Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered by the risk of developing enhanced respiratory disease (ERD) upon natural exposure to the virus. Generation of higher quality neutralizing antibodies with stabilized pre-fusion F protein antigens has been proposed as a strategy to prevent ERD. We sought to test whether there was evidence of ERD in naïve BALB/c mice immunized with an unadjuvanted, stabilized pre-fusion F protein, and challenged with RSV line 19. We further sought to determine the extent to which formulation with a Th2-biased (alum) or a more Th1/Th2-balanced (Advax-SM) adjuvant influenced cellular responses and lung pathology. When exposed to RSV, mice immunized with pre-fusion F protein alone (PreF) exhibited increased airway eosinophilia and mucus accumulation. This was further exacerbated by formulation of PreF with Alum (aluminum hydroxide). Conversely, formulation of PreF with a Th1/Th2-balanced adjuvant, Advax-SM, not only suppressed RSV viral replication, but also inhibited airway eosinophilia and mucus accumulation. This was associated with lower numbers of lung innate lymphocyte cells (ILC2s) and CD4+ T cells producing IL-5+ or IL-13+ and increased IFNγ+ CD4+ and CD8+ T cells, in addition to RSV F-specific CD8+ T cells. These data suggest that in the absence of preimmunity, stabilized PreF antigens may still be associated with aberrant Th2 responses that induce lung Eichinger et al. Protection With Th1/Th2-Balanced Prefusion Adjuvant pathology in response to RSV infection, and can be prevented by formulation with more Th1/Th2-balanced adjuvants that enhance CD4+ and CD8+ IFNγ+ T cell responses. This may support the use of stabilized PreF antigens with Th1/Th2-balanced adjuvants like, Advax-SM, as safer alternatives to alum in RSV vaccine candidates.

Together, these data suggest that quantitative and qualitative defects exist in the infant T-cell response to RSV but early, local IFN-γ exposure can increase the CD8 RSV-specific T-cell response.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, with the most severe disease occurring in very young infants. Despite half a century of research there still are no licensed RSV vaccines. Difficulties in RSV vaccine development stem from a number of factors, including: (a) a very short time frame between birth and first RSV exposure; (b) interfering effects of maternal antibodies; and (c) differentially regulated immune responses in infants causing a marked T helper 2 (Th2) immune bias. This review seeks to provide an age-specific understanding of RSV immunity critical to the development of a successful pediatric RSV vaccine. Historical and future approaches to the prevention of infant RSV are reviewed, including passive protection using monoclonal antibodies or maternal immunization strategies versus active infant immunization using pre-fusion forms of RSV F protein antigens formulated with novel adjuvants such as Advax that avoid excess Th2 immune polarization.

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Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections among infants with most infections occurring in the first year of life. Multiple RSV exposures are required for children to mount adult-like immune responses. Although adult RSV immunity is associated with less severe disease, the protection induced through natural infection is short-lived. Therefore, vaccination of RSV-experienced young children may accelerate immunity and provide long-term protection from RSV reinfection. However, the extent to which different Th-biased vaccine regimens influence pre-existing humoral and cellular immunity in RSV-experienced young children is unknown. To address this question, infant BALB/c mice were RSV-infected and subsequently immunized with the prefusion RSV F (PreF) antigen formulated with either a Th2skewing (Alum) or Th1/Th2-balanced (Advax-SM) adjuvant. These studies show that both adjuvants boosted neutralizing antibody and protected from RSV reinfection, but Advax-SM

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered due the risk of enhanced respiratory disease (ERD) following natural RSV exposure and the young age (<6 months) at which children would require protection. Risk factors linked to the development of ERD include poorly neutralizing antibody, seronegative status (never been exposed to RSV), and a Th2-type immune response. Stabilization of the more antigenic prefusion F protein (PreF) has reinvigorated hope for a protective RSV vaccine that elicits potent neutralizing antibody. While anecdotal evidence suggests that children and adults previously exposed to RSV (seropositive) are not at risk for developing vaccine associated ERD, differences in host immune responses in seropositive and seronegative individuals that may protect against ERD remain unclear. It is also unclear if vaccine formulations that skew towards Th1- versus Th2-type immune responses increase pathology or provide greater protection in seropositive individuals. Therefore, the goal of this work was to compare the host immune response to a stabilized prefusion RSV antigen formulated alone or with Th1 or Th2 skewing adjuvants in seronegative and seropositive BALB/c mice. We have developed a novel BALB/c mouse model whereby mice are first infected with RSV (seropositive) and then vaccinated during pregnancy to recapitulate maternal immunization strategies. Results of these studies show that prior RSV infection mitigates vaccine-mediated skewing by Th1- and Th2-polarizing adjuvants that was observed in seronegative animals. Moreover, vaccination with PreF plus the Th1-skewing adjuvant, Advax, increased RSV F85-93-specific CD8 T cells in both seronegative and seropositive dams. These data demonstrate the importance of utilizing seropositive animals in preclinical vaccine studies to assess both the safety and efficacy of candidate RSV vaccines.

Nanoparticle formulations have long been proposed as subunit vaccine carriers owing to their ability to entrap proteins and codeliver adjuvants. Poly(lactic-co-glycolic acid) (PLGA) remains one of the most studied polymers for controlled release and nanoparticle drug delivery, and numerous studies exist proposing PLGA particles as subunit vaccine carriers. In this work we report using PLGA nanoparticles modified with biotin (bNPs) to deliver proteins via adsorption and stimulate professional antigen-presenting cells (APCs). We present evidence showing bNPs are capable of retaining proteins through the biotin−avidin interaction. Surface accessible biotin bound both biotinylated catalase (bCAT) through avidin and streptavidin horseradish peroxidase (HRP). Analysis of the HRP found that activity on the bNPs was preserved once captured on the surface of bNP. Further, bNPs were found to have self-adjuvant properties, evidenced by bNP induced IL-1β, IL-18, and IL-12 production in vitro in APCs, thereby licensing the cells to generate Th1-type helper T cell responses. Cytokine production was reduced in avidin precoated bNPs (but not with other proteins), suggesting that the proinflammatory response is due in part to exposed biotin on the surface of bNPs. bNPs injected subcutaneously were localized to draining lymph nodes detectable after 28 days and were internalized by bronchoalveolar lavage dendritic cells and macrophages in mice in a dose-dependent manner when delivered intranasally. Taken together, these data provide evidence that bNPs should be explored further as potential adjuvanting carriers for subunit vaccines.

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