Induction of bivalent immune responses by expression of dengue virus type 1 and type 2 antigens from a single complex adenoviral vector

There are approximately 100 million new cases of dengue (DEN) virus infection each year. Infection can result in illness ranging from a mild fever to hemorrhaging, shock, or even death. There are four serotypes of dengue virus (DEN1-4), and immunity to one serotype does not cross protect from infection with other serotypes. Currently there are no approved vaccines for dengue fever. In this report, we describe the construction of a bivalent dengue virus vaccine using a complex recombinant adenovirus approach to express multiple genes of DEN1 and DEN2 serotypes. In vaccinated mice, this vector induced humoral immune responses against all four dengue serotypes as measured by enzyme-linked immunosorbent assay. However, the neutralizing antibody responses were specific for DEN1 and DEN2 serotypes. Expansion of this vaccine development platform towards the DEN3 and DEN4 serotypes can lead towards the development of an adenovirus-based tetravalent dengue vaccine.     

Dengue virus, a flavivirus, propagates in human bone marrow progenitors and hematopoietic cell lines [see comments]

Dengue and other arbovirus diseases are frequently associated with bone marrow failure. We show that dengue type 4 (DEN4) propagates in colonies derived from immature human bone marrow progenitors. DEN4 was propagated in BFU-E-derived colonies and replication was dependent on erythropoietin. DEN4 was not cytotoxic. In inoculated cultures, diffuse bursts with many clusters contained large amounts of DEN4 RNA. In contrast to dengue infection of macrophages, virus propagation in semisolid culture was sustained and not enhanced by subneutralizing amounts of antibody. DEN4 also was efficiently propagated in human hematopoietic cell lines, especially those with erythroid properties. In K562 cells, DEN4 infection persisted for months; greatly slowed cell growth, again without cytotoxicity; and resulted in cytopathic changes in cell appearance. Flaviviruses can infect human hematopoietic cells and alter their proliferative capacity.     

Cell type-specific mechanisms of interleukin-8 induction by dengue virus and differential response to drug treatment

In vitro infection with dengue virus induces interleukin (IL)-8 secretion, which increases endothelial cell permeability; this has been proposed as a mechanism for plasma leakage in dengue hemorrhagic fever. We studied the mechanisms of IL-8 induction, using luciferase reporter constructs, and the effect of pharmacological inhibitors of either IL-8 secretion or nuclear factor- kappa B (NF-kappa B) activation on IL-8 induction by dengue 2 virus (DEN2V) infection. IL-8 induction by DEN2V infection was associated with activation of NF- kappa B and activator protein-1 (AP-1) in HEK293A cells but only with activation of AP-1 in HepG2 cells. Treatment with SB203580, a mitogen-activated protein kinase inhibitor, and rolipram, a phosphodiesterase IV inhibitor, partially inhibited DEN2V-induced IL-8 secretion in HEK293A cells but increased DEN2V-induced IL-8 secretion in HepG2 cells. In contrast, treatment with dexamethasone increased DEN2V-induced IL-8 secretion in HEK293A cells but had no effect on DEN2V-induced IL-8 secretion in HepG2 cells. These results demonstrate that anti-inflammatory drugs have variable effects on IL-8 secretion in different cell types during DEN2V infection.     

免疫荧光技术检测室内感染蚊虫体内登革病毒的研究

目的　探讨检测媒介蚊虫体内登革病毒的方法。　方法　以埃及伊蚊和白纹伊蚊两种有效媒介为研究对象 ,在人工经口感染大剂量登革 2型病毒 (DEN- 2 )的基础上 ,通过蚊细胞培养病毒分离和蚊头部压片免疫荧光技术进行病毒抗原检测。　结果　埃及伊蚊感染 DEN- 2病毒 1d后 ,接种 C6 / 36细胞 ,盲传一代后第 5 d出现空斑现象 ,确证细胞发病。白纹伊蚊经口感染登革病毒后 ,取吸血雌蚊每日进行头部压片免疫荧光检测 ,结果显示 ,从蚊虫感染后第 1d～第 2 0d内均能检测到病毒抗原。　结论　用免疫荧光技术 ,可直接检测出感染蚊体内是否携带登革病毒 ,是较为简便、省时、敏感的方法     

Monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention

Infection with dengue virus (DENV) or any other flavivirus induces cross-reactive, but weakly neutralizing or nonneutralizing, antibodies that recognize epitopes involving the fusion peptide in the envelope glycoprotein. Humanized mAb IgG 1A5, derived from a chimpanzee, shares properties of cross-reactive antibodies. mAb IgG 1A5 up-regulated DENV infection by a mechanism of antibody-dependent enhancement (ADE) in a variety of Fc receptor-bearing cells in vitro. A 10- to 1,000-fold increase of viral yield in K562 cells, dependent on the DENV serotype, was observed over a range of subneutralizing concentrations of IgG 1A5. A significant increase of DENV-4 viremia titers (up to 100-fold) was also demonstrated in juvenile rhesus monkeys immunized with passively transferred dilutions of IgG 1A5. These results, together with earlier findings of ADE of DENV-2 infection by a polyclonal serum, establish the primate model for analysis of ADE. Considering the abundance of these cross-reactive antibodies, our observations confirm that significant viral amplification could occur during DENV infections in humans with prior infection or with maternally transferred immunity, possibly leading to severe dengue. Strategies to eliminate ADE were explored by altering the antibody Fc structures responsible for binding to Fc receptors. IgG 1A5 variants, containing amino acid substitutions from the Fc region of IgG2 or IgG4 antibodies, reduced but did not eliminate DENV-4-enhancing activity in K562 cells. Importantly, a 9-aa deletion at the N terminus of the CH(2) domain in the Fc region abrogated the enhancing activity.     

Ⅰ型登革病毒ns1基因克隆及其表达产物的免疫原性研究

目的克隆并表达Ⅰ型登革病毒非结构蛋白1(NS1)基因,初步鉴定其免疫原性。方法登革热Ⅰ型病毒标准株感染C6/36细胞后,抽提病毒RNA,经RT-PCR方法扩增出NS1全长基因片段,经T-A克隆后,在QIA表达系统中表达,表达产物用Ni柱亲和层析纯化后,用兔抗Ⅰ型登革病毒免疫血清及登革热患者血清对重组蛋白进行Western Blot及ELISA鉴定。结果构建的重组质粒pQE-30/DV1NS1经IPTG诱导,重组蛋白NS1高效表达并纯化成功,经Western Blot及ELISA证实重组蛋白NS1可以被免疫血清和病人血清特异识别。结论Ⅰ型登革病毒非结构蛋白NS1表达载体在大肠杆菌M15中高效表达。纯化产物具有较强的免疫原性,为进一步研究NS1的生物学特性和血清学检测奠定了基础。     

Enhancement of dengue virus infection in monocytes by flavivirus antisera

Enhanced dengue 2 virus (D2V) infection in suspension cultures of human peripheral blood mononuclear phagocytes (PBL) produced by subneutralizing concentrations of dengue antisera has been described previously. In this study, the enhancement phenomenon was found to be a general property of representative flavivirus antisera. All except one of 24 antisera, which had been raised by 1-3 injections of flaviviruses in rabbits, enhanced the growth of dengue 2 virus in human PBL. Flavivirus antisera showing the greatest level of cross-reactivity against a battery of 42 flavivirus antigens in the hemagglutination-inhibition test were most potent in enhancing dengue replication in PBL cultures. Cross-neutralizing reactivity did not relate to enhanced D2V infection. However nearly one-half of studied flavivirus antisera neutralized D2V at dilutions of 1:10 or 1:20. Heterotypic D2V neutralizing antibody could serve as a "brake" on infection enhancement in vivo. Observations should be made in the field to look for possible enhancement of dengue infection in heterotypic flavivirus immunes.     

