Lymphatic distribution considerations for subunit vaccine design and development.

Subunit vaccines are an important platform for controlling current and emerging infectious diseases. The lymph nodes are the primary site generating the humoral response and delivery of antigens to these sites is critical to effective immunization. Indeed, the duration of antigen exposure within the lymph node is correlated with the antibody response. While current licensed vaccines are typically given through the intramuscular route, injecting vaccines subcutaneously allows for direct access to lymphatic vessels and therefore can enhance the transfer of antigen to the lymph nodes. However, protein subunit antigen uptake into the lymph nodes is inefficient, and subunit vaccines require adjuvants to stimulate the initial immune response. Therefore, formulation strategies have been developed to enhance the exposure of subunit proteins and adjuvants to the lymph nodes by increasing lymphatic uptake or prolonging the retention at the injection site. Given that lymph node exposure is a crucial consideration in vaccine design, in depth analyses of the pharmacokinetics of antigens and adjuvants should be the focus of future preclinical and clinical studies. This review will provide an overview of formulation strategies for targeting the lymphatics and prolonging antigen exposure and will discuss pharmacokinetic evaluations which can be applied toward vaccine development.

Recent advances in the prevention of respiratory syncytial virus in pediatrics.

PURPOSE OF REVIEW: Respiratory syncytial virus (RSV) is a ubiquitous virus and the leading cause of pediatric hospitalization in the United States. Prevention strategies are key for reducing the burden of RSV. Several new agents aimed at preventing RSV in infants and children were FDA-approved in 2023, and many more are in the development pipeline. This review highlights new developments in RSV prevention in pediatric patients and the important safety considerations for clinical trials. RECENT FINDINGS: Two new preventive therapies were FDA approved in 2023; a maternal vaccine (Abrysvo) and a mAb (Beyfortus) have both demonstrated reduction in medically attended lower respiratory tract infections in infants and children. Evaluation of ongoing clinical trials demonstrates that the field is expanding further to include direct immunization of infants and children utilizing a variety of delivery modalities. While these developments present the optimistic prospect of RSV prevention in a range of ages, acute and long-term risks must be carefully evaluated. SUMMARY: Prevention of RSV is more accessible than ever, but careful consideration must be given to risks associated with new and developing prevention strategies. Rigor of clinical trials including longitudinal outcomes of agents in development and postmarketing surveillance of newly approved therapies will be of paramount importance to ensure long-term safety of new RSV prevention strategies.

Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection.

Clinical studies report that viral infections promote acute or chronic bacterial infections at multiple host sites. These viral-bacterial co-infections are widely linked to more severe clinical outcomes. In experimental models in vitro and in vivo, virus-induced interferon responses can augment host susceptibility to secondary bacterial infection. Here, we used a cell-based screen to assess 389 interferon-stimulated genes (ISGs) for their ability to induce chronic Pseudomonas aeruginosa infection. We identified and validated five ISGs that were sufficient to promote bacterial infection. Furthermore, we dissected the mechanism of action of hexokinase 2 (HK2), a gene involved in the induction of aerobic glycolysis, commonly known as the Warburg effect. We report that HK2 upregulation mediates the induction of Warburg effect and secretion of L-lactate, which enhances chronic P. aeruginosa infection. These findings elucidate how the antiviral immune response renders the host susceptible to secondary bacterial infection, revealing potential strategies for viral-bacterial co-infection treatment.

Exacerbated lung inflammation following secondary RSV exposure is CD4+ T cell-dependent and is not mitigated in infant BALB/c mice born to PreF-vaccinated dams.

Respiratory syncytial virus (RSV) is the leading cause of childhood hospitalizations due to bronchiolitis in children under 5 years of age. Moreover, severe RSV disease requiring hospitalization is associated with the subsequent development of wheezing and asthma. Due to the young age in which viral protection is needed and risk of vaccine enhanced disease following direct infant vaccination, current approaches aim to protect young children through maternal immunization strategies that boost neutralizing maternal antibody (matAb) levels. However, there is a scarcity of studies investigating the influence of maternal immunization on secondary immune responses to RSV in the offspring or whether the subsequent development of wheezing and asthma is mitigated. Toward this goal, our lab developed a murine model of maternal RSV vaccination and repeat RSV exposure to evaluate the changes in immune response and development of exacerbated lung inflammation on secondary RSV exposure in mice born to immunized dams. Despite complete protection following primary RSV exposure, offspring born to pre-fusion F (PreF)-vaccinated dams had exaggerated secondary ILC2 and Th2 responses, characterized by enhanced production of IL-4, IL-5, and IL-13. These enhanced type 2 cellular responses were associated with exaggerated airway eosinophilia and mucus hyperproduction upon re-exposure to RSV. Importantly, depletion of CD4+ T cells led to complete amelioration of the observed type 2 pathology on secondary RSV exposure. These unanticipated results highlight the need for additional studies that look beyond primary protection to better understand how maternal immunization shapes subsequent immune responses to repeat RSV exposure.

