Journal: Cell reports

Article Title: CD62L expression marks a functionally distinct subset of memory B cells

doi: 10.1016/j.celrep.2023.113542

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Influenza A H1N1(PR8) Nucleoprotein (11675-V08B) was purchased from Sino Biological and conjugated using the APC Conjugation Kit-Lightning Link (Ab201807) from Abcam according to manufacturer’s instructions.

Techniques: Generated, Virus, Recombinant, Cell Isolation, Conjugation Assay, TA Cloning, Software

Journal: Nature Communications

Article Title: Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria

doi: 10.1038/s41467-024-46321-9

Figure Lengend Snippet: a The solved co-crystal structure of truncated recombinant human FcRn (green) in complex with IgG1 Fc (blue). The REW amino acid substitutions (Q311R/M428E/N434W) in the Fc are indicated. The Fc residues H435 and H310, required for pH dependent binding, as well as the FcRn residues E115, E116, and L135 are also shown. The N297-linked N-glycan structure attached to the IgG1 Fc is shown in red and the β2-microglobulin subunit of FcRn is shown in yellow. The figure was made in PyMOL using crystallographic data from PDB entry 4NOU . b Illustration showing the human FcRn binding ELISA setup. c , d ELISA showing binding of NIP IgG1 WT, REW, and H310A to human FcRn at pH 6.0 and 7.4, shown as mean±s.d of duplicates. e Illustration showing the human FcRn SPR kinetics assay. f , g SPR sensorgrams showing binding of monomeric human FcRn to immobilized anti-NIP IgG1 WT or REW at pH 6.0. h Illustration outlining the HERA cellular assay. HERA showing ( i ) uptake, ( j ) rescue from intracellular degradation (recycling), ( k ) residual amounts and ( l ) the derived HERA scores for the antibodies, shown as mean±s.d of triplicates from two independent experiments. m Illustration outlining the in vivo plasma half-life experiments. Plasma clearance of ( n ) anti-NIP and ( o ) anti-SARS-CoV-2 mAb4 IgG1-WT and REW (5 mg/kg) in mice pre-loaded with 500 mg/kg IVIg, shown as mean±s.d of percent antibody remaining in plasma over time ( n = 5 animals per group). i – l Unpaired two-sided t -test, ( n , o ) RM Two-way ANOVA with Šídák’s multiple comparison test. Source data are provided as a Source Data file. b , e , h , m were created using BioRender.com.

Article Snippet: 96-well EIA/RIA plates were coated with recombinant HA (H1N1 A/PR8) protein (1 μg/mL/100 μL per well) (Sino Biological).

Techniques: Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, In Vivo, Comparison

Journal: Nature Communications

Article Title: Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria

doi: 10.1038/s41467-024-46321-9

Figure Lengend Snippet: a Illustration outlining the in vivo lung localization experiment in Tg32 mice. b Amount of NIP IgG1 WT and REW in BALF 23 days post i.v. administration of 5 mg/kg of the antibody variants in IVIg (500 mg/kg) pre-loaded mice. Shown as mean ± s.e.m ( n = 4 animals (WT) and n = 5 animals (REW). c BALF/plasma ratio of NIP IgG1 WT and REW, 23 days post i.v. administration. Shown as mean ± s.d ( n = 4 animals per group). d Illustration outlining i.n. delivery experiment in Tg32 mice. e Plasma concentration of NIP IgG1 WT and REW 24 h post i.n. delivery (2.23 mg/kg) in Tg32 mice. Shown as mean ± s.d. ( n = 4 animals (WT) or n = 6 animals (REW)). f Illustration outlining FcRn mediated transcytosis experiments in a transwell system. g Apical to basolateral (A→B) and basolateral to apical (B→A) directed transport of NIP IgG1 WT, REW and IHH across polarized human T84 cells, shown as mean ± s.d ( n = 8 independent monolayers from 2 independent experiments) (two data points was excluded from REW A→B and one data point excluded for REW (B→A) due to disrupted cell monolayers). h Apical to basolateral (A→B) and basolateral to apical (B→A) directed transport of NIP IgG1 WT, REW and IHH across MDCK-hFcRn cells, show as mean ± s.d ( n = 6 independent cellular monolayers from 2 independent experiments) (one data point were excluded from REW A→B and REW (B→A) due to disrupted cell monolayers). i Illustration outlining mucosal vaccine and challenge experiment in Tg32 mice. j Percent survival of Tg32 mice i.n. vaccinated with HA WT and REW monovalent Fc fusions or NaCl control following challenge with a lethal dose of H1N1 virus. k Illustration showing the ex vivo human placental perfusion model used to measure maternal-to-fetal transport of anti-NIP IgG1 WT and REW. l Ex vivo human placental perfusion model showing maternal/fetal (FM) transport ratio of NIP IgG1 WT and REW, shown as mean ± s.d. ( n = 4 placentas per group). b , c , e , g , h Unpaired two-tailed t -test, ( l ) Two-tailed Wilcoxon t -test. Source data are provided as a Source Data file. ( a , d , f , I , k ) were created using BioRender.com.

