postf  (Danaher Inc)

Journal: MedComm

Article Title: Combining Intramuscular and Intranasal Immunization With the MF59‐Adjuvanted Respiratory Syncytial Virus Pre‐Fusion Protein Subunit Vaccine Induces Potent Humoral and Cellular Immune Responses in Mice

doi: 10.1002/mco2.70301

Figure Lengend Snippet: IM immunization with MF59/preF vaccine induced systemic humoral and B‐cell immune responses. (A) The schematic representation of the mouse immunization and sample collection protocol. Mice were immunized intramuscularly with PBS, MF59, preF, MF59/preF‐low, or MF59/preF‐high on Days 0, 21, and 42. Sera were collected on Days 14, 35, and 56, and BALF, spleen, lung, and ILN were harvested on day 72. (B) Endpoint titers of anti‐preF/postF IgG in sera from mice intramuscularly immunized with adjuvanted preF on Days 14, 35, and 56. (C) Titers of virus neutralizing antibody (VNA) against RSV A2 and RSV B in sera collected on Day 56. (D) Endpoint titers of anti‐preF/postF IgG and IgA in BALF collected on Day 72. (E–G) The representative images and quantitative analysis of preF‐specific IgG + ASCs in bone marrow (E), spleen (F), and lung (G). Data are presented as geometric mean values ± SD in B–D. The middle line indicates the median while the whisker shows the data range in E–G. n = 6 mice per group. p values were conducted by One‐way ANOVA analysis followed by Tukey's multiple comparisons test in B, and D–G. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.

Article Snippet: Briefly, 96‐well ELISA plates (NUNC‐MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4°C with either 1 μg/mL of RSV preF or postF protein (Sino Biological, 11049‐V49H5‐B).

Techniques: Virus, Whisker Assay

Journal: MedComm

Article Title: Combining Intramuscular and Intranasal Immunization With the MF59‐Adjuvanted Respiratory Syncytial Virus Pre‐Fusion Protein Subunit Vaccine Induces Potent Humoral and Cellular Immune Responses in Mice

doi: 10.1002/mco2.70301

Figure Lengend Snippet: IN immunization with MF59/preF vaccine induced local humoral and B‐cell immune responses. (A) The schematic representation of the mouse immunization and sample collection protocol. Mice were immunized intranasally with PBS, MF59, preF, MF59/preF‐low, or MF59/preF‐high on Days 0, 21, and 42. Sera were collected on Days 14, 35, and 56, and BALF, spleen, lung, and mLN were harvested on Day 72. (B) Endpoint titers of anti‐preF/postF IgG in sera from mice intranasally immunized with adjuvanted preF on Days 14, 35, and 56. (C) Titers of virus neutralizing antibody (VNA) against RSV A2 and RSV B in sera collected on Day 56. (D) Endpoint titers of anti‐preF/postF IgG and IgA in BALF collected on Day 72. (E–J) The representative images and quantitative analysis of preF‐specific IgG + (left) and IgA + (right) ASCs in bone marrow (E, H), spleen (F, I), and lung (G, J). Data are presented as geometric mean values ± SD in B–D. The middle line indicates the median while the whisker shows the data range in E–J. n = 6 mice per group. p values were conducted by One‐way ANOVA analysis followed by Tukey's multiple comparisons test in B, and D–J. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.

Article Snippet: Briefly, 96‐well ELISA plates (NUNC‐MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4°C with either 1 μg/mL of RSV preF or postF protein (Sino Biological, 11049‐V49H5‐B).

Techniques: Virus, Whisker Assay

Journal: MedComm

Article Title: Combining Intramuscular and Intranasal Immunization With the MF59‐Adjuvanted Respiratory Syncytial Virus Pre‐Fusion Protein Subunit Vaccine Induces Potent Humoral and Cellular Immune Responses in Mice

doi: 10.1002/mco2.70301

Figure Lengend Snippet: Combination of IM and IN immunization using MF59/preF vaccine elicited both local and systemic humoral and B‐cell immune responses. (A–B) The schematic representation of the mouse immunization and sample collection protocol. BALB/c mice received two intramuscular doses of the MF59/preF vaccine followed by a single intranasal dose (IM‐IM‐IN), or one intramuscular dose followed by two intranasal doses (IM‐IN‐IN). Sera were collected on Days 14, 35, and 56, and BALF, spleen, lung, and mLN were harvested on Day 72. (C) Endpoint titers of anti‐preF/postF IgG in sera on Day 56. (D) Titers of virus neutralizing antibody (VNA) against RSV A2 and RSV B in sera collected on Day 56. (E) Endpoint titers of anti‐preF/postF IgG and IgA in BALF collected on Day 72. (F‐K) The representative images and quantitative analysis of preF‐specific IgG + (left) and IgA + (right) ASCs in bone marrow (F, I), spleen (G, J), and lung (H, K). Data are presented as geometric mean values ± SD in C–E. The middle line indicates the median while the whisker shows the data range in F–K. n = 6 mice per group. p values were conducted by One‐way ANOVA analysis followed by Tukey's multiple comparisons test in C–K. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.

