Staged induction of HIV-1 glycan–dependent broadly neutralizing antibodies

A preventive HIV-1 vaccine should induce HIV-1–specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3-glycan bnAb. Two autologous neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3-glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs

Amino acid changes in the HIV-1 gp41 membrane proximal region control virus neutralization sensitivity.

CAPRISA, 2016.Abstract available in pdf

Lymphocyte phenotypes in wild-caught rats suggest potential mechanisms underlying increased immune sensitivity in post-industrial environments

The immune systems of wild rats and of laboratory rats can been utilized as models of the human immune system in pre-industrial and post-industrial societies, respectively. In this study, lymphocyte phenotypes in wild rats were broadly characterized, and the results were compared to those obtained by us and by others using cells derived from various strains of laboratory rats. Although not expected, the production of regulatory T cells was not apparently different in wild rats compared to laboratory rats. On the other hand, differences in expression of markers involved in complement regulation, adhesion, signaling and maturation suggest increased complement regulation and decreased sensitivity in wild-caught rats compared to laboratory rats, and point toward complex differences between the maturation of T cells. The results potentially lend insight into the pathogenesis of post-industrial epidemics of allergy and autoimmune disease

Cooperation between somatic mutation and germline-encoded residues enables antibody recognition of HIV-1 envelope glycans.

Viral glycoproteins are a primary target for host antibody responses. However, glycans on viral glycoproteins can hinder antibody recognition since they are self glycans derived from the host biosynthesis pathway. During natural HIV-1 infection, neutralizing antibodies are made against glycans on HIV-1 envelope glycoprotein (Env). However, such antibodies are rarely elicited with vaccination. Previously, the vaccine-induced, macaque antibody DH501 was isolated and shown to bind to high mannose glycans on HIV-1 Env. Understanding how DH501 underwent affinity maturation to recognize glycans could inform vaccine induction of HIV-1 glycan antibodies. Here, we show that DH501 Env glycan reactivity is mediated by both germline-encoded residues that contact glycans, and somatic mutations that increase antibody paratope flexibility. Only somatic mutations in the heavy chain were required for glycan reactivity. The paratope conformation was fragile as single mutations within the immunoglobulin fold or complementarity determining regions were sufficient for eliminating antibody function. Taken together, the initial germline VHDJH rearrangement generated contact residues capable of binding glycans, and somatic mutations were required to form a flexible paratope with a cavity conducive to HIV-1 envelope glycan binding. The requirement for the presence of most somatic mutations across the heavy chain variable region provides one explanation for the difficulty in inducing anti-Env glycan antibodies with HIV-1 Env vaccination

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

Vaccine Induction of Heterologous Tier 2 HIV-1 Neutralizing Antibodies in Animal Models

The events required for the induction of broad neutralizing antibodies (bnAbs) following HIV-1 envelope (Env) vaccination are unknown, and their induction in animal models as proof of concept would be critical. Here, we describe the induction of plasma antibodies capable of neutralizing heterologous primary (tier 2) HIV-1 strains in one macaque and two rabbits. Env immunogens were designed to induce CD4 binding site (CD4bs) bnAbs, but surprisingly, the macaque developed V1V2-glycan bnAbs. Env immunization of CD4bs bnAb heavy chain rearrangement (VHDJH) knockin mice similarly induced V1V2-glycan neutralizing antibodies (nAbs), wherein the human CD4bs VH chains were replaced with mouse rearrangements bearing diversity region (D)-D fusions, creating antibodies with long, tyrosine-rich HCDR3s. Our results show that Env vaccination can elicit broad neutralization of tier 2 HIV-1, demonstrate that V1V2-glycan bnAbs are more readily induced than CD4bs bnAbs, and define VH replacement and diversity region fusion as potential mechanisms for generating V1V2-glycan bnAb site antibodies

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

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

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