Dong
Ye‡
*a,
Tina
Zimmermann‡§
b,
Victoria
Demina
c,
Sergey
Sotnikov
c,
Christian L.
Ried
d,
Harri
Rahn
d,
Marcus
Stapf
c,
Christopher
Untucht
b,
Michael
Rohe¶
b,
Georg C.
Terstappen||
b,
Karsten
Wicke
b,
Mario
Mezler
a,
Heiko
Manninga
c and
Axel H.
Meyer
*a
aAbbVie Deutschland GmbH & Co. KG, DMPK, Bioanalytical Research, Knollstraße, 67061 Ludwigshafen, Germany. E-mail: dong.ye@abbvie.com; axel.meyer@abbvie.com
bAbbVie Deutschland GmbH & Co. KG, Neuroscience Discovery, Knollstraße, 67061 Ludwigshafen, Germany
cNEUWAY Pharma GmbH, Ludwig-Erhard-Allee 2, 53175 Bonn, Germany
dAbbVie Deutschland GmbH & Co. KG, Development Sciences NBE, Knollstraße, 67061 Ludwigshafen, Germany
First published on 9th February 2021
Hollow viral vectors, such as John Cunningham virus-like particles (JC VLPs), provide a unique opportunity to deliver drug cargo into targeted cells and tissue. Current understanding of the entry of JC virus in brain cells has remained insufficient. In particular, interaction of JC VLPs with the blood–brain barrier (BBB) has not been analyzed in detail. Thus, JC VLPs were produced in this study for investigating the trafficking across the BBB. We performed a carotid artery injection procedure for mouse brain to qualitatively study JC VLPs' in vivo binding and distribution and used in vitro approaches to analyze their uptake and export kinetics in brain endothelial cells. Our results show that clathrin-dependent mechanisms contributed to the entry of VLPs into brain endothelial cells, and exocytosis or transcytosis of VLPs across the BBB was observed in vitro. VLPs were found to interact with sialic acid glycans in mouse brain endothelia. The ability of JC VLPs to cross the BBB can be useful in developing a delivery system for transport of genes and small molecule cargoes to the brain.
John Cunningham virus (JCV) is an etiological agent that can transmit from latent sites (i.e., kidney, bone marrow) to the brain, where it damages the white matter and causes a neurodegenerative disease called progressive multifocal leukoencephalopathy (PML).6–8 Similar to other polyomaviruses, JCV is a non-enveloped DNA virus, enclosed by three viral capsid proteins, VP1, VP2 and VP3. Individually, VP1 serves as a major capsid constituent to control targeting specificities,7 while minor capsid proteins VP2 and VP3 function in regulation of the viral genome.9,10 Pathogenesis of native JCV has shown its wide infectivity in human brain, gastrointestinal tract, kidney, liver or lung tissue, although the overall consequence from such infection is clinically harmless for healthy adults due to suppression by the immune system.8 The cell type-specific tropism of JCV has been connected with kidney epithelial cells, tonsillar stromal cells or bone marrow-derived cells (i.e., B lymphocyte),7,8 oligodendrocytes,11 astrocytes12 as well as brain endothelial cells.13 In light of observed PML-infections, the possibility of JCV being able to cross the BBB via JCV-infected B-cells migrating to the brain was proposed.14 However, it has not been shown that infected B-cells are indeed the critical vectors responsible for JCV infection of the central nervous system.
JCV capsid protein VP1 is known to bind to viral receptors, including α-(2,3)- and α-(2,6)-linked sialic acids15,16 or 5-hydroxytryptamine (5-HT) 2A receptors.17 Only few studies are available to investigate mechanistic interactions between VP1-assembled particles and the BBB, despite the fact that native JCV has been reported to infect brain microvascular endothelial cells, even in the absence of 5HT2A receptor in the BBB.13 The possibility to harness such pathological mechanisms as a method to deliver external biomolecules into the brain for therapeutic purposes is of great interest to explore. In fact, gene therapy via JC VLPs in treating brain tumors such as glioblastoma was already shown.18 However, such intratumoral exposure does not evidence their trafficking into the brain under healthy conditions across an intact BBB (without being compromised by tumor).
