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We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T(m)) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T(m) compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.

Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT(+) serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with Kd ∼ 10(-8)-10(-10) M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (5%) of peripheral blood plasmablast clonotypes (CD3(-)CD14(-)CD19(+)CD27(++)CD38(++)CD20(-)TT(+)) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the Kd). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.

Scientific Reports 5: Article number: 13926; published online: 10 September 2015; updated: 10 June 2016 Hidetaka Tanno and Chang-Han Lee were omitted from the author list in the original version of this Article. This has been corrected in the PDF and HTML versions of the Article. The Author Contributions section now reads

Nat. Immunol.; doi:10.1038/ni.3770; corrected online 27 June 2017 In the version of this article initially published online, the labels identifying each plot in Figure 1b were missing. The labels are as follows (left to right): CHO, FcγRI, FcγRIIaR131, FcγRIIaH131, FcγRIIb, FcγRIIIaF158 and FcγRIIIaV158.