Article Snippet: Briefly, 96‐well ELISA plates (NUNC‐MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4°C with either 1 μg/mL of RSV preF or postF protein (Sino Biological, 11049‐V49H5‐B).

Techniques: Virus, Whisker Assay

Journal: MedComm

Article Title: Intranasal Inoculation of Cationic Crosslinked Carbon Dots‐Adjuvanted Respiratory Syncytial Virus F Subunit Vaccine Elicits Mucosal and Systemic Humoral and Cellular Immunity

doi: 10.1002/mco2.70146

Figure Lengend Snippet: Intranasal immunization with the CCD/preF vaccine induced strong antibody responses. (A) A graphical illustration of the immunization schedule. Mice were immunized intranasally with CCD/preF vaccine on days 0, 21, and 42. On days 14, 35, and 56 after the first dose, postF‐ (B) and preF‐specific (C) IgG antibodies in the serum were evaluated with ELISA. (D) Site Ø‐specific IgG antibody titers were measured by D25‐competition ELISA. (E) preF‐specific IgG isotypes in the serum were assessed on day 56, including IgG1, IgG2a, IgG2b, and IgG2c. (F) Neutralization of the RSV A2 and B18537 strains’ infection into HEp‐2 cells by the immune sera on day 56. preF‐ and postF‐specific IgG (G) and IgA (H) titers in the BALF were determined with ELISA on day 72. n = 6. Data were displayed as mean ± SEM. P values were analyzed with One‐way ANOVA and Tukey's multiple comparison test. **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05. ns: not significant.

Article Snippet: preF (cat: 11049‐VNAS), Biotinylated‐preF (cat: 11049‐VNAS‐B), and postF (cat: 11049‐V08B) were obtained from SinoBiological.

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Infection, Comparison

Journal: MedComm

Article Title: Intranasal Inoculation of Cationic Crosslinked Carbon Dots‐Adjuvanted Respiratory Syncytial Virus F Subunit Vaccine Elicits Mucosal and Systemic Humoral and Cellular Immunity

doi: 10.1002/mco2.70146

Figure Lengend Snippet: The long‐term immune responses induced by the intranasal CCD/preF vaccine. In 1 year after the first immunization, mice sera and BALF were collected to evaluate postF‐ and preF‐specific IgG (A, B) and IgA antibodies (C) with ELISA. In 1 year, mice were sacrificed to obtain lung and BM tissues. (D) The frequency of preF‐specific MB cells, plasmablast cells, and plasma cells in the lung were evaluated using FCM. Lymphocytes from the lung and BM were isolated and re‐stimulated with F‐peptide pools for 24 h to assess preF‐specific ASCs that produce IgG and IgA antibodies (E, F) or determine the number of preF‐specific SFCs producing IFN‐γ (G) with ELISpot. Left: images of spot‐forming cells; Right: quantification of spot‐forming cells. (H) The proportion of CD4 + and CD8 + T RMs in the lung was evaluated with FCM. n = 5. Data were displayed as mean ± SD in (A–C). The middle line represents the median while the whisker shows the data range in (D–H). P values were conducted by Unpaired t ‐tests. **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05.

Article Snippet: preF (cat: 11049‐VNAS), Biotinylated‐preF (cat: 11049‐VNAS‐B), and postF (cat: 11049‐V08B) were obtained from SinoBiological.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Enzyme-linked Immunospot, Whisker Assay

