For the assay, 25 L of beads were were mixed with 25 L of PBS, 25 L of assay buffer and 25 L of BAL supernatant and incubated overnight at 4C. Dunnetts test, compared with PBS vaccinated control mice (*) or compared to live RSV A2 vaccinated mice (#).(TIF) pone.0074905.s002.tif (458K) GUID:?56B822A8-BE63-441D-AA92-43C4DC74B40A Abstract Nanoparticle vaccines were produced using layer-by-layer fabrication and incorporating respiratory syncytial virus (RSV) G protein polypeptides comprising the CX3C chemokine motif. BALB/c mice immunized with G protein nanoparticle vaccines produced a neutralizing antibody response that inhibited RSV replication in the lungs following RSV challenge. ELISPOT analysis showed that G nanoparticle vaccinated mice had increased levels of RSV G protein-specific IL-4 and IFN- secreting cells compared to controls following RSV challenge. Remarkably, RSV challenge of G protein nanoparticle vaccinated mice resulted in increased RSV M2-specific IL-4 and IFN- secreting T cells, and increased M2-specific H-2Kd-tetramer positive CD8+ T cells in the lungs compared to controls. Cell type analysis showed vaccination was not associated with increased pulmonary eosinophilia CNQX disodium salt following RSV challenge. These results demonstrate that vaccination of mice with the RSV G protein nanoparticle vaccines induces a potent neutralizing antibody response, increased G protein- and M2- hSNFS specific T cell responses, and a reduction in RSV disease pathogenesis. Introduction Human respiratory syncytial virus (RSV) is an important viral agent causing serious lower respiratory tract illness in infants, the elderly, and those individuals with cardiopulmonary disease or with impaired immune responses [1C4]. Natural infection with RSV provides incomplete protection from reinfection and disease as demonstrated by the recurrence of even severe RSV infections throughout life [5,6]. Despite decades of effort to develop safe and effective RSV vaccines none have been successful. The first RSV candidate vaccine, a formalin-inactivated alum-precipitated RSV (FICRSV) preparation CNQX disodium salt did not confer protection and was associated with a greater risk of serious disease with subsequent natural RSV infection [7,8]. Live attenuated and inactivated whole virus vaccine candidates have also failed to protect as they were either insufficiently attenuated or demonstrated the potential for enhanced pulmonary disease upon subsequent RSV infection [9C13]. Subunit vaccines based on the RSV F protein isolated from infected cell culture have been evaluated in adults, children over 12 months of age, and in elderly, but despite being well tolerated the F subunit vaccines were not sufficiently immunogenic [14C19]. Evidence indicates that the RSV F protein is important in inducing?protective immunity [16,20], but studies evaluating a BBG2Na?vaccine (a fusion protein that consists of the central conserved region of the RSV G protein fused to the albumin binding domain of streptococcal protein G) in combination with different adjuvants and?by different routes of administration have shown a role for?RSV G protein in protection against RSV [21C23]. Particulate vaccines e.g. virus-like particles (VLPs), nanoparticles and virosomes have been used as new vaccine strategies to potentiate immune response against RSV antigens and have shown promising results [24C30]. A recent study using VLPs demonstrated that mice immunized with VLPs carrying RSV F or G protein had higher viral neutralizing antibodies and significantly decreased lung virus loads after live RSV challenge. However, RSV G protein VLPs showed better protective efficacy than RSV F protein VLPs as determined by the level of virus load in the lungs and morbidity post-challenge . Despite the evidence that RSV G protein can induce protective immunity, G protein has also been implicated in disease pathogenesis [32C35]. One of the disease mechanisms linked to G protein is CX3C chemokine mimicry . RSV G protein has similarities to fractalkine, the only known CX3C chemokine, CNQX disodium salt and has fractalkine-like leukocyte chemotactic activity . RSV G protein acts as a fractalkine receptor antagonist modulating the immune response to infection, and inhibiting fractalkine-mediated responses including altering pulmonary trafficking of CX3CR1+ immune cells, and modifying the magnitude and cadence of cytokine and chemokine expression [37,38]. Subunit vaccination with G protein polypeptides spanning the central conserved region of the G protein induces antibodies that block G protein CX3C-CX3CR1 interaction and disease pathogenesis mediated by RSV infection . Mice vaccinated with polypeptides containing the CX3C motif generate antibodies that inhibit G protein CX3C-CX3CR1 binding and chemotaxis, reduce lung virus.