋白是感染患者免疫反应的主要目标。使用IgM和IgG特异性捕获ELISA测定抗E IgM和IgG的存在（图。

). The ELISAs were performed as per the manufacturer’s instructions. None of the samples from the 3 negative patients tested positive for anti-E IgM or IgG antibodies. IgM was detected from the onset of symptoms with detection levels increasing throughout infection, reaching 100% of patients by 7 days post onset of illness (Fig. .

). ELISAs 按照制造商的说明进行。3名阴性患者的样本均未检测出抗E IgM或IgG抗体。从症状出现开始就检测到了IgM，其检测水平在感染过程中不断增加，并在发病后7天达到100%的患者（图. 。

a). In secondary infected patients, IgM was detected from the third day post onset of symptoms (26.67%) with detection rapidly rising on day 4 (78.95%). Detection of IgM in secondary patients was maintained at ≥ 80% for the remainder of the study period. The anti-E IgM response in DENV-2 patients was delayed by 2 days compared to DENV-1 primary infected patients, however in secondary infection the anti-E IgM kinetics were similar for both DENV-1 and 2 infected patients (Fig. .

a). 在继发感染患者中，症状出现后的第三天可检测到IgM（26.67%），第四天检测率迅速上升（78.95%）。在研究期间的其余时间里，继发感染患者中IgM的检测率保持在≥80%。与DENV-1原发感染患者相比，DENV-2患者的抗-E IgM反应延迟了2天，但在继发感染中，DENV-1和DENV-2感染患者的抗-E IgM动力学相似（图。

b).

Fig. 2

图2

Anti-dengue IgM and IgG responses. (

抗登革热IgM和IgG反应。

) Daily primary patient anti-E IgM responses. (

每日初级患者抗E IgM反应。

) Daily secondary patient anti-E IgM responses. (

) 每日二次患者抗-E IgM反应。 (

) Daily primary patient anti-E IgG responses. (

每日初级患者抗E IgG反应。

) Daily secondary patient anti-E IgG responses. Blue circles: DENV-1 DF patient, Red circles: DENV-2 DF patient, Blue squares: DENV-1 DHF patient, Red squares: DENV-2 DHF patient. Limit of detection (LoD) set at 12 and 23.4 Panbio units for IgM and IgG respectively.

每日二次患者抗E IgG反应。蓝色圆圈：DENV-1 DF患者，红色圆圈：DENV-2 DF患者，蓝色方块：DENV-1 DHF患者，红色方块：DENV-2 DHF患者。IgM和IgG的检测下限（LoD）分别设为12和23.4 Panbio单位。

Full size image

全尺寸图像

As expected, anti-E IgG detection in primary patients was low in comparison with IgM with a positive response detected in only 3 patients during the acute disease phase. Anti-E IgG was detected on days 6 and 7 in 19% and 17% of patients, respectively. Higher rates of detection were observed in the follow-up samples with IgG detection in 44% of patients (Fig. .

正如预期，与IgM相比，初次感染患者中抗E型IgG的检出率较低，在急性病程中仅3名患者呈现阳性反应。在第6天和第7天，分别在19%和17%的患者中检测到抗E型IgG。在随访样本中观察到更高的检出率，44%的患者检测到IgG（图）。

c). In secondary infected patients, anti-E IgG was detected from day 3 in 6.67% of patients, with detection rapidly rising to ≥ 87% from day 5–10 (Fig.

c). 在继发感染患者中，抗E型IgG从第3天开始在6.67%的患者中被检测到，检测率迅速上升，到第5-10天达到≥87%（图。

d). Follow-up samples (day 16–45) showed that all patients mounted a detectable anti-E IgG response. In summary, primary patients infected with either DENV-1 or DENV-2 had similarly low levels of anti-E IgG responses during the acute phase of disease. In contrast, secondary responses to each DENV serotype had higher detection rates with increasing detection from day 4, reflecting a strong anamnestic antibody response to cross-reactive epitopes..

d). 随访样本（第16-45天）显示，所有患者均产生了可检测的抗E蛋白IgG反应。总之，初次感染DENV-1或DENV-2的患者在疾病急性期的抗E蛋白IgG反应水平均较低。相比之下，对每种DENV血清型的二次反应从第4天开始检测率更高，反映出对交叉反应表位的强烈回忆性抗体反应。

Discussion

讨论

In this prospective study, we examined the kinetics of appearance of NS1, anti-NS1 IgG, anti-E IgM and IgG in the serum of 51 patients infected with dengue during epidemics in Vietnam from 2005 to 2007. Patients enrolled in this study either experienced DENV-1 or DENV-2 infections (both primary and secondary), with levels of disease outcome varying from mild infection to severe disease with or without vascular leak.