Article Snippet: 96-well EIA/RIA plates were coated with recombinant HA (H1N1 A/PR8) protein (1 μg/mL/100 μL per well) (Sino Biological).

Techniques: In Vivo, Concentration Assay, Virus, Ex Vivo, Two Tailed Test

Journal: Nature Communications

Article Title: Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria

doi: 10.1038/s41467-024-46321-9

Figure Lengend Snippet: a Structural model of hexameric IgG with a close-up of the Fc:Fc interface. The REW amino acid substitutions are shown. The model was made in PyMOL using crystallographic data from PDB entry 1HZH , . b Illustration showing the human C1q binding ELISA setup. c ELISA binding of C1q from NHS to titrated amounts of antigen captured anti-NIP IgG1 WT, REW and PGLALA, shown as mean ± s.d of duplicates. d Illustration showing the C5bC9 deposition ELISA setup. e ELISA showing C5bC9 (TCC) formation from NHS as a function of titrated amounts of antigen captured anti-NIP IgG1 WT, REW and PGLALA, shown as mean±s.d of duplicates. f ELISA binding of C1q from NHS to titrated amounts of randomly immobilized anti-NIP IgG1 WT, REW, RGY and PGLALA, shown as mean ± s.d of duplicates. g Illustration outlining solution phase complement activation assays. h Amount of IgG complexes and ( i ) C4d in NHS incubated with 100 µg/mL of anti-NIP WT, REW, RGY and PGLALA or NHS only at 37 °C for 1 h, shown as mean ± s.d of duplicates. j Overview of anti-CD20 antibodies and CD20+ cell lines used in CDC assays and ( k ) Calcein-AM release assay used to measure antibody dependent CDC of cancer cells. l – n CDC activity of anti-CD20 mAb2 (low CDC), mAb1 (intermediate CDC), mAb9 (high CDC) WT and REW against WSU-NHL (low CD20), DOHH-2 (intermediate CD20) and SU-DHL-4 (high CD20) lymphoma target cells, shown as mean±s.d of duplicates. o CDC activity of recombinant forms of ofatumumab (anti-CD20, IgG1) WT, REW and P329A against Raji target cells, shown as mean ± s.d of duplicates. Source data are provided as a Source Data file. a , c , g were created using BioRender.com.

Article Snippet: 96-well EIA/RIA plates were coated with recombinant HA (H1N1 A/PR8) protein (1 μg/mL/100 μL per well) (Sino Biological).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Incubation, Release Assay, Activity Assay, Recombinant