Increased apoptosis and expression of tumor necrosis factor-alpha caused by infection of cultured human monocytes with dengue virus

Dengue (DEN) virus is responsible for one of the most significant viral diseases in tropical countries. Monocytes/macrophages (Mo/Mphi) are the major target cells for DEN virus. To determine the effects of the interaction between DEN virus and Mo/Mphi, human monocyte cultures were infected with DEN virus type 2. Apoptosis and production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide were measured in control and infected cultures. Virus was taken up by phagocytosis, but no membrane-coated pits at the virus attachment sites were observed. Increased number of apoptotic cells and increased production of TNF-a were observed in infected monocyte cultures. No increase in production of nitric oxide was observed. These results may be related to early primary viral infection, in which virus could induce apoptosis in monocytes, but monocytes may contribute to host defense mechanisms against virus by viral phagocytosis, phagocytosis of infected apoptotic cells, and the release of proinflammatory cytokines.     

广东南海市登革热病原学及血清学检测

目的　为了明确广东省南海市１９９８ 年夏、秋季一批发烧、出疹病人的诊断。方法收集疑似登革热（ＤＦ）患者血清５２份,应用逆转录 聚合酶链反应（ＲＴ ＰＣＲ）、病毒分离和间接免疫荧光技术分别进行了病原学和血清学检测。结果　从２５ 份发病早期患者血标本中分离病毒１０ 份,经用单克隆抗体间接免疫荧光检测和ＲＴ ＰＣＲ检测证实ＤＥＮ２ 型感染。ＩｇＧ抗体检测,阳性率为４６－１５％ （２４／５２） ,最高滴度达１∶１２８０,ＩｇＭ 抗体检测阳性率为６０％ （１５／２５）。部分病人双份抗体检测,抗体滴度４ 倍升高。结论　此次南海市登革热暴发流行为登革２ 型感染所致。     

Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay

Type-specific monoclonal antibodies prepared against the four dengue (DEN) virus serotypes were evaluated for their ability to identify low-passage human and mosquito isolates from Jamaica and West Africa by an indirect immunofluorescence assay. Serotyped human isolates from Jamaican dengue fever patients included 12 DEN-1, two DEN-2, and five DEN-4 viruses. Viruses from West Africa included 84 DEN-2 mosquito strains as well as two DEN-1 and one DEN-2 from humans. Results obtained using the immunofluorescence assay were consistent with virus identifications obtained using the more classical but costly and time-consuming plaque-reduction neutralization test. More viral isolates and higher virus yields were obtained using the C6/36 clone of Aedes albopictus cells rather than LLC-MK2 (monkey kidney) cells. Dengue type-specific monoclonal antibodies detected prototype viral antigens 24-48 hours postinfection in C6/36 cells. This is the first time that monoclonal antibodies have been used to serotype low-passage flavivirus isolates.     

A prospective seroepidemiologic study on dengue in children four to nine years of age in Yogyakarta, Indonesia I. studies in 1995-1996.

A prospective study on dengue (DEN) viruses was initiated in October 1995 in Gondokusuman kecamatan, Yogyakarta, Indonesia. This report presents data from the first year of the study. The studied cohort included all children 4-9 years of age living in the kecamatan. Blood samples for serology were collected from 1,837 children in October 1995 and again in October 1996. Blood samples for virus isolation and serology were collected from cohort children who were seen in municipal health clinics with febrile syndromes or admitted to hospitals with a provisional diagnosis of dengue hemorrhagic fever. Dengue serotype antibody prevalence and 1995-1996 infection rates were calculated using a single dilution (1:60) 70% plaque reduction endpoint neutralization test. Prevalence of dengue antibody at the beginning of the study was DEN 1 = 12%, DEN 2 = 16%, DEN 3 = 2%, DEN 4 = 4%, and two or more dengue infections = 22%. Total dengue antibody prevalence increased from 38% in 4-year-old children to 69% in 9-year-old children. During the observation period, primary dengue infection rates were DEN 1 = 4.8%, DEN 2 = 7.7%, DEN 3 = 4.2%, and DEN 4 = 3.4%, while two or more dengue infections occurred in 6.7% of the study population. The secondary dengue infection rate was 19.0%. From febrile cases, all four dengue viruses were isolated with DEN 3 predominating. Seven children were hospitalized, including one fatal case with a hospital diagnosis of dengue shock syndrome. Based upon presence of antibody in the initial cohort bleeding and the serologic response both weeks and several months following illness, all had secondary dengue infections. Neutralizing antibody patterns in the initial cohort bleeding and in late convalescent serum samples permitted recognition of dengue infection sequence in five patients: DEN 2-DEN 1 (3), DEN 2-DEN 4 (1), DEN 1-DEN 3 (1), and none in the sequence DEN 1-DEN 2. In the total cohort 6.5% of the observed secondary infections were of the sequence DEN 2-DEN 1, while 4.9% were DEN 1-DEN 2, a highly pathogenic sequence in previous studies. Reduced pathogenic expression of secondary DEN 2 with enhanced pathogenic expression of secondary DEN 1 infections was an unexpected finding. Further studies will be required to understand the respective contributions to pathogenicity of antibody from initial dengue infections versus the biological attributes of the second infecting dengue viruses.     

