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. Mar-Apr 2014;6(2):460-73.
doi: 10.4161/mabs.27760. Epub 2014 Jan 8.

Multiplexed Screening of Natural Humoral Immunity Identifies Antibodies at Fine Specificity for Complex and Dynamic Viral Targets

Free PMC article

Multiplexed Screening of Natural Humoral Immunity Identifies Antibodies at Fine Specificity for Complex and Dynamic Viral Targets

Krista M McCutcheon et al. MAbs. .
Free PMC article


Viral entry targets with therapeutic neutralizing potential are subject to multiple escape mechanisms, including antigenic drift, immune dominance of functionally irrelevant epitopes, and subtle variations in host cell mechanisms. A surprising finding of recent years is that potent neutralizing antibodies to viral epitopes independent of strain exist, but are poorly represented across the diverse human population. Identifying these antibodies and understanding the biology mediating the specific immune response is thus difficult. An effective strategy for meeting this challenge is to incorporate multiplexed antigen screening into a high throughput survey of the memory B cell repertoire from immune individuals. We used this approach to discover suites of cross-clade antibodies directed to conformational epitopes in the stalk region of the influenza A hemagglutinin (HA) protein and to select high-affinity anti-peptide antibodies to the glycoprotein B (gB) of human cytomegalovirus. In each case, our screens revealed a restricted VH and VL germline usage, including published and previously unidentified gene families. The in vivo evolution of paratope specificity with optimal neutralizing activity was understandable after correlating biological activities with kinetic binding and epitope recognition. Iterative feedback between antigen probe design based on structure and function information with high throughput multiplexed screening demonstrated a generally applicable strategy for efficient identification of safe, native, finely tuned antibodies with the potential for high genetic barriers to viral escape.

Keywords: broadly protective antibodies; cytomegalovirus; fusion; human antibodies; immunoglobulin germline; influenza; monoclonal antibodies; neutralizing antibodies; viral epitopes.


Figure 1. Structures of influenza A group 1 (A) and group 2 (B) hemagglutinin. Published crystal structures for H1N1 HA (pdb 3AL4) and H3N2 HA (pdb 1MQM) were compared by simplifying the trimers into “pretty” monomer formats revealing the tertiary positions of the sequential N-terminal HA1 region (orange), pre-fusion (light blue) and fusion region (yellow; with arginine 329 shown in red) loops, anti-parallel β sheet (royal blue) and finally the α-helical stalk region (green). Shown just below the pretty models are the corresponding 3D crystal structures with the discontinuous epitopes of the lead group 1 (mAb 53) and group 2 (mAb 579) antibodies, determined by PepScan methods. The identified epitope regions are colored according the fusion region domains as described above.
Figure 2. Kinetic binding and neutralizing activity of a panel of antibodies cross-reactive to the fusion region of group 1 HA. (A) Comparison of the binding kinetics of mAb 1, 8, 30, 48, 49, 52 or 53 (captured on an anti-human Fc sensors), to 200nM of HA monomer of the H1N1 A/CA/04/2009 strain. (B) Comparative fusion peptide binding (gray bars) as a percent relative to a rabbit pAb directed to and purified by the fusion peptide; and neutralizing activity of antibodies (colored bars), at a constant IgG concentration of 5 µg/ml in MDCK cells infected with the H1N1 A/CA/04/2009 virus. Plaques were counted in duplicate wells and expressed as a percent of plaques in an uninhibited infection. A comparison of the breadth and potency of HA coverage for selected neutralizing group 1 antibodies can be found in Table 4 and Figure S2.
Figure 3. Kinetic binding and limited neutralizing activity of group 1 and group 2 HA cross-reactive mAb 486. (A) Comparison of the binding kinetics of mAb 486 after capture on an anti-human Fc sensors, to 200 nM of HA trimers representative of group 1 (H1 California 07/09 and H5 Vietnam/1203/2004) and group 2 (H3 Hong Kong/8/68 and H7 Netherlands/219/03). (B) Plaque formation neutralization activity of mAb 486, rabbit pAb directed to the fusion peptide, or mAb 579 of H1 A/CA/04/09 or H3 A/Perth/16/09 infected MDCK cells, with (black) or without (blue) prior trypsin activation of viruses.
Figure 4. Development of a multiplexed strategy for discovery of high-affinity antibodies to the neutralizing site I, AD-2 region of the gB protein on human CMV. (A) A bead-based ELISA was used to track the optimization of low and high density AD-2 beads with a 10-fold binding difference to a benchmark reagent, mAb 4A2. As desired, binding of 4A2 to the low density beads was reduced when multiplexed with high density and gB beads, whereas high density beads bound 4A2 equally in single or multiplexed formats. With these bead properties we expected to be able to screen and differentiate mAb at least 10-fold improved in AD-2 binding over 4A2. (B) Digital fluorescent microscopy was used to count and quantify antibody binding to low affinity beads (high density, 20% AD-2 coating in 1% BSA), high-affinity beads (low density, 1% AD-2 coating in 1% BSA) and gB beads. Data was transformed to a bar graph format and an “affinity metric” value was assigned. A high-affinity antibody would bind equally, without bias, to both densities of AD-2 coated beads and have a calculated “affinity metric” of 1. Low-affinity antibodies would preferentially bind the high density AD-2 coated beads, and demonstrate affinity metric values < 1. Antibodies that cannot bind the peptide in the context of the native gB protein will show no signal on the gB-ECD coated beads. (C) Kinetic AD-2 binding sensorgrams for the high- (affinity metric = 0.87) and low- (affinity metric = 0.15) affinity monoclonal antibodies corresponding to these CellSpot™ phenotypes.
Figure 5. Binding of selected high-affinity human CMV antibodies to the neutralizing AD-2 epitope alone and in the context of glycoprotein B. (A) Sensorgrams of 100 nM IgG (mAb 310, 313, 338, 345 and 4A2) binding to biotinylated AD-2 peptide captured on streptavidin probes. (B) Competitive binding ELISA using a constant amount of the same panel of mAb IgG pre-incubated with increasing amounts of AD-2 peptide for 30 min prior to binding plate-coated gB. Concentrations of IgG were normalized among the antibodies to give an OD = 1 when binding 0.5 µg/ml gB for 1 h in the absence of any competing peptide. High-affinity antibodies cloned from memory B cells of naturally infected humans with HCMV, such as mAbs 310, 313 and 338, demonstrate additional contacts with gB outside of the AD-2 region causing them to bind gB more strongly in the presence of competing peptide.

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