Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.     

呼吸道合胞病毒G蛋白基因片段的克隆及高效表达

呼吸道合胞病毒(RSV)感染遍布全球,并可导致严重的疾病,但目前尚无成功的疫苗问世。为寻求可能用于RSV疫苗研制的重组蛋白抗原,我们在克隆RSV-A全长G蛋白基因的基础上,构建了多种共表达载体蛋白和G蛋白片段的表达载体,并从中筛选出能以可溶形式高效表达抗原蛋白的原核表达体系。通过亲和层析纯化了重组蛋白抗原DsbA-G101,将其免疫Balb／c小鼠后获得了相应的抗血清。经ELISA检测表明DsbA-G101具有良好的免疫原性。基于本研究所构建的系列表达载体,可以比较不同的G蛋白片段免疫原性的强弱及载体蛋白的优劣,从中发现最佳的RSV抗原蛋白。     

呼吸道合胞病毒诱导细胞凋亡与自噬

呼吸道合胞病毒感染与细胞凋亡、自噬的关系错综复杂。研究发现呼吸道合胞病毒感染细胞后,既能产生促细胞凋亡作用,也能产生抗细胞凋亡作用,还能诱导细胞发生自噬。研究这些过程机理,能帮助我们更好地认识呼吸道合胞病毒感染发病机制,为预防和治疗呼吸道合胞病毒感染提供一些新的方向。     

呼吸道合胞病毒载体疫苗研究进展

人呼吸道合胞病毒(human respiratory syncytial virus, RSV)是引起婴幼儿下呼吸道感染的最重要的病毒病原,RSV载体疫苗可在人细胞内从头合成,形成的蛋白质构象与RSV自然感染后表达的完全相同,不会导致抗原表位的丧失或变化,形成的免疫力更利于抵抗随后的自然感染;经黏膜途径免疫不会产生疾病增强作用,且能突破母传抗体的干扰,因而受到广泛关注。对近年来RSV载体疫苗的研究进展进行了综述。     

Perspective on the host response to human metapneumovirus infection: what can we learn from respiratory syncytial virus infections?

Human metapneumovirus (HMPV) is a recently discovered pathogen first identified in respiratory specimens from young children suffering from clinical respiratory syndromes ranging from mild to severe lower respiratory tract illness. HMPV has worldwide prevalence, and is a leading cause of respiratory tract infection in the first years of life, with a spectrum of disease similar to respiratory syncytial virus (RSV). The disease burden associated with HMPV infection has not been fully elucidated; however, studies indicate that HMPV may cause upper or lower respiratory tract illness in patients between ages 2 months and 87 years, may co-circulate with RSV, and HMPV infection may be associated with asthma exacerbation. The mechanisms and effector pathways contributing to immunity or disease pathogenesis following infection are not fully understood; however, given the clinical significance of HMPV, there is a need for a fundamental understanding of the immune and pathophysiological processes that occur following infection to provide the foundation necessary for the development of effective vaccine or therapeutic intervention strategies. This review provides a current perspective on the processes associated with HMPV infection, immunity, and disease pathogenesis.     

Binding and entry of respiratory syncytial virus into host cells and initiation of the innate immune response

Respiratory syncytial virus (RSV) is the most common cause of severe lower respiratory tract infection in infants and the elderly. There is currently no effective antiviral treatment for the infection, but advances in our understanding of RSV uptake, especially the role of surfactant proteins, the attachment protein G and the fusion protein F, as well as the post-binding events, have revealed potential targets for new therapies and vaccine development. RSV infection triggers an intense inflammatory response, mediated initially by the infected airway epithelial cells and antigen-presenting cells. Humoral and cell-mediated immune responses are important in controlling the extent of infection and promoting viral clearance. The initial innate immune response may play a critical role by influencing the subsequent adaptive response generated. This review summarizes our current understanding of RSV binding and uptake in mammalian cells and how these initial interactions influence the subsequent innate immune response generated.     