在这项前瞻性研究中，我们检测了2005年至2007年越南流行期间51名登革热感染患者血清中NS1、抗NS1 IgG、抗E IgM和IgG的出现动力学。本研究纳入的患者经历了DENV-1或DENV-2感染（包括初次和二次感染），病情结果从轻度感染到伴或不伴血管渗漏的严重疾病不等。

Samples were collected daily during the acute disease period then at convalescence..

在急性病期每天采集样本，然后在恢复期采集。

NS1 was detected in both primary and secondary DENV patients during the acute phase of disease from the first day of illness. In primary infections detection was retained through to at least 9 days post illness, while detection in secondary cases was found to decline by days 6–7. Peak detection for both primary and secondary patients was on days 3–4.

在疾病急性期，从发病第一天起，原发性和继发性登革热患者中均检测到NS1。在原发性感染中，检测可持续至发病后至少9天，而继发性病例的检测在第6-7天开始下降。原发和继发患者的峰值检测时间在第3-4天。

，

. NS1 was detected in ≥ 72% of samples during the first 4 days of illness ranging from undetectable to 7.8 µg/ml. By day 5, detection dropped to 50%, then decreased throughout the remainder of the disease period. These results are consistent with the findings of others who have used in-house and commercial NS1 capture ELISAs to detect NS1.

在发病的前4天内，≥72%的样本中检测到NS1，其浓度范围从无法检测到7.8 µg/ml。到第5天，检出率下降至50%，然后在整个病程的剩余时间内持续降低。这些结果与其他人使用内部和商业化的NS1捕获ELISA检测NS1的发现一致。

，

. As with the current analysis, these studies found that NS1 capture ELISAs had high sensitivity and specificity early during the acute period with detection decreasing during infection.

与当前分析一样，这些研究发现，NS1捕获ELISAs在急性期早期具有高灵敏度和特异性，而在感染过程中检测能力下降。

Primary DENV-2 patients had lower NS1 levels overall with 133 ng/ml the highest NS1 level recorded, as opposed to DENV-1 primary patients which peaked at 7.8 µg/ml. The lower levels of NS1 associated with DENV-2 infection has also been observed in other studies in Vietnam

原发性DENV-2患者的NS1水平总体较低，记录的最高NS1水平为133纳克/毫升，而原发性DENV-1患者的峰值为7.8微克/毫升。在越南的其他研究中也观察到与DENV-2感染相关的NS1水平较低。

. While the use of dengue 2 virus NS1 as the sole standard for NS1 quantification for all samples likely impacts absolute values, the trends in relative levels of NS1 within individual patients, the main focus of this study, will not be impacted. Interestingly, secondary DENV-2 patients showed comparable levels of NS1 to secondary DENV-1 patients, with peak levels detected at 5.5 µg/ml.

虽然使用登革病毒2型NS1作为所有样本NS1定量的唯一标准可能会影响绝对值，但本研究关注的重点是个体患者内NS1相对水平的趋势，这不会受到影响。有趣的是，继发性DENV-2患者的NS1水平与继发性DENV-1患者相当，峰值水平检测为5.5 µg/ml。

The lower levels of NS1 in primary DENV-2 patients may simply reflect an overall lower viral load.

原发性DENV-2患者的NS1水平较低可能仅仅反映了整体病毒载量较低。

. The higher relative levels of NS1 in secondary DENV-2 infected patients may be a consequence of antibody-dependent enhanced infection, with these higher levels shown to be a predictive marker of progression to more severe disease

二次感染DENV-2的患者中NS1的相对较高水平可能是抗体依赖性增强感染的结果，这些较高水平被证明是进展为更严重疾病的预测标志。

。

In a clinical setting, timing and choice of what diagnostic test to employ is critical, as each method described in this study has an ideal window in which it is at its most effective. The NS1 capture ELISA is at its most effective early in infection, i.e. from the onset of symptoms, whereas IgM and IgG ELISAs rely on the patients induced immune response, which in most cases appears from day 3 or 4.