Modeling the kinetics of lymph node retention and exposure of a cargo protein delivered by biotin-functionalized nanoparticles.

Generation of protective immunity through vaccination arises from the adaptive immune response developed primarily in the lymph nodes drained from the immunization site. Relative to the intramuscular route, subcutaneous administration allows for direct and rapid access to the lymphatics, but accumulation of soluble protein antigens within the lymph nodes is limited. Subunit vaccines also require immune stimulating adjuvants which may not accumulate in the same lymph nodes simultaneously with antigen. Herein we report the use of biotinylated poly (lactic-co-glycolic acid) nanoparticles (bNPs) to enhance delivery of a model protein antigen to the lymphatics. bNPs provide dual functionality as adjuvant and vehicle to localize antigens with stimulated immune cells in the same draining lymph node. Using streptavidin as a model antigen, which can be loaded directly onto the bNP surface, we evaluated the kinetics of lymph node occupancy and adaptive immune responses in wildtype C57BL/6 mice. Antigen exposure in vivo was significantly improved through surface loading onto bNPs, and we developed a working kinetic model to account for the retention of both particles and antigen in draining lymph nodes. We observed enhanced T cell responses and antigen-specific B cell response in vivo when antigen was delivered on the particle surface. This work highlights the advantage of combining intrinsic adjuvant and antigen loading in a single entity, and the utility of kinetic modeling in the understanding of particle-based vaccines. STATEMENT OF SIGNIFICANCE: Development of safe and effective subunit vaccines depends on effective formulations that render optimized exposure and colocalization of antigens and adjuvants. In this work, we utilize a nanoparticle system which features self-adjuvanting properties and allows for surface loading of recombinant protein antigens. Using in vivo imaging, we demonstrated prolonged co-localization of the antigen and adjuvant particles in draining lymph nodes and provided evidence of B cell activation for up to 21 days following subcutaneous injection. A pharmacokinetic model was developed as a step towards bridging the translational gap between particulate-based vaccines and observed outcomes. The results have implications for the rational design of particle-based vaccines.

RAGE contributes to allergen driven severe neutrophilic airway inflammation via NLRP3 inflammasome activation in mice.

Background: Asthma is a major public healthcare burden, affecting over 300 million people worldwide. While there has been great progress in the treatment of asthma, subsets of patients who present with airway neutrophilia, often have more severe disease, and tend to be resistant to conventional corticosteroid treatments. The receptor for advanced glycation endproducts (RAGE) plays a central role in the pathogenesis of eosinophilic asthma, however, it's role in neutrophilic asthma remains largely uninvestigated. Methods: A mouse model of severe steroid resistant neutrophilic airway disease (SSRNAD) using the common fungal allergen Alternaria alternata (AA) was employed to evaluate the effects of genetic ablation of RAGE and pharmacological inhibition of the NLRP3 inflammasome on neutrophilic airway inflammation. Results: AA exposure induced robust neutrophil-dominant airway inflammation and increased BALF levels of Th1/Th17 cytokines in wild-type mice, which was significantly reduced in RAGE-/- mice. Serum levels of IgE and IgG1 were increased similarly in both wild-type and RAGE-/- mice. Pharmacological inhibition of NLRP3 blocked the effects of AA exposure and NLRP3 inflammasome activation was RAGE-dependent. Neutrophil extracellular traps were elevated in the BALF of wild-type but not RAGE-/- mice and an atypical population of SiglecF+ neutrophils were identified in the BALF. Lastly, time-course studies found that RAGE expression promoted sustained neutrophil accumulation in the BALF of mice in response to AA.

In vitro and in vivo evaluation of cerium oxide nanoparticles in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis among children worldwide, yet there is no vaccine for RSV disease. This study investigates the potential of cube and sphere-shaped cerium oxide nanoparticles (CNP) to modulate reactive oxygen (ROS) and nitrogen (RNS) species and immune cell phenotypes in the presence of RSV infection in vitro and in vivo. Cube and sphere-shaped CNP were synthesized by hydrothermal and ultrasonication methods, respectively. Physico-chemical characterization confirmed the shape of sphere and cube CNP and effect of various parameters on their particle size distribution and zeta potential. In vitro results revealed that sphere and cube CNP differentially modulated ROS and RNS levels in J774 macrophages. Specifically, cube CNP significantly reduced RSV-induced ROS levels without affecting RNS levels while sphere CNP increased RSV-induced RNS levels with minimal effect on ROS levels. Cube CNP drove an M1 phenotype in RSV-infected macrophages in vitro by increasing macrophage surface expression of CD80 and CD86 with a concomitant increase in TNFα and IL-12p70, while simultaneously decreasing M2 CD206 expression. Intranasal administration of sphere and cube-CNP were well-tolerated with no observed toxicity in BALB/c mice. Notably, cube CNP preferentially accumulated in murine alveolar macrophages and induced their activation, avoiding enhanced uptake and activation of other inflammatory cells such as neutrophils, which are associated with RSV-mediated inflammation. In conclusion, we report that sphere and cube CNP modulate macrophage polarization and innate cellular responses during RSV infection.