Journal: MedComm

Article Title: Intranasal Inoculation of Cationic Crosslinked Carbon Dots‐Adjuvanted Respiratory Syncytial Virus F Subunit Vaccine Elicits Mucosal and Systemic Humoral and Cellular Immunity

doi: 10.1002/mco2.70146

Figure Lengend Snippet: Combining IN and IM immunization with the CCD/preF vaccine enhanced humoral immunity. (A) A graphical illustration of the immunization schedule. (B) A description of the detailed immunization protocol. Mice were vaccinated on days 0, 21, and 42 through three doses of intranasal (3×IN) or heterologous immunization (2×IM+1×IN and 1×IM+2×IN). (C) On day 56, mice sera were collected to determine preF‐ and postF‐specific IgG titers with ELISA. (D‐F) On day 72, mice were sacrificed. BALF was collected to assess endpoint titers of preF‐ and postF‐specific IgG (D) and IgA (E) antibodies with ELISA. (F) The proportion of Tfh cells in the mLN was evaluated with FCM. n = 6. Data were displayed as mean ± SD in (C–E). The middle line represents the median while the whisker shows the data range in f. P values were analyzed with One‐way ANOVA and Tukey's multiple comparison test. *** p < 0.001, ** p < 0.01, and * p < 0.05. ns: not significant.

Article Snippet: preF (cat: 11049‐VNAS), Biotinylated‐preF (cat: 11049‐VNAS‐B), and postF (cat: 11049‐V08B) were obtained from SinoBiological.

Techniques: Enzyme-linked Immunosorbent Assay, Whisker Assay, Comparison

Journal: Nature Communications

Article Title: Structural basis for potent neutralization of human respirovirus type 3 by protective single-domain camelid antibodies

doi: 10.1038/s41467-024-49757-1

Figure Lengend Snippet: a Cartoon representation of the RV3 preF protomer (left) colored according to the linear schematic below, and the trimer (right) with additional protomers shown as gray surface representations. DI–DIII Domains I–III, HRA–HRC Heptad Repeat A–C, FP Fusion peptide. b Three llamas were immunized with recombinant RV3 prefusion F (preF) according to the schedule shown. c Serum titers from the three immunized llamas before (day 0) and after completion of the immunization protocol (day 43), tested for binding to preF or postfusion F (postF). d After two rounds of phage-display panning, a selection of clones was tested by ELISA for binding to purified RV3 preF, postF, and a GCN4 peptide. Results are shown with a color gradient: high levels of binding indicated by yellow and low binding indicated by navy blue. e Off-rate analysis results for a selection of clones that bound preF or both pre- and postF, selected based on the results from d . 39 clones, represented as orange points, were selected for further characterization. Source data are provided within the Source Data file. Figure b created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: Biotinylated RV3 preF or postF were immobilized on a SA sensor chip (GE Healthcare, #BR-1003-98).

Techniques: Recombinant, Binding Assay, Selection, Clone Assay, Enzyme-linked Immunosorbent Assay, Purification

Journal: Nature Communications

Article Title: Structural basis for potent neutralization of human respirovirus type 3 by protective single-domain camelid antibodies

doi: 10.1038/s41467-024-49757-1

Figure Lengend Snippet: a Half-maximal inhibitory concentration (IC 50 ) values for 16 potently neutralizing VHH candidates determined by neutralization assays performed with recombinant RV3-eGFP virus. Points represent geometric means of biological replicates ( n = 2 for 2B03, 3A02, 3E06, 4E07, 5D03, 5E03, 6B02, and 6B03; n = 3 for 1A03, 1H09, 2E10, 2H02, 4C03, and 4C06; n = 4 for 1D10 and 3B04) ±geometric standard deviations. Representative curves are displayed in Supplementary Fig. and numeric IC 50 values are listed in Supplementary Table . b ELISA binding curves for the top 13 VHH candidates. VHHs were tested for binding to RV3 preF (left) and postF (right). Half maximal effective concentration (EC 50 ) values are listed in Supplementary Table . c Competition matrix indicating the level of binding observed for each of the 13 top VHH candidates to preF saturated with the same VHH or a different VHH from the same pool. Columns indicate the competition profile of each VHH. Each epitope bin is outlined based on the representative VHH that was selected for further characterization, colored as in a and b . d Sequence alignment of the representative VHH selected from each epitope bin. VHHs 4C06, 1H09, and 1D10 are aligned to the 4C03 sequence. IMGT-based residue numbers are represented by blue text. Invariant residues are indicated by black dots. Complementarity determining regions (CDRs) are indicated by boxes. e Surface plasmon resonance sensorgrams for binding of each representative VHH to preF. Binding curves are colored black. Data fit to a 1:1 binding model is colored by VHH according to a and b . Source data are provided within the Source Data file.

Article Snippet: Biotinylated RV3 preF or postF were immobilized on a SA sensor chip (GE Healthcare, #BR-1003-98).

