Developmental Pathway of the MPER-Directed HIV-1-Neutralizing Antibody 10E8

Antibody 10E8 targets the membrane-proximal external region (MPER) of HIV-1 gp41, neutralizes >97% of HIV-1 isolates, and lacks the auto-reactivity often associated with MPER-directed antibodies. The developmental pathway of 10E8 might therefore serve as a promising template for vaccine design, but samples from time-of-infection—often used to infer the B cell record—are unavailable. In this study, we used crystallography, next-generation sequencing (NGS), and functional assessments to infer the 10E8 developmental pathway from a single time point. Mutational analysis indicated somatic hypermutation of the 2nd-heavy chain-complementarity determining region (CDR H2) to be critical for neutralization, and structures of 10E8 variants with V-gene regions reverted to genomic origin for heavy-and-light chains or heavy chain-only showed structural differences >2 Å relative to mature 10E8 in the CDR H2 and H3. To understand these developmental changes, we used bioinformatic sieving, maximum likelihood, and parsimony analyses of immunoglobulin transcripts to identify 10E8-lineage members, to infer the 10E8-unmutated common ancestor (UCA), and to calculate 10E8-developmental intermediates. We were assisted in this analysis by the preservation of a critical D-gene segment, which was unmutated in most 10E8-lineage sequences. UCA and early intermediates weakly bound a 26-residue-MPER peptide, whereas HIV-1 neutralization and epitope recognition in liposomes were only observed with late intermediates. Antibody 10E8 thus develops from a UCA with weak MPER affinity and substantial differences in CDR H2 and H3 from the mature 10E8; only after extensive somatic hypermutation do 10E8-lineage members gain recognition in the context of membrane and HIV-1 neutralization

Initial sequencing and analysis of the human genome

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62798/1/409860a0.pd

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Records of selected wells in Atlantic County, New Jersey

This report contains records of 129 wells in Atlantic County. Included are 126 lithologic logs and 39 borehole geophysical logs. Information on hydrogeologic conditions at the time of well construction is included. New Jersey Department of Environmental Protection well permit numbers and U.S. Geological Survey Ground Water Site Inventory (GWSI) reference numbers are given. These logs, collected from 1891 to 1988, include the most detailed well information available for the county's 23 municipalities. Wells are shown on a 1:100,000-scale map

Atlantic City Site Report

The Atlantic City borehole was the second site drilled as part of the New Jersey coastal plain drilling project, Leg 150X. It focused on middle middle Miocene to Oligocene "Icehouse" and middle-upper Eocene "Doubthouse" sequences known from previous rotary and cable tool wells. Recovery was not as good as at Island Beach (60% vs. 87%) because of hole stability problems; however, recovery was excellent for most of the critical lower-middle Miocene interval (390-937 ft; 81%). The surficial Cape May Formation (uppermost Pleistocene-Holocene; 123 ft thick) contains nearshore gravelly sand and clay at the top and fluvial deposits at the base that apparently correlate with the Cape May Formation at Island Beach. The ?middle Miocene Cohansey Formation (96 ft thick) sand and sandy clay represents fluvial deposits not present at Island Beach. The ages of both units are uncertain. Recovery of the uppermost part of the 706-ft-thick Kirkwood Formation was poor (no recovery from 293 to 390 ft), but recovery for the Kirkwood between 390 and 937 ft was excellent. The sand, silts, and clay facies expressed in the Kirkwood Formation at Atlantic City represent diverse fluvial, nearshore, and neritic (including prodelta) environments. Several upward-coarsening sequences can be recognized on the basis of lithofacies breaks, gamma-log changes, and hiatuses, corresponding with confining units at the base and aquifer units at the top. These lithostratigraphic and geohydrologic units correspond with similar units at Island Beach, and we suggest that they correlate. Numerous shell beds in the Atlantic City borehole allow preliminary dating of these sequences with Sr-isotopic stratigraphy, including the middle middle Miocene Kirkwood 3 sequence (13.3-13.5 Ma; from at least 401.7 to at least 470 ft), the upper lower Miocene Kirkwood 2 sequence 17.0 17.9 Ma; 512-666 ft), and the uppermost Oligocene to lower Miocene Kirkwood 1 sequence (20.3- 25.8 Ma; 666-937 ft). The Kirkwood 1 sequence may be divided into several additional sequences that have dramatic shell beds at their base and distinct ages determined by Sr isotopes: 20.3-21.9 Ma, 23.6-23.7 Ma, and -25.8 Ma. A sharp lithologic and gamma-log break at 741 ft may indicate another sequence boundary between 20.3 and 20.8 Ma, although Sr-isotopic resolution is not sufficient to document this hiatus unequivocally. The upper Eocene-Oligocene may be divided further into 3 sequences based on lithologic and gamma log changes that were dated with Sr isotopes: upper Oligocene (27.4-28.7 Ma), lower Oligocene (-33.4 Ma), and upper Eocene (36.6-37.7 Ma). Biostratigraphy is consistent with the Sr-isotopic ages and indicates additional lower upper Eocene and upper middle Eocene sequences. The systems tracts are generally well developed for these middle Eocene through middle Miocene sequences, with a basal shell or glauconite sand at the base and sands at the top. Further biostratigraphic and Sr-isotopic studies should refine the ages of the sequences, whereas lithostratigraphic and benthic foraminifer biofacies studies should reveal details of the depositional environments and systems tracts of these well-developed sequences

