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.

越来越多的证据支持T细胞应答对于预防严重流感，促进病毒清除并确保长期免疫的重要性。已显示带有流感血凝素（HA）的植物来源的病毒样颗粒（VLP）疫苗可引起强烈的体液和CD4 ⁺临床前和临床研究中的T细胞反应。为了更好地了解这些疫苗的免疫原性，我们追踪了人单核细胞衍生巨噬细胞（MDM）中以VLP（H1-VLP）或可溶形式交付的HA模型（A / California / 07/2009 H1N1）在细胞内的命运。形式。与暴露于可溶性HA相比，用VLP脉冲在15分钟时会导致HA的胞内积累增加约3倍，这是由网格蛋白介导和网格蛋白非依赖性内吞作用以及巨胞饮作用/吞噬作用驱动的。在45分钟时，可溶性HA基本上消失了，这表明其主要是通过高降解内体途径来处理的。尽管在H1-VLP暴露后45分钟时，总的荧光强度/细胞下降了25％，内体的分布模式和聚集程度表明，VLP递送的HA已进入高降解晚期和低降解静态早期和/或再循环内体途径。在用VLP脉冲的细胞中，第45分钟，HA与Rab5，Rab7，Rab11，MHC II和MHC I强烈共定位。高分辨率串联质谱鉴定了H1中115种与MHC I相关的HA衍生肽VLP处理的MDM。这些数据表明，HA递送至植物来源的VLP上的抗原呈递细胞有助于抗原摄取，内体加工和交叉呈递。这些观察结果可能有助于解释这些疫苗产生的广泛和交叉反应的免疫反应。在用VLP脉冲的细胞中，第45分钟，HA与Rab5，Rab7，Rab11，MHC II和MHC I强烈共定位。高分辨率串联质谱鉴定了H1中115种与MHC I相关的HA衍生肽VLP处理的MDM。这些数据表明，HA递送至植物来源的VLP上的抗原呈递细胞有助于抗原摄取，内体加工和交叉呈递。这些观察结果可能有助于解释这些疫苗产生的广泛和交叉反应的免疫反应。在用VLP脉冲的细胞中，第45分钟，HA与Rab5，Rab7，Rab11，MHC II和MHC I强烈共定位。高分辨率串联质谱鉴定了H1中115种与MHC I相关的HA衍生肽VLP处理的MDM。这些数据表明，HA递送至植物来源的VLP上的抗原呈递细胞有助于抗原摄取，内体加工和交叉呈递。这些观察结果可能有助于解释这些疫苗产生的广泛和交叉反应的免疫反应。和交叉演示。这些观察结果可能有助于解释这些疫苗产生的广泛和交叉反应的免疫反应。和交叉演示。这些观察结果可能有助于解释这些疫苗产生的广泛和交叉反应的免疫反应。