抗呼吸道合胞病毒融合蛋白单链抗体的筛选及初步鉴定

目的：从大容量噬菌体抗体库中筛选人源性抗呼吸道合胞病毒F蛋白的单链抗体。方法：以RSV F蛋白为靶抗原,通过“吸附-洗涤-洗脱-扩增”过程从天然人源性噬菌体抗体库中筛选特异性抗F蛋白单链抗体。5轮筛选后,单克隆经ELISA检测,阳性克隆进行核酸序列分析,并将阳性克隆噬菌体感染E.coli HB2151,经IPTG诱导,制备抗RSV F蛋白的可溶性单链抗体,并进行Western及Dot blot分析。结果：经过筛选,获得了18株能与F蛋白特异性结合的阳性克隆,取OD值最高的克隆E4经测序并检索Kabat数据库分析,显示其基因与人免疫球蛋白可变区基因具有高度同源性,Western及Dot blot分析表明为单链抗体。结论：利用天然人源性噬菌体抗体库技术制备出高特异性的人源性抗RSV F蛋白单链抗体。     

Antigenic Structure of Human Respiratory Syncytial Virus Fusion Glycoprotein

New series of escape mutants of human respiratory syncytial virus were prepared with monoclonal antibodies specific for the fusion (F) protein. Sequence changes selected in the escape mutants identified two new antigenic sites (V and VI) recognized by neutralizing antibodies and a group-specific site (I) in the F1 chain of the F molecule. The new epitopes, and previously identified antigenic sites, were incorporated into a refined prediction of secondary-structure motifs to generate a detailed antigenic map of the F glycoprotein.     

Sustained Protein Kinase D Activation Mediates Respiratory Syncytial Virus-Induced Airway Barrier Disruption

Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how respiratory syncytial virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here, we studied the effect of RSV infection on the airway epithelial barrier using model epithelia. 16HBE14o- bronchial epithelial cells were grown on Transwell inserts and infected with RSV strain A2. We analyzed (i) epithelial apical junction complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to fluorescein isothiocyanate (FITC)-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pretreated or not with protein kinase D (PKD) inhibitors. UV-irradiated RSV served as a negative control. RSV infection led to a significant reduction in TEER and increase in permeability. Additionally it caused disruption of the AJC and remodeling of the apical actin cytoskeleton. Pretreatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin binding protein cortactin in a PKD-dependent manner. UV-inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potential new therapeutic target for RSV-mediated lung diseases.     

Bovine Respiratory Syncytial Virus

A large epizootic of an acute respiratory disease of cattle occurred in Japan during the months from October 1968 to May 1969. A virus was recovered in primary cultures of calf kidney and testicle cells from nasal swabs of affected cattle. Neutralization tests revealed the virus to be closely related to the Long strain of human respiratory syncytial virus. The virus induced cytopathic changes including the formation of syncytia and acidophilic-cytoplasmic inclusions in calf kidney and testicle cell cultures. A calf inoculated with the virus by the respiratory route developed an illness resembling the natural disease. Most cattle clinically diagnosed as having the disease showed significant rises of neutralizing antibody titer for the isolated virus, whereas none or only small fractions of those animals showed serological evidence for recent infection with bovine ephemeral fever virus, infectious bovine rhinotracheitis virus, Ibaraki virus, bovine diarrhea virus, bovine adenovirus Type 7 and parainfluenza virus Type 3. Neutralization tests on paired sera revealed a wide dissemination of the isolated virus among cattle in many areas of the country during the epizootic. All these findings leave no doubt that the epizootic was caused by bovine respiratory syncytial virus. This is the first study that ever shows the presence of infection of cattle with this virus in Japan.     

