Toll 样受体通路激活改善呼吸道合胞病毒疫苗诱导免疫应答的研究进展

呼吸道合胞病毒（RSV）是导致婴幼儿严重下呼吸道感染的最重要病原体,但该病毒的灭活疫苗可引起RSV疫苗增强性疾病（RVED）。RVED的机制目前仍不清楚。Toll样受体（TLR）及其信号转导对 RSV的识别和宿主免疫的激发均有重要作用,其在RVED机制中的作用也日益受到关注。本文主要介绍TLR在抗RSV天然免疫和获得性免疫中的角色及其信号通路激活状态改变对RVED免疫格局的影响,提示RVED机制可能与TLR信号通路激活不足有关,从而为RSV疫苗研制提供新的策略和方法。     

CD8 T cells inhibit respiratory syncytial virus (RSV) vaccine-enhanced disease

Vaccination of children with a formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine led to exacerbated disease including pulmonary eosinophilia following a natural RSV infection. Immunization of BALB/c mice with FI-RSV or a recombinant vaccinia virus (vv) expressing the RSV attachment (G) protein (vvG) results in a pulmonary Th2 response and eosinophilia after RSV challenge that closely mimics the RSV vaccine-enhanced disease observed in humans. The underlying causes of RSV vaccine-enhanced disease remain poorly understood. We demonstrate here that RSV M2-specific CD8 T cells reduce the Th2-mediated pathology induced by vvG-immunization and RSV challenge in an IFN-gamma-independent manner. We also demonstrate that FI-RSV immunization does not induce a measurable RSV-specific CD8 T cell response and that priming FI-RSV-immunized mice for a potent memory RSV-specific CD8 T cell response abrogates pulmonary eosinophilia after subsequent RSV challenge. Our results suggest that the failure of the FI-RSV vaccine to induce a CD8 T cell response may have contributed to the development of pulmonary eosinophilia and augmented disease that occurred in vaccinated individuals.     

呼吸道合胞病毒疫苗增强性疾病小鼠模型的建立与评价

呼吸道合胞病毒(respiratory syncytial virus,RSV)是导致婴幼儿严重下呼吸道感染的最重要病原体,但该病毒的灭活疫苗可引起RSV疫苗增强性疾病(RSV vaccine-enhanced disease,RVED),因此至今仍未研制出安全、有效的疫苗。RVED的发生机制目前仍不清楚。使用能有效模拟RVED的动物模型是探索RVED发生机制的主要手段,而本研究的目的就是建立能稳定模拟RVED表现的小鼠模型。将BALB/c小鼠分成3组,即甲醛灭活RSV疫苗接种组(FV组)、活RSV接种组(VV组)和对照组(BV组),并模拟自然感染对其攻毒。通过小鼠体重监测、肺组织苏木精-伊红染色、荧光定量聚合酶链反应(polymerase chain reaction,PCR)、流式细胞术等,观察FV组小鼠感染RSV后的病毒载量、肺部炎症和T细胞免疫状态。结果显示,与VV组和BV组相比,FV组小鼠RSV攻毒后的体重占初始体重百分比显著降低,肺部炎症最明显,且仅FV组出现支气管和毛细血管周围有大量淋巴细胞浸润及嗜酸性粒细胞浸润。荧光定量PCR显示,FV组小鼠的肺部RSV载量最低。流式细胞术显示,攻毒4d后FV组CD~(4+) T细胞数与其他两组相比显著增加,且CD~(4+) T细胞分泌的白细胞介素4(interleukin 4,IL-4)及IL-4/γ干扰素(interferonγ,IFN-γ)比值显著高于其他两组,而CD~(8+) T细胞分泌的肿瘤坏死因子α(tumor necrosis factorα,TNF-α)和IFN-γ低于VV组。结果提示,RVED小鼠呈现出Th2优势型免疫应答及CD~(8+) T细胞功能不足,模型初步建立成功,为RVED发生机制的探索和RSV疫苗的研发奠定了良好基础。     

IL-13 is required for eosinophil entry into the lung during respiratory syncytial virus vaccine-enhanced disease

