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. 2023 May 10;9(6):1190–1195. doi: 10.1021/acsinfecdis.3c00024

Generation of Synthetic Acinetobacter baumannii-Specific Nanobodies

Gregory A Knauf †, Kyra E Groover †, Angela C O’Donnell, Bryan W Davies †,‡,*
PMCID: PMC10262196  PMID: 37162304

Abstract

graphic file with name id3c00024_0004.jpg

The bacterial pathogen Acinetobacter baumannii is a leading cause of drug-resistant infections. Here, we investigated the potential of developing nanobodies that can recognize A. baumannii over other Gram-negative bacteria. Through generation and panning of a synthetic nanobody library, we identified several potential lead candidates. We demonstrate how incorporation of next-generation sequencing analysis can aid in the selection of lead candidate nanobodies. Using monoclonal phage display, we validated the binding of lead nanobodies to A. baumannii. Subsequent purification and biochemical characterization revealed one particularly robust nanobody that specifically bound select A. baumannii strains compared to other common drug-resistant pathogens. These findings support the potential for nanobodies to selectively target A. baumannii and the identification of lead candidates for future investigation.

Keywords: Acinetobacter baumannii, drug resistance, nanobodies, phage display, sequencing

Acinetobacter baumannii is a leading cause of drug-resistant bacterial disease. Its ability to quickly acquire antibiotic resistance has led to increasing numbers of multi-drug-resistant (MDR) and pan-drug-resistant infections.1−3A. baumannii has a remarkable ability to colonize medical equipment and patients, which facilitates its spread.4−7 Agents that can selectively bind A. baumannii could aid in the development of detection reagents, diagnostics, and targeted treatments to help improve clinical surveillance and therapy. Antibodies have been developed to bind different molecular components of A. baumannii. They have shown promise as stand-alone treatments, combination therapies, and detection agents.8−13 Antibody fragments have been less explored but may offer benefits including improved access to buried epitopes, simple development of fusion proteins and materials, and economical routes to production. Among antibody fragments, variable domains of heavy-chain-only antibodies, VHH (or nanobodies), are valued for their stability and adaptability for use in an array of diagnostic and treatment platforms.14−17 Nanobodies have been developed to target a specific A. baumannii protein,18 but their ability to selectively bind A. baumannii over other bacteria has not been generally investigated. Here we describe our investigation of nanobodies that selectively bind A. baumannii over other common Gram-negative bacterial pathogens.

To explore the potential for nanobodies to selectively bind A. baumannii, we developed and panned a synthetic nanobody phage display library. We panned against whole A. baumannii in a sessile community form, which we hypothesized would resemble the bacterium during colonization/contamination of tissue and medical equipment. We selected nanobodies against whole bacteria since we did not know a priori which molecular targets will prove to be the best for selective bacterial binding.

Our library design was inspired by the sdAbD10 nanobody scaffold and complementarity determining region (CDR) design from Moutel et al. (Figure 1A).19 This synthetic design produces robust nanobodies that can bind to a range of biological targets and is well suited for production in bacteria. CDR composition was biased to partially resemble naturally observed repertoires and reduce the number of poorly folded nanobodies that can abound in synthetic libraries.19 In our library design, CDR 1 and CDR 2 were 7 amino acids long and CDR 3 was 9 amino acids long. Following construction, cloning, and diagnostic sequencing we estimated diversity of the library to be approximately 7 × 107.

Figure 1.

Figure 1

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A. Schematic of the VHH scaffold used on top with the amino acid sequence below. Blue represents the framework region and red corresponds to CDR regions. The Xs in the amino acid sequence represent randomized CDR amino acids based on the Moutel et al. design. B. Flow diagram of phage panning approach to identify A. baumannii-specific nanobodies.

Our scheme to identify A. baumannii-specific nanobodies is shown in Figure 1B. In round 1, we panned against A. baumannii AB5075, which is a well-studied and clinically relevant MDR strain.20 To generate a sessile state, we grew AB5075 in 96-well plates in rich medium overnight. The medium was removed, and the bacteria were blocked with non-fat dry milk. Our phage library was first panned against an empty plate well to remove non-specific binders, then applied to AB5075-containing wells. Following washing and elution, the bound phages were amplified for a second round of panning.