Intrapatient Variation of the Respiratory Syncytial Virus Attachment Protein Gene

Intrapatient variability of the attachment (G) protein gene of respiratory syncytial virus (RSV) was examined using both population and single-genome sequencing. Samples from three patients infected with a group B virus variant which has a 60-nucleotide duplication in the G protein gene were examined. These samples were chosen because occasional mixed sequence bases were observed. In a minority of RSV genomes from these patients considerable variability was found, including point mutations, insertions, and deletions. Of particular note, the deletion of the exact portion of the gene which had been duplicated in some isolates was observed in viral RNAs from two patients.Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants and vulnerable adults (3, 9) and is unusual in that it can repeatedly reinfect individuals (5, 6). RSV isolates are classified into two groups, A and B, and the attachment (G) protein, a target for neutralizing antibodies, is the most variable of the viral proteins, showing considerable genetic and antigenic variability both within and between the groups (7, 8). The G protein is able to accommodate drastic changes, which have been observed both in culture during the selection of monoclonal antibody escape mutants (4, 12, 13) and in vivo with the emergence of new variants, including a group B strain with a duplication of 60 nucleotides (17). This strain with a 60-nucleotide duplication was first reported from Buenos Aires in 1999 (17) and then was subsequently detected in samples from 1998 in Madrid (16). The strain then became the dominant group B strain worldwide, indicating a selective advantage for this variant (16, 18). Thus, major genetic changes can be introduced into the G gene sequence while the virus replicates in its natural host, which can then be selected under favorable epidemiological conditions.Previous investigations of the genetic diversity of RSV exploited direct sequencing of PCR-amplified products (2), which represent the population average of the in vivo variants. Such sequences are derived from multiple copies of cDNA and represent the dominant sequence, and they thus do not allow detection of minority populations below about 20% prevalence (11). Information on intrapatient viral diversity during infections may therefore be missed, knowledge of which could be important in the overall understanding of the genetic diversity of this virus. We report here the analysis of individual RSV RNA molecules derived by single-genome amplification (SGA) and sequencing from clinical samples using a methodology developed for the analysis of HIV genomes (11, 14).RSV-positive samples were collected from infants admitted to Kilifi District Hospital, Kenya (10). Viral RNA extraction and cDNA synthesis were carried out as previously described (15). For population sequencing, a nested PCR was carried out on the cDNA using primers that amplified the ectodomain-coding part of the G protein gene, with the PCR product being directly sequenced. In the 2007-2008 RSV epidemic in Kilifi, group B viruses were predominant. By population sequencing of ∼100 group B samples, all were found to have the 60-nucleotide duplication observed in the Buenos Aires variant (data not shown). However, in some specimens there were some mixed bases at some positions, so the variability at the level of the single cDNA molecule was further investigated.Three samples that gave occasional mixed signals in the sequence chromatograms were further analyzed by SGA and sequencing. For SGA the cDNAs were serially diluted 3-fold up to 1:6,361. Ten nested PCRs were carried out on each dilution using Platinum high-fidelity PCR Supermix (Invitrogen) (containing Taq polymerase together with the proofreading enzyme Pyrococcus species GB-D polymerase). Based on the Poisson distribution, it has been shown that for a sample dilution yielding approximately 30% positive PCRs there is an 80% likelihood that each PCR is derived from a single cDNA molecule (11). For each of the identified endpoint dilutions, the cDNA was amplified in 80 separate nested PCRs using the high-fidelity enzyme and the positive reaction products sequenced. The nomenclature for the sequences reported in this paper is place of isolation (Kenya [Ken])/year of isolation/strain number. For SGA sequences an additional Roman number is given.The predicted length derived by population sequencing of the G proteins of the three samples examined by SGA was 310 amino acids, showing a 6-nucleotide deletion and a changed stop codon relative to the Buenos Aires strain (Fig. ​(Fig.1).1). The dominant sequences represented 60 to 88% of the sequences derived by SGA. The differences were due to point mutations, duplications, and deletions, as summarized in Table ​Table1;1; the consequences of these changes for the predicted length of the G protein are shown in Table ​Table22.Open in a separate windowFIG. 1.Nucleotide sequence alignment of part of the G protein gene (from nucleotide 400) of the sample 2 population sequence (Ken/08/80900) and a minority sequence (Ken/08/80900/ii), with the sequences of prototype group B strain CH18537 (accession number {"type":"entrez-nucleotide","attrs":{"text":"M17213","term_id":"333942","term_text":"M17213"}}M17213) and Buenos Aires strain BA/3833/99B (accession number {"type":"entrez-nucleotide","attrs":{"text":"AY333362","term_id":"33355616","term_text":"AY333362"}}AY333362). This shows the duplication of 60 nucleotides in the Kenyan and Buenos Aires viruses relative to CH18537 and the loss of the same 60 nucleotides in the Kenyan minority sequence. Termination codons are underlined.