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. Children previously vaccinated with a formalin-inactivated RSV vaccine experienced enhanced morbidity and mortality upon natural RSV infection. Histological analysis revealed the presence of eosinophils in the pulmonary infiltrate of the vaccinated children. Eosinophils are characteristic of Th2 responses, and Th2 cells are known to be necessary to induce pulmonary eosinophilia in RSV-infected BALB/c mice previously immunized with a recombinant vaccinia virus (vv) expressing the RSV G protein (vvG). Using IL-13-deficient mice, we find that IL-13 is necessary for eosinophils to reach the lung parenchyma and airways of vvG-immunized mice undergoing RSV challenge infection. IL-13 acts specifically on eosinophils as the magnitude of pulmonary inflammation, RSV G protein-specific CD4 T cell responses, and virus clearance were not altered in IL-13-deficient mice. After RSV challenge, eosinophils were readily detectable in the blood and bone marrow of vvG-immunized IL-13-deficient mice, suggesting that IL-13 is required for eosinophils to transit from the blood into the lung. Pulmonary levels of CCL11 and CCL22 protein were significantly reduced in IL-13-deficient mice indicating that IL-13 mediates the recruitment of eosinophils into the lungs by inducing the production of chemokines important in Th2 cell and eosinophil chemotaxis.     

Interleukin-27 inhibits vaccine-enhanced pulmonary disease following respiratory syncytial virus infection by regulating cellular memory responses

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract disease in young children. In the 1960s, infants vaccinated with formalin-inactivated RSV developed a more severe disease characterized by excessive inflammatory immunopathology in lungs upon natural RSV infection. The fear of causing the vaccine-enhanced disease (VED) is an important obstacle for development of safe and effective RSV vaccines. The recombinant vaccine candidate G1F/M2 immunization also led to VED. It has been proved that cellular memory induced by RSV vaccines contributed to VED. Interleukin-27 (IL-27) and IL-23 regulate Th1, Th17, and/or Th2 cellular immune responses. In this study, mice coimmunized with pcDNA3-IL-27 and G1F/M2 were fully protected and, importantly, did not develop vaccine-enhanced inflammatory responses and immunopathology in lungs after RSV challenge, which was correlated with moderate Th1-, suppressed Th2-, and Th17-like memory responses activated by RSV. In contrast, G1F/M2- or pcDNA3-IL-23+G1F/M2-immunized mice, in which robust Th2- and Th17-like memory responses were induced, developed enhanced pulmonary inflammation and severe immunopathology. Mice coimmunized with G1F/M2 and the two cytokine plasmids exhibited mild inflammatory responses as well as remarkable Th1-, suppressed Th2-, and Th17-like memory responses. These results suggested that Th1-, Th2-, and Th17-like memory responses and, in particular, excessive Th2- and Th17-like memory responses were closely associated with VED; IL-27 may inhibit VED following respiratory syncytial virus infection by regulating cellular memory responses.     

A novel inactivated intranasal respiratory syncytial virus vaccine promotes viral clearance without Th2 associated vaccine-enhanced disease

Background

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960''s led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations.

Principal Findings

In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines.

Conclusions

These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology.     

Respiratory syncytial virus (RSV) G glycoprotein is not necessary for vaccine-enhanced disease induced by immunization with formalin-inactivated RSV

Following respiratory syncytial virus (RSV) challenge, mice immunized with RSV G or with formalin-inactivated RSV (FI-RSV) exhibit severe disease associated with type 2 cytokine production and pulmonary eosinophilia. This has led to the proposal that the presence of RSV G is the factor in FI-RSV that induces disease-enhancing T-cell responses. Therefore, we evaluated the role of RSV G and its immunodominant region in the induction of aberrant immune responses during FI-RSV immunization. BALB/c mice were immunized with FI preparations of wild-type (wt) RSV or recombinant RSV (rRSV) containing deletions of (i) the entire G gene, (ii) the region of the G gene encoding amino acids 187 to 197 of the immunodominant region, or (iii) the entire SH gene. After challenge, illness, RSV titers, cytokine levels, and pulmonary eosinophilia were measured. Peak RSV titers postchallenge were significantly greater in mice immunized with FI preparations of the deletion viruses than in those immunized with FI-rRSV wt, suggesting that the absence of G or SH in FI-RSV reduced its protective efficacy. Deletion of G or its epitope did not reduce illness, cytokine production, or eosinophilia relative to that in mice immunized with FI-rRSV wt. While cytokine levels and eosinophilia were similar, illness was reduced in mice immunized with SH-deleted FI-RSV. These data suggest that G-specific immune responses may be important for vaccine-induced protection and are not solely the basis for FI-RSV vaccine-enhanced illness. These data suggest that the method of RSV antigen delivery, rather than the protein composition, influences the phenotype of the induced immune responses and that RSV G should not necessarily be excluded from potential vaccine strategies.     