Vaccine-induced immunopathology during bovine respiratory syncytial virus infection: exploring the parameters of pathogenesis

The bovine and human respiratory syncytial viruses cause severe lower respiratory tract infections. Effective vaccines against the respiratory syncytial viruses have been lacking since vaccine failures in the 1960s and 1970s. In this report, we describe a bovine respiratory syncytial virus (bRSV) challenge model in which both classical bRSV respiratory infection and vaccine-enhanced immune pathology were reproduced. The classical, formalin-inactivated (FI) bRSV vaccine that has been associated with vaccine failure was efficient in inducing high antibody titers and reducing viral loads but also primed calves for a far more serious enhanced respiratory disease after a bRSV challenge, thereby mimicking the enhanced clinical situation in FI human RSV (hRSV)-immunized and hRSV-infected infants in the 1960s. We show that immunization with FI-bRSV mainly primes a Th2-like inflammatory response that is characterized by a significant eosinophilic influx in the bronchial alveolar lung fluid and lung tissues and high levels of immunoglobulin E serum antibodies. The current model may be useful in the evaluation of new bRSV candidate vaccines for potency and safety.     

Experimental animal model for analyzing immunobiological responses following vaccination with formalin‐inactivated respiratory syncytial virus

Formalin‐inactivated respiratory syncytial virus (FI‐RSV) vaccine was developed in the 1960s. However, this vaccine does not prevent infection in RSV‐naïve recipients and has the paradoxical effect of increasing the severity of RSV illness following natural infection, which has been a major obstacle to developing RSV vaccines. Several experimental animal models for determining the cause of the severe symptoms in FI‐RSV recipients have been developed. In the present study, cotton rats immunized with FI‐RSV were challenged with RSV and histopathological findings and recovery of infectious virus were studied. Copy numbers of mRNA of Th1 and Th2 cytokines were measured in lung tissues to gain better understanding of their immune responses. Infiltration of inflammatory cells and prominent interstitial pneumonitis were observed in the FI‐RSV group, as was induction of mRNA of Th2 cytokines such as IL‐4, IL‐10, IL‐13 and RANTES. Rats immunized with recombinant measles virus expressing the RSV F protein (MVAIK/RSV/F) and those treated with anti‐RSV mAb (palivizumab) showed very mild interstitial pneumonitis. Amounts of mRNA of IL‐1β, IFN‐γ and IL‐4 were higher in the MVAIK/RSV/F group. Administration of palivizumab before RSV challenge decreased the severity of interstitial pneumonitis in the FI‐RSV group. FI‐RSV induced skewed Th2 responses, resulting in severe inflammatory responses.     

The envelope proteins from purified respiratory syncytial virus protect mice from intranasal virus challenge

A lyophilized subunit vaccine prepared from purified respiratory syncytial virus, which contained the envelope glycoproteins F and G and the nonglycosylated matrix protein VPM, was tested in SJL mice for its ability to protect the lungs of mice from intranasal viral challenge. Initially, the mice were injected subcutaneously with one, two, or three doses of 5 or 25 micrograms of vaccine in 50% complete Freund's adjuvant or with complete Freund's adjuvant or phosphate-buffered saline only. Although none of the mice produced neutralizing serum antibody, three doses of 25 micrograms elicited antibodies to F, G, and VPM. Despite the absence of detectable neutralizing antibodies, the lungs of 93% of the vaccinated mice were protected from intranasal viral challenge. Because the initial protocol did not elicit neutralizing antibodies and a few single-dose animals were not protected, a second vaccine trial was carried out. For these studies the priming dose was increased to 50 micrograms, which was followed, in half the vaccine recipients, by a second dose of 25 micrograms. Mice given the priming dose of vaccine produced antibody to G and showed no neutralizing activity, whereas the mice given two doses of vaccine produced antibodies to G, F, and VPM and also displayed neutralizing activity for respiratory syncytial virus. The lungs of 100% of the vaccine recipients in this trial were protected from intranasal challenge. Although the vaccine elicited antibody to VPM, this response did not correlate with protection. In addition, examination of the sera from unimmunized mice recovering from respiratory syncytial virus infection revealed a serum antibody profile similar to that noted for humans, lacking antibody to VPM. Thus, the data show that a combined glycoprotein subunit vaccine affords complete protection to viral challenge and offers an approach to develop a multivalent subunit vaccine.     

The 22,000-kilodalton protein of respiratory syncytial virus is a major target for Kd-restricted cytotoxic T lymphocytes from mice primed by infection.

