共生病毒组的组成及其功能

共生病毒广泛参与机体各项生理和病理过程，近年来受到高度关注。共生病毒在自身免疫性疾病、变应性疾病、肿瘤发生、抗病毒免疫反应及炎症性疾病的发生、发展过程中发挥重要作用，其可通过调控免疫细胞的分化发育和功能行使，尤其是细胞因子的分泌进而参与其中。由于二代测序技术的发展，宏基因组学研究技术进展迅速，共生病毒与疾病进展的研究进一步加深了人们对共生病毒功能的理解。由于共生病毒在机体的分布、宿主遗传背景以及共生微生物的不同，共生病毒可能诱导机体产生多种有益或不利的免疫反应。本文简要阐述了共生病毒在机体的分布、组成和作用机制，及其在疾病诊断和治疗中的潜在意义，并对共生病毒未来在疾病诊疗中的潜在应用前景做一展望。     

鳞翅目昆虫抗病毒免疫反应的研究进展

鳞翅目昆虫种类繁多,对农业生产和人类生活产生重大影响,宿主昆虫与病毒相互关系的研究对于利用病毒杀虫剂进行害虫治理和益虫病毒性疾病的预防具有重要意义.因此,鳞翅目昆虫与病毒的互作研究显得尤为重要,宿主昆虫的免疫系统在抗病毒感染过程中发挥着关键作用,对病毒产生不同程度的免疫反应.本文综述了昆虫围食膜和中肠对病毒入侵的防御作用,病毒进入体腔后昆虫所产生的细胞免疫和体液免疫反应,以及RNAi、细胞的自噬与凋亡、Toll、Imd、JAK-STAT和STING信号通路等相关的抗病毒免疫途径,并对昆虫抗病毒免疫研究的制约因素和未来鳞翅目昆虫抗病毒免疫的研究重点进行了讨论,以期为害虫的生物防治和益虫疾病的防控提供理论依据.     

抗EHFV的独特型抗体在小鼠体内诱导免疫应答的研究

本文介绍用流行性出血热（EHF）病毒J10株制备单克隆抗体（McAb）,以及用7个McAb免疫家兔,使其产生抗EHF病毒McAb的抗体即抗独特型 抗体（Ab2).Ab2能在体外同出血热病人恢复期血清和EHF病毒免疫的兔血清发生特异性结合。再经EHF－McAb亲和层析法分离提纯Ab2,免疫BALb/c小鼠,将所获得的免疫血清（Ab3）用荧光和ELISA分别加以测定。结果表明,抗－抗独特型抗体可在体外识别EHF病毒,而不能同病病人恢复期血清发生结合,从而支持免疫网络学说,可能为我们提供一种新型的抗原来源途径。     

病毒核酸定量测定及其医学意义

病毒核酸定量测定对病毒性疾病的诊断和治疗,了解病毒感染与疾病的发生、发展的关系,探讨药物对病毒的作用及抗病毒治疗效果的考核和评价,都有很和重要的作用。本文从核酸杂交技术、ＰＣＲ技术、ＮＡＳＢＡ技术及ｂＤＮＡ技术４个方面对病毒核酸定量测定的分子生物学方法以及病毒核酸定量测定在医学上的意义作一简要综述。     

抗EHFV的独特型抗体在大鼠体内诱导免疫应答的研究

本文介绍用流行性出血热（EHF）病毒J10株制备单克隆抗体（McAb),以及用7个McAb免疫家兔,使其产生抗EHF病毒McAb的抗体即独特型抗体（Ab2）。Ab2能在体外同出血热病人恢复期血清和EHF病毒免疫的兔血清发生特异性结合。再经EHF－McAb亲和层析法分离提纯Ab2,免疫BALb/c小鼠,将所获得的免疫血清（Ab3）用荧光和ELISA分别加以测定,结果表明,抗－抗独特型抗体可在体外识别EHF病毒,而不能同病病人恢复期血清发生结合,从而支持免疫网络学说,可能为我们提供一种新型的抗原来源途径。     

全球抗病毒药物研发进展与展望

病毒是危害人体健康的主要病原体之一,病毒感染和传播造成的传染性疾病严重威胁人类健康。目前,艾滋病、病毒性肝炎等发病率高、治愈率低的病毒性疾病仍在全球蔓延,流感病毒、冠状病毒等呼吸道病毒不断发生变异,2019年以来,新冠病毒引起的全球疫情对世界各国产生巨大影响,疫情走向还存在很大不确定性,开发安全有效的抗病毒药物成为应对病毒性疾病的重要手段。拟在总结全球抗病毒药物研发整体现状的基础上,分析抗艾滋病病毒、肝炎病毒、新冠病毒等重点领域的新药研发进展,提出抗病毒药物的发展建议,为未来研发更加高效的抗病毒药物提供指引和参考。     