在临床环境中，选择何时进行何种诊断测试至关重要，因为本研究中描述的每种方法都有一个最有效的理想窗口期。NS1捕获ELISA在感染早期最有效，即从症状出现开始，而IgM和IgG ELISA则依赖于患者的诱导免疫反应，这在大多数情况下从第3或第4天开始出现。

，

. By the time IgM ELISAs are performing at a high diagnostic rate, NS1 detection may be rapidly declining, particularly in those patients experiencing a secondary infection. This highlights the need for complementary testing of both NS1 and antibody levels, as this testing regime was found to cover the disease period with greater than 80% successful diagnosis (in this study day 2 was an exception with 73% detection).

当IgM ELISA以高诊断率进行检测时，NS1的检出率可能正在迅速下降，尤其是在经历二次感染的患者中。这突显了对NS1和抗体水平进行互补检测的必要性，因为该检测方案被发现在病程期间能够实现大于80%的成功诊断率（在本研究中，第2天为例外，检出率为73%）。

，

. Performing concurrent tests on patient sera for NS1 capture as well as anti-E IgM and IgG capture would allow additional determination of primary versus secondary infection

对患者血清进行NS1捕获以及抗E蛋白IgM和IgG捕获的并发测试可以进一步确定是初次感染还是再次感染。

。

Anti-NS1 IgG 50% titers were determined by indirect ELISA, with both detection and titers varying in correlation with the infection status of the patient. Previous studies have suggested that NS1 specific antibodies may play a role in the development of severe disease and vascular leak, as anti-NS1 antibodies are seldom seen in primary infection but detected in most secondary infected patients.

抗NS1 IgG 50%滴度通过间接ELISA测定，其检测结果和滴度随患者的感染状态而变化。以往的研究表明，NS1特异性抗体可能在重症和血管渗漏的发展中起作用，因为抗NS1抗体在初次感染中很少见，但在大多数二次感染患者中均可检测到。

，

. All primary infected patient titers were below the negative sample cut-off values, with the exception of a small number of patients (

所有初次感染患者滴度均低于阴性样本的临界值，只有少数患者例外 (

= 3). In secondary infected patients, specific anti-NS1 IgG was detected from day 4, with titers and sensitivity of detection rising throughout the course of the infection to ≥ 81% detection in convalescent sera. All secondary infected patients with positive anti-NS1 IgG had no or rapidly decreasing NS1 levels.

= 3）。在继发感染患者中，从第4天开始检测到特异性抗NS1 IgG，其滴度和检测敏感性在整个感染过程中不断上升，在恢复期血清中达到≥ 81%的检出率。所有抗NS1 IgG阳性的继发感染患者，其NS1水平无或迅速下降。

It was observed in most patients that decreasing levels of NS1 correlated with rising levels of anti-NS1 IgG. In other patients with no NS1 detected, high levels of anti-NS1 IgG were present. The logical interpretation of these observations is the formation of immune complexes, which render the bound NS1 unable to be detected in the capture ELISA..

在大多数患者中观察到，NS1水平的下降与抗NS1 IgG水平的上升相关。在其他未检测到NS1的患者中，存在高水平的抗NS1 IgG。这些观察结果的合理解释是免疫复合物的形成，这使得结合的NS1无法在捕获ELISA中被检测到。

One limitation of the NS1 capture ELISA employed in this study is that detection is limited to free-circulating NS1. In this study and others, it has been noted that the ability to detect NS1 diminishes from day 4–5. It has been hypothesized that this loss of NS1 detection in secondary infection is due to the presence of NS1 specific IgG removing NS1 from circulation and leading to the formation of immune complexes.

本研究中使用的NS1捕获ELISA的一个局限性在于，其检测仅限于游离循环的NS1。在本研究及其他研究中已注意到，从第4至5天起，检测NS1的能力会减弱。有假设认为，在继发感染中NS1检测能力的丧失是由于存在NS1特异性IgG，其将NS1从循环中清除并导致免疫复合物的形成。

These immune complexes are then presumed to be cleared from the circulatory system or deposited onto the surface of endothelial cells possibly contributing to vascular leak. In this study we have confirmed an inverse correlation between decreasing NS1 levels and increasing anti-NS1 IgG over the course of infection within individual patients.

这些免疫复合物随后被认为会从循环系统中被清除，或者沉积在内皮细胞表面，可能有助于血管渗漏。在这项研究中，我们已经证实，在单个患者感染过程中的 NS1 水平下降与抗 NS1 IgG 增加之间存在反比关系。

Note added in proof: while this manuscript was under review, our findings were confirmed by similar observations reported by Papa et al..