Prior respiratory syncytial virus infection reduces vaccine-mediated Th2-skewed immunity, but retains enhanced RSV F-specific CD8 T cell responses elicited by a Th1-skewing vaccine formulation.

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.

Immune Cells Activating Biotin-Decorated PLGA Protein Carrier.

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-1ß, 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.

Strategies for active and passive pediatric RSV immunization.

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.

Maternal immunization with adjuvanted RSV prefusion F protein effectively protects offspring from RSV challenge and alters innate and T cell immunity.

Respiratory syncytial virus (RSV) commonly causes severe respiratory tract infections in infants, peaking between 2 and 6 months of age; an age at which direct vaccination is unlikely to be effective. Maternal immunization can deliver high levels of antibodies to newborns, providing immediate protection. Following natural infection, antibodies targeting the prefusion conformation of RSV F protein (PreF) have the greatest neutralizing capacity and thus, may provide infants with a high degree of RSV protection when acquired through maternal vaccination. However, the influence of anti-PreF maternal antibodies on infant immunity following RSV exposure has not been elucidated. To address this knowledge gap, offspring born to dams immunized with a RSV PreF vaccine formulation were challenged with RSV and their immune responses were analyzed over time. These studies demonstrated safety and efficacy for RSV-challenged, maternally-immunized offspring but high and waning maternal antibody levels were associated with differential innate and T cell immunity.

Formulation of the prefusion RSV F protein with a Th1/Th2-balanced adjuvant provides complete protection without Th2-skewed immunity in RSV-experienced young mice.

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 Th2-skewing (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 adjuvant prevented the Th2-skewed immunity observed in RSV-experienced young mice immunized with PreF/Alum.

Prefusion RSV F Immunization Elicits Th2-Mediated Lung Pathology in Mice When Formulated With a Th2 (but Not a Th1/Th2-Balanced) Adjuvant Despite Complete Viral Protection.

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 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.

Mouse Models of Viral Infection.

Viral respiratory tract infections are common in both children and adults. Mouse models of viral infection enable the characterization of host immune factors that protect against or promote virus infection; thus, mouse models are essential for interrogation of potential therapeutic targets. Moreover, they serve as critical models for the development of novel vaccine strategies. In this chapter, we describe methods for establishing mouse models of respiratory syncytial virus (RSV) and H1N1 influenza A virus infection. Protocols are provided for viral culture and expansion, plaque-forming assays for viral quantification, and infection of mice. Alternate modifications to the models are also described, and their potential impact is discussed.

Localization of the T-cell response to RSV infection is altered in infant mice.

OBJECTIVES: Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections worldwide, causing disproportionate morbidity and mortality in infants and children. Infants with stronger Th1 responses have less severe disease, yet little is known about the infant T-cell response within the air space. Thus, we tested the hypothesis that RSV infected infant mice would have quantitative and qualitative deficiencies in CD4+ and CD8+ T-cell populations isolated from the bronchoalveolar lavage when compared to adults and that local delivery of IFN-γ would increase airway CD4+ Tbet+ and CD8+ Tbet+ T-cell responses. METHODS: We compared the localization of T-cell responses in RSV-infected infant and adult mice and investigated the effects of local IFN-γ administration on infant cellular immunity. RESULTS: Adult CD8+ CD44HI and CD4+ CD44HI Tbet+ T-cells accumulated in the alveolar space whereas CD4+ CD44HI Tbet+ T-cells were evenly distributed between the infant lung tissue and airway and infant lungs contained higher frequencies of CD8+ T-cells. Delivery of IFN-γ to the infant airway failed to increase the accumulation of T-cells in the airspace and unexpectedly reduced CD4+ CD44HI Tbet+ T-cells. However, intranasal IFN-γ increased RSV F protein-specific CD8+ T-cells in the alveolar space. CONCLUSION: 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.

Preformulation and Vaginal Film Formulation Development of Microbicide Drug Candidate CSIC for HIV prevention.

