Protective antigenic sites identified in respiratory syncytial virus fusion protein reveals importance of p27 domain
2021; Springer Nature; Volume: 14; Issue: 1 Linguagem: Inglês
10.15252/emmm.202013847
ISSN1757-4684
AutoresJee-Hyun Lee, Youri Lee, Laura Klenow, Elizabeth M. Coyle, Juanjie Tang, Supriya Ravichandran, Hana Golding, Surender Khurana,
Tópico(s)Tracheal and airway disorders
ResumoArticle8 November 2021Open Access Source DataTransparent process Protective antigenic sites identified in respiratory syncytial virus fusion protein reveals importance of p27 domain Jeehyun Lee Jeehyun Lee Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Youri Lee Youri Lee orcid.org/0000-0002-0905-5900 Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Laura Klenow Laura Klenow orcid.org/0000-0003-4170-5480 Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Elizabeth M Coyle Elizabeth M Coyle Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Juanjie Tang Juanjie Tang Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Supriya Ravichandran Supriya Ravichandran Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Hana Golding Hana Golding Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Surender Khurana Corresponding Author Surender Khurana [email protected] orcid.org/0000-0002-0593-7965 Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Jeehyun Lee Jeehyun Lee Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Youri Lee Youri Lee orcid.org/0000-0002-0905-5900 Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Laura Klenow Laura Klenow orcid.org/0000-0003-4170-5480 Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Elizabeth M Coyle Elizabeth M Coyle Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Juanjie Tang Juanjie Tang Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Supriya Ravichandran Supriya Ravichandran Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Hana Golding Hana Golding Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Surender Khurana Corresponding Author Surender Khurana [email protected] orcid.org/0000-0002-0593-7965 Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA Search for more papers by this author Author Information Jeehyun Lee1, Youri Lee1, Laura Klenow1, Elizabeth M Coyle1, Juanjie Tang1, Supriya Ravichandran1, Hana Golding1 and Surender Khurana *,1 1Division of Viral Products, Center for Biologics Evaluation and Research (CBER), FDA, Silver Spring, MD, USA *Corresponding author. Tel: +1 240 402 9632; Fax +1 301 595 1125; E-mail: [email protected] EMBO Mol Med (2022)14:e13847https://doi.org/10.15252/emmm.202013847 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Respiratory syncytial virus (RSV) vaccines primarily focused on surface fusion (F) protein are under development. Therefore, to identify RSV-F protective epitopes, we evaluated 14 antigenic sites recognized following primary human RSV infection. BALB/c mice were vaccinated with F peptides, F proteins, or RSV-A2, followed by rA2-Line19F challenge. F peptides generated binding antibodies with minimal in vitro neutralization titers. However, several F peptides (including Site II) reduced lung viral loads and lung pathology scores in animals, suggesting partial protection from RSV disease. Interestingly, animals vaccinated with peptides (aa 101–121 and 110–136) spanning the F-p27 sequence, which is only present in unprocessed F0 protein, showed control of viral loads with significantly reduced pathology compared with mock-vaccinated controls. Furthermore, we observed F-p27 expression on the surface of RSV-infected cells as well as lungs from RSV-infected mice. The anti-p27 antibodies demonstrated antibody-dependent cellular cytotoxicity (ADCC) of RSV-infected A549 cells. These findings suggest that p27-mediated immune response may play a role in control of RSV disease in vivo, and F-p27 should be considered for inclusion in an effective RSV vaccine. Synopsis This study identifies possible protective linear antigenic sites on the RSV F protein in a mouse RSV challenge model for development of RSV vaccine. We show that F-p27 peptide control viral loads and reduced RSV disease in vivo. Therefore, F-p27 should be included in an effective RSV vaccine. High F-p27 expression observed on the surface of RSV-infected cells and RSV-infected lungs. Anti-p27 antibodies does not neutralize RSV in vitro. Anti-p27 antibodies mediate antibody-dependent cellular cytotoxicity (ADCC). The paper explained Problem Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract disease in infants and young children; however, there is no vaccine against RSV. RSV vaccines