Protection against Enterovirus 71 with Neutralizing Epitope Incorporation within Adenovirus Type 3 Hexon

Enterovirus 71 (EV71) is responsible for hand, foot and mouth disease with high mortality among children. Various neutralizing B cell epitopes of EV71 have been identified as potential vaccine candidates. Capsid-incorporation of antigens into adenovirus (Ad) has been developed for a novel vaccine approach. We constructed Ad3-based EV71 vaccine vectors by incorporating a neutralizing epitope SP70 containing 15 amino acids derived from capsid protein VP1 of EV71 within the different surface-exposed domains of the capsid protein hexon of Ad3EGFP, a recombinant adenovirus type 3 (Ad3) expressing enhanced green fluorescence protein. Thermostability and growth kinetic assays suggested that the SP70 epitope incorporation into hypervariable region (HVR1, HVR2, or HVR7) of the hexon did not affect Ad fitness. The SP70 epitopes were thought to be exposed on all hexon-modified intact virion surfaces. Repeated administration of BALB/c mice with the modified Ads resulted in boosting of the anti-SP70 humoral immune response. Importantly, the modified Ads immunization of mother mice conferred protection in vivo to neonatal mice against the lethal EV71 challenge, and the modified Ads-immunized mice serum also conferred passive protection against the lethal challenge in newborn mice. Compared with the recombinant GST-fused SP70 protein immunization, immunization with the Ads containing SP70 in HVR1 or HVR2 elicited higher SP70-specific IgG titers, higher neutralization titers, and conferred more effective protection to neonatal mice. Thus, this study provides valuable information for hexon-modified Ad3 vector development as a promising EV71 vaccine candidate and as an epitope-delivering vehicle for other pathogens.     

Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.     

Structural Characterization of the Complex of SecB and Metallothionein-Labeled proOmpA by Cryo-Electron Microscopy

ProOmpA is a preprotein that is translocated across the plasma membrane by the general secretory pathway in Escherichia coli. The molecular chaperon SecB in Sec pathway can recognize and bind proOmpA for its translocation. However, the structure of the SecB/proOmpA complex remains unknown. Here, we constructed an uncleavable proOmpA fused with metallothionein at its C-terminus and labeled it with metals in vitro for the study of cryo-electron microscopy. Using single particle cryo-electron microscopy, we reconstructed 3D structure of the stable SecB/proOmpA complex. The structure shows that the major portion of preprotein locates on one side of SecB tetramer, resulting in an asymmetric binding pattern. This work also provides a possible approach to the structure determination of small protein complexes by cryo-electron microscopy.     

Selection of Peptide Mimics of HIV-1 Epitope Recognized by Neutralizing Antibody VRC01

The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.     

Conformational Plasticity of the 2A Proteinase from Enterovirus 71

The 2A proteinase (2A^pro) is an enterovirally encoded cysteine protease that plays essential roles in both the processing of viral precursor polyprotein and the hijacking of host cell translation and other processes in the virus life cycle. Crystallographic studies of 2A^pro from enterovirus 71 (EV71) and its interaction with the substrate are reported here. EV71 2A^pro was comprised of an N-terminal domain of a four-stranded antiparallel β sheet and a C-terminal domain of a six-stranded antiparallel β barrel with a tightly bound zinc atom. Unlike in other 2A^pro structures, there is an open cleft across the surface of the protein in an open conformation. As demonstrated by the crystallographic studies and modeling of the complex structure, the open cleft could be fitted with the substrate. On comparison 2A^pro of EV71 to those of the human rhinovirus 2 and coxsackievirus B4, the open conformation could be closed with a hinge motion in the bII2 and cII β strands. This was supported by molecular dynamic simulation. The structural variation among different 2A^pro structures indicates a conformational flexibility in the substrate-binding cleft. The open structure provides an accessible framework for the design and development of therapeutics against the viral target.     

