Pulmonary eosinophilia requires interleukin-5, eotaxin-1, and CD4+ T cells in mice immunized with respiratory syncytial virus G glycoprotein

Severe illness, type 2 cytokine production, and pulmonary eosinophilia are adverse immune responses resulting from respiratory syncytial virus (RSV) challenge of vvGs-immunized mice. We have shown IL-4 and IL-13 activity must be simultaneously inhibited to reduce disease severity. We now address the contributions of IL-5, eotaxin-1, and CD4+ and CD8+ T cells to the induction of disease-enhancing immune responses. Depletion of CD4+ T cells during immunization prevented IL-4, IL-13, and eotaxin-1 production, diminished eosinophilia, and reduced weight loss. Conversely, CD8+ T cell depletion did not decrease eosinophilia, weight loss, or type 2 cytokines but did dramatically reduce mucus production and increase eotaxin production. Anti-IL-5 administration at immunization or challenge significantly decreased pulmonary eosinophilia. Strikingly, there were not concomitant decreases in weight loss. Following RSV challenge eotaxin-1-deficient mice immunized with vvGs exhibited significantly less eosinophilia without decreased weight loss or type 2 cytokine production. We conclude CD4+ T cell production of IL-5 and induction of eotaxin-1 are required for vvGs-induced eosinophilia following RSV challenge, while CD8+ T cells appear to down-regulate eotaxin-1 and mucus production. In summary, we demonstrate that pulmonary eosinophilia 1) is a by-product of memory CD4+ T cell activation, 2) does not necessarily correlate with mucus production, and, most importantly, 3) is not required for the RSV G-induced illness in mice. These findings have important implications for the evaluation of candidate RSV vaccines.     

Differential immunoglobulin G subclass antibody titers to respiratory syncytial virus F and G glycoproteins in adults

Two respiratory syncytial virus glycoproteins, F and G, which differ substantially in the amount of glycosylation were used as antigens in an enzyme-linked immunosorbent assay to determine immunoglobulin G (IgG) subclass titers in 30 experimentally infected healthy adults. The titers of antibodies to the F glycoprotein achieved in postinfection sera were highest in the IgG1 subclass, whereas those to the G glycoprotein were highest and comparable in the IgG1 and IgG2 subclasses. The high IgG2 response to the G glycoprotein suggests that it is seen by the immune system as a polysaccharide antigen, a hypothesis consistent with its large carbohydrate content.     

Immunological response of mice to the bovine respiratory syncytial virus fusion glycoprotein expressed in recombinant baculovirus infected insect cells

Summary Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in calves. The BRSV genome encodes two major glycoproteins, G and F, which are the major targets for the host antibody response. We have expressed the F glycoprotein in insect cells (Sf9) using a recombinant baculovirus vector. A comparison of the F protein expressed in mammalian and insect cells by SDS-PAGE showed that only part of the baculovirus-produced protein was soluble and processed like the native protein. The antigenicity of the soluble form of the F protein expressed in insect cells was identical to that of the F protein expressed in mammalian cells. Immunization with the F protein expressed in insect cells induced neutralizing antibodies in mice. This antigenic preparation adjuvanted with Quil-A produced an increased neutralizing antibody titer and induced protection.     

Antibody responses against epitopes on the F protein of bovine respiratory syncytial virus differ in infected or vaccinated cattle

Summary.  The fusion protein F of bovine respiratory syncytial virus (BRSV) is an important target for humoral and cellular immune responses, and antibodies against the F protein have been associated with protection. However, the F protein can induce antibodies with different biological activity, possibly related to distinct antigenic regions on the protein. Therefore, epitopes were mapped on the F protein using monoclonal antibodies. Two epitopes (A and B) were identified that induced neutralizing antibodies, and one epitope (C) that did not elicit neutralizing antibodies. Subsequently, antibody responses were analysed against these epitopes in cattle sera after natural infection, experimental infection or vaccination. After natural infection or reinfection, the antibody titres against epitope A were significantly higher than those against epitope B or C. After experimental infection and after vaccination with an inactivated vaccine, antibody titres against epitope B and C were significantly higher than after natural infection. Conversely, virus neutralizing antibody titres were significantly lower in these animals with higher antibody titres against epitopes B and C than in naturally infected cattle. Because after natural infection the epitope-specific-antibody titres against epitope A, B or C differed markedly between the cattle, the magnitude of the antibody titres against epitope A, B or C in relation to the major histocompatibility complex (MHC) genes of cattle (BoLA) was studied. The magnitude of the antibody responses against epitope A of the F protein, but not against the G protein, appeared to be associated with the bovine lymphocyte antigen (BoLA) haplotype. Received February 4, 1997 Accepted July 4, 1997     