currently under clinical development are primarily focused on surface fusion (F) membrane protein for different target populations. However, there is limited information on antigenic sites within F that generate protection following vaccination. Therefore, it is important to identify all possible protective epitopes on the F protein for development of an effective RSV vaccine. Results In the current study, we evaluated the immunogenicity, neutralization potential, safety, and protective efficacy of all antigenic sites in F2 and F1 domains identified in the post-primary infection infant sera using F-GFPDL including the F-p27 antigenic site in mouse RSV challenge model. Several F peptides provided partial protection by viral loads in lung tissues with minimal lung pathology from RSV disease. Interestingly, animals vaccinated with a peptide containing F-p27 (aa 101–121 or 110–136), which is only present in unprocessed F0 protein, and not present on the mature RSV virion particles, showed significant control of viral loads with no apparent pathology in the lungs following RSV challenge. While p27 is not part of the mature F protein on virions, it is part of newly translated F0 in infected cells. We show for the first time that F-p27 is expressed on the surface of RSV-infected cells in vitro as well as lungs of RSV-infected mice in vivo, and these cells can be killed by antibody-dependent cell cytotoxicity, suggesting a possible role of p27-mediated immune response in control of RSV disease. Impact The current study evaluated the immunogenicity of multiple antigenic sites within the RSV-F protein and shows for the first time the presence of in vivo protective epitopes in the RSV F-p27 motif that did not correlate with antibody binding to mature virions or neutralization in vitro. These findings identified p27 as a potential target of protective immunity in vivo and suggest inclusion of p27 in an effective vaccine against RSV. Introduction Significant efforts are underway to develop and evaluate RSV vaccines targeted to pregnant women with hope of protecting neonates from RSV [renamed to human Orthopneumovirus (hOPV)]-induced lung disease early in life, as well as to elderly populations, who are susceptible to recurrent RSV infections (Drysdale et al, 2020). Identification of protective epitopes in RSV proteins is critical for the development of effective vaccine against RSV disease (Johnson et al, 1997; Zhu et al, 2017; Drysdale et al, 2020). In our previous study, Genome-fragment Phage Display Libraries (GFPDL) covering the RSV-A2 F and G genes were used to elucidate the epitope repertoires of serum antibodies from children at 9 and 18 months following primary RSV infection. Multiple sites were identified in the F protein, many of which are present in both the pre-fusion and post-fusion F conformations, that were confirmed by antibody binding to sera from young children (< 2 years), adolescents (14–18 years), and adults (30–45 years) (Fuentes et al, 2016). Surprisingly, the strongest binding in the youngest group was to peptide (aa 101–121), which contains part of p27. Expanding F-specific epitope diversity was observed in the older groups in addition to retaining strong binding to F-p27 (Fuentes et al, 2016). These findings suggested that during RSV infection, the immune system is exposed to unprocessed F0 on infected cells or to immature virions, and humans generate strong anti-p27 response. However, the relevance of such anti-p27 immune response in RSV disease is unknown. One RSV vaccine candidate based on a pre-fusogenic form of F containing p27 generated higher neutralizing antibodies compared with pre-fusion and post-fusion F proteins (lacking p27), and protected animals from RSV challenge in mice (Patel et al, 2019). In addition, this study demonstrated the generation of antibodies that competed with monoclonal antibodies (MAbs) to site Ø, II, IV, and VIII, but no evidence was provided to demonstrate the contribution of anti-p27 antibodies to in vitro virus neutralization or in vivo protection against RSV-A2 virus challenge (Patel et al, 2019). Moreover, there is limited information on antigenic sites within F that provide protection that are key for the development of an effective RSV vaccine. In the current study, we followed up these findings through vaccination of mice with individual F-derived peptides (representing antigenic sites identified in post-RSV infection in humans) followed by a challenge with RSV rRSV-A2-L19 strain. Serum samples were tested for virion binding, RSV neutralization titers, and ADCC activity, prior to viral