JCV VP1 can be conveniently generated by using baculovirus insect cells,19,20Escherichia coli21,22 or yeast cell23,24 expression systems. Recombinant VP1 proteins are able to self-assemble to form virus-like particles (VLPs), and thus it is possible to package drug cargo inside the capsules. For example, DNA can be loaded into JC VLPs by either osmotic shock or a dissociation–reassociation cycle, with the latter procedure reported to be more effective through adjusting chemical conditions for Ca2+ ions and disulfide bonds.19 The produced VLPs were thought to share some characteristics (i.e., morphology, cell binding) with native JCV,19 although it remains unclear whether these biological characteristics of native JCV can be matched by its derived VLP. Recombinant VP1 protein-assembled JC VLPs were successfully used for packaging of chemical substances in vitro, i.e., propidium iodide,25 suggesting suitability of the system for transport of small molecules. Also, JC VLPs were reported as efficient vehicles for delivery of genes or nucleic acids in vivo.21,22,26 Such delivery technology has been proposed for disease treatment in bone fractures26 and for vaccine generation.19
In this study, VLPs derived from JCV recombinant VP1 protein were manufactured with a defined quality, and physicochemical properties of each batch were precisely controlled. We applied JC VLPs to investigate their mechanistic interactions with the brain, specifically understanding trafficking of JC VLPs across the BBB in vitro and in vivo. In order to focus on illustrating mechanistic underpinnings of transport and providing evidence for JC VLPs crossing the BBB, we employed a range of technological approaches to conduct the BBB analysis. For some of these results this would be qualitative, instead of quantitative, owing to the remaining technical challenges. Therefore, here we aim to demonstrate that JC VLPs are able to mechanistically cross the BBB and clarify whether they can translocate to the brain.
Purified VLPs were suspended in a buffer (150 mM NaCl, 10 mM Tris–HCl, pH 7.5) and subsequently characterized for quality assurance as described in ESI Table 1.† Here, physicochemical characterization results for VLPs were shown by using three example instruments: (1) in Transmission Electron Microscopy (TEM), VLPs were mounted on carbon-coated copper grids (Plano GmbH) and stained with 2% uranyl acetate (Sigma Aldrich). Specimens were visualized with a Zeiss EM900 electron microscope, operating a voltage at 80 kV with magnifications up to 250000×; (2) in Western Blotting (WB), VLPs of total 800, 400 and 200 ng were run in SDS-PAGE gel (4–20% Criterion TGX Precast Gels) (Bio-Rad). The blotting of VP1 protein was visualized by staining with primary mouse anti-human JCV VP1 antibody (1:5000, Abcam) and secondary goat anti-mouse IgG (H + L)-HRPO (1:5000, Dianova). The blots were developed with Pierce ECL Western Blotting Substrate (ThermoFischer Scientific) and analysed using a chemiluminescence imaging system (ChemiDoc touch, Bio-Rad); (3) Dynamic Light Scattering (DLS) measurements were performed for thirty repeated runs at 23 °C using a Zetasizer Nano ZS instrument (Malvern Inc.). The VLP sizes were reported in a volume distribution curve after analysed by Zetasizer Software (version 7.11, Malvern Inc.). For additional physicochemical characterization methods, please refer to the details described in the ESI.†
For biotinylated antibody staining, sections were washed three times in TBS-T and biotinylated horseradish peroxidase was preincubated with avidin to form avidin–biotin–enzyme complexes using a Vectastain Elite ABC Kit (Peroxidase Standard) (Vector Laboratories). These complexes were transferred to the antibody-treated tissue slices for binding to biotinylated secondary antibodies for 30 min. After washing three times in TBS-T, detection of antigen was performed by adding hydrogen peroxide and 3,3′-diaminobenzidine using a DAB Peroxidase Substrate Kit (Vector Laboratories). Respective DAB times: brain, 11 min; liver, 2 min. Samples were washed in water for 5 min after immunodetection and counterstained with eosin and hematoxylin. Samples were dehydrated and coverslipped using PERTEX® medium (Histolab).
For fluorescent antibody staining, sections were washed in TBS-T, counterstained with Hoechst (Thermo Fisher) and washed with PBS. Samples were then mounted with Mowiol using a cover slip.
For image analysis, stained tissue samples were analyzed and imaged using a slide scanner Axio Scan.Z1 (Zeiss). Fluorescent signals were visualized with a Zeiss Axiovert LSM 700 (Carl Zeiss, Oberkochen, Germany) laser scanning confocal microscope, and z-stacks with optical sections of 1 μm were also performed.