我国登革2型病毒分离株E蛋白B抗原区的表达与鉴定

目的登革2型病毒(DEN2)E蛋白B抗原区的融合表达、纯化和抗原性初步分析。方法将DEN2GZ14株E蛋白B抗原区基因连接到克隆载体pGEM-T并转染E.coliDH5α,酶切后连接至表达载体pET-32a(+)并转染E.coliBL21,IPGT诱导表达,WesternBlot和间接ELISA分析表达产物的抗原性。结果含有DEN2E蛋白B抗原区基因质粒的表达菌表达了相对分子量为31kDa的融合蛋白,WesternBlot分析表明其能与小鼠多克隆抗体发生特异性反应,间接ELISA分析其与10例登革2型病毒感染者血清发生阳性反应。结论获得的DEN2GZ14株E蛋白B抗原区的基因表达蛋白有良好的抗原性。     

抗体依赖增强效应发生机制研究进展

在各类病原体特别是病毒的感染中,抗体依赖增强效应(Antibody-dependent enhancement,ADE)可使原有的感染加重,引起严重疾病。研究发现多种病原体的感染过程中均有抗体依赖增强效应ADE现象,且可能存在不同的发生机制。随着抗体依赖增强效应ADE现象产生机制研究的不断深入,有助于相应病原体疫苗的改造,从而使疫苗效用最大化,对控制包括寨卡病毒在内的病原体的感染将提供巨大帮助。本文就近年来抗体依赖增强效应ADE发生机制的研究进展进行综述,包括Fc段受体依赖、补体系统介导、非中和抗体介导、病毒表面蛋白介导及细胞活动介导的抗体依赖增强效应ADE机制,同时以登革病毒、人类免疫缺陷病毒、柯萨奇病毒、埃博拉病毒、寨卡病毒及其他病原体为例进行分类介绍。     

Absence of dengue 2 infection enhancement in human sera containing Japanese encephalitis antibodies

Sera from 52 young adults resident in a rural area in North Thailand were studied for plaque-reducing neutralizing antibodies against dengue (DEN) viruses types 1-4 and Japanese encephalitis (JE), and for DEN-2 infection-enhancing antibodies using a newly described microtest in the human monocyte cell line, U-937. Infection-enhancing antibody titers in U-937 cells using a simplified micromethod were similar to results obtained by published methods using human peripheral blood leukocytes and a macrotest using U-937 cells. In the sample, there were 23 with antibodies to one or more DEN viruses with or without accompanying JE antibodies; 16 sera demonstrated antibodies only to JE and 13 had no detectable antibodies to any flavivirus. All but two DEN antibody-containing sera enhanced DEN-2 infections in U-937 cells, often to titers of 1:10,000 or greater. By contrast, only one of 16 JE-immune sera enhanced DEN-2 infection in monocytes, and that at a dilution of 1:100. None of the flavivirus-negative sera had DEN-2 enhancing activity. The failure of human anti-JE contrasts with the ability of rabbit anti-JE to enhance DEN-2 infections, but correlates with the absence of recorded instances of dengue shock syndrome in human beings sequentially infected with JE and then a DEN virus. This report seemingly reconciles in vitro and in vivo phenomena, and may provide an opportunity to study mechanisms involved.     