Vbeta14(+) T cells mediate the vaccine-enhanced disease induced by immunization with respiratory syncytial virus (RSV) G glycoprotein but not with formalin-inactivated RSV

Mice immunized with respiratory syncytial virus (RSV) G glycoprotein or with formalin-inactivated RSV (FI-RSV) exhibit severe disease following RSV challenge. This results in type 2 cytokine production and pulmonary eosinophilia, both hallmarks of vaccine-enhanced disease. RSV G-induced T-cell responses were shown to be restricted to CD4(+) T cells expressing Vbeta14 in the T-cell receptor (TCR), and the deletion of these T cells resulted in less severe disease. We therefore examined the role of Vbeta14(+) T cells in FI-RSV-induced disease. BALB/c mice were immunized with vaccinia virus expressing secreted RSV G (vvGs) or with FI-RSV. At the time of challenge with live RSV, mice were injected with antibody to the Vbeta14 component of the TCR. vvGs-immunized mice treated with anti-Vbeta14 had reduced cytokine levels in the lung. Eosinophil recruitment to the lung was also significantly reduced. In contrast, depletion of Vbeta14(+) T cells in FI-RSV-immunized mice had little impact on cytokine production or pulmonary eosinophilia. An analysis of TCR Vbeta chain usage confirmed a bias toward Vbeta14 expression on CD4(+) T cells from vvGs-immunized mice, whereas the CD4(+) T cells in FI-RSV-immunized mice expressed a diverse array of Vbeta chains. These data show that although FI-RSV and vvGs induce responses resulting in similar immunopathology, the T-cell repertoire mediating the response is different for each immunogen and suggest that the immune responses elicited by RSV G are not the basis for FI-RSV vaccine-enhanced disease.     

Animal models of human respiratory syncytial virus disease

Infection with the human pneumovirus pathogen, respiratory syncytial virus (hRSV), causes a wide spectrum of respiratory disease, notably among infants and the elderly. Laboratory animal studies permit detailed experimental modeling of hRSV disease and are therefore indispensable in the search for novel therapies and preventative strategies. Present animal models include several target species for hRSV, including chimpanzees, cattle, sheep, cotton rats, and mice, as well as alternative animal pneumovirus models, such as bovine RSV and pneumonia virus of mice. These diverse animal models reproduce different features of hRSV disease, and their utilization should therefore be based on the scientific hypothesis under investigation. The purpose of this review is to summarize the strengths and limitations of each of these animal models. Our intent is to provide a resource for investigators and an impetus for future research.     

Cutting edge: regulatory T cells do not mediate suppression via programmed cell death pathways

Regulatory T cells (Tregs) play a critical role in the immune system to regulate peripheral tolerance and prevent autoimmunity. However, the relative importance of different mechanisms of Treg function remains obscure. In this article, we reveal a limited role for programmed cell death pathways in mediating Treg suppression of conventional T cells. We show that Tregs are able to suppress the proliferation of conventional T cells that are resistant to apoptosis (Bim(-/-), Bim(-/-)Puma(-/-), Bcl-2 transgenic) or receptor-interacting serine-threonine kinase-dependent necrosis (also referred to as regulated necrosis or necroptosis) (Ripk3(-/-)) in several in vitro and in vivo assays. These data suggest that programmed cell death pathways, such as apoptosis and receptor-interacting serine-threonine kinase-dependent necrosis, are not required for Treg-mediated suppression.     

Major histocompatibility complex-dependent cytotoxic T lymphocyte repertoire and functional avidity contribute to strain-specific disease susceptibility after murine respiratory syncytial virus infection

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vβ9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.     

Antibody response to respiratory syncytial virus structural proteins in children with acute respiratory syncytial virus infection.

