可用于辅助蛋白功能研究的人呼吸道合胞病毒双顺反子微型基因组的构建及拯救

【目的】构建可表达增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的人呼吸道合胞病毒(human respiratory syncytial virus,RSV)双顺反子微型基因组cDNA克隆及重组质粒,并进行拯救,以探讨并验证RSV的聚合酶蛋白或辅助蛋白在RSV反向遗传学操作中的作用。【方法】利用全基因合成和分子生物学相结合的方法,在获得可分别表达EGFP和无生物活性蛋白的单顺反子微型基因组质粒pUC57-RSV-EGFP和pUC57-RSV-ORF1的基础上,进一步克隆至pBR322B载体,获得编码EGFP及无生物活性蛋白的双顺反子微型基因组质粒,经限制性内切酶和核酸序列分析正确后,与可表达4种辅助蛋白的辅助质粒共转染至BHK-T7细胞,通过荧光显微镜观察EGFP的表达以及RT-qPCR对EGFP mRNA的转录水平进行定量分析。【结果】成功构建了编码EGFP和无生物活性蛋白的RSV双顺反子微型基因组质粒pBR322B-RSVⅡ-EGFP,经与编码4种辅助蛋白的辅助质粒共转染至BHK-T7细胞,发现4种辅助蛋白对EGFP的表达具有不同的功能活性。【结论】以可表达EGFP报告基因的RSV双顺反子微型基因组重组质粒,实现了对4种辅助蛋白的功能验证,其中M2-1蛋白在双顺反子微型基因组拯救过程中具有转录延长的生物学活性。     

Structural phosphoprotein M2-1 of the human respiratory syncytial virus is an RNA binding protein

The structural phosphoprotein M2-1 of human respiratory syncytial virus (HRSV) Long strain shows RNA binding capacity in three different assays that detect RNA-protein complexes: cross-linking, gel retardation, and Northern-Western assays. It is able to bind HRSV leader RNA specifically with cooperative kinetics, with an apparent K(d) of at least 90 nM. It also binds to long RNAs with no sequence specificity. The RNA binding domain has been located between amino acid residues 59 and 85, at the NH(2) terminus of the protein. This region contains the phosphorylatable amino acid residues threonine 56 and serine 58, whose modification decreases the binding capacity of M2-1 protein to long RNAs.     

Development of mRNA vaccines against respiratory syncytial virus (RSV)

Respiratory syncytial virus (RSV) is a single-stranded negative-sense RNA virus that is the primary etiologic pathogen of bronchitis and pneumonia in infants and the elderly. Currently, no preventative vaccine has been approved for RSV infection. However, advances in the characterization, and structural resolution, of the RSV surface fusion glycoprotein have revolutionized RSV vaccine development by providing a new target for preventive interventions. In general, six different approaches have been adopted in the development of preventative RSV therapeutics, namely, particle-based vaccines, vector-based vaccines, live-attenuated or chimeric vaccines, subunit vaccines, mRNA vaccines, and monoclonal antibodies. Among these preventive interventions, MVA-BN-RSV, RSVpreF3, RSVpreF, Ad26. RSV.preF, nirsevimab, clesrovimab and mRNA-1345 is being tested in phase 3 clinical trials, and displays the most promising in infant or elderly populations. Accompanied by the huge success of mRNA vaccines in COVID-19, mRNA vaccines have been rapidly developed, with many having entered clinical studies, in which they have demonstrated encouraging results and acceptable safety profiles. In fact, Moderna has received FDA approval, granting fast-track designation for an investigational single-dose mRNA-1345 vaccine against RSV in adults over 60 years of age. Hence, mRNA vaccines may represent a new, more successful, chapter in the continued battle to develop effective preventative measures against RSV. This review discusses the structure, life cycle, and brief history of RSV, while also presenting the current advancements in RSV preventatives, with a focus on the latest progress in RSV mRNA vaccine development. Finally, future prospects for this field are presented.     