challenge. Lung tissues were excised on day 5 post-challenge and evaluated for RSV viral loads, lung histopathology, and presence of CD4 and CD8 T cells. Live RSV-A2 infection (low dose) and recombinant F proteins (pre-fusion and post-fusion) were used as positive controls. Results RSV-A2 virion-binding and neutralization antibodies following vaccination of mice with F peptides Previously, an unbiased GFPDL analysis identified multiple antigenic sites recognized by sera from primary RSV-infected infants that were mapped to the exposed surface of the F protein in either the pre-fusion or post-fusion conformation (Fuentes et al, 2016). In the current study, we determined their ability as an immunogen to elicit functional, virion-binding antibodies, and the importance of these antibodies for in vivo protection against RSV challenge. To that end, RSV-F peptides were chemically synthesized, purified by HPLC, conjugated to KLH, and used for animal vaccination. BALB/c mice (n = 5 per group) were immunized intramuscularly twice with 20 μg of RSV pre-fusion or post-fusion forms of F protein (positive controls), or with the F peptides-KLH conjugates mixed with Emulsigen adjuvant, or with PBS (no vaccination control), or with 100 μg of unconjugated KLH alone (carrier control), or with a single intranasal dose of 104 pfu live RSV-A2 virus (Fig 1A). The location of the different peptides in the F protein is shown in Fig 1B. The p27 is encompassed in F (aa 110–136). After the second immunization, blood was collected from the tail veins. These mice were then challenged intranasally with 1 × 106 PFU/10 μl of RSV rA2-Line-19F-FFL. Figure 1. F protein immunization and challenge study in mice Schematic representation of mouse immunization and challenge schedule. Female BALB/c mice (n = 5 per group; 4–6 weeks old) were immunized with single dose of live RSV-A2 virus (104 pfu dose) intranasally, or two doses given i.m. with 20 μg of RSV pre-fusion or post-fusion form of F protein or F peptides from RSV strain A2 with Emulsigen adjuvant, or with KLH or with PBS as a control. After the second immunization, blood was collected from the tail veins on day 35. On day 42, mice were challenged intranasally with 106 PFU/10μl of RSV rA2-Line-19F-FFL. Mice were sacrificed on day 5 post-challenge, when lungs and blood were collected. Alignment of antigenic site peptides within the RSV-F protein used for mice vaccination in the study. Previously described antigenic sites (sites ϕ, I, II, and IV) are shown above the F protein schematic, and the antigenic site peptides used in this study are depicted below. Download figure Download PowerPoint Post-vaccination sera were tested for binding to RSV-A2 virions (Fig 2A–D). The highest virus-binding titers were observed in animals vaccinated with post-fusion F (green curve), followed by animals exposed to live RSV-A2 virus (blue curve), and animals vaccinated with the pre-fusion F proteins (purple curve) (Fig 2A). No virion binding was observed with sera from either KLH- or PBS-vaccinated animals Fig 2A). The individual peptides elicited antibodies with low to moderate virion binding at 100-fold dilution with minimal to no binding observed at 1:500 following 2nd vaccination (Fig 2B–D). The p27 sequence (aa 110–136) is uniquely found in uncleaved F0 and is excised during F protein maturation into F1/F2 complex and is expected to be absent on mature RSV-A2 virion particles. Weak antibody binding to virus particle (OD of 0.15 at 100-fold dilution) was observed for serum from mice immunized with the p27 spanning peptides 101–121 and 110–136, suggesting very minimal presence of immature or partially matured F0 proteins on virions (Fig 2B). This finding is in agreement with previous study, demonstrating that the presence of p27 peptide has a destabilizing effect on trimer formation and incorporation into virions (Krarup et al, 2015). Figure 2. RSV binding and neutralizing antibody response following mice immunization Serum samples were collected at seven days after the second immunization from individual mice (day 35 from the start of the study; n = 5 mice per group) and were tested for RSV-A2 virion binding in ELISA and neutralization by an RSV-LINT assay against RSV-A2 strain in A549 cells. A–D. The serial dilutions of post-second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2-Line19F-FFL virions. The serum samples were serially diluted, and the detection of antibodies was measured by optical density (OD) at 490 nm. End-point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control (PBS immunized animal). E. Virus neutralization titers