Journal: Nature Communications

Article Title: Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria

doi: 10.1038/s41467-024-46321-9

Figure Lengend Snippet: a Illustration of ELISA setup and binding of anti-NIP IgG1 WT, REW and low-fucose (2FF) WT and REW to ( b ) FcγRIIIa-V158 and ( c ) FcγRIIIa-F158, shown as mean ± s.d of duplicates. d Overview of anti-CD20 IgG1 mAb2 variants and B cell lines used to measure ADCC activity. e The 51 Cr release assay used to measure ADCC activity of MNCs against target cell lines in presence of the antibodies. f – h MNC mediated ADCC of WSU-NHL (low CD20), Carnaval (low CD20) and SU-DHL-4 (high CD20) lymphoma cell lines by the WT mAb2 compared with the Fc-engineered versions; REW, REW-2FF and PGLALA, shown as mean±s.e.m. from 5 replicates performed in parallel. i Experimental outline and ( j ) plasma clearance of anti-NIP IgG1 WT and REW-2FF in IVIg pre-loaded (500 mg/kg) Tg32 mice, shown as mean±s.d of percent antibody remaining in plasma over time ( n = 5 animals per group). k Illustration showing REW-engineered IgG subclasses and a recombinant form of Fc-fusion etanercept. l Cellular FcRn mediated rescue from intracellular degradation of WT and REW engineered IgG subclasses and Fc-fusion etanercept in HERA, shown as mean±s.d of triplicates from three independent experiments. m Plasma clearance of anti-NIP IgG2 WT and REW following i.v. administration (5 mg/kg) in IVIg pre-loaded (500 mg/kg) Tg32 mice, shown as mean ± s.d of percent antibody remaining in plasma over time ( n = 4 animals per group). n Plasma clearance of recombinant etanercept WT and REW following i.v. administration (5 mg/kg) in Tg32 mice, shown as mean ± s.d of percent antibody remaining in plasma over time ( n = 5 animals per group). o ELISA showing binding of human C1q to titrated amounts of antigen captured anti-NIP IgG2 WT and REW, shown as mean±s.d of duplicates. p CDC activity of mAb2 anti-CD20 IgG2 WT and REW against WSU-NHL (low CD20) using the 51 Cr release assay, shown as mean±s.d of duplicates. q ELISA showing binding of human C1q to titrated amounts of antigen captured anti-NIP IgG4 WT and REW, shown as mean±s.d of duplicates. r , s ELISAs showing relative binding of FcyRIIIa-V158 or FcyRIIIa-F158 to antigen captured anti-NIP IgG2 and IgG4 WT and REW, shown as relative binding compared to anti-NIP IgG1-WT, mean ± s.d of duplicates. f , g , h , j , m , n RM Two-way ANOVA with Šídák’s multiple comparison test. l Unpaired two-tailed t -test. Source data are provided as a Source Data file. a , d , e , I , k were created using BioRender.com.

Article Snippet: 96-well EIA/RIA plates were coated with recombinant HA (H1N1 A/PR8) protein (1 μg/mL/100 μL per well) (Sino Biological).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Release Assay, Recombinant, Comparison, Two Tailed Test

Journal: Viruses

Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

doi: 10.3390/v11100928

Figure Lengend Snippet: Effect of temperature on the polymerase activity of PR8/AA and PR8/Len. ( A ) Schematic representation of segments 1 (PB2), 2 (PB1) and 8 (NS) of PR8/WT (white, left), PR8/AA (black, middle) and PR8/Len (grey, right). Amino acid substitutions to generate PR8/AA and PR8/Len are indicated. B-C) Minigenome activity: HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of ambisense pDZ expression plasmids encoding the minimal requirements for viral genome replication and gene transcription (PB2, PB1, PA, and NP) together with 500 ng of a vRNA-like expression plasmid encoding GFP ( B ) or Gluc ( C ) under the control of the human polymerase I promoter (hPol-I-GFP and -Gluc, respectively), and 100 ng of an SV40 Cluc expressing plasmid to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and viral replication and transcription was evaluated 24 h later by GFP ( B ) and luciferase ( C ) expression. Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that obtained in the absence of pDZ NP. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P < 0.05; **, P < 0.01; NS, no statistical differences (Student t test). Scale bars, 200 μm.

Article Snippet: Briefly, 96-well plates were coated with cell lysates from mock-, PR8/WT- and X31-infected MDCK cells or with 250 ng/well of recombinant PR8 HA protein (NR-19240; BEI Resources) and incubated ON at 4 °C.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Luciferase

Journal: Viruses

Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

doi: 10.3390/v11100928

Figure Lengend Snippet: Contribution of PB2 and PB1 mutations of PR8/AA and PR8/Len in the viral polymerase activity at different temperatures. HEK293T cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were transiently co-transfected with 250 ng of the indicated combinations of ambisense pDZ expression plasmids encoding the PB2 and PB1 from PR8/WT, PR8/AA and PR8/Len together with the pDZ encoding PA and NP PR8/WT proteins; together with 500 ng of the hPol-I-GFP ( A ) and -Gluc ( B ), and 100 ng of SV40 Cluc to normalize transfection efficiencies. After transfection, cells were placed at 33 °C, 37 °C, or 39 °C, and reporter gene expression was evaluated 24 h later by GFP imaging ( A ) and Gluc expression ( B ). Gluc activity was normalized to that of Cluc. Data represent means and SDs of the triplicates. Normalized reporter expression is relative to that in the absence of pDZ NP plasmid. Data were represented as relative activity considering the activity of each polymerase complex at 33 °C as 100%. *, P < 0.05; **, P < 0.01; NS, no statistical differences (Student t test). Scale bars, 200 μm.

