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1.
Fig. 2

Fig. 2. Purification and immunoprecipitation analyses of GST-gp41 Fusion Proteins. From: Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope.

GST and three gp41 variants (-30, -64 and -100) were initially expressed and purified. Proteins were analyzed by silver stain (A), Western blot with anti-GST antibody (B), and immunoprecipitation with 2F5 (C) or 4E10 (D) followed by Western blot with anti-GST antibody. (E) Coomassie Blue-stained SDS-PAGE analyses of purified GST-gp41-142 and -172.

Adam Penn-Nicholson, et al. Virology. ;372(2):442-456.
2.
Fig. 3

Fig. 3. Characterization of antigenic properties of GST-gp41 fusion proteins. From: Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope.

Purified GST and GST-gp41 fusion proteins were analyzed by ELISA using mAbs 2F5 (A), 4E10 (B), 98-6 (C) and polyclonal HIV-Ig (D and E). Equimolar amounts (0.5 pmoles) of GST or GST-gp41 fusion proteins were coated in each well. To detect three smaller fusion proteins by HIV-Ig, higher concentrations of the antibody were used with extended enzymatic reaction time in panel (E).

Adam Penn-Nicholson, et al. Virology. ;372(2):442-456.
3.
Fig. 1

Fig. 1. Construction and expression of GST-gp41 fusion proteins. From: Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope.

(A) A schematic diagram of gp41 and the five GST-gp41 fusion proteins generated. Key features of gp41 are indicated. Linear B cell epitopes identified to date are shown above gp41, and 2F5 and 4E10 binding sites are indicated. FP = Fusion Peptide; HR = Heptad Repeat; ID = Immunodominant region; TM = Transmembrane domain. (B and C) SDS-PAGE analyses of GST-gp41 fusion protein expression in uninduced (U) and induced (I) E.coli cells. Arrows indicate GST-gp41 fusion proteins. Gels were stained with Coomassie Blue. (D) Sequence of MCON6 gp41 ectodomain with 6x-His tag. The starting residues of each construct are indicated by inverted triangle.

Adam Penn-Nicholson, et al. Virology. ;372(2):442-456.
4.
Fig 5

Fig 5. Neutralizing activity of patient plasma samples. From: Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope.

HIV-1 Env pseudotyped viruses (BAL, AD8, DH12 and 89.6) were used to assess neutralizing activity in a TZM-bl cell-based assay. Data is shown as a percentage of virus infectivity in the absence of plasma. (A) Twelve patients identified as having either high or low reactivity to GST-gp41-30 were analyzed at a single plasma dilution factor of 1:90. MAb b12 (12.5 μg/ml) and uninfected patient (HIV-ve) were used as controls. None of the plasma samples neutralized VSV-G pseudotyped virus (data not shown). (B) Titration analyses of neutralizing activity of plasma samples from six patients who exhibited strong reactivity to GST-gp41-30.

Adam Penn-Nicholson, et al. Virology. ;372(2):442-456.
5.
Fig. 4

Fig. 4. Antibody responses against gp41 in individual patients. From: Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope.

(A) ELISA was performed with plasma samples from HIV-1-infected patients at a dilution factor of 1:300. Plasma from an uninfected individual (HIV-ve) and no plasma were used as controls. Plasma samples are arranged in descending order of reactivity to GST-gp41-30. Six samples exhibiting the highest reactivity (•) and six samples with lower reactivity (*), which are further evaluated in Figs. 5 and 6, are indicated. Patients who have been infected longer than 5 years with CD4 counts >500 and never been on anti-retroviral therapy are indicated by black bars on the top. For clarity, average A450 value of GST only is shown as a line indicated as GST. Arrowheads indicate average A450 values against each fusion protein for all patients. Equimolar amounts (0.5 pmoles) of GST or GST-gp41 fusion proteins were coated in each well. (B) Dot plot analyses of ELISA data showing distribution of antibody reactivity against each fusion protein. Median values are shown.

Adam Penn-Nicholson, et al. Virology. ;372(2):442-456.
6.
Fig 7

Fig 7. Identification of immunogenic linear epitopes in gp41 by ELISA using M group consensus peptides. From: Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope.

Immunoreactivity of plasma samples from 43 patients were analyzed against seven peptides from cluster I region (a.a. 580–618), eight peptides from the MPER (a.a. 656–698), and one peptide encompassing the caveolin-1-binding site (a.a. 624–638). A negative control peptide derived from SARS-CoV S protein showed antibody reactivity profile similar to peptide 684–698 (data not shown). CWRU-5 was not included due to insufficient amount of the sample. Peptides recognized by mAbs 2F5 and 4E10 are indicated. Patient samples that reacted significantly against both peptides recognized by 2F5 or 4E10 (arbitrarily defined as A450 value greater than twice the sum of average and standard deviation of SARS peptide background reactivity) are indicated by triangles or dots, respectively. It should be noted that Y-axis for seven peptides from the MPER are in different scale.

Adam Penn-Nicholson, et al. Virology. ;372(2):442-456.
7.
Fig 6

Fig 6. Identification of immunogenic linear epitopes targeted by patients using overlapping peptide ELISA. From: Assessment of antibody responses against gp41 in HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M group consensus envelope.

(A) Plasma samples (1:100 dilution) from six patients that exhibited the strongest reactivity against GST-gp41-30 were further analyzed by peptide ELISA (20 pmoles/well). A schematic diagram of a section of gp41 is shown on the top and the key regions are indicated, including the binding sites of caveolin-1, 2F5 and 4E10, and three immunogenic clusters as defined by Binley et al. (Binley et al., 1996). Aligned amino acid sequences of peptides that are immunoreactive are shown. Peptides are numbered based on MCON6 envelope. Those recognized by mAbs 2F5 and 4E10 are indicated in red and blue, respectively (see panel C). (B) Plasma samples were further evaluated with larger peptides encompassing HR1 (N36: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL) and HR2 (C34: WMEWDREINN-YTSLIHSLIEESQNQQEKNEQELL), and 5-helix bundle. The plot shows average reactivity of six patients in either high or low GST-gp41-30-reactive groups. MAb 98-6 was used as a positive control. (C) Immunoreactivity of peptides to 2F5 (1 μg/ml) and 4E10 (20 μg/ml).

Adam Penn-Nicholson, et al. Virology. ;372(2):442-456.

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