Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs ...as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated.
The epitope sequence was genetically inserted in the αB-αB
domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure.
The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.
Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P
-type ATPases ATP13A2 and ...ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2- and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than does ATP13A2. We also compared the uptake of new green fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P
-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure-activity relationship (SAR), suggesting that isoform-specific polyamine probes can be designed.
Immune responses against pathogens require fine regulation to avoid excessive inflammation, which could be harmful to the host. Moreover, the immune system must be tolerant to nonpathogenic antigens ...to prevent allergy, autoimmunity, and transplant rejection. There is accumulating evidence that interactions between dendritic cells (DC) and regulatory T (Treg) cells play a crucial role in the balance between immune response and tolerance. Communications between these cells are complex, bidirectional, and mediated by soluble or cell surface molecules. The maturation status of DC, which may be influenced by different microenvironmental factors, is considered as an important checkpoint for the induction of peripheral tolerance through modifications of the activation status of T cells. Moreover, several lines of experimental evidence suggest that different subsets or the functional status of DC are also involved in the promotion of Treg cell differentiation. A better knowledge of the regulatory mechanisms of the immune response induced or inhibited by DC via their interactions with Treg cells could be relevant for the development of new, immunotherapeutic approaches.
Angiogenesis, extracellular matrix remodeling and cell migration are associated with cancer progression and involve at least, the plasminogen activating system and its main physiological inhibitor, ...the plasminogen activator inhibitor‐1 (PAI‐1). Considering the recognized importance of PAI‐1 in the regulation of tumor angiogenesis and invasion in murine models of skin tumor transplantation, we explored the functional significance of PAI‐1 during early stages of neoplastic progression in the transgenic mouse model of multistage epithelial carcinogenesis (K14‐HPV16 mice). We have studied the effect of genetic deletion of PAI‐1 on inflammation, angiogenesis, lymphangiogenesis and tumor progression. In this model, PAI‐1 deficiency neither impaired keratinocyte hyperproliferation or tumor development nor affected the infiltration of inflammatory cells and development of angiogenic or lymphangiogenic vasculature. We are reporting evidence for concomitant lymphangiogenic and angiogenic switches independent to PAI‐1 status. Taken together, these data indicate that PAI‐1 is not rate limiting for neoplastic progression and vascularization during premalignant progression, or that there is a functional redundancy between PAI‐1 and other tumor regulators, masking the effect of PAI‐1 deficiency in this long‐term model of multistage epithelial carcinogenesis.
If human papillomavirus (HPV) is necessary for the development of (pre)neoplastic lesions of the uterine cervix, it is not sufficient. Among the cofactors involved in the malignant transformation of ...cells infected by HPV, sex hormones may facilitate the cervical carcinogenesis by different mechanisms, including the induction of squamous metaplasia in the transformation zone of the cervix, interactions between steroid hormones and HPV gene expression and alterations of the local immune microenvironment.
Human papillomavirus (HPV) interacts, in vitro, with laminin 332 (LN332), a key component of the extracellular matrix. In this study, we performed bio-layer interferometry (BLI) and affinity ...capillary electrophoresis (ACE) to investigate the binding properties of this interaction. Virus-like particles (VLPs), composed of the HPV16 L1 major capsid protein, were used as HPV model and LN332 as the VLPs binding partner. Using BLI, we quantitatively determined the kinetics of the interaction, via the measurement of VLP binding and release from LN332 immobilized onto the surface of aminopropylsilane biosensors. We found an averaged k
of 1.74 x 10
M
s
and an averaged k
of 1.50 x 10
s
. Furthermore, an ACE method was developed to study the interaction under physiological conditions, where the interactants are moving freely in solution, without any fluorescence labeling. Specifically, a constant amount of HPV16-VLPs was preincubated with increasing LN332 concentrations and then the samples were injected in the capillary electrophoresis instrument. A shift in the migration time of the HPV16-VLP/LN332 complexes, carrying an increasing number of LN332 molecules bound per VLP, was observed. The mobility of the complexes was found to decrease with increasing LN332 concentrations in the sample. It was used to quantify stability constant. From BLI and ACE approaches, we reported an apparent equilibrium dissociation constant in the nanomolar range (8.89 nM and 17.7 nM, respectively) for the complex between HPV16-VLPs and LN332.
The reason why some cancers predominantly develop in specific sites of a given organ and, particularly in metaplastic mucosal surfaces, remains obscure. Although embryologic, microbial, chemical or ...hormonal factors are likely to have a pathogenic role, the local immune microenvironment might also facilitate tumour growth. We propose that metaplasia, which frequently occurs in some organs (uterine cervix, respiratory tract, oesophagus and stomach), is involved in cancer development by altering the expression of soluble or adhesion molecules important for cell-mediated antitumour immunity.
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