Article Snippet: Briefly, 96-well plates were coated with cell lysates from mock-, PR8/WT- and X31-infected MDCK cells or with 250 ng/well of recombinant PR8 HA protein (NR-19240; BEI Resources) and incubated ON at 4 °C.

Techniques: Activity Assay, Transfection, Expressing, Gene Expression, Imaging, Plasmid Preparation

Journal: Viruses

Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

doi: 10.3390/v11100928

Figure Lengend Snippet: In vitro characterization of PR8/AA and PR8/Len viruses. ( A ) Multicycle growth kinetics: MDCK cells (12-well plate format, 4 × 10 5 cells/well, triplicates) were infected at low MOI (0.001) with PR8/WT (white bars), PR8/AA (black bars) and PR8/Len (grey bars) viruses and placed at 33 °C, 37 °C, or 39 °C. Tissue culture supernatants were recovered at the indicated times post-infection (p.i.) and viral titers were determined by immunofocus assay (FFU/mL) using an anti-NP mAb (HB-65). Data represent the means and SDs of the results determined from triplicate wells. Dotted black lines indicate the limit of detection (200 FFU/mL). ( B ) Plaque assays: MDCK cells (6-well plate format, 1 × 10 6 cells/well) were infected with PR8/WT, PR8/AA or PR8/Len viruses and incubated at 33 °C, 37 °C, or 39 °C for 3 days. Plaque phenotype was assessed by immunostaining with the HB-65 anti-NP mAb.

Article Snippet: Briefly, 96-well plates were coated with cell lysates from mock-, PR8/WT- and X31-infected MDCK cells or with 250 ng/well of recombinant PR8 HA protein (NR-19240; BEI Resources) and incubated ON at 4 °C.

Techniques: In Vitro, Infection, Incubation, Immunostaining

Journal: Viruses

Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

doi: 10.3390/v11100928

Figure Lengend Snippet: Attenuation of PR8/AA and PR8/Len viruses in vivo. Six-to-8-week-old female C57BL/6 mice ( n = 5) were infected intranasally (i.n.) with the indicated FFU of PR8/AA ( A , B ) or PR8/Len ( C , D ) viruses and then monitored daily for 2 weeks for body weight loss ( A , C ) and survival ( B , D ). Mice that lost 25% of their initial body weight were sacrificed. Data represent the means and SDs of the results determined for individual mice ( n = 5). The viral 50% mouse lethal dose (MLD ) for PR8/AA and PR8/Len were calculated based on survival data using the method of Reed and Muench . ( A , C ) Statistical analysis was performed using one-way ANOVA test comparing % body weight loss between groups (* P < 0.0001). ( B , D ) Statistical analysis was performed using log-rank (Mantel-Cox) test (* P < 0.0001).

Article Snippet: Briefly, 96-well plates were coated with cell lysates from mock-, PR8/WT- and X31-infected MDCK cells or with 250 ng/well of recombinant PR8 HA protein (NR-19240; BEI Resources) and incubated ON at 4 °C.

