Respiratory syncytial virus suppression of the antiviral immune response: Implications for evaluation of candidate vaccines.

Respiratory syncytial virus infections recur throughout life despite induction of immunity by the first natural infection. Results of an extensive series of studies indicate that the virus adversely affects the human antiviral recall response to challenge, although subsequent infections are less severe than the initial illness. The observations suggest that candidate vaccines for respiratory syncytial virus should not be expected to prevent clinical illness upon subsequent exposure. Candidate vaccines may be considered effective if they render a subsequent natural infection less severe. This is what would be expected from an initial and commonly more severe natural infection and sensitization.

Identification of a human respiratory syncytial virus phosphoprotein domain required for virus-like-particle formation.

Perceived inefficiency and inadequate knowledge of the human respiratory syncytial virus (hRSV) assembly process present a hurdle for large-scale production of authentic hRSV virus-like particles (VLPs) for vaccine purposes. We previously established that the matrix protein, phosphoprotein (P), and fusion protein carboxy-terminus were sufficient to generate VLPs that resemble filamentous wildtype hRSV. Here, the contribution of P was examined. By co-expressing matrix, fusion, and modified P proteins, a ser/thr-rich P region (residues 39-57) was found to be critical for VLP formation, whereas the oligomerization domain was not. Substitutions throughout region 39-57 inhibited VLP formation and relevant amino acids were identified. Phosphomimetic substitutions of serines and threonines inhibited VLP formation; Phosphoblatant substitutions did not. The data show that P not only co-regulates replication and transcription but also has an important role in assembly, mediated by a separate domain that likely interacts with M and/or F and is highly regulated by phosphorylation.

Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen.

Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies.

Respiratory syncytial virus vaccine research and development: World Health Organization technological roadmap and preferred product characteristics.

The respiratory syncytial virus causes a considerable respiratory disease burden globally, most markedly in young infants, in low and middle income countries. A diverse product pipeline illustrates the recent intensification of research and development activities for vaccines and monoclonal antibodies against RSV. With the aim to ensure that product development activities are directed to address the public health needs, the World Health Organization has developed a research and development technical roadmap and articulated product characteristics preferences.

Respiratory syncytial virus mechanisms to interfere with type 1 interferons.

Respiratory syncytial virus (RSV) is a member of the Paramyxoviridae family that consists of viruses with nonsegmented negative-strand RNA genome. Infection by these viruses triggers the innate antiviral response of the host, mainly type I interferon (IFN). Essentially all other viruses of this family produce IFN suppressor functions by co-transcriptional RNA editing. In contrast, RSV has evolved two unique nonstructural proteins, NS1 and NS2, to effectively serve this purpose. Together, NS1 and NS2 degrade or sequester multiple signaling proteins that affect both IFN induction and IFN effector functions. While the mechanism of action of NS1 and NS2 is a subject of active research, their effect on adaptive immunity is also being recognized. In this review, we discuss various aspects of NS1 and NS2 function with implications for vaccine design.

Host gene expression and respiratory syncytial virus infection.

Advances in RNA interference (RNAi) and transcription studies have facilitated the application of systematic cell-based loss- or gain-of-function and cell response screening that enable genome-wide analysis of cell factors involved in viral replication and disease. Application of both experimental and computational biology approaches have led to crucial insights into virus infection, its life cycle, and host gene targets for disease intervention. A better understanding of the spatial and temporal host gene interactions during viral infection has enabled insights into mechanisms by which viral proteins co-opt host cell function, and host regulatory mechanisms that influence disease and treatment outcome. In this chapter, approaches to host gene discovery and transcriptome profiling for respiratory syncytial virus (RSV) are discussed in the context of biological relevance for disease intervention in the clinical setting and vaccine development.

Size analysis of residual host cell DNA in cell culture-produced vaccines by capillary gel electrophoresis.

Residual host cell DNA poses potential safety concerns for cell culture-derived vaccines or other biological products. In addition to the quantity of residual DNA, the size distribution is an important measure for determination of its associated risk factor. We have developed a new method for residual DNA size analysis, based on capillary gel electrophoresis (CGE) technology with sensitive laser induced fluorescence detection (LIF). The performance of this method was optimized through empirical selection of appropriate testing conditions and optimized conditions are presented. Examples are given to demonstrate the successful employment of this method for residual DNA size analysis of cell culture-produced vaccine samples.

Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice.

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (P) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines.

[Construction and effect of the recombinant pshRNA plasmid against respiratory syncystial virus M2-1 gene].

