Plasma antibodies from humans infected with zoonotic simian foamy virus do not inhibit cell-to-cell transmission of the virus despite binding to the surface of infected cells

Abstract

Zoonotic simian foamy viruses (SFV) establish lifelong infection in their human hosts. Despite repeated transmission of SFV from nonhuman primates to humans, neither transmission between human hosts nor severe clinical manifestations have been reported. We aim to study the immune responses elicited by chronic infection with this retrovirus and previously reported that SFV-infected individuals generate potent neutralizing antibodies that block cell infection by viral particles. Here, we assessed whether human plasma antibodies block SFV cell-to-cell transmission and present the first description of cell-to-cell spreading of zoonotic gorilla SFV. We set-up a microtitration assay to quantify the ability of plasma samples from 20 Central African individuals infected with gorilla SFV and 9 uninfected controls to block cell-associated transmission of zoonotic gorilla SFV strains. We used flow-based cell cytometry and fluorescence microscopy to study envelope protein (Env) localization and the capacity of plasma antibodies to bind to infected cells. We visualized the cell-to-cell spread of SFV by real-time live imaging of a GFP-expressing prototype foamy virus (CI-PFV) strain. None of the samples neutralized cell-associated SFV infection, despite the inhibition of cell-free virus. We detected gorilla SFV Env in the perinuclear region, cytoplasmic vesicles and at the cell surface. We found that plasma antibodies bind to Env located at the surface of cells infected with primary gorilla SFV strains. Extracellular labeling of SFV proteins by human plasma samples showed patchy staining at the base of the cell and dense continuous staining at the cell apex, as well as staining in the intercellular connections that formed when previously connected cells separated from each other. In conclusion, SFV-specific antibodies from infected humans do not block cell-to-cell transmission, at least in vitro, despite their capacity to bind to the surface of infected cells. Trial registration: Clinical trial registration: www.clinicaltrials.gov, https://ichgcp.net/clinical-trials-registry/NCT03225794.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Settings of the SFV cell-to-cell transmission assay.

A. Schematic description of the experiments to verify that SFV infection requires cell contact. On day 0, transmitter BHK-21 cells were infected at a moi of 0.05 for 72 h in T25 cm2. On day 3, uninfected indicator GFAB cells were seeded in 24-well plates (2 x 104 cells/well) and infected cells were added to GFAB cells on day 4. These were placed in direct contact with the indicator cell line or in the insert of transwell devices (polycarbonate membrane-insert pore size = 0.4 μm). In addition, we quantified the infectious virus present in the supernatants of infected cells. As a control, de novo infection with cell-free virus was performed using the same moi as for standard neutralization assays of cell-free virus (moi of 0.02). Plasma samples were added to the culture medium at a final dilution of 1:100 on day 4, immediately after infection. On day 7, β-galactosidase expression under control of the SFV LTR was detected by addition of its chromogenic substrate (X-gal), leading to blue-stained infected GFAB cells. Representative images of wells cultivated in the absence of plasma samples illustrate the foci of infected cells after cell-cell transmission, the efficient blockade of infection by the transwell device, the low dose of infectious virus present in the supernatant of transmitter cells on day 4, and the homogenous spread of infected cells after cell-free virus infection. B. GI-D468-infected BHK-21 cells and GI-D468 virus were used to infect GFAB cells via the four transmission modes described in panel A. On day4, we added 1:100 diluted plasma from one uninfected individual (SFVneg, MEBAK195, gray symbols) and one neutralizing plasma (anti-GI, LOBAK2, blue symbols). The number of infectious units (i.e., cells or syncytia) per well is presented for independent experiments. C. GII-K74-infected BHK-21 cells and GII-K74 virus were used to infect GFAB cells via the four transmission modes described in panel A. On day4, we added 1:100 diluted plasma from one uninfected individual (SFVneg, MEBAK195, gray symbols) and neutralizing plasma (anti-GII, MEBAK88, red symbols). The number of infected cells per well is presented for independent experiments. Panel A was created with Biorender.com.

Fig 2. The neutralization capacity of plasma samples from SFV-infected individuals depends on the infection route.

