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1.
Figure 5

Figure 5. Alterations of mAb reactivity to V3, C2 and V2 epitopes by formation of gp120/654 immune complexes. From: Improving immunogenicity of HIV-1 envelope gp120 by glycan removal and immune complex formation.

Wild type (wt) or mutant gp120s, either alone or as complexes with mAb 654, were captured on ELISA plates with Ab to the C terminus of gp120, and reacted with biotinylated mAbs specific for V3 (694) (A), C2 (1006) (B) and V2 (697) (C). Binding of the biotinylated mAbs were determined using alkaline phosphatase-conjugated streptavidin. Means and standard deviation from duplicate wells are presented. Data from one of three repeated experiments are shown. The bars in each set represent ten-fold serial dilutions of each of the biotinylated mAbs tested. *, p <0.05. **, p<0.01. ***, p<0.001. by Student's t test.

Rajnish Kumar, et al. Vaccine. ;29(48):9064-9074.
2.
Figure 4

Figure 4. Neutralizing activity of sera from mice immunized with wild type (wt) or mutant gp120/654 complexes. From: Improving immunogenicity of HIV-1 envelope gp120 by glycan removal and immune complex formation.

(A) Sera collected after the final immunization were serially diluted and tested for neutralizing activity in the single-round infection assay with TZM-bl cells against pseudovirus with HIV-1 HXB-2 Env. Similar results were obtained with the infectious LAI isolate (see panel C). (B) To detect heterologous neutralization, sera were also tested for neutralization against pseudovirus expressing HIV-1 SF162 Env. (C) To measure the contribution of anti-V3 Abs to neutralization, serially diluted sera were pre-incubated with V3HXB-2 peptide and tested for neutralization assay against LAI. A scrambled control peptide was also tested and caused no change in neutralization (not shown). (D) To evaluate the contribution of Abs against C1 and C2 to neutralization, sera from N448Q gp120/654-immunized mice were pre-incubated with C1/C2 peptides and tested for neutralization assay against LAI. Representative results from one of two experiments are shown.

Rajnish Kumar, et al. Vaccine. ;29(48):9064-9074.
3.

Figure 6. Susceptibility of V3 and C2 epitopes on gp120/654 complexes versus uncomplexed gp120 to cathepsins L, S, and D. From: Improving immunogenicity of HIV-1 envelope gp120 by glycan removal and immune complex formation.

Wild type (wt) and mutant gp120, either alone or in complex with mAb 654, were treated individually with cathepsins L, S or D for 1 hr, captured onto the microtiter plates with anti-C terminal Abs, and reacted with biotinylated mAbs specific for V3 (694) (left panel) or C2 (1006) (right panel). The binding of biotinylated mAbs were then determined by alkaline phosphatase-conjugated streptavidin. Means and standard deviation were calculated from duplicate wells. Results in (A) show the remaining mAb reactivity after digestion as compare to the respective undigested controls, which were normalized to 100%. **, p<0.01; ***, p<0.001 by Student's t-test. Examples of data showing the C2 reactivity (OD405) to wild type (wt) or N448Q gp120, as uncomplexed antigens or in immune complexes, with or without cathepsin L digestion are shown in (B).

Rajnish Kumar, et al. Vaccine. ;29(48):9064-9074.
4.
Figure 1

Figure 1. Ab responses induced in C57BL/6 mice immunized with wild type (wt) or N448Q gp120s by DNA prime-protein boost vaccination. From: Improving immunogenicity of HIV-1 envelope gp120 by glycan removal and immune complex formation.

(A) Animals received DNA prime and protein boost of the same gp120 antigens (wt or N448Q mutant). Sera from the immunized mice were collected two weeks after the last immunization and pooled, diluted serially, and tested for Ab reactivity to gp120 in ELISA. Alkaline phosphatase-conjugated secondary Abs against total IgG, IgG1, IgG2a, IgG3, and IgA were used. Data showing Ig reactivity to wild type gp120 are presented, but comparable results were obtained with N448Q gp120 (not shown). (B) Sera from immunized mice were tested for reactivity to V3 peptide in ELISA. Secondary Abs against mouse total IgG were used. (C) Sera were tested for neutralization activity in the single-round infection assay with TZM-bl target cells and pseudovirus with homologous HIV-1 HXB-2 Env. Means and standard deviations were derived from duplicate wells. Results from one of two experiments are shown.

Rajnish Kumar, et al. Vaccine. ;29(48):9064-9074.
5.

Figure 3. Reactivity of serum Abs from mice immunized with wild type (wt) gp120/654 complex or mutant gp120/654 complexes. From: Improving immunogenicity of HIV-1 envelope gp120 by glycan removal and immune complex formation.

(A) gp120, V3 peptide, gp120 core, gp120 core+V3, and gp120 outer domain (OD1) were coated on ELISA plates and reacted with serially diluted sera. Sera were collected from mice immunized with gp120/654 complexes in MPL/DDA adjuvant two weeks after the last boost and pooled. Serum IgG reactivity was detected using alkaline phosphatase-conjugated anti-mouse IgG as a secondary Ab. Sera from animals injected with PBS in the presence of MPL/DDA adjuvant were used as control. (B) Epitope mapping was done using overlapping peptides representing the entire gp120 HXB-2 sequence. Sera from animals immunized with the complexes were reacted (at a 1:200 dilution) with pools of peptides (see Material and Methods) coated on ELISA plates at 10 μg/ml. Reactivity of control sera from animals receiving no antigen (PBS) was also shown. Mean and SD values are shown. *, p<0.01. **, p<0.001. compared to wt gp120/654 and calculated by Student's t test.

Rajnish Kumar, et al. Vaccine. ;29(48):9064-9074.
6.

Figure 2. T cell responses of C57BL/6 mice immunized with wild type (wt) or N448Q gp120 by DNA prime-protein boost vaccination. From: Improving immunogenicity of HIV-1 envelope gp120 by glycan removal and immune complex formation.

Splenocytes collected two weeks after the last immunization were pooled and tested for proliferation and cytokine production. Lymphoproliferation and secretion of IFN-γ, IL-10 and IL-6 were measured after in vitro stimulation for 72 hours with wild type gp120 (1 μg/ml) (A) and peptides 40, 41 or 18 (1 μg/ml each) (B). Proliferation was measured by [3H] thymidine uptake. The results are expressed as delta counts per minute (Δ cpm), which were calculated by subtracting cpm of antigen-stimulated cells with background cpm from cells treated with medium only. The amounts of cytokines in the supernatant were measured by multiplex analyte detection. The background responses of cells in medium alone, which were below detection thresholds for most cytokines, were subtracted from the antigen-specific responses. Mean and SD values from triplicate wells are shown. The results are from one of two repeated experiments. *, p<0.05 by Student's t-test.

Rajnish Kumar, et al. Vaccine. ;29(48):9064-9074.

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