Techniques: Concentration Assay, Neutralization, Recombinant, Virus, Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Residue, SPR Assay

Journal: Nature Communications

Article Title: Structural basis for potent neutralization of human respirovirus type 3 by protective single-domain camelid antibodies

doi: 10.1038/s41467-024-49757-1

Figure Lengend Snippet: a Cryo-EM map of 1D10 and 4C06 bound to preF shown in top-down and side views, structure shown in bottom-down view. 1D10 is colored red, 4C06 is colored yellow, and each protomer of preF is colored a shade of blue. b (center) The preF trimer is represented as light colored surface, with each protomer a different shade of blue. The 1D10 epitope is opaque and colored according to domain as in Fig. . DI is colored green and DII is colored dark turquoise. Insets are zoomed-in views of the interface between 1D10 and preF. (left, top) A small interprotomer interface at the central cavity of preF is contacted by CDR3 of 1D10. Oxygen atoms are colored red, nitrogen atoms are colored blue, hydrogen bonds and salt bridges are shown as blue dashes. (left, bottom) Important residues involved at the interface between 1D10 and DI and DII of one protomer, shown as sticks. (right, top) The entire concave surface of 1D10 participates in contacts with DI. 1D10 contact residues here are shown as spheres. (right, bottom) 1D10 forms a large network of hydrogen bonds and salt bridges throughout DI of one protomer. c (left) 1D10 (red) bound to a single preF protomer, shown as cartoons colored by domain. (right) Model of 1D10 bound to a postF protomer (PDBID: 1ZTM). Modeling and calculations were performed in ChimeraX. DI of the preF protomer bound to 1D10 was aligned to DI of the postF protomer. RMSD: 1.6 Å. d Model of 1D10 bound to the postF trimer. Zoomed-in view shows the interface between 1D10 and postF, showing clashes within the DI/DII linker. Clashes are shown as purple dashes, defined by atomic distances of <0.6 Å.

Article Snippet: Biotinylated RV3 preF or postF were immobilized on a SA sensor chip (GE Healthcare, #BR-1003-98).

Techniques: Cryo-EM Sample Prep

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 Analytical SEC trace of wildtype (gray) and stabilized OnlyEcto2P (purple) in supernatant. B SEC-MALS trace of purified OnlyEcto2P. C Melting temperature as determined by DSF of purified OnlyEcto2P of ( B ). The first derivative of the fluorescence signal is plotted. BLI using kinetic Octet with PIA174 immobilized to anti-human Fc sensors (not shown), followed subsequently by capture of purified preF variants (0−900 s phase) and association of sdAb’s 4C06 ( D ) and 4C03 ( E ) (900−1800 s phase). F SEC-MALS trace of purified OnlyEcto. G Melting temperature as determined of OnlyEcto as in ( C ). H Analytical SEC trace of purified OnlyEcto after storage at either 4 °C ( left panel ) or 37 °C ( right panel ) for up to 24 weeks. I Percentage trimer recovery of ( H ). Shown is the average of two replicates. J Expression and melting temperature of OnlyEcto and back-substitutions as measured by analytical SEC and DSF on crude cell supernatant.

Article Snippet: Female cotton rats (Sigmovir Biosystems, Inc., Rockville, MD, USA; 6–8 weeks old) were immunized intramuscularly at week 0 and week 4 with indicated doses of OnlyEcto (preF), with OnlyEcto and postF protein adjuvanted with 50 µl AS01 B (a component of Shingrix, GlaxoSmithKline, London, UK) or with OnlyEcto adjuvanted with 100 µg Alum adjuvant (AlOH, Janssen) or were intranasally exposed to live RV3 (10 4 pfu/animal, VR-93 TM; ATCC) at day 0 after which animals were challenged intranasally at week 7 with 1 × 10 5 pfu per animal of RV3 virus (VR-93 TM; ATCC).

Techniques: Purification, Fluorescence, Expressing

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: Female cotton rats (Sigmovir Biosystems, Inc., Rockville, MD, USA; 6–8 weeks old) were immunized intramuscularly at week 0 and week 4 with indicated doses of OnlyEcto (preF), with OnlyEcto and postF protein adjuvanted with 50 µl AS01 B (a component of Shingrix, GlaxoSmithKline, London, UK) or with OnlyEcto adjuvanted with 100 µg Alum adjuvant (AlOH, Janssen) or were intranasally exposed to live RV3 (10 4 pfu/animal, VR-93 TM; ATCC) at day 0 after which animals were challenged intranasally at week 7 with 1 × 10 5 pfu per animal of RV3 virus (VR-93 TM; ATCC).

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