PRIMO: an interactive homology modeling pipeline

The development of automated servers to predict the three-dimensional structure of proteins has seen much progress over the years. These servers make calculations simpler, but largely exclude users from the process. In this study, we present the PRotein Interactive MOdeling (PRIMO) pipeline for homology modeling of protein monomers. The pipeline eases the multi-step modeling process, and reduces the workload required by the user, while still allowing engagement from the user during every step. Default parameters are given for each step, which can either be modified or supplemented with additional external input. PRIMO has been designed for users of varying levels of experience with homology modeling. The pipeline incorporates a user-friendly interface that makes it easy to alter parameters used during modeling

Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes on the basis of their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of advanced antibody discovery platforms

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

Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases, as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identify 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, angiotensin-converting enzyme 2 [ACE2]-blocking clone that protects in vivo) and others recognizing non-RBD epitopes that bind the S2 domain. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants. © 2021 The Author(s)Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

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

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

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