A. Schematic description of the neutralization experiments. On day 0, transmitter cells (BHK-21 or HT1080) were infected at a moi of 0.05 and seeded in 96-well microtitration plates (5 x 103 cells/well). On day 1, infected cells were incubated with serial dilutions of plasma samples for 1 h before the addition of 5 x 103 uninfected GFAB cells. On day 4, β-galactosidase expression by infected GFAB cells was detected by X-gal staining. For cell free virus neutralization, GFAB cells were seeded in 96-well microtitration plates at day 3. On day 4, the viral inoculum (moi of 0.02) was incubated with serial dilutions of plasma samples for one hour before addition to GFAB cells. At day 7, β-galactosidase expression by infected GFAB cells was detected by X-gal staining. B-E: Neutralization curves of BHK-21 SFV-infected cells (open symbols) and cell-free virus (filled symbols) are shown for four plasma samples against the matched genotype strain. Individuals BAD463 (B) and LOBAK2 (C) are infected with a GI strain and their plasma samples were tested against the GI-D468 strain; individuals BAD551 (D) and BAK232 (E) are infected with a GII strain and their plasma samples were tested against the GII-K74 strain. F-I: Plasma samples from BAD463, LOBAK2, BAD551 and BAK232 SFV-infected individuals and from one uninfected control (MEBAK189) were tested for the neutralization of BHK-21 (F, H) or HT1080 (G, I) cells infected with GI-D468 (F, G) or GII-K74 (H, I). In panels B-E, results are presented as IU/well to show that the infectious loads were comparable between cell-transmitted and cell-free virus conditions. IU/well values of 0 were replaced by 1 to allow visualization on the graph. In panels F to K, the results are expressed as the infectivity relative to that of cells cultivated in the absence of plasma sample (untreated cells) to show data from each virus–plasma pair on the same scale. Experiments have been carried out in triplicates and mean and standard deviation are shown. The mean number of IU/well transmitted by untreated cells were 24 (GI-D468 infected-BHK-21, panel F), 83 (GII-K74 infected-BHK-21, panel H), 978 (GI-D468 infected-HT1080, panel G) and 629 (GII-K74 infected-HT1080, panel I). In each panel, the inverse of the plasma sample dilution is presented on the x axis and the mean and standard errors from triplicates are shown. J-K. Twenty-two plasma samples diluted 1:80 were tested for their capacity to neutralize BHK-21 transmitter cells infected with GI-D468 (J) or GII-K74 (K). Results are expressed as the infectivity relative to that of cells cultivated in the absence of plasma samples. The reference values for cell-transmitted virus were 25 IU/well for GI-D468 and 83 IU/well for GII-K74 and ≈ 100 IU/well for cell-free virus according to the experimental design [17]. The relative infectivity of cell-transmitted virus in the presence of plasma samples is shown by open squares and labelled as “cells” on the x axis. Relative infectivity of cell-free virus in the presence of the same plasma sample is shown for comparison by filled squares, labelled as “virus” on the x axis. Data are presented for four plasma samples from uninfected controls (grey symbols), nine samples from SFV-infected individuals that neutralize the GI-D468 strain (blue symbols), five samples that neutralize the GII-K74 strain (red symbols) and four samples that neutralize both the GI-D468 and GII-K74 strains (purple symbols). P values from the paired t test are indicated in panels J and K. Panel A was created with Biorender.com.

Fig 3. The addition of plasma samples from SFV-infected individuals before the production of viral proteins does not block cell-to-cell transmission of gorilla SFV.

BHK-21 cells were infected at a moi of 0.05 with GI-D468 (A) or GII-K74 (B) for 2 h, washed and cultured in the presence of 1:100 diluted plasma samples (S2A Fig). At the last passage, cells were seeded on glass coverslips and fixed 72 h later. Cells were permeabilized with 0.5% Triton X-100 and stained with anti-SU-biotin+Streptavidin-AF488, phalloidin (actin) and DAPI (nuclei) and analyzed by brightfield microscopy at a magnification of 10x. Images covering a 37.5-mm2 square in the center of the coverslip were acquired. Nuclei and infected cells were quantified as described in Materials and Methods from 25 images per coverslip, corresponding to 48,000 to 140,000 analyzed cells/condition. Data are presented as the infection rate per image to reflect the variability of the infection rate across the coverslip. Plasma samples were derived from an uninfected individual (MEBAK189, grey symbols) and from SFV-infected individuals for whom the plasma neutralized GI-D468 (LOBAK2, blue symbols), GII-K74 (MEBAK88, red symbols), or both strains (BAK177, purple symbols). Their neutralization titers (IC50) are indicated on the figure. The Mann-Whitney test was used to compare cultures treated with samples from uninfected and SFV-infected individuals and the P values are indicated above the graphs. C. Representative images captured at magnification of 63x showing that the addition of non-neutralizing (MEBAK189, upper line) or neutralizing plasma samples (LOBAK2 and MEBAK88, bottom line) had no effect on the morphology of the BHK-21-infected cells, which consisted of either isolated elongated cells (white arrows) or multicellular infected foci (yellow arrows). Env, actin and nuclei are presented in green, red and blue, respectively; scale bar = 20 μm.

