Identifocation the B Cell Subsets Responsible for Anti-pneumococcal Response
Condition: Pneumococcal Diseases
MeSH terms: Pneumococcal Infections
Study type: Interventional

Summary: 
      The purpose of this study is to determine which B lymphocytes subsets are responsible for the
      production of IgM, IgG2 and IgA anti-pneumococcal capsular polysaccharides after vaccination
      with a 23-valent pneumococcal polysaccharide vaccine.
    

Detailed description: 
      The aim of this study is to identify which specific B cell subset(s) is/are responsible for
      the production of protective IgM, IgG2 and IgA anti-pneumococcal capsular polysaccharides
      (capPS) in response to immunization of healthy individuals with the Pneumovax, a prototypical
      T-independent type 2 vaccine. In other words, from which B cells are IgM, G2 and A-secreting
      plasma cells derived? To address this question, it will be taken advantage of the unique Ig
      heavy chain VDJ signature expressed by each B cell clone and the strategy will rely on the
      search of clonal filiations between plasmablasts (PB)/plasma cells (PC) and different B cell
      subpopulations. Therefore, healthy individuals will be vaccinated with Pneumovax, and blood
      samples will be collected at day 0, 7, 14, 28 and 56. Starting from these blood samples,
      different B cells subsets (in particular IgM+IgD+CD27+ and switched memory IgM-IgD-CD27+ B
      cells) and the PB/PC peaking at day 7 after vaccination will be isolated by cell sorting.
      CapPS-secreting PB cannot be specifically isolated, but the investigators assume that they
      will represent the majority of the isolated PB/PC at the peak of the response. Thus, day
      7-PB/PC will be sorted both in bulk or as single cells in 96-well PCR plates. RNA will be
      extracted from bulk sorted PB/PC, and VDJ-µ, -alpha and -gamma Ig transcripts will be
      amplified by RT-PCR and analyzed by the H-CDR spectratyping method in order to identify the
      sequence of dominant VDJ signatures that most probably correspond to anti-capPS-secreting
      cells. In parallel, for each PB/PC single cell, Ig heavy and corresponding Ig light chain
      gene transcripts will be amplified by nested RT-PCR, sequenced, and provided that the heavy
      chain contains a dominant VDJ signature, they will be cloned into eukaryotic expression
      vectors to produce monoclonal human Abs. The recombinant antibodies will then be tested for
      reactivity against capPS from the vaccine by ELISA. When VDJ signatures of capPS will be
      validated, with this powerful molecular tool, the investigators will look for the presence of
      these signatures through VDJ-specific PCR on cDNA prepared from the different B cell subsets.
    

Intervention name(s): pneumovax vaccination
Intervention type(s): Drug
Intervention description(s): vaccination
Other intervention name(s): pneumovax
Intervention MeSH terms: Heptavalent Pneumococcal Conjugate Vaccine
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: Immunology; human; B-lymphocytes subsets; Antibody-Producing Cells; 23 valent pneumococcal capsular polysaccharide vaccine