Protein kinase C-alpha activity is required for respiratory syncytial virus fusion to human bronchial epithelial cells

Respiratory syncytial virus (RSV) infection activates protein kinase C (PKC), but the precise PKC isoform(s) involved and its role(s) remain to be elucidated. On the basis of the activation kinetics of different signaling pathways and the effect of various PKC inhibitors, it was reasoned that PKC activation is important in the early stages of RSV infection, especially RSV fusion and/or replication. Herein, the role of PKC-alpha during the early stages of RSV infection in normal human bronchial epithelial cells is determined. The results show that the blocking of PKC-alpha activation by classical inhibitors, pseudosubstrate peptides, or the overexpression of dominant-negative mutants of PKC-alpha in these cells leads to significantly decreased RSV infection. RSV induces phosphorylation, activation, and cytoplasm-to-membrane translocation of PKC-alpha. Also, PKC-alpha colocalizes with virus particles and is required for RSV fusion to the cell membrane. Thus, PKC-alpha could provide a new pharmacological target for controlling RSV infection.     

The human respiratory syncytial virus matrix protein is required for maturation of viral filaments

An experimental system was developed to generate infectious human respiratory syncytial virus (HRSV) lacking matrix (M) protein expression (M-null virus) from cDNA. The role of the M protein in virus assembly was then examined by infecting HEp-2 and Vero cells with the M-null virus and assessing the impact on infectious virus production and viral protein trafficking. In the absence of M, the production of infectious progeny was strongly impaired. Immunofluorescence (IF) microscopy analysis using antibodies against the nucleoprotein (N), attachment protein (G), and fusion protein (F) failed to detect the characteristic virus-induced cell surface filaments, which are believed to represent infectious virions. In addition, a large proportion of the N protein was detected in viral replication factories termed inclusion bodies (IBs). High-resolution analysis of the surface of M-null virus-infected cells by field emission scanning electron microscopy (SEM) revealed the presence of large areas with densely packed, uniformly short filaments. Although unusually short, these filaments were otherwise similar to those induced by an M-containing control virus, including the presence of the viral G and F proteins. The abundance of the short, stunted filaments in the absence of M indicates that M is not required for the initial stages of filament formation but plays an important role in the maturation or elongation of these structures. In addition, the absence of mature viral filaments and the simultaneous increase in the level of the N protein within IBs suggest that the M protein is involved in the transport of viral ribonucleoprotein (RNP) complexes from cytoplasmic IBs to sites of budding.     

Respiratory syncytial virus (RSV) F, G, M2 (22K), and N proteins each induce resistance to RSV challenge, but resistance induced by M2 and N proteins is relatively short-lived.

The ability of recombinant vaccinia viruses that separately encoded 9 of the 10 known respiratory syncytial virus (RSV) proteins to induce resistance to RSV challenge was studied in BALB/c mice. Resistance was examined at two intervals following vaccination to examine early (day 9) as well as late (day 28) immunity. BALB/c mice were inoculated simultaneously by the intranasal and intraperitoneal routes with a recombinant vaccinia virus encoding one of the following RSV proteins: F, G, N, P, SH, M, 1B, 1C, or M2 (22K). A parainfluenza virus type 3 HN protein recombinant (Vac-HN) served as a negative control. One half of the mice were challenged with RSV intranasally on day 9, and the remaining animals were challenged on day 28 postvaccination. Mice previously immunized by infection with RSV, Vac-F, or Vac-G were completely or almost completely resistant to RSV challenge on both days. In contrast, immunization with Vac-HN, -P, -SH, -M, -1B, or -1C did not induce detectable resistance to RSV challenge. Mice previously infected with Vac-M2 or Vac-N exhibited significant but not complete resistance on day 9. However, in both cases resistance had largely waned by day 28 and was detectable only in mice immunized with Vac-M2. These results demonstrate that F and G proteins expressed by recombinant vaccinia viruses are the most effective RSV protective antigens. This study also suggests that RSV vaccines need only contain the F and G glycoproteins, because the immunity conferred by the other proteins is less effective and appears to wane rapidly with time.     

Regulated but not constitutive human respiratory syncytial virus (HRSV) P protein phosphorylation is essential for oligomerization

Purified human respiratory syncytial virus (HRSV) P phosphoprotein from transfected HEp-2 cells is able to oligomerize forming tetramers. The bulk of constitutive P protein phosphorylation (99. 8%) (serine residues 116, 117, 119, 232 and 237) can be removed without affecting protein oligomerization. However, dephosphorylated P protein, produced in bacteria, is unable to oligomerize. This difference can be explained by a transient P protein phosphorylation, detected in HEp-2 cells, that could be essential for P protein oligomerization.     