To evaluate expression profile of 5HT2A receptor, human iPSC-derived astrocytes were prepared via differentiation of human neural stem cells, using internally developed protocols in AbbVie. In order to validate the cell identity, the obtained iPSC astrocytes were characterized by immunocytochemistry using astrocyte markers (GFAP, CD44, S100, EEAT1 or ALDH1L1). For the experiment, the iPSC astrocytes were plated on laminin-coated 96-well plates and cultured in astrocyte differentiated media 1 (ADM1), which was prepared with advanced DMEM/F12 and Neurobasal™ media (Life Technologies), supplemented with BSA (Sigma), 5 ng mL−1 CNTF and 10 ng mL−1 BMP2 (Peprotech), 1× penicillin–streptomycin, 0.5× N-2 Supplement, 0.5× B-27-Supplement and 1 mM L-glutamine (all from Life Technologies).
For transmission electron microscopy (TEM), hCMEC/D3 monolayer was formed on a transwell filter (0.4 μm polyester, Corning Costar) and treated with 31.5 or 126 μg mL−1 VLPs for up to 4 h. The cells were fixed with 2.5% glutaraldehyde at room temperature for 1 hour in Sorensen's phosphate buffer, and postfixed in 1% osmium tetroxide for 1 hour. The cells were dehydrated by serial washes of 30%, 50%, 70%, 90% and 100% ethanol, before cells were embedded in epon resin. Ultrathin sections (80 nm) were cut with a diamond knife (Diatome, US) using a Leica EM UC7 ultramicrotome. Negative staining was performed with 2% uranyl acetate and 3% lead citrate, same as already described. TEM images were analysed under 80 kV electron beam using a Zeiss EM900 electron microscope (Zeiss Germany).
Physicochemical characterization of VLPs was performed using three bioanalytical methods: Transmission Electron Microscopy (TEM), Western Blotting (WB) and Dynamic Light Scattering (DLS). In TEM (Fig. 1a and b), VLPs maintained optimal dispersion without apparent aggregation. The VLP shape was overall spherical and similar to natural JCV capsids. The particles were measured to have a mean diameter of 40–50 nm, consistent with a previous report.25 In Fig. 1b, a magnified feature (250000×) of a single VLP is shown. The major constituent of VLPs was identified as approx. 40 kDa monomeric VP1 protein by WB as seen in Fig. 1c. The pattern of protein bands was consistent across each lane after loading of VLP dilutions (by 2 fold). In DLS, VP1-assembled VLPs showed a single peak in both intensity and volume distribution graphs, where the homogenous particle population was measured without presence of aggregation (Fig. 1d). Here, a volume distribution was included to avoid underestimation of smaller particles and to compare with an intensity distribution to show dispersity features of VLPs. The average hydrodynamic diameter of VLPs was approx. 53 nm, close to the size determined via TEM. With a low polydispersity index value (PDI = 0.07), VLPs showed superior monodispersity. A buffer formulation prepared with 10 mM Tris–HCl, 150 mM NaCl, pH 7.5 likely contributed to the VLPs' dispersion, which was used with VLPs in the following studies.
As shown in the schematic diagram (Fig. 2a), mouse external carotid artery (ECA) was surgically catheterized prior to injection of VLPs (50 μg) through ICA. After 15 and 120 min, the animals were euthanized and perfused. Brain and liver were then harvested for analysis of presence of VP1 protein using both IHC (at 15 and 120 min) and immunogold-labeling electron microscopy (IG-EM) (at 15 min). In Fig. 2b, presence of VP1 protein at brain microvascular endothelial cells was detected by anti-VP1 IHC at both time points, suggesting VLPs indeed bind to brain endothelia. As shown in Fig. 2c, extended brain exposure (from 15 min to 120 min) led to the clear distribution of anti-VP1 immunofluorescence staining from centralized brain vessel regions to the surrounding brain tissue, suggesting potential VLP transport after ICA injection. In Fig. 2d, distributed VP1 protein was co-stained with Maackia amurensis lectin II (MAL) or Sambucus nigra lectin (SNA), which binds to carbohydrate structures with α-(2,3)- or α-(2,6)-linked sialic acid, respectively. Co-localized fluorescence signals between VP1 protein and two sialic acids were observed, suggesting that both α-(2,3)- and α-(2,6)-linked sialic acids interact with VLPs during binding to the brain endothelia. Sialic acids were previously reported to mediate cell infection for native JCV.7,15,16 In that regard, our results suggest similarities between native JCV and the derived VLP.