Fig 4. Env localization in gorilla SFV-infected cells.

Cells were infected with GI-D468, GII-K74, or CI-PFV, at a moi of 0.05. At the second passage, cells were seeded on glass coverslips and fixed within 1 to 3 days, depending on the cytopathic effect (S2B Fig). Cells were permeabilized with 0.5% Triton X-100 and stained with anti-SU-biotin+Streptavidin-AF488, phalloidin (actin) and DAPI (nuclei) and analyzed by brightfield microscopy with optical sectioning. Top panels: BHK-21 cells; bottom panels: HT1080 cells (indicated as A’ to J’ for conditions matching A to J). A: Mock infected cells, B-E-H: GI-D468 infected cells, C-F-I: GII-K74 infected cells, D-G-J: CI-PFV infected cells. B-C-D: SFV Env in isolated cells is located in the cytoplasm and surrounding the nuclei and some infected cells acquired fusiform shapes. E-F-G: Intercellular connections are visible between two infected cells or between infected and uninfected cells. Env labelling is present in the intercellular connections; H-I-J: Syncytia with diffuse Env staining within a crown formed by nuclei or in the cytoplasmic space around the nuclei; both patterns were observed for the three strains and both cell lines. Representative examples of Env staining are indicated with white arrows for cytoplasmic labelling, yellow arrows for fusiform shapes, blue arrows for intercellular connections, white triangles for cytoplasmic labelling inside a crown of nuclei in syncytia and yellow triangles for cytoplasmic labelling around the nuclei in syncytia. Env, actin and nuclei are presented in green, red and blue, respectively; scale bar = 20 μm.

Fig 5. Env localization in gorilla SFV-infected cells.

The infection and labeling conditions are those described in Fig 4. Acquired serial Z-plane frames at various Z depths with the same XY position were used to build 3D views with nuclei (blue), Env (green) and actin or CD98 (red) shown in a blend-rendering mode. A. GII-K74-infected BHK-21 cells, with Env staining distributed throughout multiple vesicles and around the nuclei (3D view from the top). Yellow bars correspond to orthogonal slices showing Env staining distributed throughout multiple vesicles and around the nuclei. Grid gaps correspond to 10 μm. B. Dense network of GII-K74-infected BHK-21 cells, in which an infected cell shows an elongated intercellular connection on its right side (3D view from the side). Env staining is evenly distributed despite contacts with multiple adjacent cells. Patchy perinuclear staining is observed in one cell contacting the intercellular connection (yellow square). C. Two GI-D468-infected HT1080 cells with an intercellular connection that does not adhere to the coverslip (3D view from the top side and from the side (insert)). Yellow bars indicate the position of orthogonal slices: a. cytoplasm of a fusiform cell, b-c. Intercellular connection with Env staining (triangle), d. cytoplasm and nucleus from the second infected cell (triangle). Colocalization of Env and actin is indicated by the yellow arrows. D. GI-D468-infected HT1080 cells were permeabilized with 0.5% Triton X-100 and stained with anti-SU-biotin + Streptavidin-AF647, anti-CD98-FITC, and DAPI (nuclei) (note that the colors in the figure were matched with those of the previous panels and not the conjugated fluorochromes). Yellow bars indicate the position of orthogonal slices. Colocalization of Env and CD98 is indicated by the yellow arrows.

Fig 6. Plasma samples specifically stain SFV-infected cells.