入源中和性抗甲型肝炎病毒抗体Fab段基因的序列分析及可溶性表达

在用噬菌体表面呈现系统获得人源抗甲型肝炎(甲肝)病毒中和性基因工程Fab抗体的基础上,对所获得的4株中和性Fab抗体轻重链可变区基因进行了序列分析、可溶性表达及生物学特性鉴定.4株Fab抗体重链可变区拥有99%同源的核苷酸序列和相同的CDR区氨基酸序列,属于VHⅢ基因家族.而轻链可变区核苷酸序列同源性为95%和相似的CDR区氨基酸序列,属于VL5基因家族.这些重组抗体都能与人甲肝恢复期血清及具有中和活性的鼠抗甲肝单克隆抗体产生竞争抑制反应,表明其针对甲肝病毒结构蛋白上的主要抗原决定簇.     

Structure of IL-17A in Complex with a Potent, Fully Human Neutralizing Antibody

IL-17A is a pro-inflammatory cytokine produced by the newly identified Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-17A (CAT-2200) that can potently neutralize the effects of recombinant and native human IL-17A. We determined the crystal structure of IL-17A in complex with the CAT-2200 Fab at 2.6 Å resolution in order to provide a definitive characterization of the epitope and paratope regions. Approximately a third of the IL-17A dimer is disordered in this crystal structure. The disorder occurs in both independent copies of the complex in the asymmetric unit and does not appear to be influenced by crystal packing. The complex contains one IL-17A dimer sandwiched between two CAT-2200 Fab fragments. The IL-17A is a disulfide-linked homodimer that is similar in structure to IL-17F, adopting a cystine-knot fold. The structure is not inconsistent with the previous prediction of a receptor binding cavity on IL-17 family members. The epitope recognized by CAT-2200 is shown to involve 12 amino acid residues from the quaternary structure of IL-17A, with each Fab contacting both monomers in the dimer. All complementarity-determining regions (CDRs) in the Fab contribute to a total of 16 amino acid residues in the antibody paratope. In vitro affinity optimization was used to generate CAT-2200 from a parental lead antibody using random mutagenesis of CDR3 loops. This resulted in seven amino acid changes (three in VL-CDR3 and four in VH-CDR3) and gave an approximate 30-fold increase in potency in a cell-based neutralization assay. Two of the seven amino acids form part of the CAT-2200 paratope. The observed interaction site between CAT-2200 and IL-17A is consistent with data from hydrogen/deuterium exchange mass spectrometry and mutagenesis approaches.     

Neutralizing Antibodies Induced by Recombinant Virus-Like Particles of Enterovirus 71 Genotype C4 Inhibit Infection at Pre- and Post-attachment Steps

Background

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia–Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear.

Methods/Findings

In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs). Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC) located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment.

Conclusions

Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.     

A Keratin Antibody Recognizing a Heterotypic Complex: Epitope Mapping to Complementary Locations on Both Components of the Complex

Keratin filaments in simple epithelial cells are heteropolymers of keratin 8 (K8) and keratin 18 (K18), which can be stained by the monoclonal antibody (MAb) LE61. This antibody has been widely used to study keratin expression in normal and neoplastic tissues. In this study we have found that MAb LE61 does not react with individual keratin polypeptides either derived from natural sources or expressed as recombinant proteins inEscherichia coli.However, when K8 or K18 bound to nitrocellulose were incubated with complementary keratin they became reactive with this antibody. A mixture of K8 and K18 in solution also reacted strongly with the MAb LE61 in ELISA. These observations suggest that the antibody recognizes a discontinuous epitope on the keratin complex. The antibody also reacted with complexes of K8 and K18 with other keratins. To locate the epitope of this antibody we have expressed K8 and K18 fragments, deleted from the amino- and carboxyl-termini, as fusion proteins with glutathioneS-transferase. These fragments were able to form a heterotypic complex with the complementary keratin. Binding of the MAb LE61 to these complexes mapped the two halves of the epitope on K8, between residues 353 and 367, and on K18, between residues 357 and 385. The two halves of the epitope appear to be in close association in the heterotypic complex since deletions from the amino-terminus did not influence the antibody binding. The highly conserved nature of this epitope in both type I and type II keratins could explain the MAb LE61 reactivity with complexes of K8 or K18 with other keratins.     

