Experimental Analysis of HPV16 L1/L2 Chimeric VLP Internalization by Human Peripheral Blood Leukocytes

Human papillomavirus type 16 (HPV16) is a major etiological agent of cervical and other epithelial cancers, yet the mechanisms underlying host–pathogen interactions remain incompletely understood. In this study, we investigated the responses of human peripheral blood leukocytes to engineered HPV16 L1/L2 chimeric virus-like particles (VLPs), produced in suspension by HEK 293-F cells. These VLPs were designed to mimic native viral structures while incorporating chimeric features that enhance stability and immunogenicity. Through experimental assays, we characterized leukocyte engagement, primarily involving leukocyte phenotyping, VLP internalization, confocal colocalization, and endocytic pathway analyses. We demonstrated that recombinant L1/L2 proteins assembled into structured VLPs capable of interacting with mononuclear cells, including lymphocytes and monocytes, but not with polymorphonuclear cells, such as neutrophils, eosinophils and basophils. Uptake occurred via the CD71 transferrin receptor-mediated pathway, in addition to other endocytic routes analyzed, as confirmed by blockage assays using chlorpromazine, rCTB, filipin, nystatin, liquemine, and sodium azide. Confocal colocalization and endocytic pathway analyses further supported receptor-mediated uptake. These findings demonstrate that HPV16 L1/L2 chimeric VLPs interact with and are internalized by human peripheral blood mononuclear cells through CD71-associated and other endocytic pathways. The study provides new insights into HPV16 VLP–leukocyte interactions and contributes to a better understanding of the cellular mechanisms involved in VLP uptake, which may be relevant for future studies on HPV biology and VLP-based vaccine development.