Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

doi: 10.4049/jimmunol.1301898

Figure Lengend Snippet: A) Gelfiltration chromatography analysis of the soluble gp140 trimers from SIVmac239 and HIV-1YU2. Both complexes elute as a single symmetric peak at the expected elution volume for the trimeric species demonstrating that the gp140 trimers produced were pure and homogenous. The chromatography trace for HIVYU2 is representative for the different HIV-1 gp140 used in this study. B) Non-reducing (N/R) and reducing (Red) SDS PAGE analysis of SIVmac239 and HIV-1YU2 gp140. The SIVmac239 gp140 protein runs as a high molecular weight band under non-reducing conditions, indicating that the three polypeptides of the trimer are covalently linked by disulfide bonds between the three gp140 chains. HIV-1YU2 gp140 runs as a smear under non-reducing conditions suggesting that the three polypeptides of the trimer are not covalently linked. C) Surface plasmon resonance analysis of SIVmac239 gp140 and HIV-1 UG37 gp140 binding to human CD4 and anti-HIV-1 NAbs. SIVmac239 (right panel) or HIV-1UG37 (left panel) were immobilized and their binding to tetrameric soluble human CD4 (CD4-IgG2) as well as mAb F105, HGP68 and HR10 was measured.

Article Snippet: Overlapping linear 15-mer and circularized 15-mer peptides based on gp140 of HIV-1 CN54 were tested for reactivity against heat-inactivated rabbit sera by Pepscan Therapeutics (Netherlands).

Techniques: Chromatography, Produced, SDS Page, High Molecular Weight, SPR Assay, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

doi: 10.4049/jimmunol.1301898

Figure Lengend Snippet: A) Schematic immunization schedules of two independent studies (n= 48) and a comparative group 5 (n=4) with arrows depicting immunizations. Sera were collected before immunization (wk 0). Animals (New Zealand white rabbits) received a mix of three clade B env-DNA plasmids at weeks 0 (twice first week), 4, 8 and 12 (200 μg DNA by i.d. electroporation). Sera were collected 4 weeks after last DNA immunization (wk 16). Animals then received a heterologous protein SIVmac239 gp140 by i.d. injection and sera were collected 4 weeks after first protein boost (50 μg)(wk 20). Animals received a second protein boost with SIVmac239 gp140 4 weeks after the first protein immunization and sera were collected 2 weeks after second protein boost (50 μg) (wk 22). Three months later sera were analyzed for memory responses (wk 34). In the first study, twelve animals were included in Gr. 1 and the second study entailed six animals in this group. In the first study, Gr. 2 (n=12) received AAC adjuvant during the DNA priming phase and in the second study Gr. 2 (n=6) received a TLR5 agonist pFliC. Gr. 3 (n=6) received control DNA and albumin protein and Gr. 4 received three immunizations with SIVmac239 gp140 protein by i.d. injection (n=6). Gr.5 received the mix of three clade B env DNA immunizations followed by one i.d. injection with HIV-1ITM-1_4 gp140 (n=4). (B) Endpoint titres (log10) of IgG binding to SIVmac239 gp140 (white bars) or heterologous HIV-1UG37 gp140 (black bars) as measured by ELISA analyzing sera obtained before immunizations (wk 0) and after immunizations at wk 22 from the first study. Data are depicted as median and range. (C) Endpoint IgG titres from the second study are shown for binding to heterologous HIV-1UG37 gp140 and SIVmac239 gp140, as marked, in sera obtained at wk 0 (black bars), after the DNA priming (grey bars), and after the first gp140 boost (white bars) from the second study. Statistically significant lower endpoint IgG titres against SIVmac239 were found in Gr. 3 and against HIV-1UG37 in Groups 3 and 4 when compared with Groups 1 and 2 (One-way ANOVA using Bonferroni’s Multiple Comparison Test p<0.05).

Article Snippet: Overlapping linear 15-mer and circularized 15-mer peptides based on gp140 of HIV-1 CN54 were tested for reactivity against heat-inactivated rabbit sera by Pepscan Therapeutics (Netherlands).

Techniques: Electroporation, Injection, Adjuvant, Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

doi: 10.4049/jimmunol.1301898

Figure Lengend Snippet: Data are shown for individual animals from groups 1 and 2 combined (n=24) (A–H). Lines represent median. Sera were collected before immunization (wk 0), 4 weeks after last DNA immunization (DNA), 4 weeks after first SIVmac239gp140 boost (DNA/gp140) and 2 weeks after the second SIVmac239gp140 boost (DNA/gp140/gp140). Three months later sera were analyzed for the longevity of responses (3 mo after last boost). High ID50 neutralizing titres were detected in a TZM-bl assay against tier 1 isolates from clade B SF162.LS (A) and MN.3 (B). Purified IgGs were tested in a TZM-bl assay against HIV-1 clade B SF162 and BX08 (C and D). Percent neutralization is shown using 130 μg/ml IgG final concentration approximately corresponding to a 1/80 serum dilution and dotted line indicates ID50. Neutralizing titres in sera were detected in a TZM-bl assay against tier 1 isolates from clade C (MW965.26, E) and CRF01 AE (TH023.6, F). An arbitrary color code indicate the animals with the highest titres against SF162.LS in red (n=8), lowest in blue (n=8) and those in between in yellow (n=8). These colors are kept throughout the figure to be able to track the animals and their respective response to other viruses. ID50 titres in sera from groups 1 and 2 were tested in the A3R5.7 assay against clade B viruses RHPA.LucR (G) and SC22.3C2.LucR (H). Neutralization titres obtained in the TZM-bl assay for the control groups that received control DNA and albumin (Gr.3, displayed in green) or only SIVmac239 protein without a DNA prime (Gr.4, displayed in blue) are shown for neutralization of HIV-1 MN.3 (I) and the lab-adapted sensitive TCLA-SIVmac251 isolate (J). Significant values are indicated by *** p<0.001, **p<0.01 and *p<0.05 using One-Way ANOVA with Multiple Comparison Test.