应用噬菌体展示技术筛选人巨细胞病毒糖蛋白M新的中和抗原表位

人巨细胞病毒(HCMV)糖蛋白复合物Ⅱ包括两种蛋白,即糖蛋白M(gM)和糖蛋白N(gN)．尽管来自于HCMV阳性病人血清中的糖蛋白复合物Ⅱ的IgG抗体能够中和HCMV粒子,但迄今为止,还没有gM中和性抗原表位的相关研究．应用消减杂交技术,通过噬菌体肽库筛选获得gM抗原的一个表位,即MAD．MAD氨基酸序列与gM第32～38位序列高度同源．将MAD与钥孔血蓝蛋白偶联免疫小鼠可产生抗MAD多抗,该多抗不仅结合天然HCMV病毒粒子,而且特异结合重组表达的gM30～78多肽．ELISA结果表明MAD能够特异结合HCMV阳性的病人血清．病毒中和实验结果进一步证明抗MAD多抗能够抑制HCMV AD169株病毒感染人胚肺细胞．总之,MAD表位有可能成为HCMV病毒疫苗潜在的保护性抗原.     

Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on the Clostridium botulinum Neurotoxin Type A

Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 10³ and 10⁴ times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D⁵) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K⁵). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.     

肠道病毒71型的RT-PCR诊断及基因特征

2002～2003年从上海市和重庆市手足口病患儿的疱液标本中分离到10株病毒,其中上海9株,重庆1株.利用两对分别针对EV71和Cox.A16病毒VP1区的特异性引物,用RT-PCR方法对病毒进行初步鉴别.根据PCR所用引物及PCR扩增出的产物片段大小不同,可初步判定出EV71和Cox.A16病毒的血清型别.RT-PCR结果提示,上海分离到的9株病毒中,有2株为EV71,其余7株病毒为Cox.A16;重庆分离到的1株病毒为EV71.10株病毒的RT-PCR产物经序列测定和分析证实,PCR定型结果正确,说明PCR法具有很高的特异性,可作为EV71初步鉴定的首选方法.对所分离的3株EV71病毒进行VP1区编码基因全序列的测定和遗传学分析,通过同源性比较和构建系统发生树发现,此3株EV71病毒和中国大陆已发表的7株EV71病毒(SHZH03、SHZH98、SH-F1、SH-F2、SH-H25、SH-H26和CHN-87)全部属于C基因型,与该型代表株比较,同源性为89.3%～94.6%;与A、B基因型代表株比较,同源性为81.3%～84.0%,差异较大.在C基因型中,此3株EV71病毒和中国大陆先前分离的6株病毒(SHZH03、SHZH98、SH-F1、SH-F2、SH-H25、SH-H26)的同源性较高,在94.5%～100%范围内.在系统发生树上,这9株病毒形成一个较独立的分支.与CHN-87株的同源性在92.1%～93.6%之间.与已知的C1、C2、C3亚型代表株比较,同源性在89.3%～92.9%,差异≥7%,因此认为可将这9株病毒划分为C4亚型.上海、重庆和深圳的EV71病毒有较高的同源性,提示该病毒EV71-CA亚型在中国大陆1998～2003年间有较广泛的传播.建立中国流行的EV71病毒毒株库和基因库,对诊断、预防和控制EV71在中国的爆发有重要意义,对加强EV71的实验室诊断、病毒学监测和病毒基因型和亚型的标准命名和分子流行病学研究有重要帮助.     

TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71

Enterovirus A71(EV-A71) is one of the etiological pathogens leading to hand, foot, and mouth disease(HFMD), which can cause severe neurological complications. The neuropathogenesis of EV-A71 infection is not well understood. The mislocalization and aggregation of TAR DNA-binding protein 43(TDP-43) is the pathological hallmark of amyotrophic lateral sclerosis(ALS). However, whether TDP-43 was impacted by EV-A71 infection is unknown. This study demonstrated that TDP-43 was cleaved during EV-A71 infection. The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection. TDP-43 is cleaved by viral protease 3 C between the residues 331 Q and332 S, while mutated TDP-43(Q331 A) was not cleaved. In addition, mutated 3 C which lacks the protease activity failed to induce TDP-43 cleavage. We also found that TDP-43 was translocated from the nucleus to the cytoplasm, and the mislocalization of TDP-43 was induced by viral protease 2 A rather than 3 C. Taken together, we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection, implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71 infection.