In round 2 of phage panning, we counter selected against an empty well and then against the Gram-negative bacteria Vibrio cholerae C6706, a member of a different Order of bacteria (Vibrionales). The goal for using V. cholerae was to eliminate nanobodies that bound generically to Gram-negative bacterial features. V. cholerae is also not an M13 phage host. Following these counter selections, unbound phages were again panned against A. baumannii AB5075, washed, and eluted.

In round 3 of panning, phages eluted in round 2 were again counter selected against an empty well and then against a second control Gram-negative bacteria, Pseudomonas aeruginosa PAO1. P. aeruginosa is more closely related to A. baumannii than V. cholerae, and we hypothesized that it would help further eliminate nanobodies with general affinities for Gram-negative bacterial targets. Unbound phages were then panned against a second well-studied MDR A. baumannii strain AYE.21 We hypothesized that changing this panning step to AYE from AB5075 would help identify broader A. baumannii-binding nanobodies. Phages bound to AYE were eluted and used to infect Escherichia coli. Following plating and selection for E. coli containing a phagemid, 15 colonies were picked at random for sequencing.

In parallel with the A. baumannii panning campaign, and starting with the same input phage library, two rounds of panning were performed against green fluorescent protein (GFP). We also eluted and retained the phage bound to V. cholerae in round 2 described above. The phage pools of V. cholerae and GFP-bound phage from round 2 served as controls to improve down-selection of A. baumannii-specific binding nanobodies as described below.

Multiple rounds of phage panning can help down-select a library to potential leads, but it can also promote the dominance of a few clones that out-replicate the pool. To manage this potential issue, we selected putative A. baumannii-specific nanobodies by two means. We hypothesized that putative A. baumannii-binding nanobodies from round 2 of panning would be represented by a large sequence space. As such, phagemid libraries from round 2 of panning binding to A. baumannii, GFP, and V. cholerae were sequenced using Illumina technology. Sequences were translated to determine CDR compositions for each nanobody. The sequence results for A. baumannii-binding nanobodies were compared to GFP and V. cholerae-binding nanobodies. Nanobodies with CDRs shared between A. baumannii and GFP pools or A. baumannii and V. cholerae pools were removed as they likely represented non-specific sequences. Putative A. baumannii-binding nanobodies were then ordered by read count and the top 10 were selected for testing (Table S1, Ng1–10).

We hypothesized that the third round of panning for A. baumannii-binding nanobodies would greatly reduce the sequence pool. As such, we plated the round 3 phagemid library output and picked 15 colonies for Sanger sequencing. Several of the sequences were represented more than once, suggesting the third round of panning had significantly reduced the nanobody sequence pool. From these 15 clones there were 8 unique nanobody sequences. Of these 8 sequences, 4 had identical CDR sequences matching sequences in our GFP-binding nanobody pool, suggesting they would not be specific for A. baumannii. This left 4 putative round 3 A. baumannii-specific binding nanobodies (Table S1, R3_labeled sequences) for testing.

The 14 putative A. baumannii-binding nanobodies (10 from round 2 and 4 from round 3) were assessed using monoclonal phage display. Each nanobody was displayed on M13 phage and separately assayed by ELISA for binding to A. baumannii AB5075 or GFP under the conditions used for panning (Figure 2A). Our results showed that 11 of the 14 displayed nanobodies showed significant selectivity for AB5075 over GFP.

Figure 2.

Figure 2

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Validation of nanobody leads. A. Monoclonal phage whole-cell ELISA results for the 14 lead nanobodies. Nanobody binding to A. baumannii was assessed by comparison with binding to the control antigen GFP. Error bars represent the standard deviation of three biological replicates. Significance in binding to AB5075 over GFP was determined by unpaired t tests (*p < 0.05). B. Coomassie-stained gel of size exclusion purified nanobodies and a protein standard with the 14 kDa band of the ladder labeled. The expected size of nanobodies from our library is ∼14 kDa. C. Relative binding of purified nanobodies R3_3, Ng2, Ng3, and Ng8 at 5 μM to AB5075 by whole-cell ELISA. Each nanobody signal was normalized and compared to to the background binding of secondary antibody alone. Significance of binding was determined by one-way ANOVA with Dunnet’s multiple comparisons (****p < 0.0001). Error bars represent the standard deviation.