Recombinant Sendai virus expressing the G glycoprotein of respiratory syncytial virus (RSV) elicits immune protection against RSV

Although RSV causes serious pediatric respiratory disease, an effective vaccine does not exist. To capture the strengths of a live virus vaccine, we have used the murine parainfluenza virus type 1 (Sendai virus [SV]) as a xenogeneic vector to deliver the G glycoprotein of RSV. It was previously shown (J. L. Hurwitz, K. F. Soike, M. Y. Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, Vaccine 15:533-540, 1997) that intranasal SV protected African green monkeys from challenge with the related human parainfluenza virus type 1 (hPIV1), and SV has advanced to clinical trials as a vaccine for hPIV1 (K. S. Slobod, J. L. Shenep, J. Lujan-Zilbermann, K. Allison, B. Brown, R. A. Scroggs, A. Portner, C. Coleclough, and J. L. Hurwitz, Vaccine, in press). Recombinant SV expressing RSV G glycoprotein was prepared by using reverse genetics, and intranasal inoculation of cotton rats elicited RSV-specific antibody and elicited protection from RSV challenge. RSV G-recombinant SV is thus a promising live virus vaccine candidate for RSV.     

Nonstructural proteins 1 and 2 of respiratory syncytial virus suppress maturation of human dendritic cells

Human respiratory syncytial virus (RSV) is the most important agent of serious pediatric respiratory tract disease worldwide. One of the main characteristics of RSV is that it readily reinfects and causes disease throughout life without the need for significant antigenic change. The virus encodes nonstructural protein 1 (NS1) and NS2, which are known to suppress type I interferon (IFN) production and signaling. In the present study, we monitored the maturation of human monocyte-derived myeloid dendritic cells (DC) following inoculation with recombinant RSVs bearing deletions of the NS1 and/or NS2 proteins and expressing enhanced green fluorescent protein. Deletion of the NS1 protein resulted in increased expression of cell surface markers of DC maturation and an increase in the expression of multiple cytokines and chemokines. This effect was enhanced somewhat by further deletion of the NS2 protein, although deletion of NS2 alone did not have a significant effect. The upregulation was largely inhibited by pretreatment with a blocking antibody against the type I IFN receptor, suggesting that suppression of DC maturation by NS1/2 is, at least in part, a result of IFN antagonism mediated by these proteins. Therefore, this study identified another effect of the NS1 and NS2 proteins. The observed suppression of DC maturation may result in decreased antigen presentation and T-lymphocyte activation, leading to incomplete and/or weak immune responses that might contribute to RSV reinfection.     

Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells

Bovine respiratory syncytial virus (BRSV) escapes from cellular responses to alpha/beta interferon (IFN-alpha/beta) by a concerted action of the two viral nonstructural proteins, NS1 and NS2. Here we show that the NS proteins of human RSV (HRSV) are also able to counteract IFN responses and that they have the capacity to protect replication of an unrelated rhabdovirus. Even combinations of BRSV and HRSV NS proteins showed a protective activity, suggesting common mechanisms and cellular targets of HRSV and BRSV NS proteins. Although able to cooperate, NS proteins from BRSV and HRSV showed differential protection capacity in cells from different hosts. A chimeric BRSV with HRSV NS genes (BRSV h1/2) was severely attenuated in bovine IFN competent MDBK and Klu cells, whereas it replicated like BRSV in IFN-incompetent Vero cells or in IFN-competent human HEp-2 cells. After challenge with exogenous IFN-alpha, BRSV h1/2 was better protected than wild-type BRSV in human HEp-2 cells. In contrast, in cells of bovine origin, BRSV h1/2 was much less resistant to exogenous IFN than wild-type BRSV. These data demonstrate that RSV NS1 and NS2 proteins are major determinants of host range. The differential IFN escape capacity of RSV NS proteins in cells from different hosts provides a basis for rational development of attenuated live RSV vaccines.     

