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Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire

View Article: PubMed Central - PubMed

ABSTRACT

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition.

No MeSH data available.


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Naïve IGHV4-39-derived antibodies from naïve B cells.(a) Schematics of naïve IGHV4-39 antibody phage library generation. CD19+ CD27− IgM+ naïve B cells were isolated individually by FACS from 36 donors. Total Ig RNA was converted into cDNA using an IgM specific reverse primers, and then uniquely barcoded IGHV4-39 primers for each donor were used to selectively amplify the IGHV4-39 VH gene from the cDNA. The amplified VH genes were then pooled together and paired with the light chain variable genes from IGKV families 1–4 amplified from the same set of donors to generate the single-chain Fv library. Antibody libraries were then displayed on phage and 4 rounds of panning against recombinant IsdB NEAT1 were performed. (b) Binding characterization of IGHV4-39 encoded naïve NEAT1-binding antibodies from seven different donors. Heavy and light chain germlines usage, CDR-H3 sequence identities and number of variable heavy chain framework nucleotide mutation of the seven NEAT1 binders are shown. The observed single framework mutation in selected clones may have been introduced by the amplification process during library generation. The binding of the parental antibodies and their Y52A/Y53A variants to full-length IsdB was determined by SPR-based biosensor binding analysis at 37 °C. The binding of the parental antibodies to the full-length IsdB Y165R variant was determined by ELISA (n=2).
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f6: Naïve IGHV4-39-derived antibodies from naïve B cells.(a) Schematics of naïve IGHV4-39 antibody phage library generation. CD19+ CD27− IgM+ naïve B cells were isolated individually by FACS from 36 donors. Total Ig RNA was converted into cDNA using an IgM specific reverse primers, and then uniquely barcoded IGHV4-39 primers for each donor were used to selectively amplify the IGHV4-39 VH gene from the cDNA. The amplified VH genes were then pooled together and paired with the light chain variable genes from IGKV families 1–4 amplified from the same set of donors to generate the single-chain Fv library. Antibody libraries were then displayed on phage and 4 rounds of panning against recombinant IsdB NEAT1 were performed. (b) Binding characterization of IGHV4-39 encoded naïve NEAT1-binding antibodies from seven different donors. Heavy and light chain germlines usage, CDR-H3 sequence identities and number of variable heavy chain framework nucleotide mutation of the seven NEAT1 binders are shown. The observed single framework mutation in selected clones may have been introduced by the amplification process during library generation. The binding of the parental antibodies and their Y52A/Y53A variants to full-length IsdB was determined by SPR-based biosensor binding analysis at 37 °C. The binding of the parental antibodies to the full-length IsdB Y165R variant was determined by ELISA (n=2).

Mentions: Next, given that the majority of the IsdB NEAT domain binding was primarily driven by germline-encoded CDR-H2, we investigate if antibodies from naïve B cells can recognize IsdB in a similar manner as the one described above and asked if such antibodies could be found in additional donors. Using individually barcoded IGHV4-39 specific primers, we selectively amplified the IGHV4-39 variable heavy chain gene from the cDNA of sorted CD19+ CD27− IgM+ naïve B cells of 36 individuals; this allowed us to unequivocally match binders with their respective donors. A single-chain Fv (scFv) phage display library was then constructed by pairing the individually barcoded IGHV4-39 VH gene with the pooled naïve IGKV families 1–4 genes from all donors (schematics are shown in Fig. 6a). Sequencing of the starting phage library confirmed that the heavy chain of more than 90% of the clones was encoded by IGHV4-39, with minor contaminations from other IGHV4 family members. After four rounds of panning against IsdB NEAT1 domains, the binding of 960 individual phage clones against IsdB and its variants was evaluated by ELISA. About 90% of the clones showed specific binding to full-length IsdB and IsdB NEAT1 domain. Sequence analysis determined that three of the phage clones with unique CDR-H3 (D14-1, D15-1 and D16-1) represented the majority of the binders (96%); this could be due to their superior affinity as scFv's (not determined) or to a growth bias introduced through the phage amplification process. Despite the presence of these three dominant clones, we were able to isolate a total of 16 clones with unique CDR-H3 sequences from 13 different donors (Supplementary Fig. 17).


Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire
Naïve IGHV4-39-derived antibodies from naïve B cells.(a) Schematics of naïve IGHV4-39 antibody phage library generation. CD19+ CD27− IgM+ naïve B cells were isolated individually by FACS from 36 donors. Total Ig RNA was converted into cDNA using an IgM specific reverse primers, and then uniquely barcoded IGHV4-39 primers for each donor were used to selectively amplify the IGHV4-39 VH gene from the cDNA. The amplified VH genes were then pooled together and paired with the light chain variable genes from IGKV families 1–4 amplified from the same set of donors to generate the single-chain Fv library. Antibody libraries were then displayed on phage and 4 rounds of panning against recombinant IsdB NEAT1 were performed. (b) Binding characterization of IGHV4-39 encoded naïve NEAT1-binding antibodies from seven different donors. Heavy and light chain germlines usage, CDR-H3 sequence identities and number of variable heavy chain framework nucleotide mutation of the seven NEAT1 binders are shown. The observed single framework mutation in selected clones may have been introduced by the amplification process during library generation. The binding of the parental antibodies and their Y52A/Y53A variants to full-length IsdB was determined by SPR-based biosensor binding analysis at 37 °C. The binding of the parental antibodies to the full-length IsdB Y165R variant was determined by ELISA (n=2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120205&req=5

f6: Naïve IGHV4-39-derived antibodies from naïve B cells.(a) Schematics of naïve IGHV4-39 antibody phage library generation. CD19+ CD27− IgM+ naïve B cells were isolated individually by FACS from 36 donors. Total Ig RNA was converted into cDNA using an IgM specific reverse primers, and then uniquely barcoded IGHV4-39 primers for each donor were used to selectively amplify the IGHV4-39 VH gene from the cDNA. The amplified VH genes were then pooled together and paired with the light chain variable genes from IGKV families 1–4 amplified from the same set of donors to generate the single-chain Fv library. Antibody libraries were then displayed on phage and 4 rounds of panning against recombinant IsdB NEAT1 were performed. (b) Binding characterization of IGHV4-39 encoded naïve NEAT1-binding antibodies from seven different donors. Heavy and light chain germlines usage, CDR-H3 sequence identities and number of variable heavy chain framework nucleotide mutation of the seven NEAT1 binders are shown. The observed single framework mutation in selected clones may have been introduced by the amplification process during library generation. The binding of the parental antibodies and their Y52A/Y53A variants to full-length IsdB was determined by SPR-based biosensor binding analysis at 37 °C. The binding of the parental antibodies to the full-length IsdB Y165R variant was determined by ELISA (n=2).
Mentions: Next, given that the majority of the IsdB NEAT domain binding was primarily driven by germline-encoded CDR-H2, we investigate if antibodies from naïve B cells can recognize IsdB in a similar manner as the one described above and asked if such antibodies could be found in additional donors. Using individually barcoded IGHV4-39 specific primers, we selectively amplified the IGHV4-39 variable heavy chain gene from the cDNA of sorted CD19+ CD27− IgM+ naïve B cells of 36 individuals; this allowed us to unequivocally match binders with their respective donors. A single-chain Fv (scFv) phage display library was then constructed by pairing the individually barcoded IGHV4-39 VH gene with the pooled naïve IGKV families 1–4 genes from all donors (schematics are shown in Fig. 6a). Sequencing of the starting phage library confirmed that the heavy chain of more than 90% of the clones was encoded by IGHV4-39, with minor contaminations from other IGHV4 family members. After four rounds of panning against IsdB NEAT1 domains, the binding of 960 individual phage clones against IsdB and its variants was evaluated by ELISA. About 90% of the clones showed specific binding to full-length IsdB and IsdB NEAT1 domain. Sequence analysis determined that three of the phage clones with unique CDR-H3 (D14-1, D15-1 and D16-1) represented the majority of the binders (96%); this could be due to their superior affinity as scFv's (not determined) or to a growth bias introduced through the phage amplification process. Despite the presence of these three dominant clones, we were able to isolate a total of 16 clones with unique CDR-H3 sequences from 13 different donors (Supplementary Fig. 17).

View Article: PubMed Central - PubMed

ABSTRACT

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition.

No MeSH data available.


Related in: MedlinePlus