We next sought to purify each nanobody for further testing. Purification was carried out using His-tagged constructs expressed from pET28a in SHuffle T7 E. coli.22 This expression strain improves disulfide bond formation in the cytoplasm. Nanobodies R3_3, Ng2, Ng3, and Ng8 showed selectivity for AB5075 in monoclonal phage display (Figure 2A), expressed well in SHuffle T7, and were chosen for additional analysis. Using immobilized metal affinity chromatography and size exclusion chromatography, we purified mg quantities of each nanobody to high purity (Figure 2B).

We assayed the binding of each purified nanobody at 5 μM to A. baumannii AB5075 by ELISA using the same conditions described for panning (Figure 2C). At a concentration of 5 μM, nanobody R3_3 exhibited a substantially greater signal than Ng2, Ng3, or Ng8. This was expected, since R3_3 exhibited a stronger signal in the monoclonal phage display ELISA as well (Figure 2A). Given these results we proceeded with further characterization of nanobody R3_3.

R3_3 is derived from our synthetic library. Additional optimization of its CDR sequences will likely enable stronger binding to A. baumannii and may improve other qualities including stability and purification yield. Nevertheless, we performed an initial characterization to better understand R3_3 A. baumannii specificity and qualities that could be targeted for improvement. Thermostability is often considered a key metric for assessing the utility of proteins outside of the laboratory setting. We performed a thermal shift assay to develop a melt curve for purified R3_3 and found that it had a melting temperature of 69 °C (Figure S1). This Tm is in line with previously described well-behaved nanobodies,23−25 suggesting that R3_3 exhibits promising stability.

We assessed binding of R3_3 to additional A. baumannii strains (Table S2) and three other Gram-negative bacterial pathogens (Figure 3B). These include well-studied isolates AYE, 19606, 17978, ACICU, and SDF, and recent clinical MDR strains from the CDC AMR Isolates Bank,26 representing different lineages and capsule locus types. Non-A. baumannnii strains included Gram-negative ESKAPE pathogens Enterobacter cloacae, Klebsiella pneumoniae, and P. aeruginosa. Binding was assayed at 5 μM of R3_3. At this concentration we observed R3_3 binding to several A. baumannii strains. However, only A. baumannii 5075, AYE, and CDC 275 bound R3_3 significantly more than all three control non-A. baumannii strains (Figure 3A). Thus, while R3_3 shows selectivity for A. baumannii, it does not act as a universal A. baumannii-binding nanobody.

Figure 3.

Figure 3

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A. Relative binding of R3_3 to A. baumannii and non-baumannii strains. Nanobodies signals were normalized to R3_3 binding to AB5075. Significance was assessed via a one-way ANOVA with Tukey test. Each A. baumannii strain designated as significant was determined to be significantly different from each non-baumannii strain (*p < 0.0001). Error bars represent the standard deviation. B. Binding of R3_3 to AB5075 and isogenic itrA1::Tn capsule mutant. Significance was determined by unpaired t test (* p < 0.05). Error bars represent the standard deviation.

The antigen for R3_3 is unknown. Since capsule is a common antibody target, we tested if R3_3 binding varied between AB5075 and the isogenic capsule transposon mutant itrA1(27,28) (Figure 3B). Interestingly, we found that R3_3 binding slightly increased against the itrA1 mutant, suggesting capsule may mask the target antigen. This also indicates that R3_3 does not target capsule, which may explain why it can bind both AB5075 and AYE, which are different capsule locus types (Table S2). Phage panning was conducted against both AB5075 and AYE (Figure 1), suggesting that binding to a common epitope was selected for during screening. R3_3 was selected to bind A. baumannii in a sessile state. Thus, in addition to cell envelope proteins or lipids, R3_3 may bind secreted extracellular factors such as the biofilm polysaccharides of A. baumannii strains. Further evaluation of the specificity of R3_3 for A. baumannii compared to other bacteria and eukaryotes is also warranted.

Several studies have explored the potential of full-length antibodies to manage A. baumannii infections.8−13,29,30 These studies have shown promise in preventing infection, but also show the challenge of identifying broadly neutralizing monoclonal antibodies. The variable binding to different A. baumannii strains (Figure 3B) will limit the use of R3_3 for possible diagnostic or therapeutic purposes. Furthermore, there are no direct immune benefits for immune recognition of nanobody binding alone. Affinity maturation of R3_3 may broaden its potential to bind more diverse A. baumannii strains but will require identification of its target first. The small size and unique CDR structures available to nanobodies may allow them to access unique epitopes that are unavailable to full-length antibodies.