Techniques: In Vivo, Infection

Journal: Viruses

Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

doi: 10.3390/v11100928

Figure Lengend Snippet: Replication of PR8/AA and PR8/Len viruses in mice and induction of humoral immune responses. ( A ) Lung viral titers: Six-to-eight-week-old female C57BL/6 mice ( n = 6) were infected i.n. with 10 3 and 10 4 FFU of PR8/Len and 10 3 FFU of PR8/AA viruses. Viral titer in lungs of infected mice was evaluated at days 2 and 4 p.i. ( n = 3) by immunofocus assay (FFU/mL) in MDCK at 33 °C using an anti-NP mAb (HB-65). Data represent the means and SDs of the results determined from each group of mice ( n = 3). Dotted black line represents the limit of detection of the assay (200 FFU/mL). NS, no statistical differences (Student t test). ( B – D ) Humoral immune responses: Six-to-eight-week-old female C57BL/6 mice ( n = 5) were infected i.n. with 10 3 and 10 4 FFU of PR8/Len and 10 3 FFU of PR8/AA viruses, or mock-infected (PBS). At 14 days p.i., mice were bled and sera were collected and evaluated individually by HAI assay for hemagglutination inhibiting antibodies ( B ) and by ELISA for IgG Abs against total influenza viral proteins using cell extracts of MDCK cells infected with PR8/WT ( C ) or against recombinant purified PR8 HA protein ( D ). ( B ) HAI titers: ND, not detected. &, p < 0.05 (PR8/Len 10 3 FFU vs PR8/AA 10 4 FFU) (Student t test); NS, no statistical differences (Student t test). ( C , D ) ELISAs: OD, optical density. Data represent the means and SDs of the results for five individual mice. *, p < 0.001 (PR8/Len 10 3 FFU vs.PR8/AA 10 3 FFU); **, p < 0.05 (PR8/Len 10 4 FFU vs.PR8/AA 10 3 FFU); ***, p < 0.01 (PR8/Len 10 3 FFU vs.PR8/AA 10 3 FFU) (Student t test).

Article Snippet: Briefly, 96-well plates were coated with cell lysates from mock-, PR8/WT- and X31-infected MDCK cells or with 250 ng/well of recombinant PR8 HA protein (NR-19240; BEI Resources) and incubated ON at 4 °C.

Techniques: Infection, HAI Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Purification

Journal: Viruses

Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

doi: 10.3390/v11100928

Figure Lengend Snippet: Induction of CD8 T cell responses by PR8/AA and PR8/Len viruses: Female six-to-eight-week-old C57BL/6 mice ( n = 5) were infected (i.n.) with 10 3 and 10 4 FFU of PR8/Len and 10 3 FFU of PR8/AA viruses. Mice were also mock (PBS) infected as an internal control. Ten days p.i., lungs ( A – C ) and spleens ( D – F ) were collected and resident cells were prepared for flow cytometry. Live CD8 T cells specific for NP ( B , E ) or PA ( C , F ) tetramers were counted. Data represent the means +/− SDs of the results for 5 individual mice. *, p < 0.001 for the indicated pair wise comparisons (Student t test).

Article Snippet: Briefly, 96-well plates were coated with cell lysates from mock-, PR8/WT- and X31-infected MDCK cells or with 250 ng/well of recombinant PR8 HA protein (NR-19240; BEI Resources) and incubated ON at 4 °C.

Techniques: Infection, Control, Flow Cytometry

Journal: Viruses

Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

doi: 10.3390/v11100928

Figure Lengend Snippet: Protection efficacy of PR8/AA and PR8/Len against homologous viral challenge: Six-to-eight-week-old female C57BL/6 mice ( n = 5) were infected i.n. with 10 3 and 10 4 FFU of PR8/Len and 10 3 FFU of PR8/AA viruses, or mock vaccinated (PBS). At 15 days p.i., mice were challenged i.n. with 1,000 MLD 50 of PR8/WT and monitored daily over 10 days for body weight loss ( A ) and survival ( B ). Mice that lost 25% of their initial body weight were sacrificed. Data represent the means and SDs of the results determined for individual mice ( n = 5). Statistical analysis was performed using one-way ANOVA ( A ) and Dunnett’s ( B ) tests comparing the PBS group to the other groups (* P < 0.0001).

Article Snippet: Briefly, 96-well plates were coated with cell lysates from mock-, PR8/WT- and X31-infected MDCK cells or with 250 ng/well of recombinant PR8 HA protein (NR-19240; BEI Resources) and incubated ON at 4 °C.

Techniques: Infection

Journal: Viruses

Article Title: Comparative Study of the Temperature Sensitive, Cold Adapted and Attenuated Mutations Present in the Master Donor Viruses of the Two Commercial Human Live Attenuated Influenza Vaccines

doi: 10.3390/v11100928

Figure Lengend Snippet: Protection efficacy induced by PR8/AA and PR8/Len against heterologous virus challenge. Six-to-eight-week-old female C57BL/6 mice ( n = 5) were infected i.n. with 10 3 and 10 4 FFU of PR8/Len or 10 3 FFU of PR8/AA viruses, or mock-infected (PBS). At 15 days p.i., mice were challenged i.n. with 50 MLD 50 of X-31 and then monitored daily over 10 days for body weight loss ( A ) and survival ( B ). Mice that lost 25% of their initial body weight were sacrificed. Data represent the means and SDs of the results determined for individual mice ( n = 5). Statistical analysis was performed using one-way ANOVA ( A ) and Dunnett’s ( B ) tests comparing the PBS group to the other groups (* P < 0.0001).

