Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein

AbstractHybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

The emergence of artemisinin (ART) resistance in Plasmodium falciparum intra-erythrocytic parasites has led to increasing treatment failure rates with first-line ART-based combination therapies in Southeast Asia. Decreased parasite susceptibility is caused by K13 mutations, which are associated clinically with delayed parasite clearance in patients and in vitro with an enhanced ability of ring-stage parasites to survive brief exposure to the active ART metabolite dihydroartemisinin. Herein, we describe a panel of K13-specific monoclonal antibodies and gene-edited parasite lines co-expressing epitope-tagged versions of K13 in trans. By applying an analytical quantitative imaging pipeline, we localize K13 to the parasite endoplasmic reticulum, Rab-positive vesicles, and sites adjacent to cytostomes. These latter structures form at the parasite plasma membrane and traffic hemoglobin to the digestive vacuole wherein artemisinin-activating heme moieties are released. We also provide evidence of K13 partially localizing near the parasite mitochondria upon treatment with dihydroartemisinin. Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13. These data help define the biological properties of K13 and its role in mediating P. falciparum resistance to ART treatment

Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein

AbstractHybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection

Tissue Specificity of Human Angiotensin I-Converting Enzyme.

Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood.We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests.Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs

The structures of N and C domains of ACE with potential glycosylation sites and epitopes for mAbs.

<p>Human N domain structure was based on PDB P2C6N and C domain structure—based on PDB 1O86. The epitopes were marked on the N and C domains according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref027" target="_blank">27</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref031" target="_blank">31</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref041" target="_blank">41</a>]. The positions of the epitopes for some mAbs (12 out of 17) are shown by circles on both sides of domain globule. The potential sites of N-glycosylation, 9 on the N domain and 6 on the C domain, are marked by green; Asn494 on the N domain is not seen while Asn1196 is not present in structure of the C domain. The glycosylation sites which might be differently glycosylated in seminal fluid ACE and lung ACE are shown by arrows. Some amino acid residues are shown by numbers according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref038" target="_blank">38</a>] for orientation.</p

N-glycosylated peptides and glycan structures found by the GlycoMod tool in ACE from lung and seminal fluid using MS data.

<p>N-glycosylated peptides and glycan structures found by the GlycoMod tool in ACE from lung and seminal fluid using MS data.</p

Observed [M+H]⁺ ions of unglycosylated peptides in the mass spectra of human ACE tryptic digests.

<p>^a Acrylamide adduct on cysteine.</p><p>^b Oxidized methionine.</p><p>^c Contains one or two missed cleavage(s) by trypsin.</p><p>Peptides that contain potential N-glycosylation sites are shown in bold.</p><p>Observed [M+H]⁺ ions of unglycosylated peptides in the mass spectra of human ACE tryptic digests.</p

Amino acid sequences of the lung and seminal fluid ACEs.

<p>Peptides identified by MALDI TOF MS are shown in bold; potential sites of trypsin cleavage are underlined; potential glycosylation sites are marked by green; zinc-recognizing motives are marked by red; putative glycopeptides are shaded.</p

Effect of different additives on mAbs binding to seminal fluid and lung ACEs.

<p>ACE activity immunoprecipitated by 17 mAbs to ACE (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>) was presented as a normalized value (“binding ratio”) to highlight differences in immunoprecipitation pattern (“conformational fingerprint”) after adding of tested compounds to purified seminal fluid and lung ACEs with that without additives. <b>(A)</b> Effect of 20% of human heat-inactivated plasma. <b>(B</b>) Effect of 80% 3 kDa filtrate of human citrated plasma. <b>C</b>-<b>D</b>. Effect of bilirubin (150 ug/ml) in the absence (<b>C</b>) or presence (<b>D</b>) of human albumin at 8 mg/ml concentration (which correspond to its concentration in 20% serum). Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>.</p

Effect of human plasma, seminal fluid and albumins on mAbs binding to ACEs.

<p>ACE activity immunoprecipitated by 17 mAbs to ACE (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>) was presented as a normalized value (“binding ratio”) to highlight differences in immunoprecipitation pattern (“conformational fingerprint”) after adding heat-inactivated human citrated plasma, heat-inactivated seminal fluid, as well as human and bovine albumins to purified seminal fluid and lung ACEs with that without additives. <b>A-B</b>. Effect of 20% of heat-inactivated human plasma (<b>A</b>) and heat-inactivated seminal fluid (<b>B</b>); <b>C-D</b>. Effect of human (<b>C</b>) and bovine (<b>D</b>) albumins at concentrations of 8 mg/ml (similar to albumin concentration in 20% serum). Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>.</p