BHK-21 cells were infected with GI-D468 or GII-K74, at a moi of 0.05 for seven days, seeded onto glass coverslips and fixed within 1 to 3 days, according to the CPE (S2B Fig). Cells were either untreated (A-E) or permeabilized with 0.5% Triton X-100 (F-J) before staining with anti-GI plasma sample (LOBAK 2) diluted 1:100 and anti-human IgG-A-M-FITC and DAPI (nuclei). Cells were analyzed by brightfield microscopy with optical sectioning. A, F: Mock infected cells, B, D, G, I: GI-D468 infected cells, C, E, H, J: GII-K74 infected cells. Nuclei (blue), anti-SFV plasma (green), scale bars = 100 μm (10x magnification images) and 20 μm (63x magnification images). White arrows indicated dense SFV-specific staining localized either on the side or at the cell apex; yellow arrows indicate cytoplasmic staining.

Fig 7. Plasma antibodies stain large surfaces, long intercellular connections and cell protrusions on SFV-infected cells.

Cells were infected with GI-D468 or GII-K74 at a moi of 0.05. At the second passage, cells were seeded on glass coverslips and fixed within 1 to 3 days, according to the CPE (S2B Fig). Cells stained with anti-GI plasma sample (LOBAK2) and anti-human IgG-A-M-FITC, phalloidin (actin) and DAPI (nuclei) without incubation with Triton X-100. Labelled cells were analyzed by brightfield microscopy with optical sectioning. Acquired serial Z-plane frames at various Z depths with the same XY position were used to build 3D views with nuclei (blue), Env (green) and actin (red) shown in a blend-rendering mode. Env and actin costaining is indicated by yellow color. A. GII-K74-infected BHK-21 cell, 3D view from the side. The infected cell displays patchy SFV staining at its base, dense staining at the top and a small protrusion at the back. Yellow bars indicate the position of orthogonal slices. B. GII-K74-infected BHK-21 cells, 3D view from the top. The infected cell in the center of the image has a large cytoplasmic protrusion on its right side (yellow triangle). Yellow bars indicate the position of orthogonal slices. The protrusion has no nuclei (slices b-e), is connected to the adjacent cell (slides c and d) and does not adhere to the glass slide (slices b-e). C. GI-D468-infected HT1080 cells, 3D view from the top. An infected cell displays strong SFV staining at its surface, polarized in the direction of the intercellular connection and away from the nuclei. One densely stained protrusion is located at the center of the cell (white arrow). The long intercellular connection adheres to and wraps around the neighboring cell (yellow triangles), with extracellular SFV staining (white triangles). D. GII-K74-infected BHK-21 cells, 3D view from the top. Punctate SFV staining is observed at the basis of a syncytia (yellow triangle) and dense SFV staining at the top of cells (white triangles). Densely stained SFV protrusions (blue arrows) and several unstained cells adjacent to cells strongly expressing SFV (white arrows) are highlighted.

Fig 8. Plasma samples bind to the surface of SFV-infected cells.

BHK-21 cells were infected with GI-D468 or GII-K74 at a moi of 0.05, passed twice and stained when a CPE was visible (S2C Fig). A. Cells were permeabilized with 0.1% Triton X-100 and stained with anti-SU. Env-labelled and unlabeled cells appeared as distinct peaks and Env-labelled cells were quantified as their percentage among all cells. B. Nonpermeabilized cells were incubated with four 1:10 diluted plasma samples and anti-hIgG-BV421. Staining obtained with SFVneg (MEBAK189), anti-GI (LOBAK2), anti-GII (MEBAK88) and anti-(GI+GII) (BAK177) plasma samples are shown on the histogram overlay: mfi is presented on the x-axis and frequency is expressed as the percentage of gated events on the y-axis. Extracellular staining with plasma samples corresponds to an increase in fluorescence intensity of the whole cell population. Therefore, SFV-specific staining was quantified by the ratio of mfi from infected to uninfected cultures.

Fig 9. Plasma samples bind to Env expressed at the surface of infected cells.

A. CI-PFV-GFP-infected BHK-21 cells were stained with 1:10 diluted plasma samples from SFV-infected individuals and anti-hIgG-BV421. Staining with human plasma samples was quantified by the mfi of GFPneg and GFPpos cells and the results are expressed as mfiGFPpos/mfiGFPneg ratios (S2D Fig). B. mfiGFPpos/mfiGFPneg ratios obtained at a 1:10 dilution for the plasma from 12 individuals: n = 4 SFVneg (grey symbols), n = 8 infected with gorilla SFV belonging to genotype I (blue symbols) or genotype II (red symbols). Three to five experiments are presented per plasma sample; bars indicate median values. C. sENV-GFP-transduced BHK-21 cells were stained with 1:10 diluted plasma samples from SFV-infected individuals and anti-hIgG-BV421. Staining with human plasma samples was quantified by the mfi of GFPneg and GFPpos cells and the results are expressed as mfiGFPpos/mfiGFPneg ratios (S2E Fig). D. mfiGFPpos/mfiGFPneg ratios obtained at a 1:10 dilution for the plasma from 12 individuals: n = 4 SFVneg (grey symbols), n = 8 infected with gorilla SFV belonging to genotype I (blue symbols) or genotype II (red symbols). Three to four experiments are presented per plasma sample; bars indicate median values.

