Five decades of research have documented the mechanisms behind the detrimental immune responses associated with FI-RSV vaccines. The current studies indicate that nanoemulsion-inactivated vaccines, such as NE-RSV, may be a safe alternative to FI-RSV. Given the augmented immune responses observed with the NE-RSV vaccine, this approach could be important for vaccinating immunocompromised patients and infants with underdeveloped immune systems. The vaccine is scalable, and large quantities of virus stocks could be inactivated without the use of toxic agents, such as formaldehyde or beta-propiolactone. Since the vaccine is intrinsically microbicidal, vaccine could potentially be produced and stored without preservatives, such as thimerisol. The intranasal delivery avoids the use of needles, and, while potentially mildly unconfortable, is pain-free. Additionally, the ambient temperature stability of NE-based vaccines makes them attractive candidates for use in developing countries, where maintenance of cold storage is problematic. The functional application of the NE-RSV mucosal vaccine via a non-invasive intranasal route also allows for easy and safe approaches to vaccination, especially in children, and focuses the immune response to the proper location to control viral infections in the respiratory tract.
Our data indicates that NE-RSV vaccine-induced protection from RSV infection is associated with enhanced Th1 and Th17 responses. Though the role of Th1/Tc1 responses to RSV is well-delineated, less is known about the role Th17/Tc17-mediated responses play in RSV host defense 
. IL-17A, and other members of the IL-17 family have well-described roles in host defense against extracellular bacteria 
. Conversely, inappropriate activation of the Th17 axis of inflammation contributes to autoimmune disease 
. The role of IL-17 in pulmonary host defense against viral pathogens remains less well explored and may be dose or pathogen dependent. IL-17 clearly has effects on virus infected cells, leading to enhanced induction of IL-8 
. IL-17 producing CD4+ and CD8+ T cells protect mice against viral challenge, however cytotoxicity of virally-infected cells by Tc17 cells may depend on the viral pathogen. Prior work has supported this concept as Tc17 cells are cytolytic against vaccinia but not influenza 
. Transgenic vaccinia virus expressing IL-23, a Th17 promoting cytokine, is cleared faster than WT virus, in an IL-17-dependent manner, suggesting that IL-23 promotes clearance of vaccinia via IL-17 
. Thus, IL17 may be important in clearance of RSV from the lungs of infected individuals and our future experiments will specifically and thoroughly address whether IL-17 has a role in the protective response in this vaccination strategy.
Several mechanisms may play a role in IL-17 mediated responses to RSV. Evidence suggests that there is cross-regulation of Th2 and Th17 responses and therefore IL-17 may serve to antagonize the development of Th2 responses during RSV vaccination 
. Additionally, IL-17 may play other roles in pulmonary host defense as well, including augmentation of pulmonary antibody responses 
. The chief mechanism ascribed to IL-17 and Th17 cells in the context of bacterial infection and autoimmune disease is via the recruitment and activation of neutrophils. Neutrophil recruitment, in turn, mediates pathogen clearance or host cell damage. The role of neutrophils in antiviral immunity remains somewhat enigmatic, but neutrophils produce defensins and can participate in complement-mediated lysis of RSV infected cells 
. Other studies from our laboratories suggest that IL-17 may play a multivariate role in RSV infection. We recently showed that efferent neutralization of IL-17 in TLR7−/− mice (which produce high levels of IL-17 in response to RSV) results in attenuated mucus hypersecretion 
. Conversely, neutralization of IL-17 in RSV infected mice post-syngeneic bone marrow transplant impairs viral clearance (Lindell et al., Manuscript in Preparation). Though we do not yet know the role of IL-17 in RSV vaccination, IL-17/Th17 differentiation is negatively regulated by IFNα 
, the induction of which may be lower in the context of an inactivated virus vaccine and therefore allow the advantageous effects of IL-17 to prevail.
The coexistence of both IgG1 and IgG2a RSV-specific antibodies observed in the absence of IgE in the NE-RSV vaccinated animals was unanticipated, but both isotypes were similarly induced in the serum of non-vaccinated Balb/C mice undergoing secondary RSV infection (data not shown). These data suggest that the immunoglobulin isotypes induced by NE-RSV vaccination are similar to those induced by live viral infection. Immunoglobulin class switch to IgG2a and IgG2b in mice is promoted by IFNγ, whereas IL-4 promotes IgG1 and IgE 
; however IL-4 differentially regulates IgG1 and IgE and low levels of IL-4 can promote IgG1 isotype switch, in the absence of the high levels of IL-4 needed to drive IgE production and suppress IgG1 
. Comparatively less is known about the role of IL-17 in antibody isotype switching, although one recent study showed that Th17 cells promote Ig class switching in vitro
and in vivo 
. By using an adoptive transfer system, the latter studies demonstrate that IL-21 (also produced by Th17 cells), rather than IL-17, was critical for the induction of IgG1 
. Consistent with a role for IL-21 in promoting IgG1, we observed up-regulation of IL-21 message in NE-RSV vaccinated mice four days post-RSV challenge (624.6±265.4 fold increase, versus a 5.4±3.8 fold increase in unvaccinated controls when comparing both to uninfected mice; p<0.01).
The route of vaccination for NE-RSV (intranasal) may reflect a predominantly mucosal immune response. The use of this particular adjuvant allows the generation of a local, effective immune responses that do not lead to damaging immunopathology after RSV exposure. Recent studies suggest that the addition of TLR agonists as adjuvants can enhance the efficacy of vaccines applied to the mucosal compartment 
. While it is yet to be determined whether the nanoemulsion provided TLR signals during immunization, further studies with NE-RSV could examine this possibility.
A number of vaccine approaches to RSV have thus far been tested and/or are currently under consideration, and each of these has distinct advantages and disadvantages 
. These studies indicate that NE-RSV based vaccination promotes both humoral and cellular immune responses to RSV, and effectively improves viral clearance, while not promote detrimental, Th2-mediated immunopathology. This makes a NE-based, inactivated RSV vaccine an attractive approach to provide safe and effective protection against RSV-mediated pulmonary disease.