基于寨卡病毒复制子的抗病毒药物筛选系统

寨卡病毒(Zika virus, ZIKV)是引起成人格林巴利综合征(Guillain-Barre syndrome, GBS)以及胎儿和新生儿小头畸形疾病的重要病原体。有效的抗病毒药物筛选系统是防治ZIKV感染的有效工具。本研究构建了以海肾荧光素酶为检测靶标的寨卡病毒复制子Rluc-ZIKV-Rep,并通过突变NS5蛋白,构建了非复制型寨卡病毒复制子Rluc-ZIKV-Rep NS5△GDD。为了检测复制子的功能,将两种复制子转化至Vero细胞,结果显示随着时间延长, Rluc-ZIKV-Rep荧光素酶活性逐渐增加,寨卡病毒m RNA拷贝数逐渐增多,证明以海肾荧光素酶为检测靶标的寨卡病毒复制子构建成功。为了检测该系统是否能够用于抗病毒药物筛选,用马尼地平和西尼地平两种抗寨卡病毒的药物进行检测,结果显示Rluc-ZIKV-Rep的复制能力随着药物浓度的增加而逐渐降低,呈现一种剂量依赖的方式,说明该系统可以用于抗病毒药物的筛选。综上所述,本研究构建了一种可以用于抗病毒药物筛选的寨卡病毒复制子,为筛选有效的抗病毒药物提供了有力工具。     

IL-27：介导免疫相关疾病的新因子

白细胞介素27 (interleukin 27, IL-27)是一种多效性的细胞因子,能参与调节机体的先天性和适应性免疫。以往研究显示它能介导体内多种炎症反应,随着动物模型和技术手段的发展,多项研究表明它与自身免疫性疾病及其他免疫相关疾病密切相关,并被认为是治疗病毒性疾病、自身免疫性疾病、肿瘤及肥胖的一个重要候选因子。因此本文就IL-27在艾滋病、类风湿关节炎、肿瘤及肥胖中的作用展开综述,旨在为免疫相关疾病的治疗提供新的思路。     

人生长分化因子15单克隆抗体制备、鉴定及药用价值初探

目的:制备稳定分泌抗人生长分化因子15(GDF15)单克隆抗体(m Ab)的杂交瘤细胞系,并对其分泌的m Ab进行鉴定。方法:根据人GDF15氨基酸序列特征,设计合成了8条能够免疫产生GDF15特异性抗体的抗原多肽,与VLP载体偶联后,免疫雌性BALB/c小鼠,利用杂交瘤技术制备鼠源抗人GDF15的m Ab,用间接ELISA检测m Ab腹水效价。结果:获得针对7个抗原多肽的12株稳定分泌抗人GDF15的杂交瘤细胞系,腹水m Ab效价可达1×104~1×109。结论:获得了针对不同抗原多肽的抗人GDF15的特异性m Ab,为进一步研发以GDF15为靶点的单克隆抗体抗肿瘤药物奠定了基础。     

清除或劫持:细胞自噬与病毒感染

细胞自噬是广泛存在于真核生物中的生命现象,在进化上高度保守。作为一种防御和应激调控机制,自噬在维持细胞内稳态中发挥重要作用。近年来多项研究发现自噬能够参与机体免疫应答,产生抗病毒效应;而某些病毒则进化出一些策略劫持自噬来利于自身的增殖。通过干预自噬途径寻找药物新靶点和治疗策略逐渐成为抗病毒治疗的重要方向。本文概述了细胞自噬与病毒感染之间关系,以期为抗病毒研究提供参考。     

Detection of advanced oxidation protein products in patients with chronic kidney disease by a novel monoclonal antibody

Abstract

Advanced oxidation protein products (AOPP) as a biomarker of oxidative stress has been demonstrated in chronic kidney disease (CKD) patients; however, current methods to detect the accumulation of AOPP in serum and in tissues are limited and unreliable. This study generated a monoclonal antibody (mAb) designated 3F2, that reacts specifically with hypochlorous acid (HOCl)-modified proteins, but not with the native forms or with other types of oxidative modifications. Notably, mAb 3F2 recognizes the AOPP deposited in renal tissues of AOPP-treated rats and of patients with different kinds of CKD. Moreover, this mAb can almost completely inhibit the production of reactive oxygen species in RAW264.7 cells induced by AOPP (p < 0.001). In conclusion, mAb 3F2 can be used to detect AOPP specifically in serum and in tissues, and this antibody can potentially provide an important tool and new insight into research on diseases related to oxidative stress.     

