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MS-based sequence of anti-RSV-F monoclonal antibody 131-2a

ABSTRACT: The Respiratory Syncytial Virus (RSV) Fusion (F) protein is a major target of antiviral antibodies following natural infection and vaccination and responsible for mediating fusion between the viral envelope and the host membrane. The fusion process is driven by a large-scale conformational change in F, switching irreversibly from the metastable prefusion state to the stable postfusion conformation. Previous research has identified six distinct antigenic sites in RSV-F, termed sites Ø, I, II, III, IV, and V. Of these, only antigenic site I is fully specific to the postfusion conformation of F. A monoclonal antibody 131-2a that specifically targets postfusion F has been widely used as a research tool to probe for postfusion F and to define antigenic site I in serological studies, yet its sequence and precise epitope have remained unknown. Here we use mass spectrometry-based de novo sequencing of 131-2a to reverse engineer a recombinant product and study the epitope to define antigenic site I with molecular detail, revealing the structural basis for the antibody’s specificity towards postfusion RSV-F.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Joost Snijder

LAB HEAD: Joost Snijder

PROVIDER: PXD059427 | Pride | 2025-07-22

REPOSITORIES: Pride

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			Action	DRS
	20190722_F1_Ag5_3117030_SA_131-2a_TL.raw	Raw
	20190722_F1_Ag5_3117030_SA_131-2a_aLP.raw	Raw
	20190722_F1_Ag5_3117030_SA_131-2a_chym.raw	Raw
	20190722_F1_Ag5_3117030_SA_131-2a_elast.raw	Raw
	20190722_F1_Ag5_3117030_SA_131-2a_lysC.raw	Raw

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Project description:The discovery of hepatitis C virus (HCV) in 1989 revealed the virus as the etiology of 40%-90% of the “essential” mixed cryoglobulinemia, where immune complex forms deposit called cryoprecipitate at temperatures below 37 °C. Cryoprecipitate constitutes monoclonal IgM and polyclonal IgG, some of which has reactivity against HCV core and NS3 epitopes. Resultant immune complex is considered entrapped on microvascular endothelium via C1q receptor, leading to complement activation and organ injury presenting predominantly as dermopathy, peripheral neuropathy and nephropathy. However, currently little is known on whether auto-reactive, cold-precipitating IgG components enriched in cryoprecipitate may play some role on the deposition of immune complex and subsequent complement-mediated injury of specific organs. Recently, with the advent of high-throughput immune repertoire sequencing and mass spectrometry, technical feasibility is growing to delineate antibodies of interest and their sequences directly from serum. To date, vast majority of studies actually utilized the antigen column for affinity purification of antibodies of interest, although this strategy is not applicable to disease entity with unknown antigen involvement. In such cases, disease-specific and organ-specific immune deposits may be a good alternative source of etiological antibodies. Herein, targeting HCV cryoglobulinemic vasculitis as a model, we conducted a proof-of-concept study aiming at characterizing the IgG components most prone to cryoprecipitation. To this end, we longitudinally studied one patient with cryoglobulinemic vasculitis with chronic HCV infection. After obtaining informed consent, cryoprecipitate and supernatant were separated from peripheral blood sample. Fab fragments from Protein G-purified IgG were recovered after papain digestion for isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics. Simultaneously, total RNA was isolated from peripheral blood, and immunoglobulin heavy chain variable region (IGHV) was PCR-amplified with unique molecular identifier (UMI) strategy to construct a personal IGHV sequence library of immunoglobulin variable region. Sequencing output from MiSeq was bioinformatically converted into mass spectrometry database. Search was performed using MaxQuant software.

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MS-based sequence of anti-RSV-F monoclonal antibody 131-2a

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