Ethics and permissionsAll experiments were conducted under approved licenses and protocols, consistent with national (as authorised by the Home Office of the United Kingdom) regulations and University of Oxford guidelines.Ex vivo modification of protein with β-lactam antibioticsCarrier proteins (HEL, OVA, HSA, BSA and MSA) were purchased commercially (Merck) and dissolved in 0.1 M NaCO3 (pH = 8, unless otherwise indicated) and concentration was adjusted to 1 mg/mL. β-lactam was added to a molar ratio of 1:200 per lysine residue of carrier protein, resulting in protein concentrations for HEL and HSA of 6.8 and 60 mg/mL respectively. The mixture was rotated end-to-end at 25 °C overnight and dialysed into PBS.Denaturing MSReversed-phase chromatography was performed in-line prior to mass spectrometry using an Agilent 1100 HPLC system (Agilent Technologies inc.—Palo Alto, CA, USA). Concentrated protein samples were diluted to 0.02 mg/ml in 0.1% formic acid and 50 µl was injected on to a 2.1 mm ×12.5 mm Zorbax 5um 300SB-C3 guard column housed in a column oven set at 40 °C. The solvent system used consisted of 0.1% formic acid in ultra-high purity water (Millipore) (solvent A) and 0.1 % formic acid in methanol (LC-MS grade, Chromasolve) (solvent B). Chromatography was performed as follows: Initial conditions were 90% A and 10% B and a flow rate of 1.0 mL/min. After 15 s at 10% B, a two-stage linear gradient from 10% B to 80% B was applied, over 45 s and then from 80% B to 95% B over 3 s. Elution then proceeded isocratically at 95% B for 1 mins 12 s followed by equilibration at initial conditions for a further 45 s. Protein intact mass was determined using a 1969 MSD-ToF electrospray ionisation orthogonal time-of-flight mass spectrometer (Agilent Technologies Inc.—Palo Alto, CA, USA). The instrument was configured with the standard ESI source and operated in positive ion mode. The ion source was operated with the capillary voltage at 4000 V, nebulizer pressure at 60 psig, drying gas at 350 °C and drying gas flow rate at 12 L/min. The instrument ion optic voltages were as follows: fragmentor 250 V, skimmer 60 V and octopole RF 250 V. Obtained MS spectra were processed and deconvoluted using the Agilent MassHunter Qualitative Analysis (B.07.00) software.LC-MS/MSApproximately 5 µg protein was reduced, loaded and run on an SDS-PAGE. Gel bands were excised and washed sequentially with HPLC grade water followed by 1:1 (v/v) MeCN/H2O. Gel bands were dried (via vacuum centrifuge), treated with 10 mM dithiothreitol (DTT) in 100 mM NH4HCO3 and incubated for 45 min at 56 °C with shaking. DTT was removed and 55 mM iodoacetamide (in 100 mM NH4HCO3) was added and incubated for 30 min in the dark. All liquid was removed and gels were washed with 100 mM NH4HCO3/MeCN as above. Gels were dried and 12.5 ng/µL trypsin was added separately and incubated overnight at 37 °C. Samples were then washed and peptides were extracted and pooled with sequential washes with 5% (v/v) formic acid (FA) in H2O and MeCN. Dried samples were reconstituted in 2% MeCN 0.05% trifluoroacetic acid and run by LC-MS.Samples were analysed using an Ultimate 3000 UHPLC coupled to an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific). Peptides were loaded onto a 75 µm × 2 cm pre-column and separated on a 75 µm × 15 cm Pepmap C18 analytical column (Thermo Fisher Scientific). Buffer A was 0.1% FA in H2O and buffer B was 0.1% FA in 80% MeCN with 20% H2O. A 40-min linear gradient (0% to 40% buffer B) was used. A universal HCD identification method was used. Data was collected in data-dependent acquisition mode with a mass range 375 to 1500 m/z and at a resolution of 70,000. For MS/MS scans, stepped HCD normalized energy was set to 27, 30 and 33% with orbitrap detection at a resolution of 35,000.Raw data was first searched using the FragPipe (v19.1)59 Open Search pipeline to determine the modified mass shift caused by PenG conjugation. Approximately 2.3% of peptide spectral matches (PSM) had an unannotated mass shift of 334.099 Da. Next, to determine site specificity and occupancy, raw data was searched using Proteome Discoverer (3.0.0.757). In-house