Intranasal immunization of mice with a formalin-inactivated bovine respiratory syncytial virus vaccine co-formulated with CpG oligodeoxynucleotides and polyphosphazenes results in enhanced protection

Abstract

As respiratory syncytial virus (RSV) targets the mucosal surfaces of the respiratory tract, induction of both systemic and mucosal immunity will be critical for optimal protection. In this study, the ability of an intranasally delivered, formalin-inactivated bovine RSV (FI-BRSV) vaccine co-formulated with CpG oligodeoxynucleotides (ODN) and polyphosphazenes (PP) to induce systemic and mucosal immunity, as well as protection from BRSV challenge, was evaluated. Intranasal immunization of mice with FI-BRSV formulated with CpG ODN and PP resulted in both humoral and cell-mediated immunity, characterized by enhanced production of BRSV-specific serum IgG, as well as increased gamma interferon and decreased interleukin-5 production by in vitro-restimulated splenocytes. These mice also developed mucosal immune responses, as was evident from increased production of BRSV-specific IgG and IgA in lung-fragment cultures. Indeed, the increases in serum and mucosal IgG, and in particular mucosal IgA and virusneutralizing antibodies, were the most critical differences observed between FI-BRSV formulated with both CpG ODN and PP in comparison to formulations with CpG ODN, non-CpG ODN or PP individually. Finally, FI-BRSV/CpG/PP was the only formulation that resulted in a significant reduction in viral replication upon BRSV challenge. Co-formulation of CpG ODN and PP is a promising new vaccine platform technology that may have applications in mucosal immunization in humans. INTRODUCTIONHuman respiratory syncytial virus (HRSV) is a leading cause of respiratory disease in infants and young children worldwide (Heilman, 1990) and is responsible for significant economic loss; in the year 2000 alone, there were 86 000 HRSV infection-related hospitalizations in the USA, costing a total of $394 000 000 (Paramore et al., 2004). Nearly 98 % of these hospitalizations occurred in children under 5 years old. Between 1997 and 2000, HRSV bronchiolitis was the leading cause of infant hospitalization and, in 1999, an estimated 360 HRSV-associated postneonatal deaths (i.e. in children aged between 28 days and 1 year) occurred in the USA (Leader & Kohlhase, 2003).Like HRSV, bovine respiratory syncytial virus (BRSV) is an enveloped, non-segmented, single-stranded RNA pneumovirus of the family Paramyxoviridae and order Mononegavirales. BRSV is responsible for significant economic loss to the cattle industry (Stott & Taylor, 1985) and is one of the four known viral components of bovine shipping fever. HRSV and BRSV have similar clinical outcomes in their respective host species, ranging from asymptomatic infection to bronchiolitis and pneumonia, and sometimes death (Philippou et al., 2000). The distribution of both viruses is worldwide.Whilst there are several commercial BRSV vaccines currently available for immunizing cattle, better vaccines that are more efficacious in the face of maternal antibodies and that induce longer-lasting protection would be desirable. Also, there is currently no safe and effective vaccine against HRSV available for use in h...

Animal Models of Respiratory Syncytial Virus Pathogenesis and Vaccine Development: Opportunities and Future Directions

“…Adjuvants have potential to achieve immunogenicity. CpG oligodeoxynucleotides (CpG ODN, a TLR 9 agonist) and monophosphoryl lipid (MPL, a TLR 4 agonist) adjuvants enhance immune responses to experimental HRSV vaccines (Boukhvalova et al, 2006;Mapletoft et al, 2008;Neuzil et al, 1997;Oumouna et al, 2005). A novel oil-in-water nanoemulsion adjuvanted inactivated HRSV vaccine showed promise in mice .…”

“…Another mechanism invokes nonfunctional Ab responses in the absence of appropriate adjuvant, particularly TLR agonists. Co-formulation of ERD-causing inactivated HRSV vaccines with CpG ODN (a TLR9 agonist), MPL (a TLR4 agonist), and other adjuvants ameliorates ERD (Boukhvalova et al, 2006;Delgado et al, 2009;Mapletoft et al, 2008;Oumouna et al, 2005). …”

Formulation of bovine respiratory syncytial virus fusion protein with CpG oligodeoxynucleotide, cationic host defence peptide and polyphosphazene enhances humoral and cellular responses and induces a protective type 1 immune response in mice

“…Four to seven days following infection, cells were collected by scraping and were stored at 270 uC until used for preparation of challenge virus. Virus titres were determined by plaque assay and visualized by immunostaining as described previously (Mapletoft et al, 2008).…”

“…Four to seven days following infection, cells were collected by scraping and were stored at 270 uC until used for preparation of challenge virus. Virus titres were determined by plaque assay and visualized by immunostaining as described previously (Mapletoft et al, 2008).To prepare challenge virus, infected cell-lysate was thawed and centrifuged for 30 min at 1940 g, at 4 uC. The pellet was then resuspended in 1/100 of the original volume in serum-free DMEM and disrupted by sonication.…”

“…The serum-virus mixtures were transferred to duplicate BT cell plates and incubated for 7 days at 37 uC. BRSV plaques were visualized by immunostaining as described previously (Mapletoft et al, 2008), and virus-neutralization titres were expressed as the highest dilution of serum that resulted in ,50 % of cells displaying cytopathic effects. …”