The purified respiratory syncytial virus (RSV), Randall strain contained 10 polypeptides (72,000 molecular weight [72K], 66K, 48K, 42K, 40K, 36K, 30K, 23K, 18K, and 15K), 8 of which proved to be virus specific, and polypeptides 48K and 23K were glycosylated. In addition, a high-molecular-weight (150K), virus-specific glycopolypeptide was immunoprecipitated from RSV-infected cell lysate. The antibody response in human sera serially collected from children with primary RSV infection was mainly directed against the polypeptides 30K, 48K, and 72K. The immune response against the other viral proteins was also already detectable in the acute-phase sera. These results indicate that the immune response in RSV infection differs significantly from those for other diseases caused by paramyxoviruses.     

Cutting edge: Tissue-retentive lung memory CD4 T cells mediate optimal protection to respiratory virus infection

We identify in this article a new class of lung tissue-resident memory CD4 T cells that exhibit tissue tropism and retention independent of Ag or inflammation. Tissue-resident memory CD4 T cells in the lung did not circulate or emigrate from the lung in parabiosis experiments, were protected from in vivo Ab labeling, and expressed elevated levels of CD69 and CD11a compared with those of circulating memory populations. Importantly, influenza-specific lung-resident memory CD4 T cells served as in situ protectors to respiratory viral challenge, mediating enhanced viral clearance and survival to lethal influenza infection. By contrast, memory CD4 T cells isolated from spleen recirculated among multiple tissues without retention and failed to mediate protection to influenza infection, despite their ability to expand and migrate to the lung. Our results reveal tissue compartmentalization as a major determining factor for immune-mediated protection in a key mucosal site, important for targeting local protective responses in vaccines and immunotherapies.     

Bovine respiratory syncytial virus protects cotton rats against human respiratory syncytial virus infection.

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory infections in infancy. No vaccine against this virus has yet been protective, and antiviral drugs have been of limited utility. Using the cotton rat model of HRSV infection, we examined bovine respiratory syncytial virus (BRSV), a cause of acute respiratory disease in young cattle, as a possible vaccine candidate to protect children against HRSV infection. Cotton rats were primed intranasally with graded doses of BRSV/375 or HRSV/Long or were left unprimed. Three weeks later, they were challenged intranasally with either BRSV/375, HRSV/Long (subgroup A), or HRSV/18537 (subgroup B). At intervals postchallenge, animals were sacrificed for virus titration and histologic evaluation. Serum neutralizing antibody titers were determined at the time of viral challenge. BRSV/375 replicated to low titers in nasal tissues and lungs. Priming with 10(5) PFU of BRSV/375 effected a 500- to 1,000-fold reduction in peak nasal HRSV titer and a greater than 1,000-fold reduction in peak pulmonary HRSV titer upon challenge with HRSV/Long or HRSV/18537. In contrast to priming with HRSV, priming with BRSV did not induce substantial levels of neutralizing antibody against HRSV and was associated with a delayed onset of clearance of HRSV upon challenge. Priming with BRSV/375 caused mild nasal and pulmonary pathology and did not cause exacerbation of disease upon challenge with HRSV/Long. Our findings suggest that BRSV may be a potential vaccine against HRSV and a useful tool for studying the mechanisms of immunity to HRSV.     

Protection against respiratory syncytial virus by a recombinant Newcastle disease virus vector

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-alpha/beta) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+ T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-alpha/beta receptor.     

Building a better neonatal mouse model to understand infant respiratory syncytial virus disease

Background

Respiratory syncytial virus (RSV) is the number one cause of lower respiratory tract infection in infants; and severe RSV infection in infants is associated with asthma development. Today, there are still no vaccines or specific antiviral therapies against RSV. The mechanisms of RSV pathogenesis in infants remain elusive. This is partly due to the fact that the largely-used mouse model is semi-permissive for RSV. The present study sought to determine if a better neonatal mouse model of RSV infection could be obtained using a chimeric virus in which the F protein of A2 strain was replaced with the F protein from the line 19 clinical isolate (rA2-19F).

Methods

Five-day-old pups were infected with the standard laboratory strain A2 or rA2-19F and various immunological and pathophysiological parameters were measured at different time points post infection, including lung histology, bronchoalveolar lavage fluid (BALF) cellularity and cytokines, pulmonary T cell profile, and lung viral load. A cohort of infected neonates were allowed to mature to adulthood and reinfected. Pulmonary function, BALF cellularity and cytokines, and T cell profiles were measured at 6 days post reinfection.

