Background
Methods
Human subject and PBMC preparation
Single human plasmablast B cells sorting
PCR amplification of antibody genes
Production of recombinant antibody in mammalian cells
Polysaccharide-specific Sandwich ELISA
MOPA (multiplexed Opsonophagocytic assay) for functional polysaccharide-specific monoclonal antibodies
In-vivo evaluation of polysaccharide-specific monoclonal antibody protection from S. pneumoniae serotype 4 challenge
Passive transfer of monoclonal antibodies
Bacteria
Infection
Survival, weight change, and bacteremia
Results
Isolation of pneumococcal polysaccharide specific antibodies from plasmablast B cells nine days post pneumococcal conjugate (PCV13) vaccination
Antibody ID | Serotype(s) | VH | DH | JH | VL | JL | ||||
---|---|---|---|---|---|---|---|---|---|---|
Family | CDR3 Length | HCDR3 AA sequence | Family | Family | Family | CDR3 length | LCDR3 AA sequence | Family | ||
1A4 | 1 | 3–23*04 | 10 | DVRGSGSNSY | 3–10*01 | 4*02 | K2–30*01 | 9 | MQGTYWPPIT | KJ2*01 |
1C6 | 1 | 3–53*01 | 8 | EVDYAFDP | 3–16*01 | 5*02 | L8–61*01 | 10 | VLFMGSGTWV | LJ3*02 |
1D4 | 1 | 3–74*03 | 8 | SASGWYVN | 6–19*01 | 4*02 | K4–1*01 | 9 | QHYASVPWT | KJ1*01 |
1A6 6 total | 4 | 3–30-3*01 | 10 | DPDTSNKIDY | 2–2*02 | 4*02 | K2–30*01 | 10 | MQGTYWPPIT | KJ5*01 |
1A10 | 4 | 3–7*01 | 10 | RMFGSSFRDY | 6–6*01 | 4*02 | L2–14*01 | 10 | NSYTSSKTWV | LJ3*02 |
1B1 | 4 | 1–46*01 | 16 | GGLLPGVAGATSPFQH | 2–2*01 | 1*01 | K3–20*01 | 9 | QRYGSSPVT | KJ4*01 |
1C3 | 4 | 3–23*01 | 12 | GPVLPAPKEFDY | 2–15*01 | 4*02 | L4–69*01 | 10 | QTWDTVTNWV | LJ3*02 |
1C4 2 total | 6A, 6B | 3–7*03 | 8 | EEWYRFDY | 3–3*02 | 4*02 | L2–8*01 | 10 | SSHAGSKNVI | LJ2*01 |
1D7 | 6A, 6B | 3–7*01 | 8 | EIWFREDY | 3–10*01 | 4*02 | L2–8*01 | 10 | GSRVGSNSVV | LJ2*01 |
1A2 | 7F | 3–33*01 | 14 | EPRAIADNYYGMDV | 3–3*01 | 6*02 | L10–54*01 | 11 | SAWDSSLNAWV | LJ3*02 |
1B2 2 total | 18C | 3–7*01 | 9 | LGGWRHLDY | 3–16*02 | 4*02 | K1–39*01 | 9 | QQSYSSPYT | KJ2*01 |
Antibody ID | Number of clones | VH | VL | ||||||
---|---|---|---|---|---|---|---|---|---|
Total NT substitution (%) | R/S ratio in Frame work | R/S ratio in CDR regions | Strong Selection On HCDR | Total NT substitution (%) | R/S ratio in Frame work | R/S ratio in CDR regions | Strong Selection on LCDR | ||
1A2 | 1 | 6.08% | 4/2 | 10/2 | Yes | 0.34% | 0/0 | 1/0 | No |
1A4 | 1 | 6.08% | 3 /4 | 11/0 | Yes | 2.05% | 1/0 | 4/1 | Yes |
1A6 | 6 | 8.59% | 7/2 | 15/1 | Yes | 3.97% | 2/1 | 8/1 | Yes |
1A10 | 1 | 6.46% | 5/5 | 7/2 | Yes | 2.72% | 0/1 | 7/0 | Yes |
1B1 | 1 | 1.36% | 2/1 | 1/0 | No | 1.04% | 0/0 | 3/0 | No |
1B2 | 2 | 4.42% | 2/2 | 8/1 | Yes | 2.82% | 3/0 | 4/0 | Yes |
1C3 | 1 | 5.78% | 4/2 | 11/0 | Yes | 2.03% | 1/1 | 4/0 | Yes |
1C4 | 2 | 5.48% | 2/1 | 12/1 | Yes | 2.38% | 5/2 | 0/0 | No |
1C6 | 1 | 3.75% | 1 /2 | 8/0 | Yes | 2.37% | 0/1 | 5/1 | Yes |
1D4 | 1 | 6.16% | 5/4 | 9/0 | Yes | 3.64% | 0/3 | 8/0 | Yes |
1D7 | 1 | 7.43% | 5/3 | 14/0 | Yes | 2.74% | 0/1 | 6/1 | Yes |
Pneumococcal polysaccharide specific antibodies demonstrated functional in vitro opsonophagocytic killing (OPK) activities and in vivo protective effect
MOPA assay killing Pneumococcal serotypes | |||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Serotype | mAb | T4 | T6b | T14 | T23f | T18c | T19f | T9v | T6a | T1 | T3 | T5 | T6c | T7F | T19A | T22F | T33F |
T4 | 1A6 | 29 | – | – | – | – | – | – | – | ND | ND | ND | ND | ND | ND | ND | ND |
T4 | 1A10 | 55 | – | – | – | – | – | – | – | ND | ND | ND | ND | ND | ND | ND | ND |
T4 | 1B1 | 23 | – | – | – | – | – | – | – | ND | ND | ND | ND | ND | ND | ND | ND |
T18C | 1B2 | – | – | – | 47 | – | – | – | ND | ND | ND | ND | ND | ND | ND | ND | |
T1 | 1C6 | ND | ND | ND | ND | ND | ND | ND | ND | 14 | – | – | – | – | – | – | – |
T7F | 1A2 | ND | ND | ND | ND | ND | ND | ND | ND | – | – | – | – | 11 | – | – | – |
T6a,b | 1C4 | – | 37 | – | – | – | – | – | 100 | – | – | – | 142 | – | – | – | – |
T6a, b | 1D7 | – | 355 | – | – | – | – | – | 334 | – | – | – | 2247 | – | – | – | – |