Respiratory syncytial virus elicits enriched CD8⁺ T lymphocyte responses in lung compared with blood in African green monkeys

<div><p>Respiratory syncytial virus (RSV) is a leading cause of serious lower respiratory tract disease in young children and older adults throughout the world. Prevention of severe RSV disease through active immunization is optimal but no RSV vaccine has been licensed so far. Immune mechanisms of protection against RSV infection in humans have not been fully established, thus a comprehensive characterization of virus-specific immune responses in a relevant animal model will be beneficial in defining correlates of protection. In this study, we infected juvenile naive AGMs with RSV A2 strain and longitudinally assessed virus-specific humoral and cellular immune responses in both peripheral blood and the respiratory tract. RSV viral loads at nasopharyngeal surfaces and in the lung peaked at around day 5 following infection, and then largely resolved by day 10. Low levels of neutralizing antibody titers were detected in serum, with similar kinetics as RSV fusion (F) protein-binding IgG antibodies. RSV infection induced CD8⁺, but very little CD4⁺, T lymphocyte responses in peripheral blood. Virus-specific CD8⁺ T cell frequencies were ~10 fold higher in bronchoaveolar lavage (BAL) compared to peripheral blood and exhibited effector memory (CD95⁺CD28^-) / tissue resident memory (CD69⁺CD103⁺) T (T_RM) cell phenotypes. The kinetics of virus-specific CD8⁺ T cells emerging in peripheral blood and BAL correlated with declining viral titers, suggesting that virus-specific cellular responses contribute to the clearance of RSV infection. RSV-experienced AGMs were protected from subsequent exposure to RSV infection. Additional studies are underway to understand protective correlates in these seropositive monkeys.</p></div

RSV viral replication in AGMs following infection.

<p>Eight AGMs were infected with RSV A2 strain through intranasal and intratracheal inoculation. RSV viral shedding at nasopharyngeal (NP) mucosal surfaces and viral replication in lung (BAL) were determined at 0, 3, 5, 7, and 10 days following infection. (A). RSV viral loads in NP swab elutes were determined by RSV RT-qPCR (Geomean with 95% confidence interval (CI)). (B). RSV viral loads in BAL were determined by RSV plaque forming assay (Geomean with 95% CI). Dashed lines represent limit of detection.</p

Kinetics of RSV-specific CD8⁺ T cell responses in AGMs following infection.

<p>A second group of eight AGMs were infected with RSV A2 strain and CD8⁺ T cell responses were determined by multiparameter flow cytometry with an expanded antibody panel at 0, 7, 9, 14, 21, and 28 days following infection. (A) PBMC or cells isolated from BAL were stimulated with RSV F protein overlapping peptides for evaluation of cytokine secretions. The percentage of IFN-γ-secreting CD8⁺ T cells were used to represent the magnitude of virus-specific CD8⁺ T cell responses. (B). Frequency of IFN-γ-secreting CD8⁺ T cells in responses to RSV proteins N and M (N+M) overlapping peptides stimulation in PBMC and BAL. (C) Expression of proliferation marker Ki67 in CD8⁺ T lymphocytes from PBMC or BAL. *, p<0.05; **, p<0.01; ***, p<0.001. One-way ANNOVA compared to baseline (day 0) levels.</p

RSV cellular immune responses in AGMs following infection.

<p>RSV-specific cellular immune responses in AGMs at 28 days following infection were determined by multiparameter flow cytometry. Cytokine secretions including IFN-γ, IL-2, and TNF-α of CD4⁺ (A) and CD8⁺ (B) T cells in PBMC specific to various RSV antigen stimulations are depicted (line represents mean). (C). Cytokine secretions of CD8⁺ T cells in lung in response to RSV antigens F and N (line represents mean) were depicted. (D). Comparison of RSV F- and N-specific CD8⁺ T cell responses (represented by IFN-γ-secreting CD8⁺ T cells) in PBMC and in lung (N = 8) with p value for paired t test was depicted. (E). Polyfunctionality of RSV-specific CD8⁺ T cell responses in PBMC and BAL (N = 8) were analyzed by Boolean gating using FlowJo software and graphed using SPICE software. The percentage of RSV N-specific CD8⁺ T cells in PBMC and lung secreting one cytokine, 2 cytokines, or 3 cytokines at the same time are depicted respectively in the pie charts. The absolute magnitude of each population secreting individual cytokines, or combinations of two or three cytokines, are depicted in the scattered dot plots below the pie charts (line represent mean). *, p<0.05, one-way ANNOVA compared to the other populations.</p

La Charente

22 juillet 18831883/07/22 (A12,N5011)-1883/07/22.Appartient à l’ensemble documentaire : PoitouCh

Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein

<div><p>Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis^® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD^® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.</p></div

Neutralization of RSV by mAbs 2E1 and 3B1.