Flow cytometric analysis of dengue virus-infected cells in peripheral blood

With the development of permeabilization techniques in flow cytometry and the availability of various monoclonal antibodies (MAbs) that specifically bind with cell surface and intracellular antigens, it is now possible to use flow cytometric assay to identify dengue virus (DEN) infected cells in peripheral blood. Blood samples were analyzed using phycoerythrin (PE) labeled anti-CD3, anti-CD14, anti-CD16, and anti-CD19 antibodies and Alexa Fluor 488 labeled anti-flavivirus monoclonal antibody (MAb) 6B6C-1. The predominant DEN-infected cells were CD19+ in this study. There was dim partial to moderately bright partial expression of CD19 positive cells in the blood samples tested. Virus isolation and serotype-specific RT-PCR revealed the cells were infected with dengue serotype 3 (DEN3). Our results suggest B cells may play an important role in DEN1 and DEN3 replication, and dissemination in vivo.     

Clinical differences among PCR-proven dengue serotype infections

The objective of this study was to compare the clinical spectra of the dengue serotypes proven by the PCR technique. This retrospective study reviewed the clinical information of dengue-infected patients who were admitted to northeastern provincial hospitals in Thailand from June to September 2002. Dengue infection and viral serotypes were confirmed by polymerase chain reaction (PCR). Paired anti-dengue immunoglobulin G (IgG) and IgM from paired sera were analyzed by enzyme-linked immunosorbent assay (ELISA). Ninety-nine PCR-proven dengue-infected Thai patients were studied. Their ages ranged from 3-30 years. They were infected with DEN1, DEN2, DEN3 and DEN4 in 21, 55, 12, and 12%, respectively. Twenty-two percent had primary and 78% had secondary infections. Dengue fever was the most common presentation for both primary (77.2%) and secondary infections (46.7%). The ratios of dengue fever:dengue hemorrhagic fever (DF:DHF) and non-dengue shock syndrome:dengue shock syndrome (non-DSS:DSS) for DEN2 was the lowest of the dengue serotypes. There was no difference in the duration of fever, percentage of hepatomegaly and bleeding among the serotypes in both DF and DHF. The trends in the white blood cells, lymphocyte and atypical lymphocyte counts in DEN3 were the highest, while those of DEN1 were the lowest of the dengue serotypes.     

Replication of chimeric yellow fever virus-dengue serotype 1-4 virus vaccine strains in dendritic and hepatic cells

ChimeriVax-dengue (DEN) viruses are live attenuated vaccine candidates. They are constructed by replacing the premembrane (prM) and envelope (E) genes of the yellow fever (YF) 17D virus vaccine with the corresponding genes from wild-type DEN viruses (serotypes 1-4) isolated from humans. In this study, the growth kinetics of ChimeriVax-DEN1-4 and parent viruses (wild-type DEN-1-4 and YF 17D) were assessed in human myeloid dendritic cells (DCs) and in three hepatic cell lines (HepG2, Huh7, and THLE-3). In DC, ChimeriVax-DEN-1-4 showed similar growth kinetics to their parent viruses, wild-type DEN virus (propagated in Vero cells), or YF 17D virus (peak titers ~3-4.5 log(10) plaque-forming units (PFU)/mL at 48-72 hours post-infection). Parent wild-type DEN-1-4 viruses derived from C6/36 mosquito cells did not show any growth at a multiplicity of infection of 0.1 in DCs, except for DEN-2 virus, which grew to a modest titer of 2.5 log(10) PFU/mL at 48 hours post-infection. ChimeriVax-DEN1-4 grew to significantly lower titers (2-5 log(10) PFU/mL) than YF 17D virus in hepatic cell lines THLE-3 and HepG2, but not in Huh7 cells. These experiments suggest that ChimeriVax-DEN1-4 viruses replicate similarly to YF-VAX in DCs, but at a lower level than YF 17D virus in hepatic cell lines. The lack of growth of chimeric viruses in human hepatic cells suggests that these viruses may be less hepatotropic than YF 17D virus vaccine in humans.