curated FASTA databases were used. The digestion enzyme was set to trypsin with a maximum of 2 miss cleavages. A 10 ppm precursor mass tolerance and 0.6 Da fragment mass tolerance were allowed. Oxidation (+15.995 Da) of methionine and PenG conjugation (+334.099 Da) of lysines and protein N-termini were set to dynamic modifications. Carbamidomethylation (+57.021 Da) of cysteines was set as a static modification. Target false discovery rate (FDR) for peptide spectrum matches, peptide and protein identification was set to 1%. To approximate site-specific occupancy of PenG conjugation the total number of PSMs of peptides containing a specific lysine site in a PenG modified state were expressed as a percentage compared to the total number of PSMs containing the given lysine in both the modified and unmodified state.To estimate lysine reactivity in HSA, raw data was searched against a database of known contaminants which contains the canonical HSA sequence using FragPipe (21.1) using a standard closed search parameters with an additional variable modification confined to lysines (+334.099). IonQuant (as implemented in FragPipe) was used to calculate peptide intensities with default parameters. Intensity of each modification-specific peptide was normalised against total intensity of all HSA peptides in treated and control samples. For each position, a ratio of intensity of unmodified peptide (without missed cleavages, as present in control sample) between the treated sample and the control was calculated to estimate the extent of lysine modification at that position after treatment.LC-MS/MS raw data and search results have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository60 with the dataset identifier: PXD052026.Free primary amine ELISAAfter the drugs were conjugated to a carrier protein using the method outlined above, the relative abundance of free amines was assessed to determine the extent of lysine modification. Protein samples (5 μg) were dissolved in 10 μL of PBS and mixed with 40 μL of 0.1 M sodium bicarbonate buffer. A 5% solution of 2,4,6-trinitrobenzenesulfonic acid (TNBSA) was diluted at a ratio of 1:500 in the bicarbonate buffer, and 25 μL of this mixture was added to the protein samples. Following a 2 h incubation period at 37 °C, 25 μL of 10% SDS and 12.5 μL of 1 M HCl were added. The absorbance at 335 nm was then measured.Mice and immunisation formulationsWild-type, specific pathogen-free, sex-matched, 6–8-week-old C57BL/6 mice were purchased from Charles River. Animals were monitored daily and were provided standard chow and water ad libitum. Immunisation formulations and schedules are outlined in the results. Mice were bled periodically from the tail vein. Animals were sacrificed via a rising CO2 gradient and subsequent cervical dislocation schedule 1 procedure.ELISASerum samples were serially diluted and transferred onto an antigen-coated and blocked SpectraPlate-96 (PerkinElmer) plate. Binding was detected with an anti-mouse IgG-HRP (STAR120P, Bio-Rad). ELISAs were developed using 1-Step-Ultra TMB ELISA substrate (Life Technologies), terminating the reaction with 0.5 M H2SO4. For competition ELISAs, serial dilution of soluble ligands as preincubated with antiserum at the pre-determined EC50 concentration for 1 h. The antisera and ligand mixtures were subsequently transferred onto the antigen-coated and blocked plates and ELISA conducted, as previously outlined. Cytokine ELISAs were performed using commercially available kits (Life Technologies), screening supernatant from antigen-restimulated splenocytes.Optical densities were measured at 450 and 570 nm on a Spectramax M5 plate reader (Molecular Devices). After background subtraction, logistic dose-response curves were fitted in GraphPad Prism. Endpoint titres were determined as the point at which the best-fit curve reached an OD450-570 value of 0.01, a value which was always > 2 standard deviations above background.B cell sortingPenicilloyl-specific B cells were isolated using antigen probes. Gp120-PenG was modified with an NHS-esterified AF647 dye, as per the manufacturer’s instructions (Life Technologies). To