Monoclonal antibodies to respiratory syncytial virus: Detection of virus neutralization and other antigen-antibody systems using infected human and murine cells

Monoclonal antibodies to human respiratory syncytial (RS) virus-specific antigens can be obtained without preliminary recourse to large-scale culture and purification of the virion. Lytically infected human and persistently infected murine cultured cells expressing RS virus-specific cell surface and cytoplasmic antigens were substituted as priming immunogens and as substrates in solid-phase antibody radioimmunoassays. Seven hybridoma clones secreting murine IgG of either the γ₁ or the γ_2A subclass bearing κ light chains were isolated. Two of the antibodies were specific for cell surface viral antigens, but only one was able to neutralize RS virus infectivity. The five remaining antibodies did not neutralize virus infectivity and were specific for viral antigens associated with large cytoplasmic inclusions as judged by indirect immunofluorescence (IF) analysis on fixed infected cells. Similar IF analysis using live cells revealed that those antigens, associated with the cytoplasmic inclusions in both the human and murine infected cells, were not expressed on the cell surface of the live infected human cells, but were expressed on the cell surface of the live infected murine cells. Monoclonal antibodies generated via the present system will prove useful in the immunological analysis of viral components which are associated with specific pathogenic functions, such as infectivity, and those which may be abnormally exposed at the surface of persistently infected cells.     

Detection of asymptomatic initial herpes simplex virus (HSV) infections in animals immunized with subunit HSV glycoprotein vaccines.

The evaluation of herpes simplex virus (HSV) vaccine efficacy will require methods to detect asymptomatic acquisition of HSV infection and to assess the risk of recurrences in these patients. HSV-infected vaccinees should develop antibodies to HSV polypeptides not included in subunit vaccines. Sera from 57 HSV glycoprotein-vaccinated guinea pigs that had asymptomatic initial infections after genital HSV type 2 challenge were collected after vaccination but before HSV challenge and again 30 days after HSV challenge to determine the antibody response to HSV polypeptides. Antibodies to nonvaccine HSV polypeptides were detected in sera collected after viral challenge from 32 (56%) of these 57 animals. Twenty-six (81%) of the 32 animals with detectable antibody developed recurrent disease; however, recurrences also developed in 11 (44%) of the remaining 25 that did not show detectable antibody to nonvaccine HSV polypeptides. The magnitude of vaginal viral shedding during the initial disease period following challenge was significantly lower in animals that did not develop antibody to nonvaccine polypeptides compared with those that did develop antibody (area under the viral shedding curve, 5.2 +/- 3.2 versus 18.1 +/- 5.8; P less than 0.0001) . These data suggest that detection of antibody to nonvaccine HSV polypeptides will identify the majority (70%) of initially asymptomatic vaccinees that develop recurrent disease but that latency can be established even with markedly reduced levels of viral replication that did not induce a detectable antibody response.     

Detection of respiratory syncytial virus (RSV) antigen in the lungs of guinea pigs 6 weeks after experimental infection and despite of the production of neutralizing antibodies

Summary Infections with respiratory syncytial virus (RSV) are characterized by frequently occuring reinfections and are regarded to be responsible for bronchial hyperreactivity. In this report we describe a small-animal model suited to study RSV-induced pathogenesis and immune response. Guinea pigs are infected by inhalation of an RSV-aerosol. Lungs of infected animals show signs of a bronchiolitis at 7 days after the initial infection. Although neutralizing serum antibodies are synthesized viral proteins are still detectable at 6 weeks post infection. Therefore, the presence of neutralizing antibodies is obviously not sufficient for rapid clearance of persistent RSV-proteins from the lungs of infected guinea pigs.     

