Exploiting human immune repertoire transgenic mice for protective monoclonal antibodies against antimicrobial resistant Acinetobacter baumannii

Abstract

The use of monoclonal antibodies for the control of drug resistant nosocomial bacteria may alleviate a reliance on broad spectrum antimicrobials for treatment of infection. We identify monoclonal antibodies that may prevent infection caused by carbapenem resistant Acinetobacter baumannii. We use human immune repertoire mice (Kymouse platform mice) as a surrogate for human B cell interrogation to establish an unbiased strategy to probe the antibody-accessible target landscape of clinically relevant A. baumannii. After immunisation of the Kymouse platform mice with A. baumannii derived outer membrane vesicles (OMV) we identify 297 antibodies and analyse 26 of these for functional potential. These antibodies target lipooligosaccharide (OCL1), the Oxa-23 protein, and the KL49 capsular polysaccharide. We identify a single monoclonal antibody (mAb1416) recognising KL49 capsular polysaccharide to demonstrate prophylactic in vivo protection against a carbapenem resistant A. baumannii lineage associated with neonatal sepsis mortality in Asia. Our end-to-end approach identifies functional monoclonal antibodies with prophylactic potential against major lineages of drug resistant bacteria accounting for phylogenetic diversity and clinical relevance without existing knowledge of a specific target antigen. Such an approach might be scaled for a additional clinically important bacterial pathogens in the post-antimicrobial era.

Fig. 1. Polyclonal reactivity of Kymouse platform mouse serum raised against OMVs generated from phylogenetically selected A. baumannii.

a Phylogenetic relationship between BAL 084, BAL 191, BAL 215 and BAL 276 and the publicly available CC2, CC1-typed and reference isolates available from culture collections (ATCC and NCTC). The phylogeny was built using whole genome sequence data from the calling of 57844 single nucleotide polymorphisms from 2879 core genes using RAxML (Randomised Axelerated Maximum Likelihood- v8.12.8) using a general time reversible (GTR) model of nucleotide substitution (ASC-GTRGAMMA) and a “Lewis” method of ascertainment bias correction. One hundred bootstrap pseudo-replicate analysis was applied to measure the robustness of the ML phylogeny. The numbers on the tree branches indicate the bootstrap support values while the scale bar reveals the number of nucleotide substitutions per site. The coloured circles at the node tips represent the different capsule types (KLs). b Relative antibody titre measured by indirect ELISA of sera from Kymouse platform mice (n = 5; n defined as serum analysed from a single mouse) immunised with pooled OMVs generated from native, genetically unmodified Acinetobacter baumannii clinical isolates (nOMV) versus sera from 5 non-immunised Kymouse platform mice. c Relative antibody titre similarly measured by indirect ELISA against separate nOMVs generated from phylogenetically related individual isolates not contained in the pool used for immunisation. d Relative titres of human IgG1-4 and IgM subclasses represented in Kymouse platform mouse sera (n = 5; n defined as serum analysed from a single mouse) versus sera from 5 non-immunised Kymouse platform mice against pooled nOMVs used for immunisation. Data for (b, c, d) are presented as mean values ± SEM.

Fig. 2. Antibody sequence repertoire analysis from OMV-immunised Kymouse platform mice.

a A sunspot plot showing antibody sequence clustering, where spot colour indicates the individual mouse immunised with pooled OMVs from which the antibody sequence originated (n = 4). Individual mice (indicated by nominative code identifier in the index) are represented by colour-matched code in the index. b Sunspot plot showing antibody sequence clustering based on an individual germline IGHV and IGHJ combination utilised. Germline IGHV and IGHJ combination utilised are represented by colour-matched code in the index. c Plot of signal from indirect human IgG1 ELISAs against the OMV cocktail (x-axis) versus unfixed pooled bacteria from which the immunising OMVs were derived (y-axis). d Deposition of the C3b protein on OMVs as a surrogate for primary initiation of the complement cascade (x-axis) versus indirect human IgG1 ELISAs against unfixed pooled bacteria from which the immunising OMVs were derived (y-axis).

Fig. 3. Analysis of mAb binding by HCI to a curated collection of CC92 A. baumannii strains isolated in Vietnam between 2003 and 2018.