Article Snippet: Overlapping linear 15-mer and circularized 15-mer peptides based on gp140 of HIV-1 CN54 were tested for reactivity against heat-inactivated rabbit sera by Pepscan Therapeutics (Netherlands).

Techniques: Purification, Neutralization, Concentration Assay, Control, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

doi: 10.4049/jimmunol.1301898

Figure Lengend Snippet: (A, B). A comparative arm that received DNA clade B priming followed by the HIV-1 clade A ITM-1_4 gp140 boost (Gr.5), was tested against SF162.LS, MN.3, MW965 and TH023.6 with ID50 titres displayed in brown. (C) Magnitude-Breadth curves show pooled data from both TZM-bl and A3R5.7 assays for the viruses that were tested on all animals (HIV-1 clade B isolates MN.3, SF162.LS, 9020.A13.LucR., RHPA.LucR., SC22.3C2.LucR.). Data obtained after DNA prime and after one protein boost are shown for sera from groups 1 and 2 (left panel) as well as the HIV-1 comparative arm (group5) (right panel). A Mantel-Cox log-rank test was used for statistical comparison. **** p<0.0001; ns= not significant. (D–G) Sera were collected in another comparative study before immunization (wk 0), 4 weeks after last DNA immunization (DNA), 4 weeks after first SIVmac239gp140 boost (DNA/SIV) and 2 weeks after the second gp140 protein boost ((DNA/SIV/HIV-1) or (DNA/SIV/HIV-2)). An arbitrary color code indicate the animals with the highest titres against SF162.LS in red (n=2), lowest in blue (n=2) and those in between in yellow (n=2). These colors are kept throughout the figure to be able to track the animals and their respective response to other viruses. Significant values are indicated by *** p<0.001, **p<0.01 and *p<0.05 as well as non-significant (ns) using One-Way ANOVA, Friedman’s test with Dunns’s Multiple Comparison Test.

Article Snippet: Overlapping linear 15-mer and circularized 15-mer peptides based on gp140 of HIV-1 CN54 were tested for reactivity against heat-inactivated rabbit sera by Pepscan Therapeutics (Netherlands).

Techniques: Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

doi: 10.4049/jimmunol.1301898

Figure Lengend Snippet: (A) Selected sera (n=2 per group from the first study) from time points after HIV-1 DNA-prime (wk 16, DNA) and after the first SIVmac239 protein boost (wk 20, DNA/gp140) were subjected to epitope mapping by pepscan peptide arrays using heterologous HIV-1CN54 peptides. (B) The change in response intensity after SIVmac239 protein boosting is shown for peptides that were undetectable after DNA immunizations (white) or showed positive responses after DNA priming (grey) (C) Percentage of positive peptides detected before (wk 16, DNA) and after the boost (wk20, DNA/gp140) in the peptide array. (D) ELISA binding titres to indicated peptides from HIV-1 or SIVmac239 were determined after the second protein boost (wk 22) in animals immunized without (Gr. 1; n=6) or with a cellular adjuvant AAC during the priming phase (Gr. 2; n=6) (mean+SEM). Significant values are indicated by *p<0.05, **p<0.01 and ****p<0.0001 as well as ns=not significant using a Mann-Whitney test (B), a paired t test (C), or a one-way ANOVA of log-transformed data with a Bonferroni’s Multiple Comparison Test (D).

Article Snippet: Overlapping linear 15-mer and circularized 15-mer peptides based on gp140 of HIV-1 CN54 were tested for reactivity against heat-inactivated rabbit sera by Pepscan Therapeutics (Netherlands).

Techniques: Peptide Microarray, Enzyme-linked Immunosorbent Assay, Binding Assay, Adjuvant, MANN-WHITNEY, Transformation Assay, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

doi: 10.4049/jimmunol.1301898

Figure Lengend Snippet: IgG from animals from the first study were assessed for their capacity to neutralize HIV-1SF162 in the TZM-bl assay in the presence of blocking peptides and representative individual results are displayed. IgG were purified from sera two weeks after the second SIVmac239 gp140 boost. Percent neutralization is depicted on the y-axis and concentration of purified IgG on the x-axis.

Article Snippet: Overlapping linear 15-mer and circularized 15-mer peptides based on gp140 of HIV-1 CN54 were tested for reactivity against heat-inactivated rabbit sera by Pepscan Therapeutics (Netherlands).

Techniques: Blocking Assay, Purification, Neutralization, Concentration Assay