Respiratory syncytial virus (RSV) infection induces cyclooxygenase 2: a potential target for RSV therapy

Cyclooxygenases (COXs) are rate-limiting enzymes that initiate the conversion of arachidonic acid to prostanoids. COX-2 is the inducible isoform that is up-regulated by proinflammatory agents, initiating many prostanoid-mediated pathological aspects of inflammation. The roles of cyclooxygenases and their products, PGs, have not been evaluated during respiratory syncytial virus (RSV) infection. In this study we demonstrate that COX-2 is induced by RSV infection of human lung alveolar epithelial cells with the concomitant production of PGs. COX-2 induction was dependent on the dose of virus and the time postinfection. PG production was inhibited preferentially by NS-398, a COX-2-specific inhibitor, and indomethacin, a pan-COX inhibitor, but not by SC-560, a COX-1-specific inhibitor. In vivo, COX-2 mRNA expression and protein production were strongly induced in the lungs and cells derived from bronchioalveolar lavage of cotton rats infected with RSV. The pattern of COX-2 expression in vivo in lungs is cyclical, with a final peak on day 5 that correlates with maximal histopathology. Treatment of cotton rats with indomethacin significantly mitigated lung histopathology produced by RSV. The studies described in this study provide the first evidence that COX-2 is a potential therapeutic target in RSV-induced disease.     

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.     

Immunization of macaques with formalin-inactivated respiratory syncytial virus (RSV) induces interleukin-13-associated hypersensitivity to subsequent RSV infection

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and the elderly. RSV vaccine development has been hampered by results of clinical trials in the 1960s, when formalin-inactivated whole-RSV preparations adjuvated with alum (FI-RSV) were found to predispose infants for enhanced disease following subsequent natural RSV infection. We have reproduced this apparently immunopathological phenomenon in infant cynomolgus macaques and identified immunological and pathological correlates. Vaccination with FI-RSV induced specific virus-neutralizing antibody responses accompanied by strong lymphoproliferative responses. The vaccine-induced RSV-specific T cells predominantly produced the Th2 cytokines interleukin-13 (IL-13) and IL-5. Intratracheal challenge with a macaque-adapted wild-type RSV 3 months after the third vaccination elicited a hypersensitivity response associated with lung eosinophilia. The challenge resulted in a rapid boosting of IL-13-producing T cells in the FI-RSV-vaccinated animals but not in the FI-measles virus-vaccinated control animals. Two out of seven FI-RSV-vaccinated animals died 12 days after RSV challenge with pulmonary hyperinflation. Surprisingly, the lungs of these two animals did not show overt inflammatory lesions. However, upon vaccination the animals had shown the strongest lymphoproliferative responses associated with the most pronounced Th2 phenotype within their group. We hypothesize that an IL-13-associated asthma-like mechanism resulted in airway hyperreactivity in these animals. This nonhuman primate model will be an important tool to assess the safety of nonreplicating candidate RSV vaccines.     

Recombinant respiratory syncytial virus that does not express the NS1 or M2-2 protein is highly attenuated and immunogenic in chimpanzees

Mutant recombinant respiratory syncytial viruses (RSV) which cannot express the NS1 and M2-2 proteins, designated rA2DeltaNS1 and rA2DeltaM2-2, respectively, were evaluated as live-attenuated RSV vaccines. The rA2DeltaNS1 virus contains a large deletion that should have the advantageous property of genetic stability during replication in vitro and in vivo. In vitro, rA2DeltaNS1 replicated approximately 10-fold less well than wild-type recombinant RSV (rA2), while rA2DeltaM2-2 had delayed growth kinetics but reached a final titer similar to that of rA2. Each virus was administered to the respiratory tracts of RSV-seronegative chimpanzees to assess replication, immunogenicity, and protective efficacy. The rA2DeltaNS1 and rA2DeltaM2-2 viruses were 2,200- to 55,000-fold restricted in replication in the upper and lower respiratory tracts but induced a level of RSV-neutralizing antibody in serum that was only slightly reduced compared to the level induced by wild-type RSV. The replication of wild-type RSV in immunized chimpanzees after challenge was reduced more than 10,000-fold at each site. Importantly, rA2DeltaNS1 and rA2DeltaM2-2 were 10-fold more restricted in replication in the upper respiratory tract than was the cpts248/404 virus, a vaccine candidate that retained mild reactogenicity in the upper respiratory tracts of 1-month-old infants. Thus, either rA2DeltaNS1 or rA2DeltaM2-2 might be appropriately attenuated for this age group, which is the major target population for an RSV vaccine. In addition, these results show that neither NS1 nor M2-2 is essential for RSV replication in vivo, although each is important for efficient replication.