Journal: International Journal of Molecular Sciences

Article Title: Design and Characterization of a Peptide Mimotope of the HIV-1 gp120 Bridging Sheet

doi: 10.3390/ijms13055674

Figure Lengend Snippet: Antigenic and immunogenic characterization of GST-BS1 fusion protein. ( A ) Cloning strategy to link the four Bridging Sheet peptide mimotopes to the COOH-terminus of GST protein. The four Bridging Sheet peptide mimotopes (blue box) were cloned as cDNA into the Multiple Cloning Site (MCS) (green box) of pGEX-4T3 expression plasmid (black boxes) to be linked to the COOH-terminus of GST protein (red box). Bam HI and Sal I cloning sites are shown. GST-BS fusion proteins were purified as described in [ , ]. ( B ) Antigenicity of four GST-BS fusion proteins. ELISA assays were performed to analyze the reactivity of the four Bridging Sheet peptides as fusion proteins against IgGs purified from LTNP and AIDS patient sera. IgGs purified from a HIV-1 negative donor were used as a control. Results are reported as HIV + IgGs/HIV-IgGs ratio (fold increase) and a fold increase ≥3 was a positive result. The graph reports the mean of five independent experiments and the error bars. According to the Student’s t test result obtained by IgGs purified from LTNPs was statistically significant with a p < 0.05. ( C ) Mice antibody titers against GST-BS1 fusion protein. Bridging Sheet specific antibody titers were analyzed by performing a direct ELISA assay. The graph shows the results from three independent experiments and the antibody titers obtained immunizing the mouse 7 (black triangle) were statistically significant, by Student’s t test, with a p < 0.01. The data are reported as GST-BS1 immunized mice/GST immunized mouse (full black rhomb) ratio (fold increase) and sera dilutions with a fold increase ≥3 were positive. Error bars are indicated. The values of sera dilutions on the horizontal axis have to be incremented ×10 3 as indicated.

Article Snippet: As A→V and Y→H in β21 are essential for the co-receptor binding, we reproduced two other mimotopes BS1 and BS2 with unmodified β21 strand sequence ( ).

Techniques: Cloning, Clone Assay, Expressing, Plasmid Preparation, Purification, Enzyme-linked Immunosorbent Assay, Control, Direct ELISA

Journal: International Journal of Molecular Sciences

Article Title: Design and Characterization of a Peptide Mimotope of the HIV-1 gp120 Bridging Sheet

doi: 10.3390/ijms13055674

Figure Lengend Snippet: Antigenicity of E2-BS1. ( A ) Schematic representation of G. stearothermophylus E2 protein. E2 protein (di-hydro-lypoil-acetyl-transferase) is the central scaffold of the PDH complex. The peptide is directly bound to the core domain of E2 multi-subunit complex without altering the E2 structure. The complex includes about 20 trimeric subunits and each subunit display the BS1 mimotope. ( B ) Western blot analysis to detect E2-BS1 expression. Immunoblot of E2 wt and E2-BS1 recombinant using anti-E2 polyclonal sera (left panel) or anti mouse serum containing antibodies against BS mimotope, obtained from mice immunized by E2-BS1 (right panel). ( C ) Electron microscopy of E2-BS1 complex. Electron micrographs of E2 wild type (left panel) and E2-BS1 recombinant (right panel) stained with uranyl acetate solution. (magnification 160,000×). ( D ) Antigenicity was assessed by performing a direct ELISA assay with increasing concentrations of E2-BS1 protein. IgGs purified from LTNPs sera (marked as L1–L3) and conclamate AIDS sera (marked as A1–A3) were assessed. IgGs purified from a HIV donor serum were used as a control. Results are reported as HIV + sera/HIV serum ratio (fold increase) and a fold increase ≥3 was considered as positive result. The graph reports the mean of three independent experiments and the error bars.

Article Snippet: As A→V and Y→H in β21 are essential for the co-receptor binding, we reproduced two other mimotopes BS1 and BS2 with unmodified β21 strand sequence ( ).

Techniques: Western Blot, Expressing, Recombinant, Electron Microscopy, Staining, Direct ELISA, Purification, Control

Journal: International Journal of Molecular Sciences

Article Title: Design and Characterization of a Peptide Mimotope of the HIV-1 gp120 Bridging Sheet

doi: 10.3390/ijms13055674

Figure Lengend Snippet: End point titration against o- and m-Env of IgGs purified from mice and rabbit sera. Three indirect ELISA assays were performed to assess the cross-reactivity of mice and rabbits sera from animals immunized with E2-BS1 against monomeric (m) and oligomeric (o) gp120. The table reports end-point titers against BS1 mimotope, E2 protein carrier, m-gp120 SF162 and o-gp140ΔV2 SF162 for both mice and rabbits. Statistically significant results for the reactivity against m-gp120 SF162 and o-gp140ΔV2 SF162 are indicated.

Article Snippet: As A→V and Y→H in β21 are essential for the co-receptor binding, we reproduced two other mimotopes BS1 and BS2 with unmodified β21 strand sequence ( ).

Techniques: Titration, Purification, Indirect ELISA, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Design and Characterization of a Peptide Mimotope of the HIV-1 gp120 Bridging Sheet

doi: 10.3390/ijms13055674

Figure Lengend Snippet: Displacement of m-gp120 SF162 binding. A competitive ELISA assay was performed to analyze the specificity of rabbit immune response against m-gp120 SF162. Rabbit serum 5/76 and its relative pre-immune serum as negative control were incubated with the same increasing molar concentration of BS3 peptide mimotope ( A ) and E2-BS1 complex ( B ). Data were reported as the fold increase of rabbit 5/76/pre-immune serum ratio. The statistical significant was evaluated as the difference in binding displacement between BS3 and E2-BS1, and assessed by performing a standard Student’s t test ( p < 0.05). Error bars were indicated.

Article Snippet: As A→V and Y→H in β21 are essential for the co-receptor binding, we reproduced two other mimotopes BS1 and BS2 with unmodified β21 strand sequence ( ).

Techniques: Binding Assay, Competitive ELISA, Negative Control, Incubation, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Design and Characterization of a Peptide Mimotope of the HIV-1 gp120 Bridging Sheet

doi: 10.3390/ijms13055674

Figure Lengend Snippet: IgGs neutralization assay from rabbits immunized with E2-BS1. ( A ) Two sets of neutralization experiments with HIV-1 infected TZM-bl cells were performed to assess the ability of immunized rabbit IgGs to neutralize the infection of two clade B strain viruses: SF162.LS and NL-ADArs, along with one clade C HIV-0012466-2.52 primary isolate. ID50 (dose of virus infecting the 50% of cells) was calculated by using increasing sera dilutions. Dilutions ≥30 were positive for neutralization activity. In all the experiments the rabbit 5/76 showed a mild neutralization titer against all three viruses tested. ( B ) Evaluation of the other immunized rabbits showed no neutralization titer against the three clade B strain viruses: SF162.LS; HIV-1 MN; SIVmac239CS.23. ( p < 0.05).

Article Snippet: As A→V and Y→H in β21 are essential for the co-receptor binding, we reproduced two other mimotopes BS1 and BS2 with unmodified β21 strand sequence ( ).

Techniques: Neutralization, Infection, Virus, Activity Assay