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.

Morphological characterization of a plant-made virus-like particle vaccine bearing influenza virus hemagglutinins by electron microscopy.

Plant-made virus-like particle (VLP) vaccines that display wild-type influenza hemagglutinin (HA) are rapidly advancing through clinical trials. Produced by transient transfection of Nicotiana benthamiana, these novel vaccines are unusually immunogenic, eliciting both humoral and cellular responses. Here, we directly visualized VLPs bearing either HA trimers derived from strains A/California/7/2009 or A/Indonesia/5/05 using cryo-electron microscopy and determined the 3D organization of the VLPs using cryo-electron tomography. More than 99.9% of the HA trimers in the vaccine preparations were found on discoid and ovoid-shaped particles. The discoid-shaped VLPs presented HA trimers on their outer diameter. The ovoid-shaped VLPs contained HA trimers evenly distributed at their surface. The VLPs were stable for 12 months at 4 °C. Early interactions of the VLPs with mouse dendritic and human monocytoid (U-937) cells were visualized by electron microscopy after resin-embedding and sectioning. The VLP particles were observed bound to plasma membranes as well as inside vesicles. Mouse dendritic cells exposed to VLPs displayed classic morphological changes associated with activation including the extensive formation of dendrites. Our findings demonstrate that plant-made VLPs bearing influenza HA trimers are morphologically stable over time and raise the possibility that these VLPs may interact with and activate antigen-presenting cells in a manner similar to the intact virus.