Fig. 2 In vivo distribution of JC VLPs following carotid artery administration to mouse brain. (a) Schematic representation of catheterization of the external carotid artery (ECA) and injection of VLPs to the brain. The diagram shown here was produced with modification from a previous report.1 (b) After 15 min mouse brain sections were stained for VP1 protein by IHC. (c) With the same sections, Alex Fluor 488 fluorescence (green) was used for VP1 labeling. VP1 positive staining was observed to distribute from central brain microvascular capillary regions to surrounding cellular space (white arrows) from 15 min to 120 min after the injection. (d) Lectin histochemistry for SNA and MAL was applied to the mouse brain sections, where VP1 (green) and the lectins (red) were co-localized in the endothelial cells (blue, nuclei), as shown by the white arrows. (e) Employing electron microscopy imaging, mouse BBB presented with details for endothelial cell in close contact with its surrounding cellular entities. (f–h) A large number of intact JC VLPs (white arrows) were found to accumulate inside mouse brain endothelial cells and subsequently traverse the BBB from its lumen to the basal membrane (BM). (i–k) With pre-embedding immunogold (IG)-labeling electron microscopy, IG-labeled VP1 protein or VLPs (red arrows) were identified near or beyond the BM area of the BBB. A few magnified images showed that IG particles were clearly identified beyond the BBB (l) and inside the neuronal space (m), where some were accompanied by intact VLPs found nearby (n) (white arrow). (o) Similar to the brain, IHC of liver tissue was performed for detection of VP1 protein. (p) VP1 protein (green) was internalized by liver sinusoidal endothelial cells, as evidenced by its co-localization (white arrows) with mannose receptor (MR, red). As before, EM revealed IG-labeled VP1 or VLPs in close contact with erythrocyte (r), macrophage (s) and endothelial cell (t) in one exemplified fenestrated liver sinusoidal vessel (q). All red arrows indicate the location of IG particles. ECA, external carotid artery; CCA, common carotid artery; PPA, pterygopalatina artery; ICA, internal carotid artery; PCA, posterior cerebral artery; MCA, middle cerebral artery; VP1, JCV capsid protein; MAL, Maackia amurensis lectin II; SNA, Sambucus nigra lectin; PV, portal vein; MR, mannose receptor; E, erythrocyte; End, endothelial cell; TJ, tight junction; BM, basal membrane; P, pericyte; L, lumen; A, myelinated axon; PV, pulmonary veins; e, endosome; ly, lysosome; m, mitochondrion; F, fenestrae; M, macrophage. |
Due to the detection limit of IHC, we further used electron microscopy (EM) imaging to determine JC VLPs' distribution in the brain tissue. As shown in Fig. 2e, mouse BBB structure comprises brain endothelial cells interconnecting each other via tight junctions and vicinity support from pericytes and neurons. Erythrocytes visually occupied the partial space of the capillary lumen. As seen in Fig. 2f, a large number of intact JC VLPs were clearly visible in the lumen area of mouse brain microvascular endothelium, where the cell membrane movement was seen to endocytose the VLPs inside the cytoplasmic space. In Fig. 2g and h, VLPs were observed in the vicinity of the vascular basal membrane (BM), to penetrate the BBB and to approach its underneath area, as indicated by the presence of a pericyte. This demonstrates that JC VLPs were able to cross the BBB layer in vivo and remained intact following this transcytosis.
Due to a similar electron density of VLPs as the mouse tissue under the electron beam, it was challenging to identify distribution of intact VLPs in high tissue volumes without immunolabeling. We therefore applied a pre-embedding immunogold electron microscopy (IG-EM) approach to further detect VLPs throughout the parenchyma. Since VP1 monomers serve as building blocks for VLP particles, labeling with VP1 mouse primary antibody and 1.4 nm nanogold conjugated to anti-mouse IgG would likely lead to a unique pattern of gold particles clustered around VLPs, which therefore enables identification of VLPs and their distribution. IG-decorated VLPs were found to be distributed beyond the basal membrane (BM) of brain endothelial cells (Fig. 2i–k), confirming that VLPs had crossed the BBB. Furthermore, as shown in Fig. 2l, more IG particles were detected underneath the BM, where brain endothelial cells were still interconnected with tight junctions, indicating the BBB integrity and exclusion of paracellular transport. IG-labeled VLPs were also detected in the neuronal space, where myelinated axons were visible in vicinity (Fig. 2m). Intact VLPs also appeared alongside with IG labeling (Fig. 2n). In general, ultrasmall gold particles were clustered in an aggregation manner, mostly in rounded shapes with diameters of up to approx. 50–70 nm, dependent on silver enhancement. Individual colloid gold from those clustering could be still visible. The 1.4 nm colloid gold-labeling method enabled detection of a multi-VP1 epitope labeling feature and provided a unique way to identify JC VLPs.
