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High Inter-Individual Diversity of Point Mutations, Insertions, and Deletions in Human Influenza Virus Nucleoprotein-Specific Memory B Cells.

Reiche S, Dwai Y, Bussmann BM, Horn S, Sieg M, Jassoy C - PLoS ONE (2015)

Bottom Line: We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes.We also found several small groups of clonal relatives that were highly diversified.We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology, Faculty of Medicine, University of Leipzig, Leipzig, Germany.

ABSTRACT
The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs) originated from 26 and the kappa light chains (LCs) from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4% in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses.

No MeSH data available.


Related in: MedlinePlus

Influenza NP binding and gene amplification efficiency.A) Optical density values obtained in the influenza NP ELISA with the supernatants of cells transfected with pairs of HC and LC sequences from the same B cell vial. B) Association of gene amplification efficiency and the percentage of vials from which influenza NP-binding antibodies were obtained. Three sets of samples from individual D1 from two time points were analyzed. N is the number of tubes that gave rise to HC/LC sequence pairs. The gene-amplification efficiency varied (squares). The fraction of HC/LC pairs that yielded influenza NP-binding antibodies (circles) decreased with increasing gene amplification frequency.
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pone.0128684.g002: Influenza NP binding and gene amplification efficiency.A) Optical density values obtained in the influenza NP ELISA with the supernatants of cells transfected with pairs of HC and LC sequences from the same B cell vial. B) Association of gene amplification efficiency and the percentage of vials from which influenza NP-binding antibodies were obtained. Three sets of samples from individual D1 from two time points were analyzed. N is the number of tubes that gave rise to HC/LC sequence pairs. The gene-amplification efficiency varied (squares). The fraction of HC/LC pairs that yielded influenza NP-binding antibodies (circles) decreased with increasing gene amplification frequency.

Mentions: In total, we amplified 180 HC, 177 LCκ and 54 LCλ sequences with intact reading frames. For most HCs, one or, occasionally, two LCs were obtained from the same sample, leading to 167 HC/LC sequence pairs. Forty-seven HC/LC plasmid pairs (28.1%) gave rise to influenza NP-specific antibodies (Fig 2A). The gene amplification efficiency in the different sets of B cell samples was variable and ranged from 59 to 93%. The percentage of HC/LC pairs that gave rise to influenza virus NP-specific antibodies was negatively correlated with the percentage of isolated B cell samples from which gene sequence pairs could be amplified. Thus, a high yield of HC and LC sequences from a given sample set resulted in a low number of functional antibodies from that sample and vice versa. At an amplification efficiency of 59.7% (sample set D1_s1-NP2Z), 48% of the HC and LC yielded NP-binding antibodies (Fig 2B). We conclude from this observation that the lower than expected functional antibody yield was primarily due to mismatching heavy and light chains amplified from different B cells in vials with more than one cell.


High Inter-Individual Diversity of Point Mutations, Insertions, and Deletions in Human Influenza Virus Nucleoprotein-Specific Memory B Cells.

Reiche S, Dwai Y, Bussmann BM, Horn S, Sieg M, Jassoy C - PLoS ONE (2015)

Influenza NP binding and gene amplification efficiency.A) Optical density values obtained in the influenza NP ELISA with the supernatants of cells transfected with pairs of HC and LC sequences from the same B cell vial. B) Association of gene amplification efficiency and the percentage of vials from which influenza NP-binding antibodies were obtained. Three sets of samples from individual D1 from two time points were analyzed. N is the number of tubes that gave rise to HC/LC sequence pairs. The gene-amplification efficiency varied (squares). The fraction of HC/LC pairs that yielded influenza NP-binding antibodies (circles) decreased with increasing gene amplification frequency.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4472751&req=5

pone.0128684.g002: Influenza NP binding and gene amplification efficiency.A) Optical density values obtained in the influenza NP ELISA with the supernatants of cells transfected with pairs of HC and LC sequences from the same B cell vial. B) Association of gene amplification efficiency and the percentage of vials from which influenza NP-binding antibodies were obtained. Three sets of samples from individual D1 from two time points were analyzed. N is the number of tubes that gave rise to HC/LC sequence pairs. The gene-amplification efficiency varied (squares). The fraction of HC/LC pairs that yielded influenza NP-binding antibodies (circles) decreased with increasing gene amplification frequency.
Mentions: In total, we amplified 180 HC, 177 LCκ and 54 LCλ sequences with intact reading frames. For most HCs, one or, occasionally, two LCs were obtained from the same sample, leading to 167 HC/LC sequence pairs. Forty-seven HC/LC plasmid pairs (28.1%) gave rise to influenza NP-specific antibodies (Fig 2A). The gene amplification efficiency in the different sets of B cell samples was variable and ranged from 59 to 93%. The percentage of HC/LC pairs that gave rise to influenza virus NP-specific antibodies was negatively correlated with the percentage of isolated B cell samples from which gene sequence pairs could be amplified. Thus, a high yield of HC and LC sequences from a given sample set resulted in a low number of functional antibodies from that sample and vice versa. At an amplification efficiency of 59.7% (sample set D1_s1-NP2Z), 48% of the HC and LC yielded NP-binding antibodies (Fig 2B). We conclude from this observation that the lower than expected functional antibody yield was primarily due to mismatching heavy and light chains amplified from different B cells in vials with more than one cell.

Bottom Line: We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes.We also found several small groups of clonal relatives that were highly diversified.We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification.

View Article: PubMed Central - PubMed

Affiliation: Institute of Virology, Faculty of Medicine, University of Leipzig, Leipzig, Germany.

ABSTRACT
The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs) originated from 26 and the kappa light chains (LCs) from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4% in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses.

No MeSH data available.


Related in: MedlinePlus