Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design

Protein-RNA contacts outside of the active site.

<p>(a) The substrate strand, which leads into the active site, forms hydrogen bonds to the NP through the phosphate backbone and by contacts to G8. D426 hydrogen bonds to its nitrogenous base, and the main chain nitrogen of G392 makes hydrogen bonds to the 2′ and 3′OH of its ribose sugar. (b) The non-substrate strand forms hydrogen bonds between the 2′OH of C1 and Q425, and the 2′OH of C4 and D465. There is also a base stacking interaction between C1 and Y429.</p

Effect of mutations on exonuclease activity.

<p>(a) Wild-type, full-length NP or the immunosuppressive domain of NP (NPΔ340) containing one of several point mutants were incubated with 18 bp blunt-ended dsRNA for 15 min, and products were analyzed by PAGE. NP residues that contact the digested strand are indicated with *; NP residues that contact the non-digested strand are indicated with #.’ (b) Time course of dsRNA digestion by wild-type (WT), and Y429A and Y429L mutant NPs. (c) Digestion of 18 bp hybrid DNA:RNA double-stranded oligonucleotides. The labeled strand is denoted by *.</p

Modeling of different forms of DNA.

<p>Idealized B-form dsDNA (blue) and A-form ssDNA (magenta) were modeled and aligned with the dsRNA bound to Lassa NP (green). The 5′ terminal residue of the non-substrate strand (C1) in the B-form dsDNA clashes with Lassa NP, while A-form ssDNA does not.</p

Electron density for the dsRNA.

<p>(a) Final 2Fo-Fc, contoured at +2.0σ, of the dsRNA bound to Lassa NP. The substrate strand, which leads into the active site is colored blue and the non-substrate strand is colored yellow. (b) Stereo view of a simulated-annealing composite omit map of final RNA density, contoured at +2.0σ.</p

Location of the basic loop.

<p>Crystal structures of three DEDDh exonucleases are aligned. ε186 (PDB code 1J54 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044211#pone.0044211-Hamdan1" target="_blank">[6]</a>) is illustrated in yellow, Trex2 (PDB code 1Y97 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044211#pone.0044211-Perrino1" target="_blank">[9]</a>) in magenta, and Lassa NP in green. All of these exonucleases contain a basic loop motif. In ε186, the basic motif (K141, R142, R151) lies above and to the right of the active site. In Trex 2, the basic loop is in the same general position, but all residues except for R152 are disordered and indicated by a dotted line. In Lassa NP, no such loop exists in this location and instead strand β5 and helix α5 are directly connected by an IDIAL hydrophobic sequence (residues I482 to L486). Instead, Lassa NP has a basic loop motif in a projecting “arm” located to the left of the active site rather than above and to the right. This loop contains K516, K517, K518 and R519, although the side chains are disordered in this structure.</p

Structure of the active site.

<p>Carbons of Lassa NP residues are colored green. (a) The phosphate backbone of the terminal 3′ nucleotide (G8) is coordinated by a divalent cation (modeled here as a Mg ion, MgB) and by H528. MgB is further coordinated by D389. Due to mutation of E391 to alanine, the second catalytic divalent ion is not bound in the active site. (b) Comparison of the active site of Lassa NP E391A to the active sites of the DEDDh exonucleases ISG-20 (cyan, PDB code 1WLJ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044211#pone.0044211-Horio1" target="_blank">[5]</a>) and ε186 (yellow, PDB code 1J54 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044211#pone.0044211-Hamdan1" target="_blank">[6]</a>), which were determined in complex with mononucleotides. Both cations are apparent in the structures of ISG-20 and ε186. Amino acid numbering of the shared DEDDh motif is for Lassa NP. (c) A model of wild-type Lassa NP in complex with the terminal G8 mononucleotide. The crystallized ion MgB is drawn in grey. The position of E391 and the cation in site A are based on their positions in the unbound structure (PDB code 3Q7B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044211#pone.0044211-Hastie1" target="_blank">[3]</a>). Hydrogen bonds are colored red; coordination bonds are colored black; an arrow indicates the position of the next nucleotide in the digested strand. Expected water molecules, based on structural similarity to other DEDDh exonucleases, are drawn in pale blue.</p

Data collection, phasing and refinement statistics.

*<p>Values in parentheses are for highest-resolution shell. _ENREF_3_ENREF_16_ENREF_17_ENREF_18_ENREF_19_ENREF_11_ENREF_13_ENREF_14_ENREF_9_ENREF_15_ENREF_13_ENREF_3</p

Regulation of Indoleamine 2,3-Dioxygenase Expression in Simian Immunodeficiency Virus-Infected Monkey Brains

The human immunodeficiency virus type 1-associated cognitive-motor disorder, including the AIDS dementia complex, is characterized by brain functional abnormalities that are associated with injury initiated by viral infection of the brain. Indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme in tryptophan catabolism in extrahepatic tissues, can lead to neurotoxicity through the generation of quinolinic acid and immunosuppression and can alter brain chemistry via depletion of tryptophan. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model of AIDS, we demonstrate that cells of the macrophage lineage are the main source for expression of IDO in the SIV-infected monkey brain. Animals with SIV encephalitis have the highest levels of IDO mRNA, and the level of IDO correlates with gamma interferon (IFN-γ) and viral load levels. In vitro studies on mouse microglia reveal that IFN-γ is the primary inducer of IDO expression. These findings demonstrate the link between IDO expression, IFN-γ levels, and brain pathology signs observed in neuro-AIDS