Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer.

Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development.

Sensitive CSF ELISAs for the detection of MBL, MASP-2 and functional MBL/MASP-2.

Mannose binding lectin (MBL) mediated complement pathway is an important constituent of innate immune response in several infections including neuroinflammatory and neurodegenerative diseases. Although there are Enzyme-Linked Immunosorbent Assays (ELISAs) for estimating MBL, MBL-associated serine protease-2 (MASP-2) and functional MBL-MASP-2 (fMBL) proteins for the plasma, serum and cell supernatants there are no established methods for their estimation in the cerebrospinal fluid (CSF). We developed sensitive ELISAs for the detection of MBL, fMBL and MASP-2 in the CSF. First, we adapted standard ELISAs for the detection of these proteins in the CSF. Second, we used a biotinyl-tyramide based horseradish peroxidase (HRP) signal amplification for the sensitive detection of these proteins in the CSF. In summary, using modified ELISA and biotinyl-tyramide based HRP signal amplification, we successfully detected MBL, fMBL and MASP-2 proteins in the CSF samples with high sensitivity and reproducibility.

Mannose binding lectin mediated complement pathway in multiple sclerosis.

Role of mannose binding lectin (MBL) complement activation pathway, an arm of innate immunity in multiple sclerosis (MS) was evaluated by analyzing the expression of MBL, MBL-associated serine protease-2 (MASP-2), and functional MBL/MASP-2 mediated C4 cleavage (fMBL) in 87 plasma and cerebrospinal fluid (CSF) samples from MS patients and non-MS controls. Median fMBL and MASP-2 plasma levels were higher in MS vs. non-MS cases. These associations remained in an analysis of subtypes of MS disease. These findings suggest a potential activation of MBL complement pathway in MS that may possibly alter the risk or progression of MS disease.