单克隆抗体及其抗感染作用的研究进展

骨髓瘤细胞和单个B细胞可融合形成杂交瘤细胞,这种细胞分离并克隆后可产生针对单一表位,且结构和功能相同的抗体,即单克隆抗体(monoclonal antibody, mAb)。单克隆抗体发展历程经历了四个阶段。其制备技术也在不断革新与发展。目前单克隆抗体技术发展日益成熟,在疾病治疗与诊断中发挥着关键的作用。单克隆抗体与天然抗体不同,具有效价高、特异性强、交叉反应少、可大量制备,靶向性高等特点。mAb可用于诊断及治疗感染性疾病、自身免疫病以及癌症等。对mAb的发展历程及mAb在抗感染性疾病中的应用进行简要阐述。     

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.     

Discovery and mechanism of ustekinumab: A human monoclonal antibody targeting interleukin-12 and interleukin-23 for treatment of immune-mediated disorders

Monoclonal antibody (mAb) therapy was first established upon the approval of a mouse antibody for treatment of human acute organ rejection. However, the high incidence of immune response against the mouse mAb restricted therapeutic utility. Development of chimeric, “humanized” and human mAbs broadened therapeutic application to immune-mediated diseases requiring long-term treatment. Indeed, mAb therapeutics targeting soluble cytokines are highly effective in numerous immune-mediated disorders. A recent example is ustekinumab, a first-in-class therapeutic human immunoglobulin (Ig) G₁ kappa mAb that binds to the interleukins (IL)-12 and IL-23, cytokines that modulate lymphocyte function, including T-helper (Th) 1 and Th17 cell subsets. Ustekinumab was generated via recombinant human IL-12 immunization of human Ig (hu-Ig) transgenic mice. Ustekinumab binds to the p40 subunit common to IL-12 and IL-23 and prevents their interaction with the IL-12 receptor β1 subunit of the IL-12 and IL-23 receptor complexes. Ustekinumab is approved for treatment of moderate-to-severe plaque psoriasis and has demonstrated efficacy in Crohn disease and psoriatic arthritis. The clinical characterization of ustekinumab continues to refine our understanding of human immune pathologies and may offer a novel therapeutic option for certain immune-mediated diseases.Key words: ustekinumab, psoriasis, monoclonal antibody, interleukin-12/23p40     

Viral fitness can influence the repertoire of virus variants selected by antibodies

Minority genomes in the mutant spectra of viral quasispecies may differ in relative fitness. Here, we report experiments designed to evaluate the contribution of relative fitness to selection by a neutralizing monoclonal antibody (mAb). We have reconstructed a foot-and-mouth disease virus (FMDV) quasispecies, with two matched pairs of distinguishable mAb-escape mutants as minority genomes of the mutant spectrum. Each mutant of a pair differs from the other by 11-fold or 33-fold in relative fitness. Analysis of the mutant spectra of virus populations selected with different concentrations of antibody in infections in liquid culture medium has documented a dominance of the high fitness counterpart in the selected population. Plaque development as a function of increasing concentration of the antibody has shown that each mutant of a matched pair yielded the same number of plaques, although the high fitness mutant required less time for plaque formation, and attained a larger plaque size at any given time-point. This result documents equal intrinsic resistance to the antibody of each mutant of a matched pair, confirming previous biochemical, structural, and genetic studies, which indicated that the epitopes of each mutant pair were indistinguishable regarding reactivity with the monoclonal antibody. Thus, relative viral fitness can influence in a significant way the repertoire of viral mutants selected from a viral quasispecies by a neutralizing antibody. We discuss the significance of these results in relation to antibody selection, and to other selective forces likely encountered by viral quasispecies in vivo.     

