Computationally restoring the potency of a clinical antibody against Omicron.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3 and revealed how quickly viral escape can curtail effective options4,5. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities

Glyoxylate Carboligase: A Unique Thiamin Diphosphate-Dependent Enzyme That Can Cycle between the 4′-Aminopyrimidinium and 1′,4′-Iminopyrimidine Tautomeric Forms in the Absence of the Conserved Glutamate

Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzymes in that it lacks a conserved glutamate near the N1′ atom of ThDP (replaced by Val51) or any other potential acid–base side chains near ThDP. The V51D substitution shifts the pH optimum to 6.0–6.2 (p<i>K</i>_a of 6.2) for TSA formation from pH 7.0–7.7 in wild-type GCL. This p<i>K</i>_a is similar to the p<i>K</i>_a of 6.1 for the 1′,4′-iminopyrimidine (IP)–4′-aminopyrimidinium (APH⁺) protonic equilibrium, suggesting that the same groups control both ThDP protonation and TSA formation. The key covalent ThDP-bound intermediates were identified on V51D GCL by a combination of steady-state and stopped-flow circular dichroism methods, yielding rate constants for their formation and decomposition. It was demonstrated that active center variants with substitution at I393 could synthesize (<i>S</i>)-acetolactate from pyruvate solely, and acetylglycolate derived from pyruvate as the acetyl donor and glyoxylate as the acceptor, implying that this substitutent favored pyruvate as the donor in carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the predecarboxylation intermediate analogues derived from acetylphosphinate, propionylphosphinate, and methyl acetylphosphonate in their IP tautomeric forms notwithstanding the absence of the conserved glutamate. The role of the residue at the position occupied typically by the conserved Glu controls the pH dependence of kinetic parameters, while the entire reaction sequence could be catalyzed by ThDP itself, once the APH⁺ form is accessible

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins

<div><p>Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.</p></div

Multifunctional human monoclonal antibody combination mediates protection against Rift Valley fever virus at low doses

Abstract The zoonotic Rift Valley fever virus (RVFV) can cause severe disease in humans and has pandemic potential, yet no approved vaccine or therapy exists. Here we describe a dual-mechanism human monoclonal antibody (mAb) combination against RVFV that is effective at minimal doses in a lethal mouse model of infection. We structurally analyze and characterize the binding mode of a prototypical potent Gn domain-A-binding antibody that blocks attachment and of an antibody that inhibits infection by abrogating the fusion process as previously determined. Surprisingly, the Gn domain-A antibody does not directly block RVFV Gn interaction with the host receptor low density lipoprotein receptor-related protein 1 (LRP1) as determined by a competitive assay. This study identifies a rationally designed combination of human mAbs deserving of future investigation for use in humans against RVFV infection. Using a two-pronged mechanistic approach, we demonstrate the potent efficacy of a rationally designed combination mAb therapeutic

Antibody sequence analysis for the V gene usage and CDR3 sequence.

<p>Antibody sequence analysis for the V gene usage and CDR3 sequence.</p

Epitope binning and hMPV F cross-reactivity.

<p>(A) Epitope binning with the newly generated mAbs on post-fusion RSV A2 F protein revealed the mAbs competed for binding to antigenic site IV with the previously described mAbs 101F and 54G10. (B) Epitope binning on pre-fusion RSV A2 F SC-TM suggested binding at antigenic site IV, as competition was not observed with site II mAb motavizumab nor site Ø mAb D25. (C) Binding and neutralization curves indicate mAb 17E10 cross-reacts with hMPV F. The top graph displays ELISA binding data with post-fusion hMPV A1 F protein. Only cross-reactive mAbs 17E10, 54G10, 101F, and MPE8 show binding to the protein, while the site IV mAbs 2N6, 3M3, and 6F18 show no binding. Each data point is the average of three independent experiments, each with four technical replicates. Error bars represent the standard deviation. The bottom graph show neutralization for the cross-reactive mAbs, with D25 used as a negative control. MAbs 17E10, MPE8, and 101F neutralize hMPV F while the D25 control shows no reduction in virus. Data points are the average of three technical replicates, and error bars indicate the standard deviation. (D) Epitope binning using post-fusion hMPV F. MAbs 101F, 17E10, and 54G10 display a similar competition binding pattern to that observed with RSV F protein. The mAbs compete for a site unique from site III mAbs 25P13 and MPE8, and DS7. For epitope binning, data indicate the percent binding of the second antibody in the presence of the first antibody, compared with the second antibody alone. Cells filled in black indicate full competition, in which ≤33% of the uncompeted signal was observed, intermediate competition (gray) if signal was between 33% and 66%, and noncompeting (white) if signal was ≥66%. Antigenic sites are highlighted at the top and side based on competition-binding with the control mAbs D25 (site Ø), 131-2a (site I), palivizumab (PALI) or motavizumab (MOTA) (site II), or 101F (site IV).</p