Article Snippet: Briefly, 96-well plates were coated with cell lysates from mock-, PR8/WT- and X31-infected MDCK cells or with 250 ng/well of recombinant PR8 HA protein (NR-19240; BEI Resources) and incubated ON at 4 °C.

Techniques: Virus, Infection

Journal: Frontiers in Immunology

Article Title: Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages

doi: 10.3389/fimmu.2023.919800

Figure Lengend Snippet: SP-A inhibits IAV infection independent of its glycosylation. RAW264.7 cells were cultured overnight at a density of 2 x 10 5 cells/well in 24-well plates and infected with either IAV PR8 (A-F) or IAV Phil82 (F) . Cells were incubated with indicated concentration of virus in 1:1 (v/v) PBS/DMEM and then allowed to proceed in DMEM/10% FBS. At the endpoint of the following experiments cells were harvested, stained with NP antibodies, and analyzed by flow cytometry. (A) SP-A inhibits IAV infection in a concentration-dependent manner. RAW264.7 were pretreated with increasing concentration of 5, 10, 25 and 50 μg/mL SP-A for 5 hours and then infected with IAV PR8 at MOI=2 in the presence of respective concentration of SP-A. The percentage of infected cells was determined by flow cytometry 10-hous after infection. N=4, t-test, **p < 0.05. (B) IAV overcomes SP-A inhibition at high MOI. RAW264.7 cells were incubated in the presence or absence of 50 μg/mL of SP-A for 5 hours, then infected with IAV PR8 at MOI 1, 2, and 5 in 1:1 PBS/DMEM in the presence or absence of 50 μg/mL SP-A and infection was assessed by flow cytometry. N=6, t-test, **p < 0.05 (C) Either pretreatment of macrophages or IAV with SP-A inhibits infection. RAW264.7 cells were incubated with 50 μg/mL SP-A in indicated combinations before or during infection. N=2 independent experiments per condition. (D) De-glycosylation of SP-A was accomplished using EndoF and confirmed by visualization of molecular weight reduction of monomeric and oligomeric forms of SP-A on Western blots using a polyclonal SP-A antibody. (E) The inhibitory effect of SP-A does depend on its glycoconjugate moiety. De-glycosylation enhances the inhibitory effect SP-A on IAV infection. RAW264.7 cells were treated with either native of de-glycosylated (ΔCHO) SP-A and infected with IAV at MOI=2 and infection assessed at 6 and 12 hrs. after infection. N=4, t-test, *p < 0.05; **p < 0.01, NS, not significant. (F) SP-A delays temporal infection with both H1N1 and H3N2 strains of IAV. RAW264.7 were infected with either IAV H1N1 strain PR8 or H3N2 strain Phil82 following a 5-hour treatment with no or either 10 or 50 μg/mL SP-A. Infection was then assessed by flow cytometry at 3, 12, or 24 hours after infection. Representative histograms of n=2 independent experiments per treatment are shown.

Article Snippet: We determined the binding kinetics of the SP-A with the H1 Hemagglutinin (HA) Recombinant A/Puerto Rico/8/1934 (H1N1) HA (PR8) expressed in insect cells from a baculovirus vector with a carboxy-terminal hexa-histidine tag was obtained from BEI resources (cat no NR-19240) ( ).