following second vaccination. End-point serum dilution titer that resulted in 50% inhibition of RSV infection (ID50) in RSV-LINT assay is shown. The mean value for each group is indicated. Data information: Two independent experiments were performed for data analysis. Results were determined and presented as mean ± SEM. Statistical significances were performed by one-way ANOVA in GraphPad Prism; *P ≤ 0.05, **P ≤ 0.01. Source data are available online for this figure. Source Data for Figure 2 [emmm202013847-sup-0002-SDataFig2.xlsx] Download figure Download PowerPoint In the RSV-LINT neutralization assay (Fuentes et al, 2013), high titers were measured for the three positive control groups (ID50:103–106). Surprisingly, several F peptides elicited detectable neutralizing activity (aa 147–203, 216–244, 234–287, 310–368) encompassing sites II (234–287) and critical contact residues for the site Ø (147–203) (Fig 2E). Viral loads following RSV challenge of F-peptide vaccinated animals On day 42, mice were challenged intranasally (i.n.) with 1 × 106 PFU of rRSV-A2-L19-FFL as previously described (Fuentes et al, 2017). All animals (and KLH or PBS controls) were sacrificed on day 5 post-infection, and the lungs were used for the measurement of viral loads (in HEp-2 cells) and pathology scoring as outlined in Methods. Animals vaccinated with the live RSV-A2 or the two forms of the F protein did not show measurable lung viral loads (< 5 pfu/gm lung tissue). In comparison with the PBS (control) and KLH (control), mice vaccinated with the F peptide-KLH conjugates showed either no reduction in viral loads (aa 1–34, 23–74, and 552–572), a partial reduction in viral loads (aa 310–368, 371–400, 425–450, 443–461, 471–493, and 497–521), or significant reduction (> 90% reduction; 2 × 105 pfu/gm lung in PBS controls vs. 9 × 103–2 × 104 pfu/gm lung in mice given F peptides) (aa 101–121, 110–136, 147–203, 216–244, and 234–287) (Fig 3A). Surprisingly, animals vaccinated with peptide 101–121, which contains a portion of the p27 peptide, and the p27 110–136 peptide, also had very low viral loads, in the absence of significant virion-binding or in vitro RSV-neutralizing antibodies (Fig 2B,E). Figure 3. Lung viral load and histopathology of the lungs of animals vaccinated with the RSV-F proteins and F peptides at day 5 following RSV challenge Lung RSV titers (PFU/gram of lung tissue) were determined in individual lungs (n = 5 mice per group) collected at 5 days post-RSV infection. Results were determined and presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value, and central band represents the median value for the group. Lung tissues were collected from 5 mice per group at 5 days post-challenge and stained with hematoxylin and eosin to assess histopathology for peribronchiolar and alveolar pneumonia. Individual lungs were scored for pulmonary inflammation: bronchiolitis (mucous metaplasia of bronchioles), perivasculitis (inflammatory cell infiltration around the small blood vessels), interstitial pneumonia (inflammatory cell infiltration and thickening of alveolar walls), and alveolitis (cells within the alveolar spaces). Slides were scored blindly using a 0–4 severity scale for the pathology. The scores were subsequently converted to a combined 0–4 histopathology scales. Results are presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value, and central band represents the median value for the group. Data information: Statistical significances were performed by one-way ANOVA test in GraphPad Prism. The blue asterisks show significance compared to naïve infection (PBS) (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). Source data are available online for this figure. Source Data for Figure 3 [emmm202013847-sup-0003-SDataFig3.xlsx] Download figure Download PowerPoint Lung histopathology following RSV challenge Several studies demonstrated that vaccine candidates based on recombinant F proteins and peptides may lead to enhanced lung pathology following vaccination of mice and cotton rats (Murphy et al, 1990; Connors et al, 1992; Lee et al, 2017). Therefore, at day 5 post-RSV infection, lung tissues were histostained and subjected to blinded pathology scoring (8 fields examined from both left and right lobe per animal) as described in Methods. Lungs from animals vaccinated with live RSV-A2 virus, or purified pre- and post-fusion RSV-F proteins, that did not have any virus replication in the lungs also displayed significantly lower histopathology, including alveolitis, bronchiolitis, perivascular, and interstitial pneumonia, compared with mock-vaccinated (PBS) RSV-infected mice (Fig 3B). In addition, among the peptide-immunized animals, mice vaccinated with F peptides 101–121, 110–136 (p27), and 443–461 demonstrated significantly reduced lung pathology compared with either PBS- or KLH-vaccinated animals. The differences in pathology score between RSV-A2 intranasally immunized group vs F-p27 vaccinated group were not significant. Mean pathology scores observed for groups given other KLH-conjugated peptides were similar to or higher (aa147–203) than the mean pathology scores observed for the KLH or PBS mock-immunized group, but the differences did not reach statistical significance. Interestingly, peptides 1–34 and 23–74 elicited binding antibodies that did not neutralize the virus in vitro (Fig 1B). Yet, the lung pathology scores for these groups were highly variable and did not reach statistical significance compared with other groups (Fig 3B). Altogether, we did not find evidence for enhanced lung pathology following challenge in any of the vaccinated groups at this antigen dose. F-p27 is expressed on the surface of RSV-infected cells and in the lungs of RSV-infected mice While p27 (residues 110–136) is not part of the mature F protein on virions, some immature or unprocessed F0 may be present on virions (Krzyzaniak et al, 2013) and might not be proteolytically activated until it reaches the target cells. On the other hand, p27 is part of newly translated F0 in RSV-infected cells. As can be seen in Fig 4A, all mice vaccinated with the p27-containing peptide (110–136 residues) generated p27 peptide (110–136) antibody binding titers in ELISA but did not bind to F (1–34) peptide (negative control). Antisera from animals given "pre-F" or "post-F" proteins lacking the p27 sequence did not react to the p27 (110–136) peptide in ELISA. Figure 4. Expression of p27 on the surface of RSV-infected A549 cells Serum samples collected from individual mouse (M 1-5) immunized with F 110-136 peptide were tested for antibody binding against F-p27 (110–136) peptide or F 1-34 peptide (control) in ELISA. A549 cells were infected with RSV (MOI = 0.1) for 16 h, and fixed. Cells were treated with mock control rabbit sera (left panels), or rabbit antisera against F (center panels), or against F-p27 (110–136) peptide (right panels), followed by Alexa 594 conjugated anti-Rabbit IgG (red). Nuclei are stained with DAPI (blue). Scale bar = 10 µm. The number of cells positive for RSV-F (middle panel) and RSV F-p27 (right panel) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the 'merge' panel. The experiments were performed twice, and variation between the two independent experiments was < 6%. Source data are available online for this figure. Source Data for Figure 4 [emmm202013847-sup-0004-SDataFig4.xlsx] Download figure Download PowerPoint To determine whether the p27 peptide is expressed on the surface of RSV-infected cells, we generated rabbit antiserum against F protein and against F peptide containing residues 110–136 of p27 sequence. Immunostaining performed on non-permeabilized RSV-A2-infected A549 cells demonstrated a comparable strong cell surface staining patterns using either anti-RSV-F protein or anti-F-p27 peptide antisera. Both antisera stained similar percentages of RSV-A2-infected A549 cells (64.5% and 63.89%, respectively) (Fig 4B). Furthermore, we also observed strong F-p27 expression in sections of lung tissues from RSV-A2-infected animals using rabbit serum generated against either RSV-F protein (62.25%) or F-p27 (110–136) peptide (61.59%) (Fig 5). These observations suggest that F-p27-containing immature F0 is widely expressed on surface of RSV-infected cells in vitro and in RSV-infected lungs in vivo. Figure 5. Localization of RSV antigen expression in distal alveoli in mouse lungs following viral challenge Mice lungs were collected from PBS-(mock) vaccinated BALB/c mice at day 5 post-RSV infection, fixed, and paraffin-embedded, and slides were processed for imaging. Immunohistochemistry staining of lung sections shows RSV protein localized to distal alveoli by Control rabbit sera (left panels) or rabbit antisera against RSV-F protein (center panels), or against RSV F-p27 (110–136) peptide (right panels). Scale bar = 100 µm. The percentage of cells positive for RSV-F (middle panel) and RSV F-p27 (right panel) covering 500 × 500 µm of lung tissue for each mice for each antibody staining are shown. The experiments were performed twice, and variation between the two independent experiments was < 9%. Download figure Download PowerPoint Role of cell-mediated immunity and antibody Fc effector function in