Monoclonal antibodies demonstrate heterogeneity in the G glycoprotein of prototype strains and clinical isolates of respiratory syncytial virus

In order to study variation among prototype strains and clinical isolates of respiratory syncytial (RS) virus, four prototype strains (Long, A2, CH18537, 9320) were used to produce monoclonal antibodies to this virus. The majority of monoclonals reacted with all four prototype strains by fluorescent antibody staining. Among the non-cross-reacting monoclonals, five additional patterns of reactivity with the prototype strains were recognized. Fourteen monoclonals, including ones representative of each of the patterns of reactivity with the prototype strains, were selected to use for typing prototype strains and community isolates. All 14 were found by immunoprecipitation to recognize the RS virus G glycoprotein. These monoclonals could uniquely identify each of the prototype strains. In addition to the antigenic differences among the prototype strains detected by the monoclonals, differences were also detected in the migration of the G glycoprotein of the prototype strains in polyacrylamide gel electrophoresis. Fluorescent antibody staining with panels of monoclonals distinguished two antigenic types among 114 isolates of RS virus recovered from children in St. Louis during the period 1981-86. The predominant type (80% of isolates) had a pattern of reactivity that resembled but differed from that of either the Long or A2 strains. The second type had a pattern of reactivity identical with that of 9320. The possible significance of this heterogeneity must be considered in developing diagnostic tests as well as active or passive immunotherapy for infections caused by RS virus.     

Cytokines and chemokines in respiratory secretion and severity of disease in infants with respiratory syncytial virus (RSV) infection.

BACKGROUND: little is known about inflammatory mediators (IM); like cytokines, chemokines and receptors; in respiratory secretion as possible indicators of the severity of respiratory syncytial virus (RSV) disease. Nor have systematic studies been published on the ratios between IM as such indicators. OBJECTIVE: to define the role of IM ratios as possible indicators of the severity of RSV disease. STUDY DESIGN: about 46 infants aged 0-9 months with acute RSV infections were studied. Prematurity (PM) and/or underlying disease (UD) were present in 11 of them. The concentrations of seven different IM were measured by ELISA in samples of nasopharyngeal secretions (NPS), four cytokines; IL-1, IL-6, IL-10 and TNF-alpha; the cytokine receptor TNF-R1 and the chemokines; IL-8 and RANTES. 21 IM ratios were calculated from these concentrations. The patients were assigned a clinical score (CS) ranging from 0 to 3 according to the severity of disease. RESULTS: when 25 patients with severe disease (CS 2-3) and 21 patients with mild disease (CS 0-1) were compared with respect to different IM ratios, three ratios were related to severity of disease: IL-1/RANTES, IL-8/RANTES and TNF-R1/RANTES. When 12 patients with mild disease were compared with 16 patients with severe disease, omitting patients more than 5 months of age and patients with PM and/or UD, the following IM ratios were related to severity of disease: TNF-R1/RANTES, IL-8/RANTES and RANTES/IL-10. CONCLUSION: of 21 IM ratios studied, TNF-R1/RANTES was related to severity of disease with greatest consistency.     

呼吸道合胞病毒感染人肺上皮细胞活化Toll样受体3介导的抗病毒作用研究

目的 探讨呼吸道合胞病毒( RSV)感染人肺上皮A549细胞后,Toll样受体3(TLR3)的水平变化及其产生的Ⅰ型干扰素的抗病毒作用.方法 RSV感染体外培养的人肺上皮A549细胞,并给予TLR3特异性抗体处理,分别感染4、8、12、16、24h后收集各组细胞.未感染病毒的细胞作为对照组.RT-PCR法检测TLR3、IFN-α、IFN-β,RSV F蛋白的mRNA表达水平变化.结果 RSV感染A549细胞后,TLR3、IFN-α、IFN-β,RSV F蛋白的mRNA表达量均升高且有时间依赖性,TLR3 mRNA在24h表达量是基础表达量的5倍,IFN-α、IFN-β mRNA在24 h表达量是基础表达量的4倍多,RSVF蛋白的mRNA表达量近1.7倍.TLR3抗体预先处理以抑制TLR3受体,再行RSV感染,IFN-α和IFN-β mRNA表达量虽然升高,但较感染组均有所下降,mRNA表达在12 h后显著降低,且IFN-ββ的mRNA表达量下调更明显.但RSV F基因的mRNA表达在12 h、24 h升高有显著性差异.结论 RSV感染A549细胞后可上调TLR3表达,其活化细胞介导产生的Ⅰ型干扰素起抗病毒作用,在一定程度上可抑制病毒的增殖水平.     

Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo.

The surface glycoproteins of viruses can play important roles in viral attachment, entry, and morphogenesis. Here, we investigated the role of the attachment G glycoprotein of human respiratory syncytial virus (RSV) in viral infection. RSV G is produced both as a complete, transmembrane form and as an N-terminally truncated form that is secreted. Using reverse genetics, we created mutant recombinant RSVs (rRSV) that do not express G (DeltaG) or express either the secreted or the membrane-bound form of G only (sG and mG, respectively). In Vero cells, the DeltaG virus formed plaques and grew as efficiently as wild-type rRSV and mG. In contrast, DeltaG replicated less efficiently and did not form distinct plaques in HEp-2 cells. This defect was primarily at the level of the initiation of infection, with only a minor additional effect at the level of packaging. Replication of DeltaG in the respiratory tract of mice was very highly restricted, indicating that G is important in vivo. Although the G protein expressed by the sG virus was confirmed to be secreted, this virus grew at least as efficiently as wild-type in HEp-2 cells and was only moderately attenuated in vivo. Thus, the G protein was important for efficient replication in HEp-2 cells and in vivo, but this function could be supplied in large part by the secreted form and thus does not require the cytoplasmic and transmembrane domains. Amino acids 184-198 have been identified as the major heparin-binding domain of the G protein and were implicated in mediating binding to cells [S. A. Feldman et al., 1999, J. Virol. 73, 6610-6617]. Heparin-like glycosaminoglycans also appeared to be important for infection in vitro by direct clinical isolates of RSV. Deletion of amino acids 187-197 from rRSV did not reduce its sensitivity to neutralization in vitro by incubation with soluble heparin, did not reduce its efficiency of growth in vitro, and resulted in only a modest reduction in vivo. Thus, the putative heparin-binding domain is not the sole determinant of heparin sensitivity and is not a critical functional domain.     

Cytokine pattern is solely influenced by priming vaccine but immunity and disease by both priming and boosting vaccines in mice challenged with respiratory syncytial virus

Vaccine formulation can influence cytokine and disease patterns in mice following respiratory syncytial virus (RSV) challenge. The influence of different live and killed dual-vaccine combinations on subsequent immune responses was investigated. BALB/c mice received either killed followed by killed (KV/KV), killed followed by live (KV/LV), live followed by killed (LV/KV), or live followed by live (LV/LV) RSV vaccines intramuscularly. Mouse weight loss, viral replication, cytokine expression patterns, immunoglobulin isotype antibody profiles, neutralizing antibody responses, and cytotoxicity T lymphocyte (CTL) activities in lungs were compared on subsequent live RSV challenge. On challenge, mice vaccinated initially with KV and boosted with either KV or LV expressed significantly skewed ratios of IL-4 to IFN-gamma mRNA and IgG1 to IgG2a antibody, when compared to those vaccinated initially with LV. Low levels of RSV replication were detected in lungs of mice vaccinated with KV/KV, KV/LV, and LV/KV, but not in mice vaccinated with LV/LV. Mice vaccinated with KV/LV, LV/KV, or LV/LV had RSV-specific CTL activity in lungs six days after RSV challenge, while no CTL activity was detected in KV/KV-vaccinated mice. Mice vaccinated with KV/KV had the greatest weight loss, while LV/LV-vaccinated mice resulted in the least. Mice vaccinated with either KV/LV or LV/KV had intermediate weight loss after challenge. These data indicate that an original antigenic sin-like phenomenon was exhibited in cytokine and immunoglobulin isotype responses in mice after challenge. T helper (Th)-like immune responses were determined solely by the initial vaccination, while weight loss, viral replication, neutralizing antibody responses, and CTL activities were also influenced by boosted vaccinations.     

Comparison of the VIDAS RSV assay and the Abbott Testpack RSV with direct immunofluorescence for detection of respiratory syncytial virus in nasopharyngeal aspirates.