PURPOSE: 5-chloro-3-[phenylsulfonyl] indole-2-carboxamide (CSIC) is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 which has been shown to have a more desirable resistance profile than other NNRTIs in development as HIV prevention strategies. This work involves generation of preformulation data for CSIC and systematic development of a cosolvent system to effectively solubilize this hydrophobic drug candidate. This system was then applied to produce a polymeric thin film solid dosage form for vaginal administration of CSIC for use in prevention of sexual acquisition of HIV. METHODS: Extensive preformulation, formulation development, and film characterization studies were conducted. An HPLC method was developed for CSIC quantification. Preformulation tests included solubility, crystal properties, stability, and drug-excipient compatibility. Cytotoxicity was evaluated using both human epithelial and mouse macrophage cell lines. Ternary phase diagram methodology was used to identify a cosolvent system for CSIC solubility enhancement. Following preformulation evaluation, a CSIC film formulation was developed and manufactured using solvent casting technique. The developed film product was assessed for physicochemical properties, anti-HIV bioactivity, and Lactobacillus biocompatibility during 12-month stability testing period. RESULTS: Preformulation studies showed CSIC to be very stable. Due to its hydrophobicity, a cosolvent system consisting of polyethylene glycol 400, propylene glycol, and glycerin (5:2:1, w/w/w) was developed, which provided a uniform dispersion of CSIC in the film formulation. The final film product met target specifications established for vaginal microbicide application. CONCLUSIONS: The hydrophobic drug candidate CSIC was successfully formulated with high loading capacity in a vaginal film by means of a cosolvent system. The developed cosolvent strategy is applicable for incorporation of other hydrophobic drug candidates in the film platform.

Data describing IFNγ-mediated viral clearance in an adult mouse model of respiratory syncytial virus (RSV).

The data presented here are related to the research article entitled "Age predicts cytokine kinetics and innate immune cell activation following intranasal delivery of IFNγ and GM-CSF in a mouse model of RSV infection" (Eichinger et al., 2017) [1]. The cited manuscript demonstrated that the macrophage-stimulating cytokine, interferon gamma (IFNγ), but not granulocyte macrophage-colony stimulating factor (GM-CSF), effectively enhanced viral clearance in infant mice infected with respiratory syncytial virus (RSV) following intranasal delivery. This article describes the immune response and viral clearing effects of intranasal IFNγ in RSV-infected adult BALB/c mice demonstrating delayed production of endogenous IFNγ. The dataset is made publicly available to extrapolate the role of IFNγ in RSV-infected adult mice.

Age predicts cytokine kinetics and innate immune cell activation following intranasal delivery of IFNγ and GM-CSF in a mouse model of RSV infection.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children and is further associated with increased healthcare utilization and cost of care in the first years of life. Severe RSV disease during infancy has also been linked to the later development of allergic asthma, yet there remains no licensed RSV vaccine or effective treatment. Pre-clinical and clinical studies have shown that disease severity and development of allergic asthma are associated with differences in cytokine production. As a result, stimulation of the innate host immune response with immune potentiators is gaining attention for their prospective application in populations with limited immune responses to antigenic stimuli or against pathogens for which vaccines do not exist. Specifically, macrophage-activating cytokines such as interferon gamma (IFNγ) and granulocyte colony-stimulating factor (GM-CSF) are commercially available immune potentiators used to prevent infections in patients with chronic granulomatous disease and febrile neutropenia, respectively. Moreover, an increasing number of reports describe the protective function of IFNγ and GM-CSF as vaccine adjuvants. Although a positive correlation between cytokine production and age has previously been reported, little is known about age-dependent cytokine metabolism or immune activating responses in infant compared to adult lungs. Here we use a non-compartmental pharmacokinetic model in naïve and RSV-infected infant and adult BALB/c mice to determine the effect of age on IFNγ and GM-CSF elimination and innate cell activation following intranasal delivery.

Respiratory syncytial virus infection enhances Pseudomonas aeruginosa biofilm growth through dysregulation of nutritional immunity.

Clinical observations link respiratory virus infection and Pseudomonas aeruginosa colonization in chronic lung disease, including cystic fibrosis (CF) and chronic obstructive pulmonary disease. The development of P. aeruginosa into highly antibiotic-resistant biofilm communities promotes airway colonization and accounts for disease progression in patients. Although clinical studies show a strong correlation between CF patients' acquisition of chronic P. aeruginosa infections and respiratory virus infection, little is known about the mechanism by which chronic P. aeruginosa infections are initiated in the host. Using a coculture model to study the formation of bacterial biofilm formation associated with the airway epithelium, we show that respiratory viral infections and the induction of antiviral interferons promote robust secondary P. aeruginosa biofilm formation. We report that the induction of antiviral IFN signaling in response to respiratory syncytial virus (RSV) infection induces bacterial biofilm formation through a mechanism of dysregulated iron homeostasis of the airway epithelium. Moreover, increased apical release of the host iron-binding protein transferrin during RSV infection promotes P. aeruginosa biofilm development in vitro and in vivo. Thus, nutritional immunity pathways that are disrupted during respiratory viral infection create an environment that favors secondary bacterial infection and may provide previously unidentified targets to combat bacterial biofilm formation.