Techniques: Infection, Glycoproteomics, Cell Culture, Incubation, Concentration Assay, Virus, Staining, Flow Cytometry, Inhibition, Molecular Weight, Western Blot

Journal: Frontiers in Immunology

Article Title: Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages

doi: 10.3389/fimmu.2023.919800

Figure Lengend Snippet: SP-A binds HA in a calcium and glycosylation independent manner. SP-A binding to HA was carried out in static solid phase assays (A-C) and under flow using surface plasmon resonance (SPR) (D-F) . For static assays, recombinant PR8 HA (1 μg/well) was adsorbed onto 96-well Immobilon-2 plates in 0.05 M bicarbonate buffer (pH 9.6) overnight at 4°C. Coated plates were incubated with albumin control or SP-A at room temperature for 1 hour. Plates were washed in binding buffer and incubated with a polyclonal SP-A antibody. Bound SP-A was visualized colorimetrically using HRP-conjugated anti-rabbit antibody and tetramethylbenzidine. Albumin was used as non-specific control. (A) SP-A binding to HA does not depend on the presence of calcium. HA and albumin control coated plates were washed and incubated with increasing concentration of 10, 25, 50, and 100 μg/mL of SP-A in the presence or absence of 5 mM EDTA. N=4 of 2 independent experiments. (B) De-glycosylation (ΔCHO) of HA does not prevent SP-A binding. Plates coated with ΔCHO recombinant HA, or albumin were washed, and then incubated with increasing concentration of 10, 50, and 100 μg/mL of SP-A. N=2. (C) De-glycosylation (ΔCHO) of SP-A does not prevent binding to HA. Recombinant HA or albumin control coated plates were incubated with increasing concentration of 10, 50, 75, and 100 μg/mL of SP-A or ΔCHO SP-A. N=4 of 2 independent experiments. (D-F) Calcium does not impact kinetic and binding parameters of SP-A binding to HA. For SPR assays, recombinant HA with a carboxy-terminal hexa-histidine tag was immobilized onto Nicoya nitrilotriacetic acid (NTA) sensor chips. Increasing concentration of 10, 20, 40, or 80 μg/mL of SP-A analyte in the presence (D) or absence (E) of 0.5 mM CaCl 2 was injected at 20 μL/min for 300 sec to obtain on-rates and switched to buffer without SP-A for an additional 600 sec to obtain off-rates. Binding sensograms were acquired using a 2-channel Nicoya OpenSPR instrument. Curves were fitted according to 1:1 interaction model to obtain kinetic and binding parameters (F) . Data shown are representative of N=3 independent experiments.

Article Snippet: We determined the binding kinetics of the SP-A with the H1 Hemagglutinin (HA) Recombinant A/Puerto Rico/8/1934 (H1N1) HA (PR8) expressed in insect cells from a baculovirus vector with a carboxy-terminal hexa-histidine tag was obtained from BEI resources (cat no NR-19240) ( ).

Techniques: Glycoproteomics, Binding Assay, SPR Assay, Recombinant, Incubation, Control, Concentration Assay, Injection

Journal: Frontiers in Immunology

Article Title: Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages

doi: 10.3389/fimmu.2023.919800

Figure Lengend Snippet: Differential reduction in uptake of endocytic cargo and binding of IAV PR8 to cell membrane in the presence of SP-A. Endocytic and binding experiments were performed using RAW264.7 cells (A-E) or isolated RAW264.7 membrane (F) . Raw264.7 cells were incubated with Dextran 10,000 or transferrin conjugated with either the pH sensitive dye pHRodo (A, B) or FITC (C, D) in 1:1 DMEM in the presence or absence of SP-A, for 15 minutes at room temperature. The media were replaced with dye-free 1:1 DMEM and cells incubated with PHRodo- and FITC-labeled molecules were chased at 37°C for 10, 20, 30, and 45-minutes and 5, 10, 15, 30, and 45-minutes at 37°C to monitor rates of endosomal acidification and uptake, respectively. (A, B) SP-A does not alter acidification rate for either Dextran (A) or transferrin (B) tracked endosomes, N=6. (C) SP-A reduced level of FITC-Dextran over the first 5-minutes, whereas SP-A appeared to delay uptake of FITC-transferrin between 5-20-minutes reaching a similar plateau after 30-minutes. Differences were significant for FITC-Dextran only. N=6, *p<0.05. (D) and (E) SP-A suppresses uptake rate of Alexa Fluor488-labeled PR8. RAW264.7 cells were incubated with IAV with SP-A throughout infection or pre-treatment only. Cells were infected with Alexa Fluor488-labeled PR8 at MOI=15. The uptake of the labeled virus was monitored for 5, 15, 30, and 45-minutes at 37°C by flow cytometry. N=6 per condition per time point, **p < 0.005 and ****p < 0.001 for vehicle vs SP-A throughout; ++, p < 0.005 and +++, p<0.0005 for vehicle vs SP-A pretreatment; ## , p < 0.05 for SP-A throughout vs SP-A. (F) To assess differences in membrane binding, immulon-2 flat bottom plates were coated overnight at 4°C with 12.5 μg/ml of Raw264.7 cell membranes and then washed and incubated with PBS, 5 x 10 6 ffc of PR8, or 50 μg/ml SP-A for 2-hours at room temperature. The membranes were then washed, and incubated for an additional 2-hours with PR8, pre-formed PR8+SP-A complex, or SP-A to evaluate PR8 binding in a combination of conditions shown on the x-axis on panel F graph. Plates were washed and bound PR8 visualized spectrophotometrically using a polyclonal anti-HA antibody and HRP-conjugated anti-rabbit antibody as described in Materials and Methods. N=8, ****p < 0.00001, ***p < 0.001. Statistical differences were assessed by 2-way ANOVA.