On the other hand, JC VLPs were also detected in peripheral liver tissue (Fig. 2o). A co-staining against VP1 protein and mannose receptor (MR) showed a high degree of co-localization (Fig. 2p). Normally, MR is recruited as a scavenger receptor in liver sinusoidal endothelial cells (LSECs) for degradation of foreign materials from the blood.33 This result indicated that LSECs remained exerting an effect on mechanistic clearance after VLPs being injected via ICA. A cross section of liver sinusoidal blood vessel was visualized by EM imaging. The section featured fenestrated endothelial cells as demonstrated in Fig. 2q. Immunogold particles were found to accumulate near the vessel lumen as well as the plasma membrane of endothelial cells (Fig. 2t). Erythrocytes and macrophages were also seen in association with IGs (Fig. 2r and s). In liver sections IG-labeled VLPs were also detected.
Overall, our carotid artery injection demonstrated that VLPs reached the mouse brain, while the peripheral clearance of VLPs was associated with mouse liver and kidney (data not shown).
In Fig. 3, continuous uptake and export experiments are shown. Concentration-dependent kinetics are presented in Fig. 3a, where hCMEC/D3 cells were treated for 24 h with serial dilutions of VLPs. The uptake showed a progressive increase with VLP concentration in a linear fashion, implicating such uptake process was not saturable with the given concentrations. VLPs' effective exposure concentration (31.5 μg mL−1) was calculated based on our in vivo parameters, factored by mouse body weight (approx. 25 g), blood volume (approx. 58.5 mL kg−1) as well as the volume and dose of injected VLPs (approx. 50 μg per animal). With this concentration hCMEC/D3 cells were incubated with VLPs for 1–6 h. Again, a linear increase was shown, indicating a time-dependent VLP uptake (Fig. 3b). There was a three-fold uptake change observed in between 1 and 6 h. Finally, to validate VLP transport we performed a pulse-and-chase study over time. After 24 h of continuous exposure to VLPs (pulse time), hCMEC/D3 cells were re-cultured in fresh media for 0, 1, 2 or 4 h (chase time). As demonstrated in Fig. 3c, the cells pre-loaded with VLPs showed a steady decrease of fluorescence intensity from the cells. In particular, by 4 h chase time VLP content in the cells was reduced by approximately 50%, which was significantly different from 0 h chase. The diminishing quantity of cellular VLPs might be a result of the exocytosis of BBB cells. In addition, under fluorescence imaging (Fig. 3d), immunofluorescence from VP1 and Lysosomal Associated Membrane Protein 1 (LAMP1) staining was apparently separated, and lysosomal co-localization was hardly observed in between VLPs and lysosomes at chase of 0 and 4 h. This again indicates that VLPs were exocytosed from the BBB cells without involvement of lysosomal degradation.
By electron microscopy cross sections of in vitro BBB monolayer were imaged to substantiate the overall transport process for VLPs. For this study, concentration- or time-dependent experiments were performed in order to obtain optimal imaging conditions for VLPs crossing the BBB (data not shown). We found that use of a higher concentration of 126 μg mL−1 (compared to 31.5 μg mL−1) was helpful to capture more cellular activities from transport of VLPs. We first observed that VLPs contacted cell membranes in large quantity (Fig. 4c). A clathrin-coated pit was found opening in the vicinity of VLPs while they were approaching the apical cell membrane. VLPs adhered to the cell surface like “pearls” and subsequently were engulfed via membrane movement (Fig. 4d). VLPs were also seen to traffic with a range of subcellular structures, which involved vesicular transport (Fig. 4e) or endosomal sorting (Fig. 4f and g). Importantly, VLPs seemed to remain intact in morphology during subcellular sorting. Lastly, as an evidence of transcytosis, an image of VLPs exiting the basal membrane is presented (Fig. 4h), where a vesicle was docking with opening cavity and VLPs of 40–50 nm in size were discharged towards the transwell membrane. VLP particles were visibly spherical in shape after traversing the BBB layer.
