Reduced protection of RIPK3-deficient mice against influenza by matrix protein 2 ectodomain targeted active and passive vaccination strategies

Abstract

RIPK3 partially protects against disease caused by influenza A virus (IAV) infection in the mouse model. Here, we compared the immune protection of active vaccination with a universal influenza A vaccine candidate based on the matrix protein 2 ectodomain (M2e) and of passive immunization with anti-M2e IgG antibodies in wild type and Ripk3^-/- mice. We observed that the protection against IAV after active vaccination with M2e viral antigen is lost in Ripk3^-/- mice. Interestingly, M2e-specific serum IgG levels induced by M2e vaccination were not significantly different between wild type and Ripk3^-/- vaccinated mice demonstrating that the at least the humoral immune response was not affected by the absence of RIPK3 during active vaccination. Moreover, following IAV challenge, lungs of M2e vaccinated Ripk3^-/- mice revealed a decreased number of immune cell infiltrates and an increased accumulation of dead cells, suggesting that phagocytosis could be reduced in Ripk3^-/- mice. However, neither efferocytosis nor antibody-dependent phagocytosis were affected in macrophages isolated from Ripk3^-/- mice. Likewise following IAV infection of Ripk3^-/- mice, active vaccination and infection resulted in decreased presence of CD8+ T-cells in the lung. However, it is unclear whether this reflects a deficiency in vaccination or an inability following infection. Finally, passively transferred anti-M2e monoclonal antibodies at higher dose than littermate wild type mice completely protected Ripk3^-/- mice against an otherwise lethal IAV infection, demonstrating that the increased sensitivity of Ripk3^-/- mice could be overcome by increased antibodies. Therefore we conclude that passive immunization strategies with monoclonal antibody could be useful for individuals with reduced IAV vaccine efficacy or increased IAV sensitivity, such as may be expected in patients treated with future anti-inflammatory therapeutics for chronic inflammatory diseases such as RIPK inhibitors.

Fig. 1. Ripk3-deficient mice are not protected against IAV lethal infection following active vaccination with M2e-VLP despite similar serum levels of M2e-specific antibodies and viral clearance.

A Active vaccination with M2e-VLP particles was administered with Alhydrogel® adjuvant intraperitoneally. Age-matched Ripk3^+/+ and Ripk3^−/− mice received either 5 µg/mouse M2e-VLP with Alhydrogel^® adjuvant vaccination or just the Alhydrogel® adjuvant dissolved in PBS (vehicle) 3 weeks and 6 weeks before infection. Lethal challenge was done with the mouse-adapted influenza X47 with either 2 × LD₅₀ (virus batch 1) or 0.5 × LD₅₀ (virus batch 2). The experiment was repeated four times independently and data were pooled. Survival curves were plotted for indicated groups and evaluated statistically according to Kaplan–Meier. A log-rank test verified significant differences between Ripk3^+/+ and Ripk3^−/− mice post-vaccination; ****p-value< 0.0001 (GraphPad Prism 8). Similar numbers of female and male mice were used comparing both genotypes (19 females, 3 males Ripk3^+/+ versus 21 females, 6 males Ripk3^−/−). The body weight readout of this experiment is provided in Supplementary Fig. 2A. B RIPK3-deficient mice effectively produce specific antibodies against M2e: Age-matched Ripk3^+/+ and Ripk3^−/− mice were primed (6 weeks before infection) then immunized (3 weeks before infection) 10 μg M2e-VLP with Alhydrogel® adjuvant or received PBS with Alhydrogel^® adjuvant (vehicle). Serum samples were prepared two weeks after each immunization. Titers of IgG1 IgG2a IgG2b and total IgG against M2e were measured after the immunization were determined by ELISA. The legend shows the M2e-specific antibody titers obtained for individual mice (dots) of each group from three independent experiments. C Virus clearance from the lungs post vaccination is independent from RIPK3-deficiency: On day 6 post-infection, lungs were harvested and lung homogenates were assessed for viral titers by TCID₅₀. Data for pooled lung homogenates from different mice (two independent experiments) of the same group are shown. The means for TCID₅₀ are shown for each group as indicated in the legend. Error bars represent SD. D Overview of the active vaccination strategy and the results obtained in Ripk3^+/+ and Ripk3^−/− mice.

Fig. 2. Immune cells infiltration in the lungs is required for protection against IAV lethal infection.

