sequenced wild-type strain  (NCIMB Ltd)

Journal: Nature Structural & Molecular Biology

Article Title: Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

doi: 10.1038/s41594-023-01062-z

Figure Lengend Snippet: a , Effect of trimer opening on the neutralization potency of bnD.8 and bnD.9. Single JR-CSF alanine mutants known to confer trimer opening were compared for sensitivity to bnDs and antibodies. Bars indicate fold change of IC 50 relative to wild-type JR-CSF; depicted ratios are means from two to four independent experiments. mut, mutant; wt, wild-type. b , Preattachment and postattachment inhibition activity of bnDs and bnAbs against BG505 and SF162 pseudoviruses as deferred in an adapted TZM-bl pseudovirus assay where total activity corresponds to the conventional neutralization setup with inhibitor present at all steps and consisting of an activity before attachment and after attachment. Through measuring postattachment activity in a setup where inhibitors are added after virus binding to cells, preattachment activity can be deferred. This total activity measured at a fixed antibody (6.4 nM) or bnD (10 µM) concentration is set to 100%, and preattachment and postattachment activity are calculated relative to it. Values are means of three or four independent experiments.

Article Snippet: Codon-optimized sequences of strain JR-FL gp120 wild-type and the V3 and V1V2 loop deletion mutants , were custom synthesized (GeneArt), fused to a C-terminal AviTag and cloned into the CMV/R expression vector .

Techniques: Neutralization, Mutagenesis, Inhibition, Activity Assay, Virus, Binding Assay, Concentration Assay

Journal: Nature Structural & Molecular Biology

Article Title: Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

doi: 10.1038/s41594-023-01062-z

Figure Lengend Snippet: a , Cryo-EM structure at 3.9 Å resolution of bnD.9 in complex with BG505.SOSIP.664 trimer induced by sCD4. For one protomer, gp41 (yellow), gp120 (orange), bnD.9 (light blue) and sCD4 (light green) are indicated. b , Helical V3 segment as seen in the X-ray structure of the bnD.9–V3 (BG505) peptide complex. V3 interface residues that are within 4 Å distance to bnD.9 are shown as sticks. c , Comparison of X-ray structures of bnD.8 in complex with V3 (BF520) and bnD.9 in complex with V3 (BG505) peptide. The structures were superimposed on V3 in PyMOL. bnD.9 is shown in blue, with the bound V3 peptide in orange. bnD.8 is depicted in green, with its bound V3 peptide in gray. d , Superposition of bnD.9–V3 (BG505) X-ray structure and bnD.9–BG505.SOSIP–sCD4 cryo-EM structure (superposition based on V3). e , Comparison of bnD.9-bound V3 with published V3 structures. PDB IDs are indicated above the respective structures. From left to right: closed, unliganded trimer ; HIV-1 YU2 gp120 in complex with 412D and sCD4 (ref. ); HIV-1 JR-FL gp120 in complex with sCD4 and X5 antibody ; gp120 in complex with the CCR5 and sCD4 (ref. ); not fully resolved V3 on open trimer in complex with 17b and sCD4 (ref. ); bnD.9-bound V3 on sCD4-triggered open trimer (this work); 10A37-bound peptide ; bnD.2-bound peptide ; bnD.3-bound peptide ; 447-52D-bound peptide ; and 268-D-bound MN peptide .

Article Snippet: Codon-optimized sequences of strain JR-FL gp120 wild-type and the V3 and V1V2 loop deletion mutants , were custom synthesized (GeneArt), fused to a C-terminal AviTag and cloned into the CMV/R expression vector .