improve true antigen-specific cell sorting efficiency, a negative backbone-specific probe was synthesised, wherein biotinylated gp120 was tetramerised with PE-conjugated strepdavidin (Biolegend).Single cell suspensions were stained with LIVE/DEAD Fixable Blue and Fc receptors blocked. Surface staining was performed using anti-mouse F4/80-PE (1:200, BM8, Biolegend), anti-mouse Gr-1 (1:200, RB6-8C5, Biolegend), anti-mouse CD3-PE (1:200, 17A2, Biolegend), anti-mouse CD4-PE (1:200, RM4-5, Biolegend), anti-mouse CD8-PE (1:200, RPA-T8, Biolegend), anti-mouse B220-eFluor450 (1:100, RA3-6B2, BD Biosciences), anti-mouse IgD-AF700 (1:200, 11-26 c.2a, Biolegend), anti-mouse IgM-PE/Cy7 (1:200, R6-60.2, BD Biosciences), anti-mouse IgG1-FITC (1:200, A85-1, BD Biosciences), anti-mouse IgG2a/2b-FITC (1:200, R2-40, BD Bioscience) and antigen probes (10 μg/mL). Cells were stained on ice for 1 h, washed and stored on a BD FACSAriaFusion (BD Biosciences). Single cells were sorted into MicroAmp Optical 96-well PCR plates (Life Technologies), isolating LIVE/DEAD-DUMP-B220+IgD-gp120-gp120-PenG+ events. Cells were sorted directly into 5 µL if 1X TCK buffer supplemented with 1% 2-ME and stored at −80 °C until use.Thymidine incorporationWhole splenocytes were stimulated in vitro with 10 µg/mL antigen in cRMPI for 16 h in a flat-bottom 96-well plate. During the final 6 h stimulation, each well was spiked with 0.037 mBq tritiated thymidine (Perkin Elmer). Cells were transferred and lysed on glass filter mats (Perkin Elmer) using a Micro 96 Harvester (Skatron Instruments). Tritium incorporation was measured using Betaplate Scint and a Microbeta Trilux Scintilation counter (Perkin Elmer).Intracellular cytokine stainingWhole splenocytes were stimulated in vitro with 10 µg/mL antigen in cRPMI for 16 h. For the final 6 h, 5 µg/mL brefeldin A (Biolegend) was added to suspend ET–Golgi trafficking and block cytokine secretion. Cells were stained with TruStain mouse FcX Plus (Biolegend) and LIVE/DEAD Fixable Blue in PBS with 2 mM EDTA for 30 mins. Surface markers were subsequently stained: PE-conjugated anti-mouse CD3 (dilution: 1:200, clone: 17A2, manufacturer: Biolegend), APC-conjugated anti-mouse CD4 (1:200, RM4-5, Biolegend), AF700-conjugated anti-mouse CD8 (1:200, RPA-T8, Biolegend). Following fixation and permeabilization (Biolegend), cells were stained with PE/DAZZLE-conjugated anti-mouse IFN-γ (1:100, XMG1.2, Biolegend). Cells were washed and data was acquired on the BD Fortessa X-20 (BD Biosciences), collecting 500,000 events per sample.Variable region cloning and antibody expressionB cell receptor variable regions were recovered, as previously described29. Briefly, RNA was captured on RNAClean XP beads (Beckman Coulter) and washed with 70% ethanol. RNA was eluted and cDNA was synthesised using SuperScript III (Life Technologies) with random primers (Life Technologies). VH and VK regions were recovered43 and Q5 polymerase (New England Bioscience), sequencing the amplicons via Sanger. VH amplicon sequences were used to determine B cell clonality.To validate that the sequences were specific to the penicilloyl adducts, antibodies were recombinantly expressed. The VH/VK amplicons were incorporated into expression vectors: vector-overlapping adapters were incorporated via PCR42, and the V regions were inserted into pre-cut recombinant FAb expression44 vector via Gibson reaction (New England Bioscience). Vector products were transiently transfected into HEK 293Freestyle cells and FAb was purified from supernatant using Ni-NTA resin.Immunogenetic analysesAnalyses were performed using VH regions. Sequences were aligned to the murine reference genome using the Immunogenetics Information System (IMGT; https://www.imgt.org/IMGT_vquest/input), as described previously29. Sequence outputs of poor quality or those unproductive were excluded from our analyses. Alignments of CDRs were visualised using WebLogo61. Clonal lineages were evaluated using GCTree62.SPRSPR was performed using a Biacore T200 instrument. Details of chip design, synthesis and testing, refer to Document S1. FAb binding was evaluated by sequentially injecting serial