Journal: Nature Communications

Article Title: Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein

doi: 10.1038/s41467-024-48059-w

Figure Lengend Snippet: A Schematic of the RV3 F ectodomain used to screen for stabilizing substitutions in the head domain. The domain organization is color-coded, with start and end residue numbers indicated. Location of N-linked glycosylation sites are shown. B , C Expression of preF protein variants in supernatant as measured by BioLayer Interferometry (BLI) using immobilized PIA174. The initial slope, V0, at the start of binding is plotted as the average of three independent transfections; shown is the mean with error bars representing the standard deviation (SD). B Individual substitutions were tested and are colored by design feature. The dotted red line indicates the binding rate of the backbone construct RV3 F-GCN4. C Combinations (orange) of promising stabilizing substitutions of ( B ) were tested and compared to the individual substitutions (green and blue).

Article Snippet: BALB/c mice (Charles River Laboratories; female, 8 weeks) were immunized at week 0 and 4 via the intramuscular route with preF (OnlyEcto) or postF proteins with or without 100 μg Alum adjuvant per animal (Adju-Phos®; Invivogen) or formulation buffer or were intranasally exposed to 10 5 pfu RV3 virus (VR-93 TM; ATCC) and intramuscularly immunized 19 weeks later with preF protein.

Techniques: Residue, Glycoproteomics, Expressing, Binding Assay, Transfection, Standard Deviation, Construct

Journal: Nature Communications

Article Title: Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein

doi: 10.1038/s41467-024-48059-w

Figure Lengend Snippet: A Cartoon representation of HRB from PDB ID 6MJZ. The heptad register is shown in sticks, with the serines at positions 470 and 477 highlighted in red. B The RV3 F HRB heptad register indicating the suboptimal residues at positions 470 and 477. C Analytical SEC trace of F variants with a minimally stabilized head domain (Q89M, Q222I and L168P) and with or without HRB stabilization in supernatant. The trimer (T) and monomer (M) peaks are indicated. D Analytical SEC-MALS trace of F variants with a stabilized head domain with or without HRB stabilization in supernatant. The molar mass as determined by MALS at peak max of the trimer are indicated. A cartoon to visualize the presumed opening of HRB is shown. E Stability of indicated F variants using analytical SEC of supernatants after 15 min incubation at 4 °C (blue line), 50 °C (orange line), or 60 °C (red line). The trimer (T) and aggregate (A) peaks are indicated.

Article Snippet: BALB/c mice (Charles River Laboratories; female, 8 weeks) were immunized at week 0 and 4 via the intramuscular route with preF (OnlyEcto) or postF proteins with or without 100 μg Alum adjuvant per animal (Adju-Phos®; Invivogen) or formulation buffer or were intranasally exposed to 10 5 pfu RV3 virus (VR-93 TM; ATCC) and intramuscularly immunized 19 weeks later with preF protein.

Techniques: Incubation

Journal: Nature Communications

Article Title: Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein

doi: 10.1038/s41467-024-48059-w

Figure Lengend Snippet: Cell-cell fusion assay using HEK293T cells transiently transfected with the F protein of the RV3 JS strain, or variants thereof, in a 15:1 ratio to HN carrying a H552Q substitution, to allow fusion activation, and mScarlet to mark the cytosol of transfected cells in red. Cell nuclei were visualized with Hoechst in blue. Syncytia can be recognized by the dilution of the mScarlet signal after fusion with non-transfected cells, and the clustering of nuclei. Scale bar represents 100 μm. Experiment was performed twice with similar outcome, data shown are from the same, representative experiment.

Article Snippet: BALB/c mice (Charles River Laboratories; female, 8 weeks) were immunized at week 0 and 4 via the intramuscular route with preF (OnlyEcto) or postF proteins with or without 100 μg Alum adjuvant per animal (Adju-Phos®; Invivogen) or formulation buffer or were intranasally exposed to 10 5 pfu RV3 virus (VR-93 TM; ATCC) and intramuscularly immunized 19 weeks later with preF protein.

Techniques: Cell-Cell Fusion Assay, Transfection, Activation Assay

Journal: Nature Communications

Article Title: Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein

doi: 10.1038/s41467-024-48059-w

Figure Lengend Snippet: A Sequence alignment of the HRB heptad register of selected F proteins. The a and d positions are highlighted in cyan, with the suboptimal residues at positions 470 and 477 highlighted in red (numbering according to RV3). B Neighbor joining phylogenetic tree of representative paramyxovirus F proteins. C , D Expression of RV1 F protein variants in supernatant as measured by BLI using immobilized 3 × 1 antibody. The initial slope, V0, at the start of binding is plotted as the average of three independent transfections; error bars represent the SD. RV1 F substitutions A44P, E170P, Q171P, S473V, and A480V substitutions are the equivalents of the following substitutions in RV3, respectively: S41P, N167P, L168P, S470V, and S477V. E Analytical SEC trace of wildtype and stabilized (A44P + E170P + Q171P + S473V + A480V) RV1 F in cell supernatant. The trimer (T) peak is indicated. F Analytical SEC-MALS of purified stabilized RV1 preF. Expression of NiV F protein variants in supernatant as measured by BLI using immobilized 5B3 antibody ( G , I , K ), or by analytical SEC ( H , J , L ). For BLI, the initial slope, V0, at the start of binding is plotted as the average of three independent transfections; error bars represent the SD. NiV F substitutions K49P, K167P, S470V, and A477V are the equivalents of the following substitutions in RV3, respectively: S41P, N167P, S470V, and S477V.