On the other hand, clathrin-mediated endocytosis (CME) was also reportedly responsible for the infectivity of JCV in brain glial cells.35 Considering that clathrin-coated pits are the predominant type of vesicles at the BBB,36 we further investigated the involvement of CME during VLP uptake into BBB cells. Chlorpromazine (CPZ) and Pitstop2™ inhibitors were reported to deplete or block CME from cell plasma membrane or cytosol,37 therefore we applied them for inhibiting VLP endocytosis. As shown in Fig. 5c, hCMEC/D3 cells exhibited a concentration-dependent decrease in uptake of VLPs for 1.5 h, after 1 h pre-treatment with CPZ. VLP uptake was reduced by approx. 17% at 1 μM CPZ and 47% at 100 μM CPZ, both significantly different from the untreated control. It was observed that a higher CPZ dose at ∼300 μM affected cell viability, but not the concentrations used here (data not shown). On the other hand, we pre-treated hCMEC/D3 cells with Pitstop2 inhibitor for 30 min, and the VLP uptake for 1.5 h was assessed to be reduced by 36%, 32% and 34%, in comparison to the untreated, DMSO, Pitstop2 negative control, respectively. The differences between Pitstop2 and controls were statistically significant (Fig. 5d). In conclusion, we confirmed that clathrin-mediated endocytosis contributed to VLP endocytosis in human brain endothelial cells, similar to a previous report for native JCV.35
It was previously reported that liver sinusoid vessels served as a sink for clearance of blood-borne JC VLPs from intravenous injection via the tail vein.32 In our independent IV pilot study we also found a similar result for VLPs in peripheral organs. Moreover, production quality and bioanalytical control for these viral particles would improve VLPs' in vivo distribution outcomes. For example, an abnormal endotoxin level from a non-GMP production process may aggravate an innate immune response to VLPs and promote a peripheral clearance, which therefore needs to be carefully controlled.40 Also, elevated endotoxins could induce neuroinflammation and exert an effect on the disruption of the BBB.41 Therefore, it is necessary to implement a range of bioanalytical tests as proposed in ESI Table S1† to circumvent emerging stability and integrity issues during in vivo testing so that the results are comparable in follow-up studies.
As a proof of concept, we used in vivo carotid artery injection to demonstrate delivery outcome of JC VLPs into mice. Our qualitative analysis revealed modest translocation of JC VLPs into brain, although a more quantitative assessment of JC VLP delivery into brain will be required in future studies for understanding the brain entry efficiency. With carotid artery injection we found that if given the chance to bind, VLPs could be enriched in brain vasculature before liver clearance occurs. Anti-VP1 IHC and IF results together showed that VLPs were indeed binding to the endothelia throughout the whole brain. From 15 to 120 min post injection, the brain capillary binding persistently occurred, and IF staining showed increased signals spreading from central brain capillary regions to the nearby brain tissue. According to Simon-Santamaria, J. et al.,32 negligible brain distribution was observed in an IV study using radiolabeled JC VLPs with normal or mutant binding properties for sialic acids. However, it remains unclear whether the radioactivity fully represented characteristics of JC VLPs, particularly regarding their interacting nature with sialic acids from brain tissue. Based on evidence provided in our study VLPs are enriched on brain capillary walls, which seems to be the result of receptor interaction with sialic acids. Our IHC data showed that α-(2,3)- and α-(2,6)-linked sialic acids were co-localized with VP1 protein and indeed required for VLP binding to mouse brain, while they were reportedly not required for liver binding due to its scavenging mechanism.32 The exact nature of interactions between JC VLPs and sialic acids remains under debate in the literature. α-(2,6)-Linked sialic acid was reported as a primary binding site for recombinant JCV VP1 pentamer.42 However, α-(2,3)- and α-(2,6) linkages reportedly shared similar functions in mediating multivalent JCV infection to brain cells.15,16 In particular, lactoseries tetrasaccharide C (LSTc) was reported in literature as a specific JCV receptor motif, which was found to mediate JCV attachment and infectivity in human glial cells.42 In an attempt to validate the presence of LSTc-mediated VLP uptake mechanism in human BBB cells, we had tried to use LSTc as an inhibitor to block VLP binding and entry in hCMEC/D3 cells. However, no clear LSTc-dependent effect was observed to influence VLP uptake, suggesting such mechanism is not present with hCMEC/D3 cells (ESI Fig. S1†).