Article Snippet: We determined the binding kinetics of the SP-A with the H1 Hemagglutinin (HA) Recombinant A/Puerto Rico/8/1934 (H1N1) HA (PR8) expressed in insect cells from a baculovirus vector with a carboxy-terminal hexa-histidine tag was obtained from BEI resources (cat no NR-19240) ( ).

Techniques: Binding Assay, Membrane, Isolation, Incubation, Labeling, Infection, Virus, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages

doi: 10.3389/fimmu.2023.919800

Figure Lengend Snippet: Disruption of SP-A improves recovery and clearance of IAV infection in mice. Sftpa +/+ (n=14), Sftpa -/- (n=31), Csf2 -/- (n=9) and Sftpa -/- , Csf2 -/- (n=27) mice were infected with 1000 ffc (1 LD50) of IAV PR8 via the intranasal route. Body weight and survival were monitored daily. Total RNA was extracted from whole lung to measure viral RNA and IFNβ. (A) Lack of SP-A in Sftpa -/- mice enhances body weight recovery of Sftpa -/- mice compared to Sftpa +/+ , Csf2 -/- and Sftpa -/- ,Csf2 -/- mice (*p < 0.05, Sftpa -/- vs. Sftpa +/+ days 9-14, ***p < 0.001 Sftpa -/- vs. Csf2 -/- and Sftpa -/- ,Csf2 -/- mice), reduces body weight loss and mortality in the absence of GM-CSF (*p<0.05 Sftpa -/- vs. Sftpa +/+ and ***p<0.001, Csf2 -/- vs. Sftpa -/- , Csf2 -/- ) (A, B) , enhances IAV clearance from the lung (***p < 0.001 Sftpa -/- vs. Sftpa +/+ on day 7, # p < 0.05 Sftpa -/- day 7 vs. day 3 (C) and enhances expression of IFNβ as measured on day 3 after infection, n=6 mice, *p < 0.05 (D) . The histopathology (E) and histopathology scores (F, G) Sftpa -/- and Sftpa +/+ consisting of perivascular cuffing (yellow arrows) and interstitial inflammation 14 days after infection were similar. Representative hematology and eosin stained tissue sections for Sftpa +/+ (upper panel) and Sftpa -/- (lower panel) lungs in (E) were captured at 20x magnification. For (F, G) n=6 mice per group. Percent lung affected (F) and inflammation severity (G) scores shown are the mean observation from 5 microscopic fields from each lobe per mouse.

Article Snippet: We determined the binding kinetics of the SP-A with the H1 Hemagglutinin (HA) Recombinant A/Puerto Rico/8/1934 (H1N1) HA (PR8) expressed in insect cells from a baculovirus vector with a carboxy-terminal hexa-histidine tag was obtained from BEI resources (cat no NR-19240) ( ).

Techniques: Disruption, Infection, Expressing, Histopathology, Staining

Journal: STAR Protocols

Article Title: Protocol for building an in vivo model for observation and evaluation of neutrophil extracellular trapping nets in influenza virus-infected mice

doi: 10.1016/j.xpro.2024.103436

Figure Lengend Snippet:

Article Snippet: Influenza A virus strain A/PR/8/34 (H1N1) , ATCC, USA , Cat#VR-95.

Techniques: Control, Virus, Recombinant, Staining, Lysis, Bicinchoninic Acid Protein Assay, Marker, Software, Flow Cytometry, Laser-Scanning Microscopy