Article Snippet: BALB/c mice (Charles River Laboratories; female, 8 weeks) were immunized at week 0 and 4 via the intramuscular route with preF (OnlyEcto) or postF proteins with or without 100 μg Alum adjuvant per animal (Adju-Phos®; Invivogen) or formulation buffer or were intranasally exposed to 10 5 pfu RV3 virus (VR-93 TM; ATCC) and intramuscularly immunized 19 weeks later with preF protein.

Techniques: Sequencing, Expressing, Binding Assay, Transfection, Purification

Journal: Nature Communications

Article Title: Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein

doi: 10.1038/s41467-024-48059-w

Figure Lengend Snippet: A − D Mice ( n = 5 or 8) were immunized with 15 µg adjuvanted preF or postF protein or formulation buffer (Mock) at week 0 and 4. Two weeks later, serum samples were taken and preF ( A ) and postF ( B ) binding antibody titers were measured by ELISA or virus neutralization titers (VNT) in preF, postF or Mock pooled serum samples were determined with an VNA (virus neutralization assay) on Vero cells ( C ) or on differentiated human airway epithelial cell cultures ( D ) using the RV3 JS strain equipped with a GFP reporter gene. E , F Mice ( n = 5 or 8) were immunized with a dose range of 1.5, 5 or 15 µg non-adjuvanted OnlyEcto or formulation buffer (Mock) at week 0 and 4. Two weeks later serum samples were taken and preF binding antibody titers were measured by ELISA ( E ) or RV3 neutralizing antibody titers by RV3-GFP VNA on Vero cells were determined ( F ) . G , H Mice ( n = 3 or 6) were intranasally exposed to RV3 or formulation buffer as control and immunized 19 weeks later with OnlyEcto or formulation buffer. Six weeks later, serum samples were taken and preF binding antibody titers were measured by ELISA ( G ) or RV3 neutralizing antibody titers by RV3-GFP VNA on Vero cells were determined ( H ). Analysis of variance (ANOVA; 2-sided t-test) was used for statistical comparisons between groups. Tukey-Kramer ( A , B and G , H ) or Dunnett adjustments. E , F for multiple comparisons were applied. I − L Cotton rats ( n = 5 or n = 6) were immunized with OnlyEcto with or without adjuvant or postF with adjuvant or formulation buffer (Mock) at week 0 and 4. Three weeks after the final immunization, cotton rats were challenged intranasally with RV3. Four days later viral loads in nose and lung tissue were determined ( I , J ). RV3 neutralizing antibody titers by plaque reduction neutralization test (PRNT) were determined in pre-challenge sera ( K ) and the correlation between nose viral loads and VNA titers were calculated with a spearman correlation analysis ( L ). Mean responses per group are indicated with horizontal lines. Statistical comparisons were performed across dose levels using a Tobit model ( I − L ). P < 0.05 were considered statistically significant.

Article Snippet: BALB/c mice (Charles River Laboratories; female, 8 weeks) were immunized at week 0 and 4 via the intramuscular route with preF (OnlyEcto) or postF proteins with or without 100 μg Alum adjuvant per animal (Adju-Phos®; Invivogen) or formulation buffer or were intranasally exposed to 10 5 pfu RV3 virus (VR-93 TM; ATCC) and intramuscularly immunized 19 weeks later with preF protein.

Techniques: Formulation, Binding Assay, Enzyme-linked Immunosorbent Assay, Virus, Neutralization, Control, Adjuvant, Plaque Reduction Neutralization Test

Journal: Nature Communications

Article Title: Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein

doi: 10.1038/s41467-024-48059-w

Figure Lengend Snippet: A The prefusion structure (OnlyEcto2P) compared with the postfusion structure of RV3 F (PDB ID 1ZTM ). HRA and HRB, including the preceding loop, are colored in blue and purple respectively. DI and DII in the prefusion structure are indicated with an orange outline. B Cross-section indicated in ( A ) for the prefusion (left) and postfusion (right) structures. C-alpha of residue 41 has been indicated as yellow sphere at the corner of the triangle and the distances between the C-alpha’s of the three monomers are indicated below. C Fusion peptide (green) pocket (yellow) for the preF and postF structures were plotted in the context of the full protein (upper panels) and without it (lower panels). D left panel Translation of DI and DII during the conformational change, with the preF structure indicated in orange and postF in cyan. Middle and right panel Aligned on the beta sheet around residue 41, with three residues in green to indicate the rotation of the domain during the refolding. E Comparison of the conformational change of the DI-DIII β-sheet structure and helical bundle (HB) between RV3 F and RSV F, with the preF structure indicated in green and postF in red. The structures were aligned based on the top part of the DI-DIII β-sheet.

Article Snippet: BALB/c mice (Charles River Laboratories; female, 8 weeks) were immunized at week 0 and 4 via the intramuscular route with preF (OnlyEcto) or postF proteins with or without 100 μg Alum adjuvant per animal (Adju-Phos®; Invivogen) or formulation buffer or were intranasally exposed to 10 5 pfu RV3 virus (VR-93 TM; ATCC) and intramuscularly immunized 19 weeks later with preF protein.

Techniques: Residue, Comparison

Journal: Molecular cell

Article Title: Altered m 6 A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection

doi: 10.1016/j.molcel.2019.11.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: n-Dodecyl-β-D-maltoside (DDM) , Chem-Impex , Cat# 21950.

Techniques: Virus, Recombinant, Protease Inhibitor, Purification, SYBR Green Assay, Luciferase, Reporter Assay, Cell Viability Assay, Clone Assay, Transfection, Plasmid Preparation, Infection, Functional Assay, Software