Techniques: Cryo-EM Sample Prep, Comparison

Journal: Nature Structural & Molecular Biology

Article Title: Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

doi: 10.1038/s41594-023-01062-z

Figure Lengend Snippet: A) Representative cryo-EM micrograph of bnD.9 in complex with BG505.SOSIP.664 and sCD4. Representative micrograph, of 2813 collected micrographs, is shown. B) Representative 2D class averages of bnD.9 in complex with BG505.SOSIP.664 and sCD4. C) The gold-standard Fourier shell correlation resulted in a resolution of 3.87 Å for the overall map using non-uniform refinement (left panel); the orientations of all particles used in the final refinement are shown as a heatmap (right panel). D) The gold-standard Fourier shell correlation resulted in a resolution of 3.66 Å for the locally refined map obtained using a mask that included bnD.9 and the interacting region of gp120 (left panel); the orientations of all particles used in the final refinement are shown as a heatmap (right panel). E) The local resolution of the final overall map is shown, generated through ResMap. F) Representative density is shown for the interface between bnD.9 and gp120; the contour level is 0.48 (1.2σ). BnD.9 is colored in light blue, gp120 subunit is colored in orange. Electron density of the V3 helix is shown at different contour levels: 0.25 (7.8 s, left), 0.3 (9.4 s, middle), 0.35 (10.9 s, right). The flexible region not visible in the map is represented as a dashed line.

Article Snippet: Codon-optimized sequences of strain JR-FL gp120 wild-type and the V3 and V1V2 loop deletion mutants , were custom synthesized (GeneArt), fused to a C-terminal AviTag and cloned into the CMV/R expression vector .

Techniques: Cryo-EM Sample Prep, Generated

Journal: Nature Structural & Molecular Biology

Article Title: Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

doi: 10.1038/s41594-023-01062-z

Figure Lengend Snippet: A) Entropy of the bnD.9 binding area. Sequence diversity was calculated as normalized entropy based on the 42-virus panel and plotted on one protomer. Dark purple indicates high diversity. Binding residues were defined as residues with non-zero buried surface area (calculated by NACCESS) and the average entropy of the bnD.9 binding area on gp120 calculated as 0.16, the entropy of sCD4 binding area as 0.19. B) Normalized sequence entropy on helical V3 as defined by X-Ray structure of the bnD.9:V3 (BG505) complex across the 42-virus panel colored for diversity as in (A). V3 interface residues that are in 4 Å distance to bnD.9 are shown as sticks. C) Normalized sequence entropy of surfaces on V3 recognized by CCR5 co-receptor, bnDs and antibodies across the 42-cross-clade virus panel (Supplementary Table ) is indicated by a color gradient as in A. V3-binding areas of ligands are encircled in cyan. For structures describing epitopes of antibodies and CCR5 extending beyond V3 the average entropy was calculated for the binding area on V3 only. D) Hydrophobicity of bnD.9 and V3 surfaces. Analysis was performed based on the cryo-EM structure of bnD.9:BG505.SOSIP:sCD4. Hydrophobicity scores are based on the Eisenberg hydrophobicity scale and depicted as a gradient, with hydrophobic residues shown in white. E) Surface charge of DARPin and V3 on gp120. Analysis was performed based on the cryo-EM structure of bnD.9:BG505.SOSIP.664:sCD4. Surface charge was calculated using the APBS electrostatics PyMOL plugin and visualized using a color gradient.

Article Snippet: Codon-optimized sequences of strain JR-FL gp120 wild-type and the V3 and V1V2 loop deletion mutants , were custom synthesized (GeneArt), fused to a C-terminal AviTag and cloned into the CMV/R expression vector .

Techniques: Binding Assay, Sequencing, Virus, Cryo-EM Sample Prep

Journal: Nature Structural & Molecular Biology

Article Title: Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

doi: 10.1038/s41594-023-01062-z

Figure Lengend Snippet: A) Principal component analysis (PCA) of αV3C dynamics. Each point represents a conformation sampled during the MD simulation starting from the bnD.9:BG505.SOSIP.664:sCD4 cryo-EM structure (0µs, red) up to 2μs of simulation (blue) in absence of sCD4. Contour lines depict the frequency of adopted αV3C conformations. Green crosses indicate the centroids defined by a k-means clustering, and the corresponding V3 structures are shown as cartoons. B) Stick representation of the salt bridges stabilizing the helical V3 during the MD simulation. C) Glycan-density (green surface) covering the Env surface during the 2µs MD simulation time on one protomer (gp120 in light grey, gp41 dark grey, V3 dark blue and sCD4 in red). The simulation was done in absence of bnD.9. For display, bnD.9 (orange) was docked to helical V3 of the final frame of the simulation. D)Molecular docking of bnD.8 onto one frame of an accessible conformation of the helical V3 sampled during the MD simulation.

Article Snippet: Codon-optimized sequences of strain JR-FL gp120 wild-type and the V3 and V1V2 loop deletion mutants , were custom synthesized (GeneArt), fused to a C-terminal AviTag and cloned into the CMV/R expression vector .