Detection of advanced oxidation protein products in patients with chronic kidney disease by a novel monoclonal antibody

Advanced oxidation protein products (AOPP) as a biomarker of oxidative stress has been demonstrated in chronic kidney disease (CKD) patients; however, current methods to detect the accumulation of AOPP in serum and in tissues are limited and unreliable. This study generated a monoclonal antibody (mAb) designated 3F2, that reacts specifically with hypochlorous acid (HOCl)-modified proteins, but not with the native forms or with other types of oxidative modifications. Notably, mAb 3F2 recognizes the AOPP deposited in renal tissues of AOPP-treated rats and of patients with different kinds of CKD. Moreover, this mAb can almost completely inhibit the production of reactive oxygen species in RAW264.7 cells induced by AOPP (p < 0.001). In conclusion, mAb 3F2 can be used to detect AOPP specifically in serum and in tissues, and this antibody can potentially provide an important tool and new insight into research on diseases related to oxidative stress.     

GM-CSF and IL-2 as adjuvant enhance the immune effect of protein vaccine against foot-and-mouth disease

Background

The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported.

Results

In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae.

Conclusion

The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex.     

Substitution at Aspartic Acid 1128 in the SARS Coronavirus Spike Glycoprotein Mediates Escape from a S2 Domain-Targeting Neutralizing Monoclonal Antibody

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941–50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111–1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.     

Improved in vitro and in vivo activity against CD303-expressing targets of the chimeric 122A2 antibody selected for specific glycosylation pattern

Plasmacytoid dendritic cells (pDCs) play a central role for both innate and adaptive antiviral responses, as they direct immune responses through their unique ability to produce substantial concentrations of type I interferon (IFNs) upon viral encounter while also activating multiple immune cells, including macrophages, DCs, B, natural killer and T cells. Recent evidence clearly indicates that pDCs also play a crucial role in some cancers and several auto-immune diseases. Although treatments are currently available to patients with such pathologies, many are not fully efficient. We are proposing here, as a new targeted-based therapy, a novel chimeric monoclonal antibody (mAb) that mediates a strong cellular cytotoxicity directed against a specific human pDC marker, CD303. This antibody, ch122A2 mAb, is characterized by low fucose content in its human IgG1 constant (Fc) region, which induces strong in vitro and in vivo activity against human pDCs. We demonstrated that this effect relates in part to its specific Fc region glycosylation pattern, which increased affinity for CD16/FcγRIIIa. Importantly, ch122A2 mAb induces the down-modulation of CpG-induced IFN-α secretion by pDCs. Additionally, ch122A2 mAb shows in vitro high pDC depletion mediated by antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis. Remarkably, in vivo ch122A2 mAb efficacy is also demonstrated in humanized mice, resulting in significant pDC depletion in bloodstream and secondary lymphoid organs such as spleen. Together, our data indicates that ch122A2 mAb could represent a promising cytotoxic mAb candidate for pathologies in which decreasing type I IFNs or pDCs depleting may improve patient prognosis.     

A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy

Antiviral monoclonal antibodies (mAbs) represent promising therapeutics. However, most mAbs-based immunotherapies conducted so far have only considered the blunting of viral propagation and not other possible therapeutic effects independent of virus neutralization, namely the modulation of the endogenous immune response. As induction of long-term antiviral immunity still remains a paramount challenge for treating chronic infections, we have asked here whether neutralizing mAbs can, in addition to blunting viral propagation, exert immunomodulatory effects with protective outcomes. Supporting this idea, we report here that mice infected with the FrCas^E murine retrovirus on day 8 after birth die of leukemia within 4–5 months and mount a non-protective immune response, whereas those rapidly subjected to short immunotherapy with a neutralizing mAb survive healthy and mount a long-lasting protective antiviral immunity with strong humoral and cellular immune responses. Interestingly, the administered mAb mediates lysis of infected cells through an antibody-dependent cell cytotoxicity (ADCC) mechanism. In addition, it forms immune complexes (ICs) with infected cells that enhance antiviral CTL responses through FcγR-mediated binding to dendritic cells (DCs). Importantly, the endogenous antiviral antibodies generated in mAb-treated mice also display the same properties, allowing containment of viral propagation and enhancement of memory cellular responses after disappearance of the administered mAb. Thus, our data demonstrate that neutralizing antiviral mAbs can act as immunomodulatory agents capable of stimulating a protective immunity lasting long after the end of the treatment. They also show an important role of infected-cells/antibody complexes in the induction and the maintenance of protective immunity through enhancement of both primary and memory antiviral T-cell responses. They also indicate that targeting infected cells, and not just viruses, by antibodies can be crucial for elicitation of efficient, long-lasting antiviral T-cell responses. This must be considered when designing antiviral mAb-based immunotherapies.