Journal: Cell reports

Article Title: Boost immunizations with NA-derived peptide conjugates achieve induction of NA inhibition antibodies and heterologous influenza protections.

doi: 10.1016/j.celrep.2023.112766

Figure Lengend Snippet: Figure 3. Titration of rtN1- and rtN2-spe- cific serum IgG in ELISA The sera samples collected 2 weeks after (A and D) prime immunization, (B and E) first booster, and (C and F) second booster were titrated for (A–C) rtN1 and (D–F) rtN2. Each dot represents the serum antibody titer from one mouse (n = 5 per group). Bars represent the mean values of serum antibody titers. The dash line represents the limit of detection, corresponding to the initial serum dilution. The positive wells were determined as OD values above mean +2 3 SD of the OD value of the initial pre-immune sera dilution. Statistical signifi- cance was analyzed by t test for (B), (C), (E), and (F). *p < 0.05, **p < 0.01, and ****p < 0.001. All experiments were repeated twice using the same serum samples and showed similar results.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-63His tag Sino Biological Cat#105327-MM02T-H Rabbit polyclonal anti-N1 Sino Biological Cat#11058-R001 Rabbit polyclonal anti-N2 Sino Biological Cat#40017-RP01 HRP-conjugated goat anti-human IgG Proteintech Group Cat#SA00001-17 HRP-conjugated goat anti-mouse IgG Proteintech Group Cat#SA00001-1 HRP-conjugated goat anti-mouse IgG1 Proteintech Group Cat#SA00012-1 HRP-conjugated goat anti-mouse IgG2a Proteintech Group Cat#SA00012-2 HRP-conjugated goat anti-mouse IgG3 Proteintech Group Cat#SA00012-5 HRP-conjugated goat anti-mouse IgM Proteintech Group Cat#SA00012-6 HRP-conjugated peanut agglutinin Sigma-Aldrich Cat#L7759 Bacterial and virus strains E.coli DH5a competent cells Lab stock N/A E.coli DH10Bac competent cells Lab stock N/A A/Hong Kong/8/1968 (H3N2) ATCC Cat#VR544TM A/New Jersey/8/1976 (H1N1) ATCC Cat#VR897TM A/Puerto Rico/8/1934 (H1N1) Haiyan Chang, Hunan Normal University N/A A/reassortant/NYMC X179A (H1N1) Haiyan Chang, Hunan Normal University N/A A/Guizhou/54/1989 (GZ89, H3N2) Haiyan Chang, Hunan Normal University N/A A/Puerto Rico/8/1934 (H1N1) Haiyan Chang, Hunan Normal University N/A Biological samples Human convalescent sera samples Yao-Qing Chen’s laboratory stock N/A Chemicals, peptides, and recombinant proteins N1P1 Top-peptide N/A N1P2 Top-peptide N/A N1P3 Top-peptide N/A N1P4 Top-peptide N/A N2P1 Top-peptide N/A N2P2 Top-peptide N/A N2P3 Top-peptide N/A N2P4 Top-peptide N/A rtN1 This paper N/A rtN2 This paper N/A KLH Solarbio Cat#K8160 Receptor destroying enzyme Denka Seiken Cat#340122 Fetuin from fetal bovine serum Sigma-Aldrich Cat#F2379 Incomplete Freund’s adjuvant Sigma-Aldrich Cat#F5506 Critical commercial assays ReadiLinkTM KLH Conjugation Kit AAT Bioquest Cat#5502 Mouse IFNg ELISPOT Kit BD Biosciences Cat#551083 Mouse IL-4 ELISPOT Kit Dakewe Cat#DKW22-2040-500 (Continued on next page) Cell Reports 42, 112766, July 25, 2023 11

Techniques: Titration, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (Clone # 535919) (R&D systems, FAB9332R), AF-488 mouse anti-human ACE2 (Clone # 171607) (R&D systems, FAB9333G) (0.4 μg/10 6 cells), AF-647 isotype control mouse IgG2b (Clone # 20102) (R&D systems, IC003R) or AF-488 isotype control mouse IgG2bAF488 (Clone # 20102) (R&D systems, IC003G) for 45 min on ice, followed by washing twice with 2% EV-free FBS/PBS.

Techniques: Clinical Proteomics, Negative Control, Expressing, Control, Over Expression, Derivative Assay, Western Blot, Membrane, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation