A Robust Protocol to Isolate Outer Membrane Vesicles from Nontypeable <i>Haemophilus influenzae</i>

Outer membrane vesicles (OMVs) are lipid structures containing various biomolecules in their native environment and are spontaneously shed by gram-negative bacteria. OMVs perform several biological functions critical to both bacterial physiology and pathogenicity. Scientific research on OMV function and biogenesis requires a standardized and robust method of isolating these vesicles from bacterial cultures that reliably provide high-purity OMVs. Herein, we describe an optimized protocol to isolate OMVs from overnight cultures of three different strains of nontypeable Haemophilus influenzae (NTHi) for use in different downstream applications. Involving mainly differential centrifugation of the culture supernatant, the procedure described is relatively simple, efficient, and generates high-quality OMV preparations from each strain tested with sufficient yields, while preserving the native outer membrane composition

Evaluation of HSV-2 gE Binding to IgG-Fc and Application for Vaccine Development

Glycoprotein E (gE) and glycoprotein I (gI) are expressed as a heterodimer on the surface of Herpes simplex virus (HSV). Glycoprotein E binds Fc domain of immunoglobulin G (IgG) and inhibits activities mediated by the IgG Fc domain, contributing to immune evasion by HSV. It has been reported that HSV type 1 gE (gE-1) is capable of binding IgG Fc as a monomer and in a heterodimeric complex with gI, with the heterodimer having 50- to100-fold greater affinity for Fc than gE alone. We report the production of both a soluble form of HSV type 2 gE (gE-2) and a soluble HSV-2 gE/gI heterodimer (gE-2/gI-2). Characterization of soluble gE-2 by surface plasmon resonance (SPR) demonstrates that it is incapable of binding human IgG or the IgG Fc domain. Co-expression with HSV-2 gI (gI-2) and purification of the gE-2/gI-2 heterodimer enable gE-2 to bind human IgG through its Fc domain. We hypothesize that functional epitopes of wildtype gE-2 may be masked by plasma IgG Fc and affect the immunogenicity of the gE-2/gI-2 heterodimer as a vaccine antigen. A series of gE-2 mutations within the surface-exposed Fc:gE-2 interface was designed, and gE-2 mutants were co-expressed with gI-2. Evaluation of twelve gE-2 mutant heterodimers by SPR assay identified nine gE-2 mutations which abrogated or reduced Fc binding while maintaining heterodimer formation with gI. Vaccinating rabbits with the four most Fc-binding deficient gE-2/gI-2 heterodimers elicited comparable anti-heterodimer binding antibody titers and statistically significantly higher serum neutralization antibody levels than wildtype heterodimers. Taken together, these data support the concept of rational antigen design for improved vaccine candidates

Dissecting the heterogeneous glycan profiles of recombinant coronavirus spike proteins with individual ion mass spectrometry

Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2 play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we have utilized individual ion mass spectrometry (I2MS), a form of charge detection mass spectrometry (CDMS) that uses a commercially available Orbitrap analyzer, to directly produce heterogeneous glycan mass profiles of these three coronavirus spike protein trimers under native-like conditions. Analysis by I2MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques. This highlights the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I2MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N-glycans followed by I2MS indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts and differences in glycan removal are correlated with solvent accessibility. I2MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional protein mass analysis methods such as size exclusion chromatography-multiple angle light scattering (SEC-MALS) and field flow fractionation-multiple angle light scattering (FFF-MALS). An added advantage of I2MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I2MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development

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

<div><p>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.</p></div

Analysis of RSV F proteins by differential scanning fluorimetry.

<p>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.</p

Monitoring DS-Cav1 stability with 4D7.

<p>(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).</p

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

<p>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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164789#pone.0164789.ref023" target="_blank">23</a>] (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.</p

RSV F prefusion and postfusion structures and antigenic sites.

<p>Surface representation of prefusion (left panel) and postfusion RSV F (right panel) trimers are shown in gray [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164789#pone.0164789.ref013" target="_blank">13</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164789#pone.0164789.ref020" target="_blank">20</a>]. 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.</p