SARS-CoV-2 multi-antigen protein microarray for detailed characterization of antibody responses in COVID-19 patients.

Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target multiple epitopes on different domains of the spike protein, and other SARS-CoV-2 proteins. We developed a SARS-CoV-2 multi-antigen protein microarray with the nucleocapsid, spike and its domains (S1, S2), and variants with single (D614G, E484K, N501Y) or double substitutions (N501Y/Deletion69/70), allowing a more detailed high-throughput analysis of the antibody repertoire following infection. The assay was demonstrated to be reliable and comparable to ELISA. We analyzed antibodies from 18 COVID-19 patients and 12 recovered convalescent donors. The S IgG level was higher than N IgG in most of the COVID-19 patients, and the receptor-binding domain of S1 showed high reactivity, but no antibodies were detected against the heptad repeat domain 2 of S2. Furthermore, antibodies were detected against S variants with single and double substitutions in COVID-19 patients who were infected with SARS-CoV-2 early in the pandemic. Here we demonstrated that the SARS-CoV-2 multi-antigen protein microarray is a powerful tool for detailed characterization of antibody responses, with potential utility in understanding the disease progress and assessing current vaccines and therapies against evolving SARS-CoV-2

Structure-based design of a single-chain triple-disulfide-stabilized fusion-glycoprotein trimer that elicits high-titer neutralizing responses against human metapneumovirus.

The Pneumoviridae family of viruses includes human metapneumovirus (HMPV) and respiratory syncytial virus (RSV). The closely related Paramyxoviridae family includes parainfluenza viruses (PIVs). These three viral pathogens cause acute respiratory tract infections with substantial disease burden in the young, the elderly, and the immune-compromised. While promising subunit vaccines are being developed with prefusion-stabilized forms of the fusion glycoproteins (Fs) of RSV and PIVs, for which neutralizing titers elicited by the prefusion (pre-F) conformation of F are much higher than for the postfusion (post-F) conformation, with HMPV, pre-F and post-F immunogens described thus far elicit similar neutralizing responses, and it has been unclear which conformation, pre-F or post-F, would be the most effective HMPV F-vaccine immunogen. Here, we investigate the impact of further stabilizing HMPV F in the pre-F state. We replaced the furin-cleavage site with a flexible linker, creating a single chain F that yielded increased amounts of pre-F stabilized trimers, enabling the generation and assessment of F trimers stabilized by multiple disulfide bonds. Introduced prolines could increase both expression yields and antigenic recognition by the pre-F specific antibody, MPE8. The cryo-EM structure of a triple disulfide-stabilized pre-F trimer with the variable region of antibody MPE8 at 3.25-Å resolution confirmed the formation of designed disulfides and provided structural details on the MPE8 interface. Immunogenicity assessments in naïve mice showed the triple disulfide-stabilized pre-F trimer could elicit high titer neutralization, >10-fold higher than elicited by post-F. Immunogenicity assessments in pre-exposed rhesus macaques showed the triple disulfide-stabilized pre-F could recall high neutralizing titers after a single immunization, with little discrimination in the recall response between pre-F and post-F immunogens. However, the triple disulfide-stabilized pre-F adsorbed HMPV-directed responses from commercially available pooled human immunoglobulin more fully than post-F. Collectively, these results suggest single-chain triple disulfide-stabilized pre-F trimers to be promising HMPV-vaccine antigens

Thermal stability of HMPV F variants.

The melting temperature of variants was determined by (A) Nano Differential Scanning Fluorimetry (NanoDSF) and (B) Differential scanning calorimetry (DSC). (C) Summary of melting temperature of HMPV F variants. (TIF)</p

Pro mutations can increase yield and enhance recognition by the prefusion specific antibody, MPE8.

(A) Structure-based design of proline substitutions based on prefusion-postfusion conformation change structure of HMPV F (PDB ID 5WB0) (B) Analysis interprotomer disulfide-stabilized HMPV F prefusion variants by size exclusion chromatography (C) SDS-PAGE analysis of interprotomer disulfide-stabilized HMPV F prefusion variants. (D) Additional proline substitutions increase the expression yield of HMPV F, the yield of v3B_D12 is show as dotted line. (E) The binding affinity of HMPV designs to MPE8, the affinity of v3B_D12 is show as dotted line.</p

Additional disulfide-bonds can stabilize HMPV F trimer in a prefusion state.

(A) Structure-based design of interprotomer disulfides based on the prefusion structure of HMPV F (PDB ID 5WB0); additional designs are shown in S1 Table. (B) Properties of HMPV Fs. SDS-PAGE analysis of interprotomer disulfide-stabilized HMPV F prefusion variants. (C) Analysis of HMPV F prefusion variants by size exclusion chromatography SEC and expression level (top row) and binding affinity (bottom row) of HMPV designs with additional disulfide bonds. (D) Negative-stain electron micrographs of HMPV F trimer variants. 2D classes indicate the F proteins are in prefusion conformation. Scale bar = 200 Å.</p

Human globulin analysis indicates prefusion conformation stabilized F glycoprotein absorbs neutralizing responses more efficiently than the postfusion form.

(A) Experimental scheme for the depletion of Flebogamma. (B) Depletion of Flebogamma with prefusion- and postfusion-stabilized HMPV(top panel), RSV (middle panel), and PIV3 F (bottom panel) glycoproteins.</p

Serum neutralization of HMPV subtype A2 and B2 strain.

Serum neutralization of HMPV subtype A2 and B2 strain.</p

Prefusion-stabilized HMPV F variants induce high titer neutralizing responses in mice.

(A) Immunization regimen for CB6F1J mice (n = 10 /group) with two HMPV F immunizations followed by serum analysis at week 5. (B) Elicited HMPV F neutralization by prefusion F immunogens. Limit of detection (LOD) is shown as an arrow.</p

Octet Bio-Layer interferometry profiles for binding of MPE8, MPF5 and MPE33 Fabs to HMPV prefusion-stabilized F variants.

Octet Bio-Layer interferometry profiles for binding of MPE8, MPF5 and MPE33 Fabs to HMPV prefusion-stabilized F variants.</p