Results

The rA2-19F strain in neonatal mice caused substantial lung pathology including interstitial inflammation and airway mucus production, while A2 caused minimal inflammation and mucus production. Pulmonary inflammation was characterized by enhanced Th2 and reduced Th1 and effector CD8⁺ T cells compared to A2. As with primary infection, reinfection with rA2-19F induced similar but exaggerated Th2 and reduced Th1 and effector CD8⁺ T cell responses. These immune responses were associated with increased airway hyperreactivity, mucus hyperproduction and eosinophilia that was greater than that observed with A2 reinfection. Pulmonary viral load during primary infection was higher with rA2-19F than A2.

Conclusions

Therefore, rA2-19F caused enhanced lung pathology and Th2 and reduced effector CD8⁺ T cell responses compared to A2 during initial infection in neonatal mice and these responses were exacerbated upon reinfection. The exact mechanism is unknown but appears to be associated with increased pulmonary viral load in rA2-19F vs. A2 infected neonatal lungs. The rA2-19F strain represents a better neonatal mouse model of RSV infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0244-0) contains supplementary material, which is available to authorized users.     

Hyperresponsiveness to inhaled but not intravenous methacholine during acute respiratory syncytial virus infection in mice

Background

To characterise the acute physiological and inflammatory changes induced by low-dose RSV infection in mice.

Methods

BALB/c mice were infected as adults (8 wk) or weanlings (3 wk) with 1 × 10⁵ pfu of RSV A2 or vehicle (intranasal, 30 μl). Inflammation, cytokines and inflammatory markers in bronchoalveolar lavage fluid (BALF) and airway and tissue responses to inhaled methacholine (MCh; 0.001 – 30 mg/ml) were measured 5, 7, 10 and 21 days post infection. Responsiveness to iv MCh (6 – 96 μg/min/kg) in vivo and to electrical field stimulation (EFS) and MCh in vitro were measured at 7 d. Epithelial permeability was measured by Evans Blue dye leakage into BALF at 7 d. Respiratory mechanics were measured using low frequency forced oscillation in tracheostomised and ventilated (450 bpm, flexiVent) mice. Low frequency impedance spectra were calculated (0.5 – 20 Hz) and a model, consisting of an airway compartment [airway resistance (Raw) and inertance (Iaw)] and a constant-phase tissue compartment [coefficients of tissue damping (G) and elastance (H)] was fitted to the data.

Results

Inflammation in adult mouse BALF peaked at 7 d (RSV 15.6 (4.7 SE) vs. control 3.7 (0.7) × 10⁴ cells/ml; p < 0.001), resolving by 21 d, with no increase in weanlings at any timepoint. RSV-infected mice were hyperresponsive to aerosolised MCh at 5 and 7 d (PC₂₀₀ Raw adults: RSV 0.02 (0.005) vs. control 1.1 (0.41) mg/ml; p = 0.003) (PC₂₀₀ Raw weanlings: RSV 0.19 (0.12) vs. control 10.2 (6.0) mg/ml MCh; p = 0.001). Increased responsiveness to aerosolised MCh was matched by elevated levels of cysLT at 5 d and elevated VEGF and PGE₂ at 7 d in BALF from both adult and weanling mice. Responsiveness was not increased in response to iv MCh in vivo or EFS or MCh challenge in vitro. Increased epithelial permeability was not detected at 7 d.

Conclusion

Infection with 1 × 10⁵ pfu RSV induced extreme hyperresponsiveness to aerosolised MCh during the acute phase of infection in adult and weanling mice. The route-specificity of hyperresponsiveness suggests that epithelial mechanisms were important in determining the physiological effects. Inflammatory changes were dissociated from physiological changes, particularly in weanling mice.     

Immunoprophylaxis of respiratory syncytial virus: global experience

In sarcoidosis, host genetic factors are discussed as contributing to disease susceptibility and course. Since tumor necrosis factor (TNF)-α is a central mediator of granuloma formation and since elevated TNF-α levels are found during active phases of sarcoidosis, genetic polymorphisms correlating with influences on TNF-α levels are of special interest. The complete sequencing of the MHC region and the increase in the number of identified gene polymorphisms in this locus associated with TNF-α production offer the opportunity of detecting new genes associated with sarcoidosis and perhaps of defining disease-associated haplotypes that bear the potential of serving as predictive markers for this disease.