Techniques: Cryo-EM Sample Prep

Journal: Nature Structural & Molecular Biology

Article Title: Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

doi: 10.1038/s41594-023-01062-z

Figure Lengend Snippet: A) Molecular model showing an IgG antibody engaging with the HIV-1 Env and CD4 complex as seen from the side (top) and from the virus (bottom left). All proteins are represented by their molecular surface, with gp41 colored in yellow, gp120 in orange, CD4 in green, and IgG in blue. The hydrophobic core of the viral and T-cell membranes is shown with pink and light grey sticks, respectively, and the phosphate groups are highlighted with gold spheres. B) Neutralization capacity of size-increased variants of bnD.8 and bnD.9. DARPin-Fc fusion constructs are bivalent constructs of DARPin fused at its C-terminus to the constant region of huIgG1. bnD + α-His Ab are complexes of the bnD with an anti-His antibody bound non-covalently to the DARPin’s N-terminal His-Tag. Bars on the left depict IC50 against the indicated Env pseudotyped virus strains. The dotted line indicates the maximum assay concentration, empty bars indicate that 50% inhibition was not reached at this concentration. Dots on the right depict percent inhibition reached at the maximum concentration of each inhibitor. Values from one of two independent experiments are shown. Inhibitors were titrated from 2.15 μM (bnD.8_trc + α-His Ab), 2.07 μM (bnD.9_opt + α-His Ab), 0.45 μM (bnD.8 + α-His Ab) and 10 μM for all other inhibitors.

Article Snippet: Codon-optimized sequences of strain JR-FL gp120 wild-type and the V3 and V1V2 loop deletion mutants , were custom synthesized (GeneArt), fused to a C-terminal AviTag and cloned into the CMV/R expression vector .

Techniques: Virus, Neutralization, Construct, Concentration Assay, Inhibition

Journal: Nature Structural & Molecular Biology

Article Title: Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

doi: 10.1038/s41594-023-01062-z

Figure Lengend Snippet: a , Mutational antigenic profiling of bnD.8 and bnD.9 resistance. b , Line plots (top panels) depicting results of mutational profiling. Lines indicate the differential selection, which is the logarithm of the relative enrichment of mutations in the BF520 mutant library upon neutralization with bnD.8 (0.56 µM) or bnD.9 (3 µM) averaged across all mutations at each site (Extended Data Fig. ). The V3 region is highlighted by a yellow shaded area. Bottom panels depict V3 profiles as sequence logo plots. The height of the letters is proportional to the differential selection of respective amino acids. Mean differential selection of two independently generated and selected BF520 mutant libraries is shown. c , Escape mutations localize along the DARPin–V3 helix interface. Visualization of highly selected resistance sites defined in b on the X-ray structure of bnD.9 in complex with V3 (BG505) peptide. Zoomed images depict selected residues and their surrounding areas. d , Continuous escape cultures of BF520 mutant library and BF520 wild-type virus on PBMC in the presence of escalating doses of bnD.8 (Extended Data Fig. ). V3 sequence of escape strains isolated aligned to the BF520 wild-type sequence. e , Resistance testing of BF520 mutants defined in b , c and d in the pseudovirus TZM-bl assay against bnD.8 and bnD.9. Data depict means from two independent experiments.

Article Snippet: Codon-optimized sequences of strain JR-FL gp120 wild-type and the V3 and V1V2 loop deletion mutants , were custom synthesized (GeneArt), fused to a C-terminal AviTag and cloned into the CMV/R expression vector .

Techniques: Selection, Mutagenesis, Neutralization, Sequencing, Generated, Virus, Isolation

Journal: Nature Structural & Molecular Biology

Article Title: Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization

doi: 10.1038/s41594-023-01062-z

Figure Lengend Snippet: a , Mutational antigenic profiling of bnD.8 and bnD.9 resistance as outlined in Fig. focusing on regions outside V3. Line plots (left panel) indicate differential selection. Env regions shaded in gray are shown as logo plots in the right panel. Mean differential selection of two independently selected BF520 libraries is shown. b , Comparison of mutational antigenic profiling of bnD.8 and bnD.9 shown in Fig. and a based on site differential selection readout (circles). Sites with differential selection >3.5 shared by bnD.8 and bnD.9 within V3 (blue), C1 (green), HR1 (orange) or other regions (purple) are highlighted. c , Localization of differentially selected sites shared by bnD.8 and bnD.9 (shown in b ) on JR-FL Env (PDB 5FUU). Clusters in V3 (blue), C1 (green) and HR1 (orange) are highlighted. Other sites are shown in purple. d , Functional analysis in the conventional TZM-bl assay of BF520 resistance sites outside V3 detected in mutational scanning ( a – c ) shows heightened sensitivity to bnD.8 and bnD.9. e , Env mutations analyzed in d predominantly increase sensitivity to neutralizing antibodies underlining effects on Env stability and quaternary structure. The ratio of IC 50 values derived in the conventional TZM-bl assay for mutant/wild-type BF520 virus is shown. f , Modified TZM-bl assay recapitulating bnD.8 and bnD.9 resistance patterns defined during deep mutational scanning after harmonization of culture conditions. Removing inhibitors and unbound pseudovirus (BF520 wild-type and mutants) after 3 h co-incubation with cells (analogous to conditions during mutational antigenic profiling) shows decreased sensitivity of mutants to bnD.8 and bnD.9. g , Induction of gp120 shedding from JR-FL Env-pseudotyped virions by bnAb 2F5 (black), the V3 crown-directed bnD.3 (orange), and V3-CD4i bnD versions (blue). Shedding activity is depicted as percent gp120 shedding relative to the mock-treated control, normalized to p24 levels. Concentration of inhibitors was adjusted to 20× above the respective IC 50 . 2F5 was tested in addition at 100 µg ml −1 . Bars depict mean values from two (bnD.9_L15D, bnD.8_trc, bnD.3) or three (all other inhibitors) independent experiments shown as open circles. bnD.8_trc is a more potent variant of bnD.8 that lacks the N cap; bnD.9_L15D is a more potent point mutation variant of bnD.9 (see and Supplementary Fig. ).

Article Snippet: Codon-optimized sequences of strain JR-FL gp120 wild-type and the V3 and V1V2 loop deletion mutants , were custom synthesized (GeneArt), fused to a C-terminal AviTag and cloned into the CMV/R expression vector .

Techniques: Selection, Comparison, Functional Assay, Derivative Assay, Mutagenesis, Virus, Modification, Incubation, Activity Assay, Concentration Assay, Variant Assay

Journal: Frontiers in Immunology

Article Title: Functional Profiling of Antibody Immune Repertoires in Convalescent Zika Virus Disease Patients

doi: 10.3389/fimmu.2021.615102

Figure Lengend Snippet: Repertoire-scale functional data for mAb lineages targeting flavivirus antigens. (A) Computational interrogation of yeast display NGS data after 2 rounds of sorting revealed the functional profile of anti-flavivirus antibodies in convalescent donors. Antibodies were screened against three antigens: ZIKV wild-type VLPs [ZIKV (wt)], ZIKV immature VLPs [ZIKV (pr-mut)], and YFV wild-type VLPs [YFV (wt)]. NGS data from Round 2 was used to calculate enrichment ratio and determine antigen specificity while antibody affinity was predicated based on enrichment in Round 3 affinity gates. Antibody isotype was determined from natively paired heavy:light sequence data. (B) Number of isolated mAb lineages and their antigen reactivity profiles for each donor.

Article Snippet: The cDNA sequence encoding the wild-type prM-E protein sequence of ZIKV “H/PF/2013” strain (Genbank #KJ776791) was codon optimized, synthetized, and cloned into plasmid VRC8400 (VRC/NIAID/NIH, Bethesda, MD, USA) at Genscript (Piscataway, NJ, USA) for expression of ZIKV (wt).

Techniques: Functional Assay, Sequencing, Isolation

Journal: Scientific Reports

Article Title: High mannose-specific lectin Msl mediates key interactions of the vaginal Lactobacillus plantarum isolate CMPG5300

doi: 10.1038/srep37339

Figure Lengend Snippet: Strains and plasmids used in this study.

Article Snippet: WCFS1 , Sequenced wild-type strain; single colony isolate of NCIMB 8826 from human saliva , .

Techniques: Isolation, Mutagenesis, Expressing, Bacteria