<p>Li-Cor based RSV micro-neutralization assay was performed. Neutralization curves against RSV A Long strain (A) and RSV B Washington strain (B) for 2E1, 3B1, and palivizumab are plotted with error bars representing the standard deviation from replicate data. 2E1 bivalent Fab is shown in red, full length IgG version of 2E1 shown in green, 3B1 bivalent Fab shown in purple, full length IgG version of 3B1 shown in orange, and palivizumab shown in blue.</p

Epitope mapping of 2E1 by hydrogen/deuterium-exchange mass spectrometry.

<p>(A) Heat map plot showing the difference in deuteration levels of the RSV prefusion F protein alone compared to RSV prefusion F protein in the presence of the 2E1 monovalent Fab at five time points (15, 50, 150, 500, and 1500 sec). Slower deuterium exchange indicates regions containing the binding sites. White areas are ‘gaps’ for which there was no sequence coverage, and thus no HDX-MS information was obtained. Dashed greyed-out areas represent sequences of the signal and P27 peptides which are not present in the mature purified protein. (B) Uptake plots of several RSV F peptides spanning the conformational epitope region. Red curves show the deuteration levels of peptides of RSV F protein alone, while blue curves show the peptides of the RSV F / 2E1 complex. Peptides containing the residues of antibody epitope (417–434, 441–448, and 457–467) showed decreased deuteration level upon 2E1 binding. In contrast, peptides with no significant decrease in the deuteration level upon 2E1 exposure represent non-epitope sequences (435–440 and 449–457). The residues identified as critical for binding by shotgun mutagenesis are indicated in red font.</p

Anti-RSV F antibodies identified from Morphosys HuCAL GOLD^® phage display libraries.

<p>Heavy chain C-terminal 6xHis tagged antigen specific bivalent Fabs were purified with Ni-NTA column and then tested in ELISA binding to RSV prefusion and postfusion F proteins. (A) Antibodies preferentially binding to RSV postfusion F protein; (B) Antibodies binding to both RSV postfusion and prefusion F proteins; (C) antibody binding specifically to RSV prefusion F protein. Full-length human IgG1 D25 (prefusion F specific) and palivizumab (binding to both prefusion and postfusion F) were used as control antibodies in the above experiments.</p

Characterization of mAbs 2E1 and 3B1.

<p>(A-B) ELISA analysis of 2E1 IgG (A) and 3B1 IgG (B) binding to RSV pre- (red circle) and postfusion F (blue square) proteins. (C-H) Surface plasmon resonance (SPR) analysis of 2E1 and 3B1 Fabs binding to pre- and postfusion RSV F proteins. RU = Resonance Units. Monovalent Fab antibody fragments were captured on the surface of a Series S Sensor Chip CM5 previously functionalized with Human Fab Binder. Prefusion or postfusion F protein diluted 2-fold serially starting at 100 nM or 200 nM was then injected over captured 2E1 (C) or 3B1 (F), respectively. To determine steady-state affinity, response levels at equilibrium were plotted over concentration of pre- (D) or postfusion F (G) protein. 50 nM of post- (E) or prefusion F (H) was injected to demonstrate the specificity of 2E1 binding to prefusion F and 3B1 to postfusion F. (I-K) SPR based competition analysis of 2E1 and 3B1 against palivizumab (I), D25 (J) and MPE8 (K) in binding to RSV prefusion F protein. RU = Resonance Units. Palivizumab and D25 were amine coupled to the surface of separate flow channels of a CM5 chip. A third flow channel was subjected to amine coupling activation without an antibody and used for reference subtraction. Prefusion F (40 μg/mL) was then injected over all surfaces. After a brief stabilization period, 2E1, 3B1, or running buffer was injected to measure binding to sites not occupied by the capturing antibody (palivizumab or D25). To assess competition to MPE8, the MPE8 antibody was captured (6000 RU, not shown) to flow channel 2 of a Biacore Sensor Chip Protein A. Prefusion F (40 μg/mL) was passed over channels 1 and 2 followed by 2E1 Fab, 3B1 Fab, D25 Fab and buffer to measure binding to sites not occupied by MPE8. (L) Bio-Layer Interferometry (BLI) based competition experiment of 3B1 IgG against site I antibody 131-2a. Palivizumab (blue) is able to bind to postfusion F protein in an Octet sandwich competition assay using 131-2a as the capture antibody, but antibody 3B1 (red) does not bind.</p