校对时添加的注释：在本文稿审阅期间，Papa 等人报告的类似观察结果证实了我们的发现。

。

Speculation on the role of antibodies directed against NS1 has, at times provided a somewhat confusing and contradictory narrative. Historically, NS1 has been viewed as a potential vaccine candidate that would give rise to a protective immune response that does not have the same intrinsic ADE risks associated with E-based vaccines.

关于针对NS1的抗体作用的推测，有时提供了一个有些令人困惑且矛盾的叙述。历史上，NS1一直被视为一种潜在的疫苗候选者，能够引发保护性免疫反应，同时不会带来与基于E蛋白的疫苗相同的内在ADE风险。

. Our group identified a role for an anti-dengue NS1 monoclonal antibody in protection of mice from dengue virus or Zika virus challenge

我们的团队发现了一种抗登革热NS1单克隆抗体在保护小鼠免受登革病毒或寨卡病毒攻击方面的作用。

and there is a wealth of literature demonstrating that NS1 vaccination elicits protective antibodies by targeting dengue-infected cells for destruction via complement-mediated lysis and other mechanisms

并且有大量的文献表明，NS1疫苗接种通过补体介导的裂解和其他机制，靶向登革热感染细胞进行破坏，从而引发保护性抗体。

，

. However, NS1 vaccination has also been shown to produce inappropriate immune responses with antibodies that are host reactive via self-mimicry apparently enhancing disease

然而，NS1疫苗接种也被证明会产生不适当的免疫反应，通过自我模拟产生对宿主有反应的抗体，似乎会加重疾病。

，

. Furthermore, the protective efficacy of the anti-NS1 response needs to be balanced against its role in pathogenesis. As demonstrated in our manuscript and others, NS1 is rapidly cleared from circulation during a secondary infection by anti-NS1 IgG. The loss of free circulating NS1 through the formation of immune complexes aligns with the onset of severe dengue disease.

此外，抗NS1反应的保护效力需要与其在发病机制中的作用相平衡。正如我们在手稿中及其他研究中所展示的，NS1在继发感染期间被抗NS1 IgG迅速从循环中清除。通过免疫复合物形成导致游离循环NS1的减少，与重症登革热病的发作相吻合。

We speculate that the formation of immune complexes and deposition onto endothelial cell walls rather than clearance, likely plays a role in the vascular leak characteristic of severe dengue. Based on our observations, this hypothesis is limited by the relatively small number of patients experiencing secondary dengue and severe disease.

我们推测，免疫复合物的形成并沉积在内皮细胞壁上，而不是被清除，可能在严重登革热的血管渗漏特征中起作用。基于我们的观察，这一假设受到经历继发性登革热和重症患者数量相对较少的限制。

Therefore, the sample size needs to be expanded in future studies to conclusively determine the role that immune complexes may be playing in severe disease..

因此，未来的研究需要扩大样本量，以最终确定免疫复合物在重症疾病中可能发挥的作用。

While the kinetics of dengue biomarker appearance has been intensively investigated, to our knowledge, this is the first prospective study to examine the kinetics of NS1, anti-NS1, anti-E IgM and IgG in a single dengue patient cohort. Overall, our findings confirm what has otherwise been inferred by numerous previous studies reporting the kinetics of dengue biomarkers in various patient cohorts.

虽然登革热生物标志物出现的动力学已经被广泛研究，但据我们所知，这是首个前瞻性研究，旨在单一登革热患者群体中检测NS1、抗NS1、抗-E IgM和IgG的动力学。总体而言，我们的研究结果证实了先前众多研究对不同患者群体中登革热生物标志物动力学的推断。

The identification of a viral toxin activity for NS1 that impacts vascular integrity of the endothelial barrier 3 days post intravenous injection in mouse studies further highlights the importance of early NS1 detection.

在小鼠研究中，静脉注射三天后，NS1的病毒毒素活性对内皮屏障的血管完整性产生影响，这进一步凸显了早期检测NS1的重要性。

，

. Taken together, these results support the suggestion that both soluble NS1 and its antibody bound immune complex may be contributing factors in the pathogenesis of severe disease.

综上所述，这些结果支持了这样的建议，即可溶性NS1及其抗体结合的免疫复合物可能是重症发病机制中的促成因素。

Materials and methods

材料与方法

Clinical samples and study objectives

临床样本和研究目标

We selected 303 serum samples from 52 dengue patients (confirmed positive by qPCR) enrolled in research studies at the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam (reviewed by the ethics committee and approved by the internal review board of the hospital), during outbreaks that occurred between 2005 and 2007.