The sensitivity and accuracy of the VIDAS RSV assay in testing fresh specimens were 82.7 and 87.1%, respectively, whereas specimens previously frozen at -70 degrees C gave a sensitivity of 96.2% and an accuracy of 95.4%. The sensitivity and accuracy of Abbott Testpack RSV were 92.6 and 91.3% for fresh specimens and 86.8 and 88.1% for frozen specimens. The advantages and drawbacks of the two assays are discussed.     

Alteration of leukocyte populations in calves concurrently infected with bovine respiratory syncytial virus and bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) infection altered leukocyte populations in calves that were reflected by depression of T, BoCD4+, and BoCD8+ lymphocytes in the thymus and depression of B lymphocytes in Peyer's patches (PP). The present study was based on mononuclear leukocyte preparations from eighteen 9- to 12-month-old crossbred calves that were each exposed to either bovine respiratory syncytial virus (BRSV), BVDV, or BRSV and BVDV concurrently, or served as mock-infected controls. Peripheral blood leukocytes were collected on postinfection days (PID) 0, 2, 4, 6, and 8, and cell populations from thymus, spleen, mesenteric lymph node, and PP were collected at necropsy on PID 9. The leukocytes were analyzed using flow cytometry for lymphocyte subpopulations expressing antigens specific for BoCD2, BoCD4, BoCD8, BoWC1, lambda light chain of bovine immunoglobulin, BoCD11b and major histocompatibility complex (MHC) class II. Concurrent BRSV and BVDV infections caused exaggerated alterations in leukocyte populations with a greater percentage of T-lymphocytes harvested from the PP. Alterations in the leukocyte populations in lymphatic tissues and in peripheral circulation due to BVDV infection may be an important mechanism for causation of clinically severe diseases of the respiratory and digestive tracts during concurrent BRSV and BVDV infections.     

Antigen-dependent proliferation and cytokine induction in respiratory syncytial virus-infected cotton rats reflect the presence of effector-memory T cells

Respiratory syncytial virus (RSV) is a major cause of lower airway disease in infants and children. Immunity to RSV is not long lasting, resulting in re-occurring infections throughout life. Effective long-lived immunity results when central-memory T cells that proliferate vigorously and secrete IL-2 are present. In contrast, effector-memory T cells that mainly produce IFN-gamma, facilitate virus clearance but are not long lived. To identify the type of memory response induced after RSV-A (Long) infection, we characterized the kinetics of the antigen-specific immune response and identified the types of cytokines induced. RSV-specific lymphocytic proliferation following primary and secondary infection was similar, and in both cases responses waned within a short period of time. In addition, mRNA for IFN-gamma but not IL-2 was induced in RSV-specific CD4(+) T cells. This supports the idea that the presence of effector-memory rather than central-memory T cells contributes to the ineffectiveness of the immune response to RSV.     

北京地区正常人群血清中呼吸道合胞病毒F和G蛋白特异性IgG抗体水平的观察

以表达呼吸道合胞病毒（ＲＳＶ）Ｆ和Ｇ蛋白的重组痘苗病毒作为抗原，用间接免疫荧光法检测北京地区不同年龄组正常儿童及成人血清中ＲＳＶＦ和Ｇ蛋白特异性ＩｇＧ抗体。其结果表明：成人及产妇静脉血中有中等滴度的ＲＳＶ抗体及较低水平的Ｆ和Ｇ蛋白特异性ＩｇＧ抗体。初生婴儿脐带血中的ＲＳＶＦ和Ｇ蛋白ＩｇＧ抗体水平同母亲静脉血中的一致，表明属母传抗体。婴幼儿ＲＳＶ抗体水平在生后６个月内迅速降低，６个月以后抗体的滴度随年龄消长，７－１４岁时水平最高。Ｆ和Ｇ蛋白特异性ＩｇＧ抗体滴度在生后６个月内无明显降低，但低于大年龄组儿童。６个月后Ｆ和Ｇ蛋白抗体滴度显著升高并随年龄波动，Ｆ较Ｇ蛋白更为显著，分别在３～７岁（Ｆ蛋白）和７－１４岁（Ｇ蛋白）组中达是高滴度。从ＲＳＶ，Ｆ和Ｇ蛋白三种特异性ＩｇＧ抗体间的相关性来看：ＲＳＶ分别同Ｆ和Ｇ蛋白抗体呈现明显的相关性，但Ｆ和Ｇ蛋白特异性ＩｇＧ抗体间无明显相关性。