Journal: Frontiers in immunology

Article Title: Signal peptide exchange alters HIV-1 envelope antigenicity and immunogenicity.

doi: 10.3389/fimmu.2024.1476924

Figure Lengend Snippet: FIGURE 3 Changes in antigenicity of signal peptide (SP) DNA immunogens. (A) Schematic representation of WT and SP swapped gp160 Env. Dashes (-) indicate missing residues. Differences between the SP are shown in bold and are underlined. (B) Mouse muscle cell line C2C12 were transfected with gp160 expressing plasmids. Cells were probed with mAbs, 24 hours post-transfection followed by detecting the ligand binding by flow cytometry. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by 2-way ANOVA, p>0.05 was left unmarked. (C) Summary of the changes in antigenicity of gp160 proteins upon SP swapping. Fold changes (MFI) of SP swapped/WT were calculated for each mAb tested from (B). Significant increases or decreases are marked with blue (fold change of >1 with p<0.05) or red (fold change of <1 with p<0.05), respectively.

Article Snippet: Twenty four hours later, cells in each dish were transfected with 20 mg gp160 expression plasmid (WT or SP swaps) using TransfeXTM Transfection Reagent (ATCC) following the manufacturer’s instructions.

Techniques: Transfection, Expressing, Ligand Binding Assay, Cytometry

Journal: Frontiers in immunology

Article Title: Signal peptide exchange alters HIV-1 envelope antigenicity and immunogenicity.

doi: 10.3389/fimmu.2024.1476924

Figure Lengend Snippet: FIGURE 4 Changes in glycosylation of Env upon signal peptide (SP) swapping. (A) Relative amounts of unoccupied, complex and oligomannose/hybrid glycans at each glycosites on SP-swapped vs WT gp120 proteins as determined by LC-MS/MS. ND: glycosites not detected. (B) WT and SP swapped gp120 proteins were coated onto ELISA plates (2 mg/ml) and probed with serially diluted biotinylated lectins. Representative data are shown depicting the binding of biotinylated lectins specific to mannose, fucose, and sialic acid sugars on the N-linked Env glycans. (C) Mouse muscle cell line C2C12 were transfected with gp160 expressing plasmids. Cells were probed with lectins, 24 hours post-transfection followed by detecting the ligand binding by flow cytometry. Comparisons were made between AA05 versus AC02, AA05 versus AA05-02 and AC02 versus AC02-05. *p < 0.05; ***p < 0.001; ****p < 0.0001 by 2-way ANOVA, p>0.05 was left unmarked. nd, not detected.

Article Snippet: Twenty four hours later, cells in each dish were transfected with 20 mg gp160 expression plasmid (WT or SP swaps) using TransfeXTM Transfection Reagent (ATCC) following the manufacturer’s instructions.

Techniques: Glycoproteomics, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection, Expressing, Ligand Binding Assay, Cytometry

Journal: Frontiers in immunology

Article Title: Signal peptide exchange alters HIV-1 envelope antigenicity and immunogenicity.

doi: 10.3389/fimmu.2024.1476924

Figure Lengend Snippet: FIGURE 7 Antibody responses induced by vaccination with gp120 and gp160 DNA. (A) BALB/c mice were immunized with gp120 and gp160 DNA (1:1) (WT and swapped SP). (B) Serum from individual animals collected 2 weeks after the last immunization was tested with autologous proteins using multiplex Luminex assay. The serum IgG were diluted four-fold starting at 1:100. Pooled serum from unimmunized normal mouse (NMS) served as negative control (Ctrl). Geometric mean fluorescence intensity (MFI) are shown. Cut-off (167.8) shown in red dotted line is defined as the mean value of the MFI of Neg control at highest dilution tested plus 3SD value. (C) Area under titration curve (AUC) values were calculated from Figure 9B. Horizontal line: median; p>0.05 was left unmarked. (D) Cross-reactivity of serum IgG from animals immunized with gp120 and gp160 DNA. Sera from individual mice were tested with SOSIP/gp140/gp120 proteins using multiplex Luminex assay. The serum IgG were diluted four-fold starting at 1:100. Area under titration curve (AUC) values were calculated and are colored as indicated. Cut-off value (497) is defined as the mean value of the AUC of Neg control + 3SD value. (E) The AUC values for each group from (D) were plotted as scatter-plots. Mean + SEM are shown. #p= 0.06, ****p <0.0001 by non-parametric Mann-Whitney test, p>0.05 was left unmarked. x, mice died.

Article Snippet: Twenty four hours later, cells in each dish were transfected with 20 mg gp160 expression plasmid (WT or SP swaps) using TransfeXTM Transfection Reagent (ATCC) following the manufacturer’s instructions.

Techniques: Multiplex Assay, Luminex, Negative Control, Control, Titration, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: Signal peptide exchange alters HIV-1 envelope antigenicity and immunogenicity.

doi: 10.3389/fimmu.2024.1476924

Figure Lengend Snippet: FIGURE 9 Vaccine-Induced antibodies mediate antibody-dependent cellular phagocytosis (ADCP) and virus neutralization. Serially diluted samples from protein, DNA+ Protein, and DNA immunizations were tested for (A) ADCP using phagocytic THP-1 cells and fluorescent beads coated with AA05_02 (upper panel) and REJO gp120 proteins (lower panel). Pooled sera from unimmunized animals was tested in parallel as negative control. Titration curves were plotted for ADCP scores and area under titration curve (AUC) were calculated. AUC values for negative controls were subtracted for both ADCP and IgG binding. ADCP potency of each sera was calculated as the ratio of DADCP/DIgG against AA05_02 and REJO gp120 and shown. (B) Serum samples collected from mice immunized with gp120 and gp160 DNA immunogens were tested for neutralization of Tier 1 (pseudovirus HXB2) and Tier 2 viruses (pseudovirus 398F1, TRO.11, CH119 and RHPA infectious virus) in a standard assay using the TZM.bl target cells. Pooled serum from unimmunized normal mouse served as negative control (Ctrl). Mouse sera neutralization titers are reported as serum dilution required to inhibit 50% of virus infection (ID50). Reciprocal serum ID50 values that can be measured above the cut-off (1:20) are indicated in graded green. x, animal died. **p < 0.01; p>0.05 was left unmarked by ANOVA.

Article Snippet: Twenty four hours later, cells in each dish were transfected with 20 mg gp160 expression plasmid (WT or SP swaps) using TransfeXTM Transfection Reagent (ATCC) following the manufacturer’s instructions.

Techniques: Virus, Neutralization, Negative Control, Titration, Binding Assay, Infection