TABLE 1.

Diversity in the SGA-derived sequences

抗呼吸道合胞病毒的免疫预防

呼吸道合胞病毒(RSV)感染是一个影响婴幼儿健康的全球性的问题,目前尚未有令人满意的治疗药物,免疫预防就显得尤为重要。近年研究表明,免疫预防在疫苗、单克隆抗体以及免疫球蛋白等领域均取得较大进展。     

抗人呼吸道合胞病毒融合糖蛋白单克隆抗体的制备及体外鉴定

目的:获得能稳定分泌抗人呼吸道合胞病毒(human respiratory syncytial virus, RSV)融合糖蛋白(fusion glycoprotein, F)单克隆抗体(monoclonal antibody, mAb)的杂交瘤细胞株,以期用于RSV感染的早期诊断和被动免疫治疗研究。方法:通过杂交瘤技术制备可特异性识别RSV F的单抗,体外鉴定生物学特性。结果:获得了可分泌抗RSV F蛋白的杂交瘤细胞株F8,体外连续传代培养2个月,能稳定分泌抗体F8,培养上清效价为1∶1000,亲和常数(Ka)为6.8×10⁸ L/mol。F8属IgG1型抗体,可特异性识别RSV F1亚单位的AA 205-222。免疫酶法蚀斑减少中和实验证实F8具有体外中和活性及融合抑制活性。结论:获得具有中和活性的抗RSV F蛋白的单克隆抗体,为RSV感染的早期诊断及被动免疫治疗等奠定了基础。     

Temperature-sensitive Mutants of Respiratory Syncytial Virus

Four conditional-lethal temperature-sensitive mutants of RS virus were detected among the progeny of 454 plaques derived from virus grown in the presence of 10(-4)m 5-fluorouridine. These mutants were stable (reversion frequency, 10(-5.0) or less and failed to produce plaques at 38 or 39 C. Plaquing efficiency was depressed 100-fold or more at 37 C. Variable suppression of growth at the restrictive temperature of 39 C was observed, ranging from 16-fold to complete suppression. The temperature-sensitive defect of three of the mutants appeared to affect functions which were expressed late in the replicative cycle. One of the mutants produced atypical nonsyncytial plaques.     

The Respiratory Syncytial Virus Fusion Protein Targets to the Perimeter of Inclusion Bodies and Facilitates Filament Formation by a Cytoplasmic Tail-Dependent Mechanism

The human respiratory syncytial virus (HRSV) fusion (F) protein cytoplasmic tail (CT) and matrix (M) protein are key mediators of viral assembly, but the underlying mechanisms are poorly understood. A complementation assay was developed to systematically examine the role of the F protein CT in infectious virus production. The ability of F mutants with alanine substitutions in the CT to complement an F-null virus in generating infectious progeny was quantitated by flow cytometry. Two CT regions with impact on infectious progeny production were identified: residues 557 to 566 (CT-R1) and 569 to 572 (CT-R2). Substitutions in CT-R1 decreased infectivity by 40 to 85% and increased the level of F-induced cell-cell fusion but had little impact on assembly of viral surface filaments, which are believed to be virions. Substitutions in CT-R2, as well as deletion of the entire CT, abrogated infectious progeny production and impaired viral filament formation. However, CT-R2 mutations did not block but rather delayed the formation of viral filaments, which continued to form at a low rate and contained the viral M protein and nucleoprotein (N). Microscopy analysis revealed that substitutions in CT-R2 but not CT-R1 led to accumulation of M and F proteins within and at the perimeter of viral inclusion bodies (IBs), respectively. The accumulation of M and F at IBs and coincident strong decrease in filament formation and infectivity upon CT-R2 mutations suggest that F interaction with IBs is an important step in the virion assembly process and that CT residues 569 to 572 act to facilitate release of M-ribonucleoprotein complexes from IBs.