Pairing of heavy and light 10E8 intermediates.

<p>(<b>A</b>) Heavy (top) and light (bottom) phylogenetic trees with pairing of inferred intermediates indicated by dashed lines. Intermediate pairing based on tree structure and considerations from polyreactivity (see text and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.ref018" target="_blank">18</a>]). (<b>B</b>) Heavy chain UCA, intermediates and mature sequences. (<b>C</b>) Light chain UCA, intermediates and mature sequences.</p

Functional characteristics of 10E8 UCA and maturation intermediates.

<p>(<b>A</b>) Neutralization on eight diverse HIV-1 isolates (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.s012" target="_blank">S6 Table</a> for details on HIV-1 isolate panel). (<b>B</b>) Sensogram profiles shown represent two-fold serial dilutions of Fab analyte starting at top concentrations of 500 nM for UCA, pI1, and 17b, or 125 nM for pI2, pI3 and 10E8 mature, through to final concentrations of 3.9–31.25 nM. (<b>C</b>) ELISA assessment of MPER-liposome recognition. Shown are bare liposome (left) and MPER proteoliposome (right) competiton ELISA assays for antibody recognition of a soluble MPER peptide captured on a plate. Binding of 10E8 mature and pI3 antibodies to soluble MPER was effectively competed by MPER proteoliposomes in this assay. (<b>D</b>) Hep2 cell assessment of autoreactivity. (<b>E</b>) Structural models of 10E8 showing location and degree of somatic hypermutation by intermediate. Structural models of 10E8 intermediate antibodies shown in ribbon representation showing the location of resides mutated from the UCA as spheres and colored according to paired intermediate. The degree of somatic mutation for each 10E8 intermediate is given below each structure model with % nucleotide and % amino acid change from the germline VH gene and also for the calculated UCA gene.</p

Crystallographic data collection and refinement statistics.

<p>Crystallographic data collection and refinement statistics.</p

Analysis of 10E8 somatic hypermutation identifies critical K52T mutation.

<p>(<b>A</b>) Summary of CDR H2 somatic hypermutation and 10E8 neutralization. Total mutations from germline are indicated for both heavy and light chains. The CDR H2 germline sequence is shown colored blue with somatic mutations indicated in black. Viruses neutralized with an IC₅₀ < 50 μg/ml or < 1 μg/ml are indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.s007" target="_blank">S1 Table</a> for details on HIV-1 isolate panel). (<b>B</b>) Superposition of mature 10E8 complex structure and 10E8 gHv/gLv. Coloring is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157409#pone.0157409.g001" target="_blank">Fig 1</a>. (<b>C</b>) Expanded view of CDR H2. Residues that were mutated and gave improved 10E8 gHv/gLv neutralization levels are shown in stick representation. Residues Phe 673 (MPER) and Lys 52 (10E8 gHv/gLv) overlap and are not compatible with binding due to steric clashing.</p