Recombinant vaccinia viruses containing the 22-kilodalton protein (matrixlike or 22K protein) or phosphoprotein gene from respiratory syncytial virus were constructed. These recombinant viruses expressed proteins which were immunoprecipitated by appropriate respiratory syncytial virus antibodies and comigrated with authentic proteins produced by respiratory syncytial virus infection. The new recombinant viruses (and others previously described containing the attachment glycoprotein, fusion, or nucleoprotein genes of respiratory syncytial virus) were used to infect target cells for cultured polyclonal cytotoxic T lymphocytes generated from the spleens of BALB/c or DBA/2 mice primed by intranasal infection with respiratory syncytial virus. Respiratory syncytial virus-specific cytotoxic T lymphocytes (CTL) showed strong Kd (but not Dd)-restricted recognition of the 22K protein. As previously reported, the fusion protein and nucleoprotein were both seen by CTL, but recognition of these proteins was comparatively weak. There was no detectable recognition of other respiratory syncytial virus proteins tested (including phosphoprotein). 22K protein-specific splenic memory CTL persisted for at least 11 months after infection of BALB/c mice. Priming BALB/c mice with recombinant vaccinia virus containing the 22K protein gene induced respiratory syncytial virus-specific memory CTL at lower levels than that previously reported following infection with a similar recombinant containing the fusion protein gene. These data identify the 22K protein as a major target antigen for respiratory syncytial virus-specific CTL from H-2d mice primed by respiratory syncytial virus infection.     

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.     

Heparin-Like Structures on Respiratory Syncytial Virus Are Involved in Its Infectivity In Vitro

Addition of heparin to the virus culture inhibited syncytial plaque formation due to respiratory syncytial virus (RSV). Moreover, pretreatment of the virus with heparinase or an inhibitor of heparin, protamine, greatly reduced virus infectivity. Two anti-heparan sulfate antibodies stained RSV-infected cells, but not noninfected cells, by immunofluorescence. One of the antibodies was capable of neutralizing RSV infection in vitro. These results prove that heparin-like structures identified on RSV play a major role in early stages of infection. The RSV G protein is the attachment protein. Both anti-heparan sulfate antibodies specifically bound to this protein. Enzymatic digestion of polysaccharides in the G protein reduced the binding, which indicates that heparin-like structures are on the G protein. Such oligosaccharides may therefore participate in the attachment of the virus.     

人呼吸道合胞病毒活疫苗研究进展

人呼吸道合胞病毒是引起婴幼儿支气管炎和肺炎的主要原因,也可导致免疫缺陷病人及老年人群显著发病和死亡.人呼吸道合胞病毒疫苗已被世界卫生组织(World Health Organization,WHO)列为全球最优先发展的疫苗之一.经过50多年的研究,尤其是随着重组技术和反向遗传学的出现,对RSV疫苗的研究取得了重要进展,...     

人呼吸道合胞病毒M蛋白非复制型重组腺病毒的构建和鉴定

人呼吸道合胞病毒（human respiratory syncytial virus, RSV)基质蛋白（matrix protein,M）在RSV形态发生上具有重要作用,因含有CTL抗原表位,在疫苗研究上具有一定意义。为此,应用RT-PCR 方法从感染RSV的HEp-2 细胞中扩增获得M蛋白基因,构建了含M基因的非复制型重组腺病毒并进行表达和鉴定。基因序列分析显示RSV M基因仅有一处碱基发生错义突变。非复制型重组腺病毒DNA分子FGAd/RSVM转染293细胞,观察到细胞出现CPE,RT-PCR发现M基因有转录,Western blotting及间接免疫荧光分析检测到M蛋白。成功克隆A亚型RSV Long株M基因,并获得一株可表达A亚型RSV M蛋白的非复制型重组腺病毒FGAd/RSVM,可用于体内研究观察其免疫效果及免疫保护作用。     

反向遗传学在呼吸道合胞病毒减毒活疫苗研究中的应用

人呼吸道合胞病毒(human respiratory syncytial virus, RSV)是引起婴幼儿下呼吸道感染的最重要的病毒病原,减毒RSV活疫苗能模拟自然感染充分活化机体固有免疫系统,并诱导产生体液免疫和细胞免疫,不会产生疾病增强作用,经黏膜途径应用,能突破母传抗体的干扰,因而受到广泛关注,反向遗传学(reverse genetics)在减轻野生型RSV毒力和增强其免疫原性等方面具有传统减毒技术不可比拟的优势,所以综述了反向遗传学在RSV减毒活疫苗研究中的应用。     

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.     

