Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein

Abstract

Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7.

Fig 1. RSV F prefusion and postfusion structures and antigenic sites.

Surface representation of prefusion (left panel) and postfusion RSV F (right panel) trimers are shown in gray [13, 20]. Antigenic sites are highlighted in different colors: site Ø, red; site I, magenta; site II, orange; site III, yellow. Sequences contained in the overlapping antigenic sites IV and V are green, and additional residues that contribute to site V antibody AM14 binding are shown in cyan.

Fig 2. Identification of critical residues for monoclonal antibody 4D7 binding using shotgun mutagenesis epitope mapping.

A shotgun mutagenesis alanine scanning library was constructed for the RSV F protein. The library contains 368 individual mutations at residues identified as surface exposed on the prefusion and postfusion forms of RSV F proteins. Each well of the mutation array plate contained one mutant with a defined substitution. (A) Human HEK293T cells expressing the RSV F mutation library were tested for immunoreactivity with 4D7, measured on an Intellicyt high-throughput flow cytometer. Clones with reactivity of <15% relative to that of wildtype RSV F (horizontal line) yet >70% reactivity for a control monoclonal antibody were initially identified to be critical for 4D7 binding (red dots), and were verified using algorithms described elsewhere [23] (U.S. patent application 61/938,894). (B) Mutation of three individual residues reduced 4D7 binding (red bars) but not the binding of D25 and palivizumab (gray bars). Error bars represent range (half of the maximum minus minimum values) of at least two replicate data points. (C) Comparison of 4D7 binding epitope on prefusion and postfusion RSV F structures. Prefusion RSV F structure is shown in magenta and postfusion F structure shown in green. Residues 384 to 392 are shown in stick representation highlighting both main chain and side chain atoms, and the rest of the structure is shown in line representation with only main chain bonds depicted.

Fig 3. Characterization of monoclonal antibody 4D7 binding to RSV F.

Surface plasmon resonance was used to assess the ability of 4D7 to bind DS-Cav1 and postfusion forms of RSV F. (A) Various concentrations of DS-Cav1 or postfusion F (two-fold serial dilutions from 40–0.08 μg/mL) were flowed over the surface of a 4D7-coated sensor chip, and response units were plotted over time, in seconds. Each line represents the results from a single concentration of postfusion F (left panel) or DS-Cav1 (right panel). (B) DS-Cav1 was flowed over the surface of a 4D7-coated sensor chip (grey shaded area), followed by palivizumab (red line) or D25 (blue line). Response units were plotted over time, in seconds.

Fig 4. Monitoring DS-Cav1 stability with 4D7.

(A) DS-Cav1 was stored at -70°C or at 4°C for either 14 or 102 days, and surface plasmon resonance was used to assess protein binding to 4D7 (left panel) and D25 (right panel). DS-Cav1 stored at -70°C and thawed immediately before use (cyan line) or stored at 4°C for 14 (blue line) or 102 (green line) days was flowed over the surface of 4D7- or D25-coated sensor chip channels, and response units over time, in seconds, were plotted. (B) Sandwich ELISA. DS-Cav1 stored for approximately 5 months at 4°C was captured on an ELISA plate coated with 4D7. The captured 4D7-reactive protein was bound by palivizumab (orange line), which recognizes both prefusion and postfusion forms of RSV F. In contrast, the captured 4D7-reactive protein was not bound by any of the prefusion-specific monoclonal antibodies tested, such as D25 (site Ø), MPE8 (site III), or AM14 (site V).

Fig 5. Analysis of RSV F proteins by negative stain transmission electron microscopy and 2D class averaging.

(A) Representative averages for DS-Cav1 stored at -70°C and thawed immediately prior to analysis, indicating that mostly globular particles (left) or particles that contained a slightly tapered, short tail portion (middle and right) were observed. A characteristic indentation was visible in some of the head domains (middle). (B) Representative averages for postfusion RSV F protein stored at -70°C and thawed immediately prior to analysis, indicating that particles with distinct head and tail portions were primarily observed. A characteristic indentation was also visible in some of the head domains (left). (C) Representative averages for DS-Cav1 protein after long-term storage at 4°C. The lack of detail of the averages suggests that the conformation of 4°C-stored DS-Cav1 is more heterogeneous and does not resemble the postfusion form.

Fig 6. Analysis of RSV F proteins by differential scanning fluorimetry.

First derivative of F350/F330 DSF unfolding curves for freshly thawed DS-Cav1 stored at -70°C (red line), DS-Cav1 stored for 90 days at 4°C (black line) and postfusion F protein (blue line). Transition midpoints are shown as vertical lines. (A) Protein concentration analyzed was 15 μM. (B) Protein concentration analyzed was 1 μM. (C) DSF transition midpoints of freshly thawed DS-Cav1, DS-Cav1 stored for 90 days at 4°C, and postfusion F. Mean values and standard deviations are calculated from measurements taken at protein concentrations between 35 μM and 0.27 μM. (*) Tm1 is observed only in preparations of freshly thawed DS-Cav1. The intensity of this transition increases with lower concentration.