Individual bacteria were stained and segmented using DAPI which stains intracellular bacterial DNA (a), antibody binding was measured by the indirect signal from binding human IgG1 mAb using an Alexa Fluor 647 labelled secondary anti-human IgG1 (b) overlay of both signals (c). d Bacterial surface mAb binding at 1 μg/ml across the entire phylogenetically characterised CC92 collection. Binding was defined as positive where the Alexa Fluor 647 mean intensity per well was >500 relative fluorescent units (RFU).

Fig. 4. Functional binding of mAbs targeting LOS (OCL-1), Oxa-23 and KL49 typed capsule on live A. baumannii.

a Analysis of mAb binding to live bacterial cells by indirect ELISA. Heat map indicates background corrected signal as relative fluorescent units (RSU) from an indirect ELISA evaluating mAb binding against live bacteria at 1 μg/ml. Signal intensity expressed as the mean of independent assay replicates (n = 3). b Ability of selected mAbs at 1 μg/ml and 100 pg/ml concentrations to trigger C3b deposition when binding live BAL 276 bacteria as measured by indirect ELISA targeting C3b. Relative intensity of fluorescent signal shows mean, and SEM (n = 4; n is a single biological replicate for each bacterial isolate analysed in a single experiment). Statistical analysis was performed using Dunnett’s one-way ANOVA to compare multiple groups with p shown as a numerical value when p ≤ 0.05. c Transmission electron microscopy of ultrathin sections of KL49 producing strain CM10420 and (d) KL-3 producing strain ATCC1798, labelled with mAb 1416 and gold-conjugated anti-human IgG. Labelling via mAb binding to KL49 was observed for CM10420 in (c), but not for ATCC17978. e Uptake of strain CM10420 by THP-1 cells and the alternative KL49 expressing strain BAL 186, in the presence or absence of mAb 1416 as measured by High Content Imaging (n = 6; n is a single biological replicate for each bacterial isolate analysed in a single experiment). In both cases percentage THP-1 cells per well analysed containing bacteria increased in the presence of mAb 1416. Bar represents median with interquartile range. Statistical analysis was performed using an ordinary one-way ANOVA with p shown as a numerical value when p ≤ 0.05.

Fig. 5. Ability of mAbs to protect in vivo against A. baumannii BAL 191.

Adult BALB/c mice were intravenously dosed with 10 mg/kg of 1042, 1349, isotype control mAb or left untreated on day -1 and day 0, challenged on day 0 with 5 × 10⁷ CFU A. baumannii isolate BAL 191 and culled 24 h later. Bacterial loads at 24 h in lungs (a), nasal wash (b) and spleen (c) were enumerated to evaluate protection. In a subsequent study, mice intravenously or intranasally dosed with 10 mg/kg of 1416 or isotype control were similarly evaluated for protection using the KL49 isolate BAL 191. Bacterial loads at 24 h in lungs and spleen of intravenously dosed (d, e, f) and intranasally dosed mice (g, h, i) were evaluated. Statistical analysis was performed using a Mann–Whitney test with p shown as a numerical value when p ≤ 0.05. The data presented in (a–c) are a single study (n = 5; n defined as an individual mouse); the data presented in (d–f) represent 2 experiments combined (n = 5 per study, n = 10 total); (g–i) represent a single n = 5 study. Bar represents mean value for each data set.

Fig. 6. Ability of mAbs to protect mice against neonatal sepsis causing strains of A. baumannii.

a Temporal phylogenetic characterisation of carbapenem-resistant A. baumannii strains isolates from Vietnamese healthcare settings between 2003 and 2018. Phylogeny was built using whole genome sequence data from the calling of single nucleotide polymorphisms from core genes using RAxML (Randomised Axelerated Maximum Likelihood). b Adult C57BL6 mice were intravenously dosed with 10, 5 or 1 mg/kg of 1416 and 10 mg/kg isotype control mAb on day -1 and day 0, challenged on day 0 with 5 × 10⁷ CFU A. baumannii isolate CM10420. Bacterial loads at 24 h in lungs were enumerated as CFU/gram of lung tissue (CFU/g) to evaluate protection. Statistical analysis was performed using a Mann–Whitney test with p shown as a numerical value when p ≤ 0.05. The data presented are from a single study (n = 5; n defined as an individual mouse). c Sunspot plot showing antibody sequence clustering for different bacterial antigen targets. Colour of symbol in index indicates target bound by individual mAb. Symbol shape in index indicates individual mouse from which corresponding VH/VL sequence pair was derived (indicated by nominative code identifier in the index). The mAb 1416 is indicated in a large cluster (indicated by enclosed region with blue Asterix).