Despite VLP clearance was observed in mouse peripheral organs, employing EM imaging we showed evidence that VLPs were capable of crossing the BBB and entering the neuronal space in vivo. It is likely that the binding properties of VLPs to brain vasculature could be a prerequisite for their transmigration through the BBB.
With regard to viral transport, native JCV was postulated in literature to utilize its etiological pathway transmitting from its peripheral latency sites to the brain. During this stage, B lymphocytes reportedly served as a reservoir of viral genome and a dissemination vehicle for JCV crossing the BBB.14 The role of JC viral capsid during BBB transmigration remained unclear despite suggested infectivity of JCV to the brain endothelia.13 Moreover, the lack of 5HT2AR in the BBB in vitro suggested other potential mechanisms for VLP transport across the brain endothelium. Due to mechanistic inaccessibility from in vivo study, here we addressed this question by using in vitro approaches.
VLP uptake was investigated by employing hCMEC/D3 cells and quantified with anti-VP1 immunofluorescence staining. The overall uptake progressively increased with VLP exposure time and concentration, similar to kinetics of other forms of nanoparticles.43,44 Also, in a pulse-and-chase experiment we pre-loaded the BBB cells with VLPs for 24 h, and a steady export was observed over time as soon as the cells were re-cultured in fresh media. By fluorescence imaging we confirmed that the VLP export was not influenced by a “diminishing” factor from lysosomal degradation in hCMEC/D3 cells, a process otherwise commonly seen in case of polymeric nanoparticles.45
Further, we grew hCMEC/D3 cells into polarized cell monolayer in vitro to validate an apical-to-basal transport of VLPs by employing confocal and electron microscopy. After a pulse of 10 minutes only, VLPs already showed binding to the surface of the hCMEC/D3 monolayer without immediate localization in endosomes. Preceded by a 110 min chase the surface-bound VLPs were further translocated to the intracellular space, where endosomal co-localization concomitantly emerged. Thus, we clearly demonstrated the binding and transportation of VLPs throughout the BBB in vitro, consistent with the carotid injection study as discussed above. By electron microscopy we studied the VLP transport process in more detail. Overall, our EM data confirm that VLPs could be endocytosed by the hCMEC/D3 monolayer. Active cell membrane interactions were captured post VLP treatment, including the presence of clathrin-coated pits, membrane invagination or engulfment. Trafficking of VLPs was identified by their localization in vesicles or endosomes but rarely in lysosomes. We further observed that the transcytosis of VLPs indeed occurred in the BBB model in vitro, in accordance with IG-EM findings from the in vivo BBB study. Collectively, these results suggest that VLPs can transmigrate across the BBB using a pathway which does not involve lysosomal accumulation.
Our in vivo results suggested the involvement of sialic acids during JC VLP binding to brain endothelia, which were known to promote clathrin-mediated endocytosis (CME) of biologics across the BBB.46 Also, clathrin-coated pits (CCPs) are vesicles predominately present at the BBB,36 where their endocytosis is reportedly restricted by expression of Mfsd2a.47 According to Pho et al.35 JCV entered human glial cells via CME. Also, since 5HT2AR is not expressed in the BBB and hence cannot facilitate VLP transport, we further explored the involvement of CME during JC VLP transport across the BBB. CPZ was previously used to block infection of JCV in brain cells through CME.17,35,48 Pitstop2 was also reported to interfere with CME for viral uptake (i.e., swine fever virus,49 HIV31). Applying both inhibitors in our study resulted in a significant decrease of VLP uptake into BBB cells. One schematic diagram is provided to summarize the current understanding of JC VLP transport pathway across the BBB (Fig. 6). This also led to our conclusion that – although VLPs were only assembled with capsid protein VP1 and generally a simpler version compared to its native viral form – they preserved similar functionality to exploit CME for uptake and further transport across the BBB.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0na00879f |
‡ Authors contributed equally. |
§ Current address: Boehringer Ingelheim Pharma GmbH & Co. KG, CardioMetabolic Diseases Research, 88397 Biberach an der Riß, Germany. |
¶ Current address: H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark. |
|| Current address: Cambrian Biopharma, New York, USA. |
This journal is © The Royal Society of Chemistry 2021 |