我们选择了来自52名登革热患者（经qPCR确认为阳性）的303份血清样本，这些患者参与了越南胡志明市热带病医院在2005年至2007年期间爆发的研究项目（经伦理委员会审查并由医院内部审查委员会批准）。

Daily serum samples were taken from patients on presentation to healthcare facilities during the course of disease development (1–9 days) and convalescent serum samples were taken several weeks after illness resolution. The patient cohort contained primary and secondary patients experiencing DENV-1 and 2 infections, with symptoms ranging from mild dengue fever through to severe dengue involving haemorrhage and shock (Table .

在疾病发展过程（1-9天）中，患者就诊于医疗机构时每日采集血清样本，并在病愈后数周采集恢复期血清样本。患者群体包含经历DENV-1和DENV-2感染的初次及二次感染患者，症状范围从轻度登革热到涉及出血和休克的重度登革热不等（表。

). Patients were classified as having a primary vs. secondary infection based on RT-qPCR results coupled with IgM and IgG analysis. A negative IgG ELISA result in the first 72 h with no increase in dengue-reactive IgG by day 7 defined a primary infection, and a positive indirect ELISA result within 72 h and/or an equivocal response, with a clear increase in dengue-reactive IgG greater than the IgM response by day 7, defined a secondary infection.

). 根据RT-qPCR结果结合IgM和IgG分析，将患者分为原发感染和继发感染。在前72小时内IgG ELISA结果为阴性且第7天登革病毒特异性IgG无增加的被定义为原发感染；而在72小时内间接ELISA结果为阳性和/或反应不确定，但到第7天登革病毒特异性IgG明显增加且高于IgM反应的，则被定义为继发感染。

This study examines the kinetics of NS1 production, as well as the appearance of anti-NS1 IgG, anti-dengue IgM and anti-dengue IgG in patients with primary and secondary infections..

本研究检查了原发感染和继发感染患者中NS1产生的动力学，以及抗NS1 IgG、抗登革热IgM和抗登革热IgG的出现。

Table 1 Patient cohort breakdown based on infection and disease status.

表1 基于感染和疾病状态的患者队列细分。

Full size table

全尺寸表格

Study design

研究设计

Diagnostic ELISAs: NS1, anti E IgM and anti-E IgG

诊断用ELISAs：NS1、抗E IgM和抗E IgG

Evaluation of NS1, anti-dengue IgM and anti-dengue IgG levels were assessed using the Panbio Early dengue ELISA, Panbio Dengue IgM capture ELISA and Panbio IgG capture ELISA IgG kits as per manufacturer’s instructions (Abbott). Quantitation of NS1 in patient samples was made possible by the addition of a baculovirus expressed recombinant dengue virus 2 NS1 protein standard curve from which quantitative values were determined using GraphPad Prism.

按照制造商（Abbott）的说明，使用Panbio早期登革热ELISA、Panbio登革热IgM捕获ELISA和Panbio IgG捕获ELISA IgG试剂盒评估NS1、抗登革热IgM和抗登革热IgG的水平。通过添加杆状病毒表达的重组登革病毒2 NS1蛋白标准曲线，实现了对患者样本中NS1的定量，定量值使用GraphPad Prism确定。

The authors note that the use of a single serotype NS1 will likely impact absolute protein values attributed to other serotypes. However the findings of this study relate to the relative rise and fall in levels of circulating NS1 within patients..

作者指出，使用单一血清型的NS1可能会影响归因于其他血清型的绝对蛋白值。然而，本研究的发现与患者体内循环NS1水平的相对升降有关。

Indirect anti-NS1 IgG

间接抗NS1 IgG

Nunc maxisorp plates were coated with 50 µl of 100 ng/ml of NS1 in carbonate buffer pH 9.6 and incubated at 4 °C overnight. After coating, the plates were blocked with 150 µl of a solution of 1% milk powder, 1% sucrose, 0.3% Tween 20 in PBS at room temperature for 2 h. Following blocking, plates were washed and 50 µl of 1 in 10 dilutions of patient plasma samples were serially diluted in a 1:10 series in blocking solution and incubated for 1 h at 37 °C.

