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6 protocols using igg2b

1

IgG Subclass Profiling by ELISA

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The profiles of specific IgG subclasses in antisera were determined by ELISA. Briefly, microtitre plates were coated with Polypeptides or the individual immunodominant epitope 1 μg/well overnight at 4 °C. After blocking with 2% BSA (v/v), Polypeptides or the individual immunodominant epitope-specific antisera were added at a dilution of 1:500 (data at the factor of 1:250, 1:1000 and 1:2000 not shown) and incubated for 1 h at 37 °C. Normal mouse sera (pre-immune sera) served as a negative control. After washing, IgG isotype-specific primary antibodies (goat anti-mouse IgG1, IgG2a, IgG2b and IgG3, purchased from AbD Serotec) were added to the wells at 1:3,000 dilution and incubated for 1 h at 37 °C. After extensive washing, tetramethyl benzidine (TMB) substrate was added for 10 min at room temperature, and the reaction was stopped by addition of 100 μl 2 M sulfuric acid. Endpoint absorbances were read at 450 nm using a microplate reader (Bio-Rad).
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2

Quantifying CII-Specific IgG Levels

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Levels of CII-specific IgG in serum, taken at indicated time points after CII immunization, were determined by ELISA. Low binding plates (NUNC, Fisher Scientific, Gothenburg, Sweden) were coated with rat CII (1 μg/ml). Serum samples were diluted (1/6000, 1/18 000, 1/54 000 and 1/162 000) and after incubation, CII-specific IgG was detected by using biotinylated rat anti-mouse IgG, IgG1, IgG2a or IgG2b at 0.5 μg/ml (Serotec, Oxford, UK). The assay was developed using extravidin-horseradish peroxidase (HRP) and tetramethylbenzidine substrate. The reactions were stopped with H2SO4 and read in Spectra Max 340PC (Molecular Devices) at 450 nm and correction at 650 nm.
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3

Dendritic Cell Phenotype Analysis

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Unless stated otherwise, antibodies for phenotype analysis were purchased from BD Pharmingen (San Diego, CA, USA) and were the following: fluorescein isothiocyanate-conjugated HLA-DR, CD80, IgG2A, CD52 (Bio-Rad, Hercules, CA, USA) and IgG2B (Bio-Rad); phycoerythrin-conjugated CD1a, CD86, IgG1 and CD83 (Beckman Coulter, Brea, CA, USA); phycoerythrin-Cy7- conjugated CD14 (eBioscience, San Diego, CA, USA), ILT-3 (Beckman Coulter) and IgG1 (eBioscience); PercPCy5.5-conjugated CD209 and IgG2B; and allophycocyanin-conjugated IgG1, CD3, CD25, PD-L1 (eBioscience) and CD40 (eBioscience). DCs were incubated with a mix of monoclonal antibodies for 30 min on ice. Cells were washed in fluorescence-activated cell sorting (FACS) buffer containing 1% fetal bovine serum and 0.05% sodium azide (Sigma-Aldrich) and analyzed using FACSCanto or Fortessa (BD Biosciences). Data were analyzed using FACSDiva 8 (BD Biosciences) and FlowJo 10 software (Ashland, Oregon, USA).
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4

Dendritic Cell Phenotype Analysis

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Unless stated otherwise, antibodies for phenotype analysis were purchased from BD Pharmingen (San Diego, CA, USA) and were the following: fluorescein isothiocyanate-conjugated HLA-DR, CD80, IgG2A, CD52 (Bio-Rad, Hercules, CA, USA) and IgG2B (Bio-Rad); phycoerythrin-conjugated CD1a, CD86, IgG1 and CD83 (Beckman Coulter, Brea, CA, USA); phycoerythrin-Cy7- conjugated CD14 (eBioscience, San Diego, CA, USA), ILT-3 (Beckman Coulter) and IgG1 (eBioscience); PercPCy5.5-conjugated CD209 and IgG2B; and allophycocyanin-conjugated IgG1, CD3, CD25, PD-L1 (eBioscience) and CD40 (eBioscience). DCs were incubated with a mix of monoclonal antibodies for 30 min on ice. Cells were washed in fluorescence-activated cell sorting (FACS) buffer containing 1% fetal bovine serum and 0.05% sodium azide (Sigma-Aldrich) and analyzed using FACSCanto or Fortessa (BD Biosciences). Data were analyzed using FACSDiva 8 (BD Biosciences) and FlowJo 10 software (Ashland, Oregon, USA).
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5

Quantifying Thawed LNP-RNA Vaccine Efficacy

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To quantitatively assess the efficacy of thawed LNPs compared with freshly prepared samples using repRNA encoding for the HIV env trimer, we vaccinated healthy Balb/C mice by injecting 1 μg RNA doses of the LNP-RNA i.m. in each of the left and right gastrocnemius muscle. At weeks 2 and 4 post-i.m. injection, the mice underwent retro-orbital bleeding; blood was collected in Z-gel PP tubes for blood serum collection (CAT#41.1500.005; Sarstedt). Serum was collected by centrifuging blood at 10,000 ×g for 4 min, and stored at −80 °C prior to use. To conduct ELISAs, NUNC MaxiSorp plates were coated overnight with 1 μg/mL purified HIV antigen in PBS, then blocked for 2 h with 10% BSA in PBS. Mouse sera were initially diluted 50× in blocking buffer, followed by 3× serial dilutions. Diluted sera were transferred to blocked plates and incubated for 2 h. HRP-conjugated immunoglobulins (e.g., IgG, IgG1, IgG2a, IgG2b, IgG3, IgM; Bio-Rad) were used as detection antibodies at 1:5000 for endpoint titer assessments, with gp120-specific monoclonal antibody VRC01 used as a positive control. 3,3′,5,5’-Tetramethylbenzidine (TMB) signal was read using a microplate reader by subtracting the absorbance at 450 nm by that at 550 nm.
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6

Multicolor Flow Cytometry Immunophenotyping

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Cells were harvested by treatment with 0.05% trypsin-EDTA or by gentle scraping in cold PBS containing 2 mM EDTA (for CD80 and CD86 labeling) and washed twice with FACS buffer. For the labeling of surface markers, the following mouse primary antibodies were used: anti-MHCII (CAT82A, IgG1, Kingfisher), anti-CD11c (BAQ153A, IgM, Biorad), anti-CD209 (209MD26A, IgG2a, Kingfisher), anti-CD11b (hybridome IAH CC104, IgG2b, kindly provided by Dirk Werling), anti-CD172a (CC149 conjugated to RPE-Cy5, Biorad), anti-CD14 (TUK4 conjugated to Alexa Fluor 647), anti-CD80 (IL-A159 conjugated to FITC), and anti-CD86 (IL-A190 conjugated to RPE). The following antimouse secondary antibodies were used when necessary: anti-IgG1 conjugated to Alexa 488 (A211121, Invitrogen), anti-IgM conjugated to PE-Cy7 (406513, Biolegend), anti-IgG2a conjugated to RPE (115-115-206, Jackson ImmunoResearch), and anti-IgG2b conjugated to BV510 (743175, BD Biosciences). After the antibody incubations, dead cells were labeled using the Fixable Viability Dye eFluor 450 (eBioscience). Cells were examined by flow cytometry using a BD LSR Fortessa cytometer and data were analyzed with the Kaluza software (Beckman Coulter). Dead cells were excluded from the analysis and gates were set according to appropriate isotype/control staining (Supplementary Figures 1, 3).
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