Sequence and binding characteristics of HIV-1 Env gp41-reactive <i>IGHV</i>1-69 B-CLL mAbs.

<p>(A) The gp41-reactive <i>IGHV</i>1-69 B-CLL mAbs were unmutated and preferentially used <i>IGHD</i>3-3 and <i>IGHJ</i>6 gene segments. The aa sequences of the HCDR3 regions of 5 gp41-reactive <i>IGHV</i>1-69 B-CLL IgMs were aligned to that of CLL526 IgM. Each sequence was aligned independently to CLL526 (pairwise alignment) using ClustalW and final adjustment was made manually. Gaps are indicated as dashes. The aa conserved between the sequences of CLL526 and the other IgMs are highlighted in red. The number of aa shared with CLL526 over the total aa is reported on the right for each IgM. The CLL1296 IgM was used as a negative control. (B) Binding characteristics of the B-CLL mAbs expressed as recombinant IgG₁ with HIV-1, HCV, and intestinal commensal bacterial antigens. Serial dilutions ranging from 100 µg/ml to 0.004 µg/ml of each IgG were tested in ELISA for binding to ADA AT-2-inactivated virion, MN gp41, HIV-1 BAL gp41 immunodominant region peptide SP400 (RVLAVERYLRDQQLLGIWGCSGKLICTTAVPWNASWSNKSLNKI), and HCV E2, or in Luminex assay for aerobic and anaerobic intestinal commensal bacterial whole-cell lysates. Data are expressed in OD for ELISA or mean fluorescence intensity (MFI) for Luminex assay. The dotted lines indicate the cut-off value ≥100 MFI used to denote positivity. Data are representative of at least two separate experiments.</p

B-CLL cases with anti-viral reactivity correlate with poor clinical outcomes.

<p>The Kaplan-Meier plots are shown for the time to first treatment (TFT, in months) in all samples (A) and <i>IGHV</i>1-69 samples (B). The p values for Mantel-Cox test in groups A and B are 0.011 and 0.217, respectively. The Kaplan-Meier plots are shown for overall patient survival (in months) in all samples (C) and <i>IGHV</i>1-69 samples (D). The p values for Mantel-Cox test in groups C and D are <0.0001 and 0.012, respectively. Virus+ group represents B-CLL samples with ≥10 wells out of 20 wells tested showing a specific anti-viral reactivity (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090725#pone.0090725.s001" target="_blank">Figure S1</a></b>). The results for virus-binding activity of 2 B-CLL samples (CLL821 and CLL1296) were obtained from the purified IgM paraproteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090725#pone-0090725-g001" target="_blank"><b>Figure 1</b></a>).</p

HCDR3 alignment of CLL1324 to gp41-reactive <i>IGHV</i>1-69 antibodies isolated from HIV-1-infected patients.

<p>The aa sequences of the HCDR3 regions of gp41-reactive <i>IGHV</i>1-69 antibodies isolated from HIV-1-infected patients were aligned to that of CLL1324. Each sequence was aligned independently to CLL1324 (pairwise alignment) using ClustalW and final adjustment was made manually. Gaps are indicated as dashes. The aa conserved between the sequences of CLL1324 and the other antibodies are highlighted in red. The number of aa shared with CLL1324 over the total aa is reported on the right for each antibody. Only the gp41 antibody sequences with HCDR3 % similarity ≥50% are reported. The CLL1296 IgM was used as a negative control. ¹Previously published sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090725#pone.0090725-Morris1" target="_blank">[51]</a>; ²CLL1296, HIV-1-negative control mAb; ³<i>IGHV</i>1-69 antibodies with an F₅₄ allelic variant. D RF, D gene reading frame; AA₅₄, aa in position 54.</p

Reactivity of IgM paraproteins produced by B-CLL hetero-hybridomas and the corresponding recombinant IgG₁ mAbs.

<p>Values are representative endpoint concentrations (in μg/ml) from at least two separate experiments. ¹Deglycosylated JRFL gp140; ²HIV-1 gp41 HR-1 region peptide, DP107 sequence (NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ); ³HIV-1 envelope clade C gp41 HR-2 region peptide, MPR.03 sequence (KKKNEQELLELDKWASLWNWFDITNWLWYIRKKK); ⁴HIV-1 BAL gp41 immunodominant region, SP400 sequence (RVLAVERYLRDQQLLGIWGCSGKLICTTAVPWNASWSNKSLNKI); Aerobic/anaerobic intestinal commensal bacterial whole-cell lysates. A HIV-1 gp41 antibody, D5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090725#pone.0090725-Luftig1" target="_blank">[26]</a> and an influenza HA antibody, CR6261 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090725#pone.0090725-Throsby1" target="_blank">[22]</a> were included as positive controls. “-” denotes no binding.</p

<i>IGHV1</i>-69 allelic variants used by gp41 mAbs upon HIV-1 infection.

1<p>All sequences are available in the IMGT and GenBank databases. ²p<0.0001 versus gp41-reactive <i>IGHV1</i>-69 antibodies isolated from HIV-1-infected subjects. Nd, not determined.</p

Binding of recombinant B-CLL IgG1 mAbs to MN gp41 and HCV E2 proteins in surface plasmon resonance binding assays.

<p>MN gp41 (A) or HCV E2 (B) protein was captured on a sensor chip surface and test mAbs were injected over each of the test antigens. Test mAbs preincubated with either MN gp41 or HCV E2 proteins were injected over MN gp41 immobilized on a sensor chip surface (C). Data are expressed in response unit of binding to MN gp41.</p