Fig 10. CI-PFV-GFP cell-to-cell transmission visualized by live-cell imaging.

CI-PFV-GFP infected BHK-21 cells were mixed with uninfected cells to obtain a GFPpos cell frequency of 5% and cultivated in 96-well plates (3000 cells/well) inside an Incucyte device for 96 h (S2F Fig). Hourly acquisition was started within 30 min after seeding. We selected sequentially acquired images (one per hour) showing representative events in CI-PFV-GFP-infected cultures. A. Acquisition between 0D-14H and 0D-20H, 250 μm x 250 μm images with a superimposed 5 x 5 grid (50 μm between lines). A GFPbright syncytium has established contact with uninfected cells; its orientation frequently shifted with the lamellipodium, often oriented towards an adjacent cell. The contacts between the syncytium and cellsto fusion (white triangles indicate cells that will be fused to the syncytium in the subsequent time frame) or not (blue triangles). B. Acquisition between 1D-0H to 1D-3H, 400 μm x 400 μm images with a superimposed 5 x 5 grid (80 μm between lines). A round GFPbright cell is in contact with a layer of uninfected cells at 1D-0H. One hour later, one adjacent cell expressed GFP (white triangle) and the cell cluster has split (arrow). Three hours later, among the cluster which left the GFPpos cells, one cell becomes GFPlow (yellow triangle). C. Acquisition between 1D-8H and 1D-15H, 250 μm x 250 μm images with a superimposed 5 x 5 grid (50 μm between lines). Morphology of a migrating infected mesenchymal, with a large lamellipodium that often splits, leading to changes in direction. The uropod appears to remain anchored, either to a cell that was in contact hours before (white dot) or to the substratum (pink dot). The former interaction can generate structures that resemble intercellular connections. One cell has remained in contact with the syncytium without fusion nor productive infection (blue triangle).

Fig 11. Number and length of intercellular connections in SFV-infected BHK-21 and HT1080 cells.

CI-PFV-GFP-infected BHK-21 and HT1080 cells were mixed with uninfected cells to obtain a GFPpos cell frequency of 5% and cultivated in 96-well plates (3000 cells/well) inside an Incucyte device for 96 h. Uninfected cells were seeded at the same density. For each condition, one 57 mm2 field acquired at 20x magnification, at 4-h intervals over 96 h, was randomly selected to manually count intercellular connections and measure their size. Cell confluence in the same wells was quantified by Incucyte software hourly from 0 to 96 h and expressed as the percentage of the total well surface. Intercellular connections counted per field (A-B), their mean ± SD length (μm, C-D), and cell confluence (percentage, E-F) are presented for BHK-21 (A, C, E) and HT1080 cells (B, D, F). Mock infected cultures are presented in grey and SFV infected cultures in blue.

Fig 12. Hypotheses to explain the resistance of SFV cell-to-cell transmission to plasma antibodies.

Schematic representation of the five hypotheses; cell-free virus is presented on the left side in red and cell-associated virus on the right side in orange. A. Env molecules bind all neutralizing antibodies, leaving sufficient free Env to mediate cell-to-cell fusion. Cell-to-cell virus transmission occurs at a higher multiplicity of infection than infection with cell free particles [58]. B. Viral particle entry and cell-to-cell spread rely on distinct Env properties: Env-mediated fusion depends upon an acidic pH [32] and cell fusion is restricted at physiological pH [33]. Cell-to-cell transfer of viral capsids and genomes is independent of the fusogenic activity of Env [62]. C. Env expression at the plasma membrane is restricted: Intracellular retention of Env probably acts as an immune escape mechanism because it affects the syncytium formation but not infectivity of viral particles [34,86]. D. Altered Env conformation or interference from host molecules [58,60]. E. Uncharacterized cell structures protect viruses from neutralizing antibodies. Images were created using biorender.com.