anti-p27-mediated protection from RSV disease The observed reduction in lung viral titers and lung pathology after challenge of mice vaccinated with F-p27 suggested that mechanisms other than neutralizing antibodies may play a role in protection. To evaluate number of CD4 or CD8 T cells in lungs following RSV infection, we stained sections of lungs collected from mice at day 5 post-RSV challenge and determined CD4 and CD8 T cells in both airway and distal lung tissues. As shown in Fig 6A, in all RSV challenged mice there was influx of both CD4 and CD8 T cells compared with uninfected control animals. When compared with the PBS (unvaccinated) or KLH controls, some vaccinated animals showed higher number of either CD4 or CD8 cells, but these differences did not reach statistical significance for the p27 vaccinated group. Figure 6. CD4/CD8 T cells in the airways and distal alveoli of mouse lungs following viral challenge and ADCC activity of post-immunization serum Lung tissues from individual mice (n = 5) at day 5 post-RSV challenge (or uninfected control) were used to evaluate CD4/CD8 T cells separately for airway and distal lungs. Lung slides were fixed and stained with DAPI to visualize nuclei (blue) and either anti-CD4 (green) or anti-CD8 (red) T cells. The number of CD4/CD8 T cells were determined in each lung airway and distal region. Individual CD4+/CD8+ T cells' data are presented as number of positive cells per 174 × 174 µm of lung tissue for each mice. Results are presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value, and central band represents the median value for the group. RSV-specific antibody-dependent cellular cytotoxicity (ADCC) activity of post-second immunization serum was evaluated using RSV-infected A549 cells or mock A549 cells that served as target cells. After incubation with serum, Jurkat/NFAT-luc+ FcγRIV that expresses a luciferase reporter were added as the effector cells. The amount of luciferase was then measured as relative luciferase units (RLU). The RLU readings of ADCC induction from serum of each group were normalized to assay control (no serum) background. The RSV-specific ADCC activity (RLU measured) fold-change induced is shown as the ratio of normalized RLU from RSV-infected target cells to normalized RLU from uninfected target cells. The experiments were performed twice and variation between the two independent experiments (two biological replicates) was < 12%. Results are presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value, and central band represents the median value for the group. Data information: Statistical significances were performed by one-way ANOVA in GraphPad Prism; ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Source data are available online for this figure. Source Data for Figure 6 [emmm202013847-sup-0005-SDataFig6.xlsx] Download figure Download PowerPoint Antibody-dependent cellular cytotoxicity (ADCC) may play a role in control of RSV-mediated pathology. Therefore, to determine whether anti-p27 antibodies mediate ADCC, post-second immunization serum was assessed with a Promega ADCC Reporter Bioassay. A549 target cells were either mock treated or infected with RSV-A2 virus, and incubated with post-second immunization sera, followed by the addition of genetically engineered Jurkat T effector cells that expresses mouse FcγRIV along with a luciferase reporter driven by an NFAT-response element (NFAT-RE). The RSV-specific ADCC activity induced by the serum antibodies was calculated against RSV-infected target cells compared with mock A549 cells as baseline. Sera from uninfected mice did not induce effector cell activation above the baseline (Fig 6B). The highest level of ADCC activity was observed with sera from mice immunized with live A2 virus prior to viral challenge. Importantly, sera from two out of four mice vaccinated with p27 peptide showed ADCC activity that was higher compared with sera from mice vaccinated with pre-fusion F, which does not contain p27 sequence (Fig 6B). Together, these studies suggest that p27 immunization induces non-neutralizing protection mechanisms against RSV that includes Fc-mediated killing of infected cells via ADCC and possibly T cell-mediated effector functions. Discussion Primary RSV infection in children identified antigenic sites in the pre-fusion and post-fusion conformation of RSV-F protein (Fuentes et al, 2016). Earlier studies using F peptide-based vaccine generated strong antibody-binding response, but those antibodies failed to bind native F proteins or offer any protection against RSV challenge (Jaberolansar et al, 2
Referência(s)