dilutions at a flow rate of 10 µL/min.uSTASamples for uSTA were prepared by buffer-exchange of purified FAb fragments with D2O PBS using Amicon 30 K MWCO. All NMR experiments were conducted on Bruker Avance Neo 600 MHz spectrometer at 25 oC equipped with QCIF cryoprobe and a SampleJet, running TopSpin 4.2.0. The uSTA experiments were recorded using a pseudo-3D pulse sequence based on stddiffesgp.2 from the standard Bruker library as described previously45. The following saturation times were used: 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 1.7, 1.9, 2, 2.5, 3, 3.5, 4 and 5 s. Low-power gaussian excitation pulse was applied at 9 ppm and 37 ppm for the on- and off-resonance spectra respectively, where the specific choice of excitation at 9 ppm minimised artefacts in a ligand-only spectrum45 (Fig. S1). All experiments were recorded with 16 scans per transient, 32768 complex points and sweep width of 16.01 ppm for a total acquisition time of 7 h 41 min. These were acquired on a protein-only, a ligand-only and a mixed protein/ligand sample. Data were processed using the nmrPipe module embedded within the uSTA workflow, where the protein-only data is subtracted from the mixture to give a uSTA transfer efficiently, from which values from the ‘ligand only’ sample are then subtracted to account for residual ligand excitation, as previously reported45. The ‘heatmaps’ were generated by mapping the uSTA transfer efficiency from the detectable protons onto heteroatoms (carbon, sulphur, oxygen and nitrogen) using a 1/r6 dependency, before rendering in pymol as described previously45.X-ray crystallographyMIL-3 FAb was loaded onto a gel filtration Superdex 200 column 10/30 (GE Healthcare) in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl. Co-crystals appeared at 20 C after a week from a hanging drop of 0.1 μL of protein solution (15 mg/mL with 2.5 mM PenG-Lys with 0.1 μL of reservoir solution containing 20% (w/v) PEG 6000, 0.1 M MES pH 6, 0.2 M ammonium chloride in vapor diffusion with reservoir. Crystals were frozen with the same solution containing 16% glycerol. Data were collected at the Diamond light source oxfordshire (beamlines I04). Data were processed with XIA263,64,65,66. Structure has been solved by molecular replacement using PHASER and pdb file 7bh8 for VL, CH and CL domains and 7n09 for VH domain. The structure was builded with Autobuild program, refined with REFINE of PHENIX with NCS restraints67 and adjusted with COOT68. Coordinates and topologies of ligands were generated by AceDRG69.Microbiological assaysThe attenuated, unencapsulated lab strain Streptococcus pneumoniae D39 (Δcps2A’-Δcps2H’)46 was routinely grown in tryptic soy broth (TSB) (BD Biosciences) at 37 °C (standing incubation) in a 5% CO2 atmosphere.Microbroth dilutions of S. pneumoniae D39 revealed a PenG MIC of 0.01 μg/mL (that is, the lowest antibiotic concentration that prevented bacterial growth) (data not shown). For the liquid antibiotic rescue assay, 2 ng of PenG in 10 μL PBS were pre-incubated with 10 μL of antisera for 2 h in a flat-bottom 96-well plate. 180 μL of exponentially growing S. pneumoniae cells (OD600 = 0.2) were added and incubated overnight (such that the final PenG concentration was 0.01 μg/mL). The following day, bacterial growth was measured using a Spectramax M5 plate reader (Molecular Devices), evaluating the OD600 nm as a proxy for bacterial density.For disk diffusion assays, 0.1 μg of PenG and mAb (1:50 molar ratio) were spotted onto paper disks. The disk was placed onto a blood agar plate (Merck), carrying a 5 ml nutrient soft agar overlay with 200 μL exponentially growing S. pneumoniae cells (OD600 = 0.2). Plates were incubated overnight. The following day, the kill zone diameter was manually measured. PenG-only, PenG-raised antibody clones were tested, as well as an irrelevant HIV-1-specific antibodies were tested.Data and statisticsFlow cytometry data was evaluated using FlowJo V.10.8.2 for Mac. Statistical analyses were conducted in either GraphPad Prism V.10.0.1 or in RStudio V.4.1. Statistical test details are provided in the results, figures and associated figure legends.