将50 µl的100 ng/ml NS1溶液用碳酸盐缓冲液（pH 9.6）包被于Nunc maxisorp板上，并在4°C下孵育过夜。包被后，用含1%脱脂奶粉、1%蔗糖和0.3% Tween 20的PBS溶液150 µl在室温下封闭2小时。封闭完成后，将板洗涤，并将患者血浆样本以1:10稀释，每孔加入50 µl，随后在封闭液中进行1:10系列梯度稀释，在37°C下孵育1小时。

After antibody binding, the plates were washed 3 times and anti-human IgG HRP at 1:100 in 0.3% Tween 20 in PBS (PBST-20) was added to the plate for 1 h at 37 °C, then washed a further 6 times. Fifty µl of K-Blue TMB substrate (ELISA systems) was added, and the colour reaction was developed for 5 min in the dark.

抗体结合后，将板洗涤3次，并加入1:100稀释的抗人IgG HRP（用含0.3% Tween 20的PBS溶液，即PBST-20稀释），在37°C孵育1小时，然后进一步洗涤6次。加入50 µl K-Blue TMB底物（ELISA系统），在暗处显色反应5分钟。

The reaction was stopped by the addition of 25 µl of 2 M H.

通过加入25 µl的2 M H终止反应。

因此

, with plates read spectrophotometrically at 450 nm.

，用酶标仪在450 nm处读取吸光度。

Data availability

数据可用性

The authors declare that all data supporting the findings of this study are available within the paper.

作者声明支持本研究发现的所有数据均可在论文中获得。

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Funding

资金

This research was funded by Australian Research Council Linkage grant, (LP0668437).

本研究由澳大利亚研究理事会联动资助（LP0668437）。

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作者信息

Author notes

作者笔记

Catriona McElnea

凯特里奥娜·麦克尔内亚

Present address: AnteoTech, Eight Mile Plains, Brisbane, QLD, 4113, Australia

当前地址：AnteoTech，八英里平原，布里斯班，昆士兰州，4113，澳大利亚

Authors and Affiliations

作者与所属机构

Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, 4072, Australia

澳大利亚昆士兰大学化学与分子生物科学学院，澳大利亚传染病研究中心，布里斯班，昆士兰州，4072，澳大利亚

David A. Muller, Jovin J. Y. Choo, Catriona McElnea & Paul R. Young

大卫·A·穆勒，乔文·J·Y·周，卡特里奥娜·麦克尔内亚，保罗·R·杨

Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam

越南胡志明市热带病医院牛津大学临床研究部

Huynh T. L. Duyen & Bridget Wills

黄氏莲·杜莹 & 布里吉特·威尔斯

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David A. Muller

大卫·A·穆勒

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Jovin J. Y. Choo

朱乔文

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Catriona McElnea

卡特里奥娜·麦克尔内亚

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Huynh T. L. Duyen

黄氏莲惠

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Bridget Wills

布里吉特·威尔斯

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Paul R. Young

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Contributions

贡献

Conceptualization, D.A.M. and P.R.Y.; methodology, D.A.M. and P.R.Y.; investigation, C.M., H.T.L.D.; formal analysis, D.A.M.; writing—original draft preparation, D.A.M.; writing—review and editing, D.A.M., P.R.Y., C.M., J.J.Y.C., and B.W.; visualization, D.A.M.; funding acquisition, D.A.M. and P.R.Y.

概念化，D.A.M. 和 P.R.Y.；方法论，D.A.M. 和 P.R.Y.；调查，C.M., H.T.L.D.；正式分析，D.A.M.；撰写—原稿准备，D.A.M.；撰写—审阅与编辑，D.A.M., P.R.Y., C.M., J.J.Y.C., 和 B.W.；可视化，D.A.M.；资金获取，D.A.M. 和 P.R.Y.

All authors have read and agreed to the published version of the manuscript..

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Corresponding author

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Correspondence to

致函

Paul R. Young

保罗·R·杨

。

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Competing interests

竞争利益

The authors declare no competing interests.

作者声明没有竞争性利益冲突。

Institutional review board statement

机构审查委员会声明

The study was conducted in accordance with the Declaration of Helsinki, and the use of serum samples routinely taken from patients as part of ongoing research projects was approved by the Ethics Committee of the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.

本研究按照《赫尔辛基宣言》进行，作为正在进行的研究项目的一部分，从患者身上常规采集的血清样本的使用已获得越南胡志明市热带病医院伦理委员会的批准。

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知情同意声明

Informed consent was obtained from all subjects involved in the study.

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Muller, D.A., Choo, J.J.Y., McElnea, C.

穆勒，D.A.，周，J.J.Y.，麦克尔内亚，C.