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

Quantitative measurement of <i>IGHG</i>, <i>IGHM</i>, <i>IGHA</i>, <i>IGJ</i> and <i>PIGR</i> mRNA in <i>P. alecto</i> tissues.

<p>mRNA transcripts were measured by SYBR Green qPCR and normalised to 18 s ribosomal RNA. Results show the mean ± standard deviation of n = 3 individual healthy wild-caught bats. Abbreviations: L.N., lymph node; S.I., small intestine; S.G., salivary gland; PBMC, peripheral blood mononuclear cells.</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

Purification of <i>P. alecto</i> IgG and generation of Fc and Fab fragments.

<p>Panel A; Protein G purification. Panel B; Protein A purification. Lane 1 (all panels); Mark 12 standard. FT; flow-through, W; wash, E; elution. Panel C; papain digestion containing Fab and Fc fragments was fractionated on immobilised Protein A column. Flow through (FT) fraction contained the Fab fragment (a doublet of approximately 25 kDa).</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

Electrophoretic characterisation of <i>P. alecto</i> and human IgG and IgM before/after deglycosylation.

<p>Proteins were visualised with Coomassie blue (panel A) or silver nitrate (panel B). Bands with asterisks indicate neuraminidase and hashes indicate PNGaseF used for deglycosylation. Lane 1, See Blue plus 2 markers; lanes 2–4, <i>P. alecto</i> IgG; lanes 5–7, human IgG; lanes 8–10, <i>P. alecto</i> IgM; lanes 11–13, human IgM; lanes 3, 6, 9 and 12, PNGaseF treatment; lanes 4, 7, 10 and 13, neuraminidase treatment.</p

Purification strategy of <i>P. alecto</i> IgG, IgM and IgA.

<p><i>Light blue</i> boxes represent those components which polyclonal antiserum was raised against. The <i>green</i> box represents <i>P. alecto</i> anti-Fab-specific antibody. <i>Red dashed</i> lines represent Protein G or Protein A affinity chromatography, <i>blue dashed</i> lines represent heavy chain gel elution, <i>solid green</i> lines represent specific affinity chromatography. Crosses represent unsuccessful purification of IgA. Abbreviations: SEC; size exclusion chromatography.</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

Glycoengineering HIV-1 Env creates ‘supercharged’ and ‘hybrid’ glycans to increase neutralizing antibody potency, breadth and saturation

<div><p>The extensive glycosylation of HIV-1 envelope (Env) glycoprotein leaves few glycan-free holes large enough to admit broadly neutralizing antibodies (bnAb). Consequently, most bnAbs must inevitably make <i>some</i> glycan contacts and avoid clashes with others. To investigate how Env glycan maturation regulates HIV sensitivity to bnAbs, we modified HIV-1 pseudovirus (PV) using various glycoengineering (GE) tools. Promoting the maturation of α-2,6 sialic acid (SA) glycan termini increased PV sensitivity to two bnAbs that target the V2 apex and one to the interface between Env surface gp120 and transmembrane gp41 subunits, typically by up to 30-fold. These effects were reversible by incubating PV with neuraminidase. The same bnAbs were unusually potent against PBMC-produced HIV-1, suggesting similar α-2,6 hypersialylated glycan termini may occur naturally. Overexpressing β-galactosyltransferase during PV production replaced complex glycans with hybrid glycans, effectively 'thinning' trimer glycan coverage. This increased PV sensitivity to some bnAbs but ablated sensitivity to one bnAb that depends on complex glycans. Other bnAbs preferred small glycans or galactose termini. For some bnAbs, the effects of GE were strain-specific, suggesting that GE had context-dependent effects on glycan clashes. GE was also able to increase the percent maximum neutralization (i.e. saturation) by some bnAbs. Indeed, some bnAb-resistant strains became highly sensitive with GE—thus uncovering previously unknown bnAb breadth. As might be expected, the activities of bnAbs that recognize glycan-deficient or invariant oligomannose epitopes were largely unaffected by GE. Non-neutralizing antibodies were also unaffected by GE, suggesting that trimers remain compact. Unlike mature bnAbs, germline-reverted bnAbs avoided or were indifferent to glycans, suggesting that glycan contacts are acquired as bnAbs mature. Together, our results suggest that glycovariation can greatly impact neutralization and that knowledge of the optimal Env glycoforms recognized by bnAbs may assist rational vaccine design.</p></div