Binding and neutralization values for isolated RSV F-specific mAbs.

<p>Binding and neutralization values for isolated RSV F-specific mAbs.</p

Binding curves determined by biolayer interferometry for mAbs targeting antigenic site IV.

<p>Streptavidin biosensors were incubated with biotinylated peptides, and then mAbs were tested for binding in real-time. The first dashed line indicates the end of the peptide loading step, and the second dashed line indicates the beginning of the mAb binding step. After binding, mAbs were allowed to dissociate in real-time. The data are representative chromatograms from one experiment. Wild-type (WT) and mutant peptides consist of RSV F residues 422–436. The 30-mer A peptide includes RSV F residues 407–436, and the 30-mer B peptide includes RSV F residues 422–451.</p

Characterization of antigenic site IV mutations.

<p>(A) Alanine-scanning mutagenesis binding values for the generated site IV mAbs, compared with palivizumab and mAb D25 controls. The mAb reactivity for each RSV F variant was calculated relative to that of wild-type RSV F. Error bars indicate the measurement range of two independent experiments. (B) Overlay of RSV F and hMPV F sequences and crystal structures (PDB IDs: 3RRR, 5L1X, overlaid at chain H for each structure) at antigenic site IV, with RSV F residues from the alanine-scanning mutagenesis shown. Conserved residues between RSV F and hMPV F are displayed in red font. In the crystal structure overlay, RSV F residues are shown in cyan and hMPV F residues are shown in blue. (C) ELISA EC₅₀ values for recombinant post-fusion or pre-fusion (SC-TM) mutant proteins for the site IV mAbs or controls. Neutralization IC₅₀ values also are displayed for the RSV strain A2 F variant R429A.</p

Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies

Hantaviruses are high-priority emerging pathogens carried by rodents and transmitted to humans by aerosolized excreta or, in rare cases, person-to-person contact. While infections in humans are relatively rare, mortality rates range from 1 to 40% depending on the hantavirus species. There are currently no FDA-approved vaccines or therapeutics for hantaviruses, and the only treatment for infection is supportive care for respiratory or kidney failure. Additionally, the human humoral immune response to hantavirus infection is incompletely understood, especially the location of major antigenic sites on the viral glycoproteins and conserved neutralizing epitopes. Here, we report antigenic mapping and functional characterization for four neutralizing hantavirus antibodies. The broadly neutralizing antibody SNV-53 targets an interface between Gn/Gc, neutralizes through fusion inhibition and cross-protects against the Old World hantavirus species Hantaan virus when administered pre- or post-exposure. Another broad antibody, SNV-24, also neutralizes through fusion inhibition but targets domain I of Gc and demonstrates weak neutralizing activity to authentic hantaviruses. ANDV-specific, neutralizing antibodies (ANDV-5 and ANDV-34) neutralize through attachment blocking and protect against hantavirus cardiopulmonary syndrome (HCPS) in animals but target two different antigenic faces on the head domain of Gn. Determining the antigenic sites for neutralizing antibodies will contribute to further therapeutic development for hantavirus-related diseases and inform the design of new broadly protective hantavirus vaccines