OBJECTIVE: Respiratory syncystial virus (RSV) is the most common cause of lower respiratory infections in infants worldwide. There is no reliable vaccine or antiviral drug against RSV at present. RNA interference (RNAi) technology is a potent method to degrade expression of the cognate mRNA. In order to inhibit the replication of RSV at gene level, the effects of specific RNAi against M2-1 gene of RSV on inhibition of viral replication in cell culture system was observed in this study. METHODS: RSV M2-1 gene, which plays a key role in RSV transcription, was chosen in this study and was used as target gene and recombinant plasmid pshRNA7816 targeting the mRNA of RSV M2-1 gene coding sequence was constructed. The pshRNA7816 was transfected into Hep2 cells. The effects of the pshRNA7816 on changes of cytopathogenic effect (CPE) of Hep2 cell induced by RSV infection were observed microscopically. Viral plaque forming assay and MTT assay were used to detect the viral titer change and protective function of the pshRNA7816 on RSV infected Hep2 cell. RESULTS: The recombinant RNAi plasmid pshRNA7816 which targets the mRNA of RSV M2-1 gene was successfully constructed. The pshRNA7816 significantly reduced CPE of RSV infected Hep2 cells, reduced the viral titer of RSV in the cells (P < 0.001). The pshRNA7816 raised the survival rate of RSV infected Hep2 cells (P < 0.001). Non-specific pshRNA plasmid did not show anti-RSV effects (P > 0.05). CONCLUSION: The recombinant pshRNA7816 plasmid which targeted the mRNA of RSV M2-1 gene showed a significant and specific anti-RSV effect.

[Plasmid construction, expression, immunogenicity and protective efficacy of recombinant protein candidate vaccine of respiratory syncytial virus].

To construct plasmid of recombinant protein candidate vaccine of respiratory syncytial virus, express it in E. coli, and to investigate its immunogenicity and protective efficacy. A CD8+ T cell epitope from respiratory syncytial virus (RSV) M2 protein F/M2:81 - 95 and the G:125-225 (G1) gene fragments from RSV-G protein containing B cell epitopes were amplified by PCR method and then inserted into the prokaryotic expression vector pET-DsbA after bonding to a linker. The fusion protein DsbA-G1-Linker-F/M2:81-95 (D-G1LF/M2) was expressed successfully in E. coli BL21 (DE3). The product was proved to be RSV-specific by Western-blot. After purified by affinity chromatography on Ni+ Sepharose and renatured by gradient dialysis. D-G1LF/M2 was used to immune BALB/c mice. D-G1LF/M2 induced high anti-D-G1LF/M2 IgG, anti-RSV IgG and neutralizing antibody titers in serum and lung of BALB/c mice, and elicied RSV-specific CTL responses. The IgG subclass distribution revealed that IgG1/IgG2a ratio was 2.66. Viral titration indicated that D-G1LF/M2 could protect BALB/c mice against RSV challenge in lung.

Shifting immunodominance pattern of two cytotoxic T-lymphocyte epitopes in the F glycoprotein of the Long strain of respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T-lymphocyte (CTL) and antibody responses. Two murine H-2K(d)-restricted CTL epitopes (F85-93 and F92-106) are known in the F protein of the A2 strain of RSV. F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators were generated, and it was found that in this strain only the F85-93 epitope is conserved. Motif based epitope prediction programs and an F2 chain deleted F protein encoded in a recombinant vaccinia virus enabled identification of a new epitope in the Long strain, F249-258, which is presented by K(d) as a 9-mer (TYMLTNSEL) or a 10-mer (TYMLTNSELL) peptide. The results suggest that the 10-mer might be a naturally processed endogenous K(d) ligand. The CD8(+) T-lymphocyte responses to epitopes F85-93 and F249-258 present in the F protein of RSV Long were found to be strongly skewed to F85-93 in in vitro multispecific CTL lines and in vivo during a secondary response to a recombinant vaccinia virus that expresses the entire F protein. However, no hierarchy in CD8(+) T-lymphocyte responses to F85-93 and F249-258 epitopes was observed in vivo during a primary response.

The immunogenicity, protective efficacy and safety of BBG2Na, a subunit respiratory syncytial virus (RSV) vaccine candidate, against RSV-B.

Respiratory syncytial virus (RSV) is divided into subgroups A and B, based primarily on variation within the G glycoprotein. A safe vaccine that protects against both would be the ideal. BBG2Na is a recombinant subunit RSV vaccine candidate derived in part from the G protein of RSV-A. Interestingly, BBG2Na formulated in alum protected against RSV-B challenge at early time points following vaccination in mice. Over 6 months, however, BBG2Na-induced immunogenicity and protective efficacy progressively diminished, such that few animals were considered protected at the end. To study the safety of BBG2Na relative to RSV-B challenge, we established a novel enhanced immunopathology mouse model. We confirmed that RSV-B challenge of formalin-inactivated RSV-A (FI-RSV-A)-immunized BALB/c mice results in enhanced pulmonary pathology. Therefore, this phenomenon is neither subgroup-specific nor dependent on a previously incriminated Th epitope in the RSV-A G protein. In stark contrast, BBG2Na did not induce any signs of enhanced pulmonary pathology. In conclusion, our data indicate that BBG2Na, formulated in alum, induces safe and protective immune responses against RSV-B challenge in mice. However, the duration of protective immunity will probably be insufficient to prevent RSV-B infection for the duration of the RSV epidemic season.