Pneumovirus Biology And Vaccine Development

人呼吸道合胞病毒（HRSV）是世界范围内儿童呼吸道疾病最重要的病毒病原体，在成人、老年人和骨髓移植受者中也很重要。疫苗开发的障碍包括病毒在细胞培养中生长不良，在大多数动物模型中感染是半容许性的，难以在免疫原性和衰减之间取得适当的平衡，以及非常年幼的婴儿的免疫反应效率低下。我们之前开发了一种完全由cDNA克隆产生传染性RSV的方法，通过这种方法可以通过cDNA中间体将定义的变化引入传染性病毒。这种方法正在被用于构建通过引入已知的点突变和/或基因缺失而减毒的重组候选疫苗。其中一些病毒正在进行临床评估。

Human respiratory syncytial virus (HRSV) is the most important viral agent of pediatric respiratory tract disease worldwide and also is important in adults in general and in the elderly and bone marrow transplant recipients in particular. Obstacles to vaccine development include the poor growth of the virus in cell culture, the semi-permissive nature of infection in most animal models, the difficulty of achieving an appropriate balance between immunogenicity and attenuation, and the inefficiency of the immune response in the very young infant. We previously developed a method for producing infectious RSV entirely from cDNA clones, whereby defined changes can be introduced into infectious virus via the cDNA intermediate. This is being used on an ongoing basis to construct recombinant vaccine candidates that are attenuated by the introduction of known point mutations and/or gene deletions. Some of these viruses are under clinical evaluation. As a strategy to increase the immunogenicity of a live virus vaccine, we moved the G and F genes, encoding the major neutralization and protective antigens, from their natural positions downstream in the gene order to positions immediately following the major viral promoter. This resulted in a 3- to 4-fold increase in the expression of each protein and provided a modest increase in immunogenicity. It thus represents a useful modification that should be included in a live engineered vaccine virus, and is a strategy that should be applicable to any mononegavirus. Another vaccine strategy is to use recombinant human parainfluenza virus (HPIV) such as HPIV serotype 3 (HPIV3) as a vector to express the major HRSV protective antigens, namely the G and F proteins. HPIV3 resembles HRSV in that it efficiently infects the respiratory tract and induces local and systemic immunity, and does not cause disease outside of the respiratory tract. This strategy has several advantages: (1) the vector itself is a needed vaccine, and thus makes a single vaccine against the two most important agents of pediatric respiratory tract disease, (2) HPIV3 can be grown to a substantially higher titer than HRSV in vitro, facilitating vaccine manufacture, and (3) whereas HRSV infectivity is notoriously unstable, that of HPIV3 is not. The recombinant vector used was a chimeric virus called rB/HPIV3 that consists of a bovine PIV3 (BPIV3) backbone bearing the F and HN protective antigen genes from HPIV3. This virus is attenuated due to a natural host range restriction conferred by the BPIV3 backbone and is in itself a promising candidate to be a HPIV3 vaccine. The G and F genes of subgroups A and B were introduced singly or as subgroup-matched pairs into the promoter-proximal position of the B/HPIV3 vector. Evaluation in rodents and rhesus monkeys showed that the rB/HPIV3/RSV viruses retained the attenuation phenotype and were equivalent to rB/HPIV3 and HRSV in immunogenicity against HPIV3 and RSV, respectively. We extended this strategy by engineering the B/HPIV3 vector (lacking the HRSV inserts) to express granulocyte macrophage colony stimulating factor (GM-CSF) from an added gene insert. We had previously shown that the expression of GM-CSF by recombinant HRSV during pulmonary infection of mice stimulated a very substantial increase in the accumulation and activation of pulmonary dendritic cells and macrophages compared to control virus with a heterologous insert. This provides a basis for increased antigen presentation and increased immunogenicity. The recombinant B/HPIV3 virus expressing GM-CSF was administered intranasally to Rhesus monkeys and evaluated for replication and immunogenicity. The B/HPIV3-GM-CSF virus induced three- to six-fold higher levels of HPIV3-specific serum antibodies compared to the control, and also induced several-fold more interferon-gamma-secreting T lymphocytes in the peripheral blood, a measure of stimulation of cellular immunity. A comparable increase in a pediatric vaccine would be anticipated to confirm a very substantial increase in resistance to wild type HPIV3 infection and disease. Thus, it is possible to substantially increase the immunogenicity of an attenuated vaccine virus administered to the respiratory tract. Apoptosis, or programmed cell death, is a host defense mechanism that can reduce virus replication. Apoptosis can also be an important factor in augmenting antigen presentation and the host immune response. We examined apoptosis in response to RSV infection of primary small airway cells, primary tracheal-bronchial cells, and A549 and HEp-2 cell lines. The primary cells and the A549 cell line gave generally similar responses, indicating their appropriateness as models in contrast to HEp-2 cells. RNAse protection assays with probes representing 33 common apoptosis factors provided evidence of strong transcriptional activation of both pro- and anti-apoptotic factors in response to RSV infection, which were further studied at the protein level and by functional assays. In particular, RSV infection strongly up-regulated the expression of Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) and its functional receptors DR4 and DR5. Consistent with the up-regulation of TRAIL receptors, RSV-infected cells became highly sensitive to apoptosis induced by exogenous TRAIL. These findings suggest that RSV-infected cells in vivo are susceptible to killing through the TRAIL pathway by immune cells such as natural killer and CD4+ cells that bear membrane-bound TRAIL. RSV infection also induced several pro-apoptotic factors of the Bcl-2 family and caspases-3, -6, -7, -8, -9, and --10, representing both the death receptor and mitochondrial-dependent apoptotic pathways. RSV also mediated the strong induction of anti-apoptotic factors of the Bcl-2 family, especially Mcl-1, which might account for the delayed induction of apoptosis in RSV-infected cells in the absence of exogenous induction of the TRAIL pathway. Thus, this study showed that RSV-infection triggers counter-balancing pathways of pro- and anti-apoptotic factors, and identified the TRAIL pathway as a mechanism for destroying RSV-infected cells in vivo.