Current progress on development of respiratory syncytial virus vaccine

Human respiratory syncytial virus (HRSV) is a major cause of upper and lower respiratory tract illness in infants and young children worldwide. Despite its importance as a respiratory pathogen, there is currently no licensed vaccine for prophylaxis of HRSV infection. There are several hurdles complicating the development of a RSV vaccine: 1) incomplete immunity to natural RSV infection leading to frequent re-infection, 2) immature immune system and maternal antibodies of newborn infants who are the primary subject population, and 3) imbalanced Th2-biased immune responses to certain vaccine candidates leading to exacerbated pulmonary disease. After the failure of an initial trial featuring formalin-inactivated virus as a RSV vaccine, more careful and deliberate efforts have been made towards the development of safe and effective RSV vaccines without vaccine-enhanced disease. A wide array of RSV vaccine strategies is being developed, including live-attenuated viruses, protein subunit-based, and vector-based candidates. Though licensed vaccines remain to be developed, our great efforts will lead us to reach the goal of attaining safe and effective RSV vaccines in the near future.     

Immunogenicity of recombinant measles vaccine expressing fusion protein of respiratory syncytial virus in cynomolgus monkeys

Respiratory syncytial virus (RSV) is a common cause of respiratory infections in infants. Effective vaccines are currently being sought, but no vaccine is thus far available. In our previous study, recombinant AIK‐C measles vaccine expressing the RSV fusion protein (MVAIK/RSV/F) was developed and protective immunity against RSV demonstrated in cotton rats. In the present study, the immunogenicity and protective effects were investigated in three cynomolgus monkeys immunized with MVAIK/RSV/F. Neutralizing test antibodies against RSV were detected and no infectious virus was recovered from the lungs of monkeys immunized with MVAIK/RSV/F after challenge. MVAIK/RSV/F has the potential to inhibit RSV infection.

     

Interactions between respiratory syncytial virus and the host cell: opportunities for antivirus strategies?

At the start of the 21st century, respiratory syncytial virus (RSV) remains a serious global health concern. Although RSV has traditionally been acknowledged as a leading cause of morbidity and mortality in the paediatric population, the elderly and people with suppressed immune systems are now also recognised as being at risk from serious RSV infection. This problem is currently exacerbated by the lack of an effective vaccine to prevent RSV infection. Although the virus proteins play a variety of roles during the virus replication cycle, in many cases these tasks are performed via specific interactions with host-cell factors, including proteins, carbohydrates and lipids. The way in which RSV interacts with the host cell is currently being examined using a battery of different techniques, which encompass several scientific disciplines. This is providing new and interesting insights into how RSV interacts with the host cell at the molecular level, which in turn is offering the hope of new strategies to prevent RSV infection.     

Recombinant baculovirus-based vaccine expressing M2 protein induces protective CD8<Superscript>+</Superscript> T-cell immunity against respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is an important cause of acute lower respiratory tract disease in infants, young children, immunocompromised individuals, and the elderly. However, despite ongoing efforts to develop an RSV vaccine, there is still no authorized RSV vaccine for humans. Baculovirus has attracted attention as a vaccine vector because of its ability to induce a high level of humoral and cellular immunity, low cytotoxicity against various antigens, and biological safety for humans. In this study, we constructed a recombinant baculovirus- based vaccine expressing the M2 protein of RSV under the control of cytomegalovirus promoter (Bac_RSVM2) to induce CD8⁺ T-cell responses which play an important role in viral clearance, and investigated its protective efficacy against RSV infection. Immunization with Bac_RSVM2 via intranasal or intramuscular route effectively elicited the specific CD8⁺ T-cell responses. Most notably, immunization with Bac_RSVM2 vaccine almost completely protected mice from RSV challenge without vaccine-enhanced immunopathology. In conclusion, these results suggest that Bac_RSVM2 vaccine employing the baculovirus delivery platform has promising potential to be developed as a safe and novel RSV vaccine that provides protection against RSV infection.