Effects of partially dismantling the CD4 binding site glycan fence of HIV-1 envelope glycoprotein trimers on neutralizing antibody induction

Previously, VLPs bearing JR-FL strain HIV-1 Envelope trimers elicited potent neutralizing antibodies (nAbs) in 2/8 rabbits PLoS Pathog 11(5): e1004932) by taking advantage of a naturally absent glycan at position 197 that borders the CD4 binding site (CD4bs). In new immunizations, we attempted to improve nAb responses by removing the N362 glycan that also lines the CD4bs. All 4 rabbits developed nAbs. One targeted the N197 glycan hole like our previous sera. Two sera depended on the N463 glycan, again suggesting CD4bs overlap. Heterologous boosts appeared to reduce nAb clashes with the N362 glycan. The fourth serum targeted a N362 glycan-sensitive epitope. VLP manufacture challenges prevented us from immunizing larger rabbit numbers to empower a robust statistical analysis. Nevertheless, trends suggest that targeted glycan removal may improve nAb induction by exposing new epitopes and that it may be possible to modify nAb speciUcity using rational heterologous boosts

Infrared based saliva screening test for COVID-19

Abstract: Severe acute respiratory syndrome coronavirus 2 has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 patients testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Here, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening

A point-of-care lateral flow assay for neutralising antibodies against SARS-CoV-2

Background: As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing. Methods: We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques. Findings: We show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test. Interpretation: Taken together, the COVID-19 NAb-testTM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care

International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist

Purification and Characterisation of Immunoglobulins from the Australian Black Flying Fox (<em>Pteropus alecto</em>) Using Anti-Fab Affinity Chromatography Reveals the Low Abundance of IgA

<div><p>There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, <em>Pteropus alecto</em>. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture <em>P. alecto</em> IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy <em>P. alecto</em> bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.</p> </div

Purification of <i>P. alecto</i> IgM from IgG depleted serum on immobilised anti-Fab-specific antibody.

<p>Panel A; purification of IgM from IgG depleted serum. Immobilised Protein G column was used to deplete IgG from whole serum. The immobilised anti-Fab-specific antibody column was then used to purify IgM. Lane 1; See Blue plus 2 markers. FT; flow-through, E; elution. Panel B; purified IgM fractions from serum (lane 1) and plasma (lane 3). Lane 2; Mark 12 standard. Selected protein bands (labelled 1 to 8) were excised from gels for LC-MS/MS analysis.</p

Reactivity of rabbit antibodies to <i>P. alecto</i> IgG_H and IgM_H in <i>P. alecto</i> serum.

<p>Panel A. <i>P. alecto</i> serum samples (neat and 1∶5) were separated by reducing SDS-PAGE and probed with rabbit antiserum against <i>P. alecto</i> IgG_H (lanes 1 and 2), IgM_H (lanes 3 and 4) and Fab fragment (lanes 5 and 6). Panel B. 2-DE separation of <i>P. alecto</i> serum sample (silver stain). Panel C and D, 2-DE separation of <i>P. alecto</i> serum sample probed with rabbit antiserum against <i>P. alecto</i> IgM_H (panel C) and IgG_H (panel D).</p

Detection of immunoglobulin heavy chains by LC-MS/MS of gel-purified tissue lavages, extracts and secretions.

<p>Samples were run on separate gels and bands were excised from the region predicted to comprise the immunoglobulin heavy chains (50–80 kDa). Gel images are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052930#pone.0052930.s002" target="_blank">Figure S2</a>.</p

LC-MS/MS analysis of major gel bands from Fab-purified <i>P. alecto</i>.

<p>Serum bands 1–8 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052930#pone-0052930-g003" target="_blank">Figure 3B</a> are denoted MS1 to MS8, plasma bands 1–8 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052930#pone-0052930-g003" target="_blank">Figure 3B</a> are denoted MP1 to MP8.</p