Structural Analysis Of Neutralizing Epitopes Of The SARS-CoV-2 Spike To Guide Therapy And Vaccine Design Strategies

Coronavirus research has gained tremendous attention because of the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (nCoV or SARS-CoV-2). In this review, we highlight recent studies that provide atomic-resolution structural details important for the development of monoclonal antibodies (mAbs) that can be used therapeutically and prophylactically and for vaccines against SARS-CoV-2. Structural studies with SARS-CoV-2 neutralizing mAbs have revealed a diverse set of binding modes on the spike’s receptor-binding domain and N-terminal domain and highlight alternative targets on the spike. We consider this structural work together with mAb effects in vivo to suggest correlations between structure and clinical applications. We also place mAbs against severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses in the context of the SARS-CoV-2 spike to suggest features that may be desirable to design mAbs or vaccines capable of conferring broad protection

Structural basis for llama nanobody recognition and neutralization of HIV-1 at the CD4-binding site

Nanobodies can achieve remarkable neutralization of genetically diverse pathogens, including HIV-1. To gain insight into their recognition, we determined crystal structures of four llama nanobodies (J3, A12, C8, and D7), all of which targeted the CD4-binding site, in complex with the HIV-1 envelope (Env) gp120 core, and determined a cryoelectron microscopy (cryo-EM) structure of J3 with the Env trimer. Crystal and cryo-EM structures of J3 complexes revealed this nanobody to mimic binding to the prefusion-closed trimer for the primary site of CD4 recognition as well as a secondary quaternary site. In contrast, crystal structures of A12, C8, and D7 with gp120 revealed epitopes that included portions of the gp120 inner domain, inaccessible on the prefusion-closed trimer. Overall, these structures explain the broad and potent neutralization of J3 and limited neutralization of A12, C8, and D7, which utilized binding modes incompatible with the neutralization-targeted prefusion-closed conformation of Env

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

Thermal stability of HMPV F variants by Nano Differential Scanning Fluorimetry (NanoDSF).

Variants with proline substitution were shown in panel (A) and variants with disulfide bonds were shown in (B). (C) SDS-PAGE and antigenic analysis of disulfide and 3P stabilized HMPV F variants after incubation at 37°C for the indicated time. (TIF)</p

Modification of F2-F1 linkage further stabilize HMPV F trimer in a prefusion state.

(A) Structure-based design of a single chain pre-F HMPV F based on PDB ID 5WB0; additional designs are shown in S1 Table; D is length change compared to the v3B. (B) SDS-PAGE analysis of interprotomer disulfide-stabilized HMPV F prefusion variants; (C) Analysis of single chain HMPV F prefusion variants by size exclusion chromatography with yields (top row) and immunogen binding affinity to antibodies (bottom row); (D) Negative-stain electron micrographs of v3B single chain variants. 2D classes indicate that most trimers are in prefusion conformation which lack the long tail (pseudo-colored in cyan), whereas some F trimers are in postfusion conformation which appear as elongated torpedo shape with a tail (pseudo-colored in red). Scale bar = 200 Å.</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

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

Analysis of HMPV F elicited responses in mice.

(A) Correlation of antigenicity and immunogenicity of HMPV F variants. (B) Comparison of titers elicited by single chain prefusion stabilized with and without 3P mutations. (TIF)</p