Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates.

Human respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5' to 3') a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

High-throughput screening of active compounds against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is one of the predominant pathogens causing lower respiratory tract infection in infants and young children worldwide, whereas there is so far no vaccine or drug against RSV infection for clinical use. In this work, we developed and validated a fluorescence-based high-throughput screening (HTS) assay to identify compounds active against RSV, using RSV-mGFP, a recombinant RSV encoding enhanced green fluorescent protein (EGFP). Thereafter, among 54,800 compounds used for our screen, we obtained 62 compounds active against RSV. Among these hits, azathioprine (AZA) and 6-mercaptopurine (6-MP) were identified as RSV inhibitors with half maximal inhibitory concentration (IC50) values of 6.69 ±â€¯1.41 and 3.13 ±â€¯0.98 µM, respectively. Further experiments revealed that they functioned by targeting virus transcription or/and genome replication. In conclusion, the established HTS assay is suitable to screen anti-RSV compounds, and the screened two hits of AZA and 6-MP, as potential anti-RSV agents targeting RSV genome replication/transcription, are worthy of further investigation on their anti-RSV activity in vivo.

Construction and Characterization of a Recombinant Human Respiratory Syncytial Virus Encoding Enhanced Green Fluorescence Protein for Antiviral Drug Screening Assay.

Human respiratory syncytial virus (RSV) is the single most important cause of lower respiratory tract disease in infants and young children and a major viral agent responsible for respiratory tract disease in immunosuppressed individuals and the elderly, but no vaccines and antiviral drugs are available. Herein the recombinant RSV (rRSV) encoding enhanced green fluorescence protein (EGFP, rRSV-EGFP) was constructed and the potential for screening anti-RSV drugs was investigated. The recombinant plasmid of pBRATm-rRSV-EGFP, containing T7 transcription cassette composed of T7 promoter, RSV antigenomic cDNA with EGFP gene, HDV ribozyme (δ), and T7 terminator in the order of 5' to 3', was constructed and cotransfected into BHK/T7-9 cells together with helper plasmids encoding N, P, L, and M2-1 gene, respectively. The rescued rRSV-EGFP was confirmed by increasing expression of EGFP over blind passages and by RT-PCR. rRSV-EGFP was comparable to the other two recombinant RSVs encoding red fluorescent protein (RFP, rRSV-RFP) or luciferase (Luc, rRSV-Luc) in the growth kinetic, and there was a difference in sensitivity between them for screening anti-RSV agents based on infection of HEp-2 cells. The EGFP-encoding rRSV has been constructed and rescued successfully and has the potential for high-throughput anti-RSV drug screening in vitro.

A Built-In CpG Adjuvant in RSV F Protein DNA Vaccine Drives a Th1 Polarized and Enhanced Protective Immune Response.

Human respiratory syncytial virus (RSV) is the most significant cause of acute lower respiratory infection in children. However, there is no licensed vaccine available. Here, we investigated the effect of five or 20 copies of C-Class of CpG ODN (CpG-C) motif incorporated into a plasmid DNA vaccine encoding RSV fusion (F) glycoprotein on the vaccine-induced immune response. The addition of CpG-C motif enhanced serum binding and virus-neutralizing antibody responses in BALB/c mice immunized with the DNA vaccines. Moreover, mice vaccinated with CpG-modified vaccines, especially with the higher 20 copies, resulted in an enhanced shift toward a Th1-biased antibody and T-cell response, a decrease in pulmonary pathology and virus replication, and a decrease in weight loss after RSV challenge. This study suggests that CpG-C motif, cloned into the backbone of DNA vaccine encoding RSV F glycoprotein, functions as a built-in adjuvant capable of improving the efficacy of DNA vaccine against RSV infection.

A single intranasal administration of virus-like particle vaccine induces an efficient protection for mice against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is an important pediatric pathogen causing acute viral respiratory disease in infants and young children. However, no licensed vaccines are currently available. Virus-like particles (VLPs) may bring new hope to producing RSV VLP vaccine with high immunogenicity and safety. Here, we constructed the recombinants of matrix protein (M) and fusion glycoprotein (F) of RSV, respectively into a replication-deficient first-generation adenoviral vector (FGAd), which were used to co-infect Vero cells to assemble RSV VLPs successfully. The resulting VLPs showed similar immunoreactivity and function to RSV virion in vitro. Moreover, Th1 polarized response, and effective mucosal virus-neutralizing antibody and CD8+ T-cell responses were induced by a single intranasal (i.n.) administration of RSV VLPs rather than intramuscular (i.m.) inoculation, although the comparable RSV F-specific serum IgG and long-lasting RSV-specific neutralizing antibody were detected in the mice immunized by both routes. Upon RSV challenge, VLP-immunized mice showed increased viral clearance but decreased signs of enhanced lung pathology and fewer eosinophils compared to mice immunized with formalin-inactivated RSV (FI-RSV). In addition, a single i.n. RSV VLP vaccine has the capability to induce RSV-specific long-lasting neutralizing antibody responses observable up to 15 months. Our results demonstrate that the long-term and memory immune responses in mice against RSV were induced by a single i.n. administration of RSV VLP vaccine, suggesting a successful approach of RSV VLPs as an effective and safe mucosal vaccine against RSV infection, and an applicable and qualified platform of FGAd-infected Vero cells for VLP production.

Enhanced humoral and CD8+ T cell immunity in mice vaccinated by DNA vaccine against human respiratory syncytial virus through targeting the encoded F protein to dendritic cells.

