Plant-derived virus-like particle vaccines drive cross-presentation of influenza A hemagglutinin peptides by human monocyte-derived macrophages

A growing body of evidence supports the importance of T cell responses to protect against severe influenza, promote viral clearance, and ensure long-term immunity. Plant-derived virus-like particle (VLP) vaccines bearing influenza hemagglutinin (HA) have been shown to elicit strong humoral and CD4+ T cell responses in both pre-clinical and clinical studies. To better understand the immunogenicity of these vaccines, we tracked the intracellular fate of a model HA (A/California/07/2009 H1N1) in human monocyte-derived macrophages (MDMs) following delivery either as VLPs (H1-VLP) or in soluble form. Compared to exposure to soluble HA, pulsing with VLPs resulted in ~3-fold greater intracellular accumulation of HA at 15 min that was driven by clathrin-mediated and clathrin-independent endocytosis as well as macropinocytosis/phagocytosis. At 45 min, soluble HA had largely disappeared suggesting its handling primarily by high-degradative endosomal pathways. Although the overall fluorescence intensity/cell had declined 25% at 45 min after H1-VLP exposure, the endosomal distribution pattern and degree of aggregation suggested that HA delivered by VLP had entered both high-degradative late and low-degradative static early and/or recycling endosomal pathways. At 45 min in the cells pulsed with VLPs, HA was strongly co-localized with Rab5, Rab7, Rab11, MHC II, and MHC I. High-resolution tandem mass spectrometry identified 115 HA-derived peptides associated with MHC I in the H1-VLP-treated MDMs. These data suggest that HA delivery to antigen-presenting cells on plant-derived VLPs facilitates antigen uptake, endosomal processing, and cross-presentation. These observations may help to explain the broad and cross-reactive immune responses generated by these vaccines.


Electron microscopy (EM)
For negative stain preparation, the VLP samples were diluted with PBS pH 7.4 -100 µg/mL based on estimated influenza hemagglutinin (HA) content. 5 µL of the diluted VLP solution was placed for 45 sec on 200 Hex grids (EMS Inc., Hatfield, PA), formerly carbon coated and glow-discharged. The grids were washed twice with 5µL of distilled water for 45 sec followed by 45 sec incubation with 2% uranyl acetate (EMS Inc.). Excess fluid was removed by blotting with a filter paper and samples were left to air dry. The grids were imaged commonly on a Tecnai T12 (FEI Inc., Hillsboro, OR). The MDM samples processing for EM is described in the manuscript, Methods section. Scientific, Roskilde, Denmark) at 5 x 10 4 cells per well. The following day, B10R cells in triplicate wells were exposed to DiD-labelled VLPs in the medium (HA concentration 15.0 µg/mL); plates were kept at 4°C for one hour and then washed with ice-cold medium twice.

Toxicity assessment of endocytosis inhibitors
B10R cell culture was maintained in the medium supplemented with 10% FBS until reaching cell confluency. B10R cells were detached from plastic flask surface using 0.25% trypsin/2.21 mM EDTA in HBSS and plated on 96-well Nunclon Delta black flat-bottom plates at 5 x 10 4 cells per well. The following day, endocytosis inhibitors (see the endocytosis inhibitors screening section and the Supplemental Table 1) in the medium supplemented with 10% FBS were applied to B10R cells in triplicate wells in a volume 100 µL/well (5% CO2, 37°C for 2 h).
Chlorpromazine hydrochloride was applied at concentrations 10 and 100 µg/mL. Pitstop 2 was also tested in serum-free medium. Triton X-100 0.01% solution served as a positive control. The effects of endocytosis inhibitors on B10R cells viability were evaluated in parallel experiments with the CytoTox-ONE™ homogeneous membrane integrity assay and the CellTiter-Glo ® 2.0 assay (both from Promega, Madison, WI). Cell membrane integrity was assessed by lactate dehydrogenase (LDH) release. CytoTox-ONE™ reagent (100 µL/well) was applied for 10 min at RT. Stop solution (50µl) was then added to each well, and the fluorescence was measured on Infinite 200 PRO spectrophotometer at excitation and emission wavelengths 560 and 600 nm, respectively. The results were reported as a % of the maximum LDH release caused by adding lysis solution to the control wells. The effects of endocytosis inhibitors on metabolically active cells were quantitated by the amount of ATP with CellTiter-Glo ® luminescent cell viability assay. CellTiter-Glo ® 2.0 reagent (100 µl/well) was applied for 10 min at RT. Luciferase luminescence was measured on Infinite 200 PRO spectrophotometer. The results were reported as a % of ATP level reduction compared to control wells unexposed to any endocytosis inhibitor.

Conventional image analysis
Confocal microscopy images were analyzed with ImageJ software 1 for the purpose of evaluation the fluorescence intensity of immunolabelled HA or fluorophore-conjugated transferrin, or for the assessment of colocalization of HA with endosomal markers Rab5, Rab7 and Rab11. 2 In brief, to analyze the HA or transferrin fluorescence intensity per cell area we identified cellular boundaries on the brightfield channel, and used them to establish the regions of interest (ROIs) on the fluorescent channel(s). Then we determined the background fluorescence intensity in each experiment by averaging the values obtained from cells in the control sample (for HA: monocyte-derived macrophages (MDMs) unexposed to HA but stained with anti-HA primary and fluorescent secondary antibody; for transferrin: MDMs unexposed to transferrin). The average background fluorescence intensity was subtracted from the fluorescence values measured from HA or transferrin-exposed cells. For "cell-based" colocalization analysis, both "green" and "red" channels were denoised with ImageJ PureDenoise plugin, 3 then image background was subtracted with the "rolling ball" algorithm. 4 HA colocalization with endosomal markers was determined within cell boundaries-defined ROIs (see above) using Colocalization Threshold plugin; 5 Costes thresholding approach was applied. 6 Pearson correlation coefficient R, Pearson coefficient for pixels whose intensity falls above a threshold value R (>t), and Manders above threshold colocalization coefficients tM1 and tM2 were analyzed. 2,7,8

Statistical analysis
Statistical analysis was performed using GraphPad Prism 6.0 software. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons post-test, two-way ANOVA followed by Tukey's multiple comparisons post-test or Mann-Whitney test were used to examine the differences between samples. P values < 0.05 were considered statistically significant.

Supplemental tables
Supplemental Table 1 Endocytosis inhibitors used in screening experiments and their suggested mechanism of action

Suggested mechanism of action Toxicity
Dynamin-dependent endocytosis Dynasore 50 µM Non-competitive and reversible inhibitor of GTPase activity of dynamin. 9 Dynasore suppresses both CME and CIE. 10,11 Non-toxic Clathrin-mediated endocytosis (CME) Chlorpromazine 10 -100 µg/mL Not well understood. It has been suggested that chlorpromazine causes AP-2 and clathrin relocation from plasma membrane to endosomal membranes and therefore depletes AP-2 and clathrin from the plasma membrane and prevents clathrin-coated endocytic vesicles formation. 12 Chlorpromazine probably affects dynamin activity. 13 Non-toxic at 10 µg/mL.  Peripheral (towards the plasma membrane) re-distribution (recycling) of non-degraded HA can be seen. Green: fluorescently labeled HA, blue: nuclei stained with DAPI. Scale bar -25 µm.