The protective immunity induced by intranasally inoculated serotype 63 chimpanzee adenovirus vector expressing human respiratory syncytial virus prefusion fusion glycoprotein in BALB/c mice.

Human respiratory syncytial virus (RSV) is a ubiquitous pediatric pathogen causing serious lower respiratory tract disease worldwide. No licensed vaccine is currently available. In this work, the coding gene for mDS-Dav1, the full-length and prefusion conformation RSV fusion glycoprotein (F), was designed by introducing the stabilized prefusion F (preF) mutations from DS-Cav1 into the encoding gene of wild-type RSV (wtRSV) F protein. The recombinant adenovirus encoding mDS-Cav1, rChAd63-mDS-Cav1, was constructed based on serotype 63 chimpanzee adenovirus vector and characterized in vitro. After immunizing mice via intranasal route, the rChAd63-mDS-Cav1 induced enhanced neutralizing antibody and F-specific CD8+ T cell responses as well as good immune protection against RSV challenge with the absence of enhanced RSV disease (ERD) in BALB/c mice. The results indicate that rChAd63-mDS-Cav1 is a promising mucosal vaccine candidate against RSV infection and warrants further development.

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.

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.

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.

A prime-boost vaccination strategy using attenuated Salmonella typhimurium and a replication-deficient recombinant adenovirus vector elicits protective immunity against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV), for which no clinically approved vaccine is available yet, is globally a serious pediatric pathogen of the lower respiratory tract. Several approaches have been used to develop vaccines against RSV, but none of these have been approved for use in humans. An efficient vaccine-enhancing strategy for RSV is still urgently needed. We found previously that oral SL7207/pcDNA3.1/F and intranasal FGAd/F were able to induce an effective protective immune response against RSV. The heterologous prime-boost immunization regime has been reported recently to be an efficient vaccine-enhancing strategy. Therefore, we investigated the ability of an oral SL7207/pcDNA3.1/F prime and intranasal (i.n.) FGAd/F boost regimen to generate immune responses to RSV. The SL7207/pcDNA3.1/F prime-FGAd/F boost regimen generated stronger RSV-specific humoral and mucosal immune responses in BALB/c mice than the oral SL7207/pcDNA3.1/F regimen alone, and stronger specific cellular immune responses than the i.n. FGAd/F regimen alone. Histopathological analysis showed an increased efficacy against RSV challenge by the heterologous prime-boost regimen. These results suggest that such a heterologous prime-boost strategy can enhance the efficacy of either the SL7207 or the FGAd vector regimen in generating immune responses in BALB/c mice.

Intranasal vaccination with a helper-dependent adenoviral vector enhances transgene-specific immune responses in BALB/c mice.

Helper-dependent adenoviral (HDAd) vectors were developed primarily for genetic disease therapy by deleting all coding regions for attenuating the host cellular immune response to adenovirus (Ad) and long-lasting gene expression. Recently Harui et al. reported that HDAd vaccine could stimulate superior transgene-specific cytotoxic T lymphocyte (CTL) and antibody responses via the intraperitoneal route, compared to first-generation adenoviral (FGAd) vaccine. This prompted us to explore the potential of HDAd as a vaccine vector administrated intranasally. In this study, we prepared HDAd and FGAd vectors expressing enhanced green fluorescent protein (EGFP), respectively, and compared their efficacy in mice. Mice were immunized intranasally with 5x10(9) vp HDAd or FGAd vector particles. Despite stimulating similar anti-Ad antibody responses with FGAd vaccine in the prime/boost strategy, HDAd vector expressing EGFP displayed superior transgene-specific serum IgG, mucosal IgA and cellular immune response, with the characterization of balanced or mixed Th1/Th2 CD4+ T-cell responses. Meanwhile, a single dose of intranasal (i.n.) vaccine of HDAd-EGFP induced a serum IgG response with more efficacy than FGAd-EGFP. In addition, i.n. boost immunization enhanced transgene-specific humoral and cellular responses, compared to single i.n. HDAd-EGFP immunization. Our results suggest that HDAd has potential for a mucosal vaccine vector via i.n. route, which will be useful for the development of vaccines against respiratory viruses, such as respiratory syncytial virus and influenza virus.

[Expression and purification of a secreted form of fusion glycoprotein of human respiratory syncytial virus encoded by recombinant baculovirus].

OBJECTIVE: To study the expression and purification of a secreted form of fusion glycoprotein (sF) of human respiratory syncytial virus (RSV) encoded by recombinant baculovirus. METHODS: According the ORF of F protein, a pair of specific primers was designed and PCR technique was exploited to amplify the gene of sF in which the gene sequence of the transmembrane and cytoplasmic tail domains were replaced by a C-terminal six-histidine tag. Then, a recombinant baculovirus encoding sF-His was constructed, and transfected into sf9 insect cells by Lipofectamine cellfectine reagent. Finally, the expressed sF was purified by Ni2+ -affinity chromatograph. RESULTS: The gene encoding sF-His was obtained. The resulting construct of recombinant baculovirus is capable of expressing sF protein. The concentration of Ni2+ -affinity chromatograph purified sF is 1.084 mg/ml with the purity of no less than 90%. CONCLUSION: Baculovirus expression system is a good method for large scale of preparation of sF. The purified F paves the way for the development of potential RSV vaccine and diagnostic kit, etc.

[Construction and identification of replication deficient recombinant adenovirus encoding F gene of subgroup A human respiratory syncytial virus].

OBJECTIVE: A strain of replication deficient recombinant adenvirus encoding fusion glycoprotein (F) of subgroup A human respiratory syncytial virus (RSV) was constructed and the expression of F was identified. METHODS: The F gene was obtained from pGEM3zf-F with Xho I and Hind III, cloned into adenoviruse shuttle vector pShuttle-CMV,and then the resulting pShuttle-CMV/F was transformed into E. coli BJ5183/p with pAdeasy-1 to produce pre-adenoviral plasmid encoding F by homologous recombination. This resultant plasmid was linearized by digestion with Pac I and transfected into 293 packaging cells to generate FGAd-F. Finally, the expression of F protein was identified by Western Blot analysis. RESULTS: FGAd/F was successfully constructed, and the expression of RSV F protein was identified by Western Blot. CONCLUSION: We have obtained a strain of replication-defective adenovirus FGAd/F encoding RSV F protein, which can be used further to investigate its protective efficacy against RSV infection in vivo.