Human respiratory syncytial virus (RSV) is the most important cause of serious lower respiratory tract infection in infants, the elderly, and the immunocompromised population. There is no licensed vaccine against RSV until now. It has been reported that targeting antigen to DEC205, a phagocytosis receptor on dendritic cells (DCs), could induce enhanced CD4+ and CD8+ T cell responses in mice. To develop RSV DNA vaccine and target the encoded antigen protein to DCs, the ectodomain of fusion glycoprotein (sF, amino acids: 23-524) of RSV was fused with anti-DEC205 single-chain Fv fragment (scDEC) and designated scDECF. Following successful expression from the recombinant plasmid of pVAX1/scDECF, the recombinant protein of scDECF was found capable of specifically binding to DEC205 receptor on CHOmDEC205 cells, and facilitating uptake of RSV F by DC2.4 cells in vitro. Furthermore, the higher levels of RSV-specific IgG antibody responses and neutralization antibody titers, as well as RSV F-specific CD8+ T cell responses were induced in mice immunized intramuscularly by pVAX1/scDECF than by the control plasmid of pVAX1/scISOF encoding sF protein fused with isotype matched control single-chain Fv fragment (scISO). Compared with pVAX1/scISOF, both the ratio of IgG2a/IgG1, >1, and the enhanced IFN-γ cytokine were induced in mice following pVAX1/scDECF immunization, which exhibited a Th1 dominant response in pVAX1/scDECF vaccinated mice. Notably, the elevated efficiency of RSV F protein bound by DCs in vivo could also be observed in mice inoculated by pVAX1/scDECF. Collectively, these results demonstrate the enhanced IgG and CD8+ T cell immune responses have been induced successfully by DNA vaccine against RSV by targeting F antigen to DCs via the DEC205 receptor, and this DC-targeting vaccine strategy merits further investigation.

DNA vaccine encoding central conserved region of G protein induces Th1 predominant immune response and protection from RSV infection in mice.

Human respiratory syncytial virus (RSV) can cause serious infection in the lower respiratory tract, especially in infants, young children, the elderly and the immunocompromised population worldwide. Previous study demonstrated the polypeptide (amino acids 148-198) of RSV attachment (G) glycoprotein, corresponding to the central conserved region and encompassing CX3C chemokine motif, could induce antibodies and protection from RSV challenge in mice [1,2]. In this study, we evaluated the immune efficacy of the recombinant DNA vaccine of pVAX1/3G148-198 encoding RSV G protein polypeptide. RSV specific serum IgG antibodies with neutralizing activity were stimulated following prime-boost immunization of pVAX1/3G148-198 intramuscularly, and the ratio of IgG2a/IgG1 was 4.93, indicating a Th1 biased immune response. After challenged intranasally with RSV Long, the vaccinated mice showed both decreased lung RSV titers, pulmonary inflammation and body weight loss. The results suggest that pVAX1/3G148-198 DNA vaccine may be an effective RSV vaccine candidate, and deserves further exploration.

Single chain variable fragment against aß expressed in baculovirus inhibits abeta fibril elongation and promotes its disaggregation.

Alzheimer's disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-ß (Aß) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aß N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aß fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aß aggregation or disaggregation in vitro. Here, a model of cell-free Aß "on-pathway" aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of "on-pathway" aggregation and Aß fibril maturation. The effect of A8 scFv on Aß fibrillogenesis was markedly more significant when administered at the start of the Aß folding reaction. Furthermore, the results also showed that pre-formed Aß fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aß aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free "on-pathway" Aß aggregation and will assist in the development of therapeutic strategies for AD.

Sublingual administration of a helper-dependent adenoviral vector expressing the codon-optimized soluble fusion glycoprotein of human respiratory syncytial virus elicits protective immunity in mice.

Sublingual (s.l.) immunization has been described as a convenient and safe way to induce mucosal immune responses in the respiratory and genital tracts. We constructed a helper-dependent adenoviral (HDAd) vector expressing a condon-optimized soluble fusion glycoprotein (sFsyn) of respiratory syncytial virus (HDAd-sFsyn) and explored the potential of s.l. immunization with HDAd-sFsyn to stimulate immune responses in the respiratory mucosa. The RSV specific systemic and mucosal immune responses were generated in BALB/c mice, and the serum IgG with neutralizing activity was significantly elevated after homologous boost with s.l. application of HDAd-sFsyn. Humoral immune responses could be measured even 14weeks after a single immunization. Upon challenge, s.l. immunization with HDAd-sFsyn displayed an effective protection against RSV infection. These findings suggest that s.l. administration of HDAd-sFsyn acts as an effective and safe mucosal vaccine against RSV infection, and may be a useful tool in the prevention of RSV infection.

Intranasal immunization with a helper-dependent adenoviral vector expressing the codon-optimized fusion glycoprotein of human respiratory syncytial virus elicits protective immunity in BALB/c mice.

BACKGROUND: Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract. Currently, there is no clinically approved vaccine against RSV infection. Recent studies have shown that helper-dependent adenoviral (HDAd) vectors may represent effective and safe vaccine vectors. However, viral challenge has not been investigated following mucosal vaccination with HDAd vector vaccines. METHODS: To explore the role played by HDAd as an intranasally administered RSV vaccine vector, we constructed a HDAd vector encoding the codon optimized fusion glycoprotein (Fsyn) of RSV, designated HDAd-Fsyn, and delivered intranasally HDAd-Fsyn to mice. RESULTS: RSV-specific humoral and cellular immune responses were generated in BALB/c mice, and serum IgG with neutralizing activity was significantly elevated after a homologous boost with intranasal (i.n.) application of HDAd-Fsyn. Humoral immune responses could be measured even 14 weeks after a single immunization. Immunization with i.n. HDAd-Fsyn led to effective protection against RSV infection on challenge. CONCLUSION: The results indicate that HDAd-Fsyn can induce powerful systemic immunity against subsequent i.n. RSV challenge in a mouse model and is a promising candidate vaccine against RSV infection.