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

Figures

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  Figure 1. The Th2-biased humoral immunity of PreF/Alum and the Th1 skewed-humoral immunity of PreF/Advax-SM protect from RSV challenge. At 7–8 weeks of age, female adult BALB/cJ mice were immunized with phosphate buffered saline vehicle control (naïve & PBS), prefusion RSV F protein alone (PreF), or PreF formulated with Advax-SM (PreF/Advax-SM) or Alum (PreF/Alum) as depicted in (A). Six weeks post-boost, pre-challenge serum was collected and mice were subsequently intranasally challenged with vehicle (naïve) or 5 × 105 PFU/gram of RSV Line 19. Animals were culled for sample collection at 4 or 8 days post infection (dpi) (A). Pre-challenge serum was analyzed for PreF-specific IgG2a (B), IgG1 (C), IgG2a to IgG1 ratios (D) and neutralizing antibody titers (E). At 4 dpi, left lungs were harvested and virus quantified using standard H&E plaque assays (F). Data are represented as mean ± SEM (n = 6-8 mice per group);*p < 0.05, **p < 0.01, and ****p < 0.0001. Due to a lack of pre-existing PreF-specific antibody, PBS was not included in the analysis in (D).

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  Figure 2. PreF and PreF/Alum immunization elicited enhanced pulmonary inflammation characterized by robust airway eosinophil recruitment. Naive mice were immunized and challenged with RSV as described in Figure 1. At 4 dpi, left lungs were formalin filled, paraffin embedded and sectioned for staining with H&E. Each panel represents an individual mouse from the indicated group (Scale bar 100 μm) (A–E). Two blinded, independent pathologists scored all slides as described in the methods. Scores between the two investigators were averaged and data is represented as mean ± SEM (n = 3 mice) (F). At 4 dpi, BAL was collected and eosinophils (G), neutrophils (H), and monocytes (I) were identified via flow cytometry. Data are represented as mean ± SEM (n = 6–8 mice per group);*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Yellow arrows highlight perivascular inflammation and blue arrows highlight peribronchial inflammation.

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  Figure 3. Increased mucus production parallels enhanced inflammation in mice immunized with PreF alone or PreF/Alum. Mice were immunized and challenged with RSV as described in Figure 1. At 4 dpi, lungs were formalin filled, paraffin embedded and sectioned for staining with PAS. Each panel represents an individual mouse from the indicated group (Scale bar 100 μm) (A–E). To quantify the extent of PAS staining, lungs were scored as previously described in the methods. Scores were averaged and the total percentage of PAS+ airways were graphed (n = 3) (F); **p < 0.01 and ***p < 0.001. A more detailed breakdown of scores for each cohort is provided, calculated as a proportion i.e., number of airways of each severity score (0–4) divided by the total number of airways, according to the methods (G–K).

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  Figure 4. PreF/Advax-SM immunization produced Th1-dominant immunity with increased RSV F-specific CD8+ T cells. At 4 dpi, first wash samples were harvested from the airways for quantification of IFNγ (A), IL-5 (B), and IL-13 (C) by Luminex. Right lungs were harvested and homogenized for quantification of ILC2s (D) and ILC2 intracellular cytokine staining of IL-5 (E) and IL-13 (F) by flow cytometry. Intracellular production of IFNγ (G) and IL-5 (H) by CD4+ T cells from lung homogenate were quantified by flow cytometry and the ratio of IFNγ/IL-5-producing CD4+ T cells was calculated (I). Total CD8+ T cells (J) and RSV F85−93-specific CD8+ T cells (K) were measured from BAL. Data are represented as mean ± SEM (n = 6–8 mice per group);*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

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  Figure 5. Inflammation worsens over time in unimmunized compared to immunized groups. At 8 dpi, left lungs were formalin fixed and stained with H&E to assess inflammation. Each panel represents an individual mouse from the indicated group (Scale bar 100 μm) (A–E). Two blinded, independent pathologists scored all slides and scores were averaged. Data is represented as mean ± SEM (n = 3–4 mice) (F). BAL was collected at 8 dpi for quantification of eosinophils (G), neutrophils (H), and monocytes (I) via flow cytometry. Data are represented as mean ± SEM (n = 6–8 mice). Inflammatory scores from 4 and 8 dpi were compared for each immunization groups over time (J). Data are represented as mean ± SEM (n = 3–4 mice); *p < 0.05 and **p < 0.01.

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  Figure 6. Mucus production persisted in the lungs of PreF/Alum-immunized mice at 8 dpi and remained low in mice immunized with PreF/Advax-SM. At 8 dpi, left lungs were formalin fixed and stained with PAS to assess mucus production. Each panel represents an individual mouse from the indicated group (Scale bar 100 μm) (A–E). A more detailed breakdown of scores for each cohort is provided, calculated as a proportion i.e., number of airways of each severity score (0–4) divided by the total number of airways, according to the methods (F–J). To quantify the extent of PAS staining, lungs were scored as previously described in the methods. Scores were averaged and the total percentage of PAS+ airways were graphed. Total percentage PAS+ airways from 8 dpi are shown in (K) (n = 3–4) and are further compared over time between 4 and 8 dpi in each group in (L); **p < 0.01.

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  Figure 7. Th2-dominant immunity of PreF/Alum and the Th1-dominant immune responses of PreF/Advax-SM persisted through 8 dpi. At 8 dpi, BAL samples collected for analysis of IFNγ+ (A), IL-5+ (B), and IL-13+ (C) CD4+ T cells, and Granzyme B+ (D) and IFNγ+ (E) CD8+ T cells via flow cytometry. Right lungs were also harvested at 8 dpi and processed for analysis of ILC2 populations (F), IL-5+ ILC2s (G), and IL-13+ ILC2s (H), as well as, IFNγ+ (I), IL-5+ (J) CD4+ T cells by flow cytometry; the ratio between IFNγ+/IL-5+ CD4+ T cells was also calculated (K) (n = 3–5); *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.