A Vaccinated and non-vaccinated Ripk3^+/+ and Ripk3^−/− mice challenged with lethal IAV dose challenge (2 × LD₅₀; virus batch 1) were sacrificed at 6 days post-infection and their lungs were collected and stained with hematoxylin-eosin. Representative images of individual mice revealed less inflammation in the vaccinated Ripk3^−/− mice compared to their Ripk3^+/+ control. B Vaccinated and non-vaccinated Ripk3^+/+ and Ripk3^−/− mice challenged with lethal IAV were sacrificed at 6 days post-infection and their lungs were collected. C Representative images displaying TUNEL positivity (red) and cells nuclei (blue) are shown in (B) and CD8 positivity (red) and cell nuclei (blue) is shown in (C). A tendency to accumulate more dying-cells in the lungs of Ripk3^−/− mice compared to their Ripk3^+/+ controls is observed. D The extent of lung inflammation and injury score was blindly scored (0 - no inflammatory cell infiltration; normal alveolar septa; 1 - mild peribronchial/perivascular inflammatory cell infiltration in parts of the lung; normal alveolar septa; 2 - moderate peribronchial/perivascular inflammatory cell infiltration in parts of the lung; mild thickening of alveolar septa; 3 - severe alveolar and interstitial inflammatory cell infiltration in parts of the lung; moderate thickening of alveolar septa; 4 - very severe alveolar and interstitial inflammatory cell infiltration in parts of the lung; severe thickening of alveolar septa; 5 - very severe alveolar and interstitial inflammatory cell infiltration throughout the lung; severe thickening of alveolar septa). TUNEL positivity and for CD8 positivity was scored with a software 0.2.3. and QuPath software 0.3.0. respectively. Graphics and statistical analysis were done with GraphPad Prism 8. Comparisons were done with Mann–Whitney test in GraphPad Prism 8.

Fig. 3. RIPK3-deficient macrophages are competent to perform efferocytosis and antibody-dependent cellular phagocytosis.

A Primary peritoneal macrophages isolated from Ripk3^+/+ and Ripk3^−/− mice were co-incubated in 1:5 ratio with apoptotic (dexamethasone-killed, DEX) or non-apoptotic (no DEX) thymocytes stained with pH-sensitive dye, CypHER 5E. Live cell imaging was performed with IncuCyte^® S3 (Sartorius). Representative images of macrophages which engulfed DEX-treated or no DEX-treated thymocytes become positive for CypHER 5E (red) can be seen. The difference between Ripk3^+/+ and Ripk3^−/− at 8 h of co-incubation is shown as fold change relative to the no DEX control (three independent experiments). B M2e-coated fluorescent polystyrene beads were incubated in 2.5:1 ratio with primary alveolar macrophages isolated form Ripk3^+/+ or Ripk3^−/− mice in the absence or presence of 0.1 μg/mL of isotype control antibody, Anti-SHE or an anti-M2e mouse IgG2a monoclonal antibody (Mab65) specific antibody, Anti-M2e. Live cell imaging was performed with Operetta high content imaging system and analysis was done with Harmony software. The difference between Ripk3^+/+ and Ripk3^−/− at 8 h of co-incubation is shown as fold increase in antibody-mediated phagocytosis relative to no antibody control (three independent experiments). Significance was determined using one-way ANOVA with Tukey correction (GraphPad Prism 8). Error bars represent SEM.

Fig. 4. Increased doses of passive immunization with Anti-M2e monoclonal antibodies can completely protect Ripk3-deficient mice.

A The administration of a standard dose Anti-M2e (0.5 mg/kg) and challenge with lethal IAV (5 × LD₅₀; viral batch 3) (four independent experiments, total number of mice indicated between brackets). The passive immunization with monoclonal Anti-M2e was done i.p. one day before the i.n. infection with IAV. Survival was monitored daily for up to 18 days post-challenge. Survival curves were plotted for indicated groups and evaluated statistically according to Kaplan–Meier (GraphPad Prism 8). Similar numbers of female and male mice were used comparing both genotypes (18 females, 15 males Ripk3^+/+ versus 18 females, 17 males Ripk3^−/−). The body weight readout of this experiment is provided in Supplementary Fig. 2B. Cox regression did not reveal any significant difference (p = 0.517) in survival between female and male mice. B The combination of passive immunization with a standard Anti-M2e dose (0.5 mg/kg) and a decreased (but still lethal) IAV dose (2.4 × LD₅₀; viral batch 3) was performed three times (total number of mice indicated between brackets). The combination of passive immunization with an increased dose of Anti-M2e (2.5 mg/kg) and lethal IAV dose (5 × LD₅₀; viral batch 3) in three independent experiments (total number of mice indicated in the legends). The passive immunization with monoclonal Anti-M2e was done i.p. one day before the i.n. infection with IAV. Survival was monitored daily for up to 18 days post-challenge. Survival curves were plotted for indicated groups and evaluated statistically according to Kaplan–Meier (GraphPad Prism 8). The body weight readout of this experiment is provided in Supplementary Fig. 2C. C Scheme of passive immunization and main conclusions. Overview of the results in terms or survival of Ripk3^+/+ and Ripk3^−/− mice are shown in approximative percentages based on the results in panels (A and B). Similar numbers of female and male mice were used comparing both genotypes (27 females, 14 males Ripk3^+/+ versus 26 females, 13 males Ripk3^−/−).