While R3_3 showed the strongest binding to A. baumannii compared to the other tested nanobodies (Ng2, Ng3, Ng8), this difference cannot be immediately ascribed to a better affinity for its antigen. Binding differences could be a result of differences in the amount of antigen produced or antigen accessibility. Further investigation of additional nanobodies discovered here (Figure 2A) could prove valuable to uncover additional components of A. baumannii that can be targets for species-specific nanobody binding. Our results further support the use of next-generation sequencing for aiding in identification of nanobodies. While we did not proceed with A. baumannii round 2 nanobodies, the sequencing results from GFP panning helped to down-select leads in round 3. In conclusion we have identified a lead nanobody, R3_3, that can selectively recognize certain A. baumannii strains. R3_3 may serve as a starting point for the development of an A. baumannii-specific nanobody for detection and therapeutic development.

Methods

Library Construction and Phage Panning

The nanobody library was based on the design of Moutel et al.’s 2016 library using trimer synthesis.19 The G3P attachment protein was used for display on M13 Phage. Phage libraries were produced and suspended in PBS prior to panning using M13KO7 Helper Phage from New England Biolabs (catalog no. N0315S). Panning was conducted as follows and performed in triplicate at each step. A. baumannii was grown overnight in LB in a 96-well polystyrene plate wells. GFP at 1 μg/mL in PBS was incubated overnight to absorb the polystyrene wells. The wells were then blocked at room temperature using PBS–tween (PBST, 0.1%) plus 5% non-fat dry milk. The phage library was added 1:1 to PBST plus 10% milk to reach a final milk concentration of 5%. The phage–milk mixture was then incubated in non-blocked wells for 1 h at room temperature to assist in removing non-specific nanobodies. Following blocking, wells for panning were washed with PBST three times. The phages were then transferred to the washed wells and incubated at room temperature for 1.5 h. The bound phages were washed 5 times with PBST before acid elution. The eluted phages from the triplicate wells for each target were neutralized, combined, and used to infect E. coli to amplify the phagemid library for the next round. The output phages from each round were titered to determine the efficiency of the washing and quality of the library.

For rounds 2 and 3, an additional step was added prior to binding phage to A. baumannii to select for specific nanobodies. In these rounds, phages were removed from the unblocked wells and incubated in blocked wells containing Vibrio cholerae C6706 (round 2) or Pseudomonas aeruginosa PAO1 (round 3) as a counter selection. The unbound phages from these wells were then transferred to A. baumannii wells and the round was completed as described above.

Nanobody Identification

Output phage libraries were sequenced from round 2 using MiSeq v3 PE 300. The resulting sequences were tallied based on nucleotide sequence. Nucleotide sequences identified only once were discarded as possible sequencing errors. The reads were translated into an amino acid sequence to acquire CDR sequences. CDR sequences were compared between A. baumannii, GFP, and V. cholerae sets to identify A. baumannii-specific nanobodies. Random picking of output colonies was used to collect nanobodies from round 3. Sixteen colonies were picked, resulting in 8 unique nanobodies being identified.

ELISA

Monoclonal phage was produced for ELISAs. Target bacteria were grown as above in 96-well plates. The culture was removed, and the wells were blocked with PBST plus milk for 2 h at room temperature. Following blocking, phage in PBST milk mixture (5% milk final concentration) was added, incubated, and washed five times with PBST. HRP anti-M13 phage antibody (ab50370) was added at a dilution of 1:100 in PBST plus 5% milk. This was incubated for 45 min before washing five times. TMB-ELISA substrate solution (ThermoFischer, 34029) was added to each well and incubated for 20 min before measuring optical absorbance at 652 nm. All assays were performed in biological triplicate.

The purified nanobody ELISAs were performed using the above method with the following changes. After blocking with 10% milk PBST the wells were washed once with PBST. Purified nanobodies were added at 5 μM, incubated for 45 min, and washed 5 times with PBST after incubation. An anti-nanobody HRP antibody (Genescript, A01861) was used for nanobody detection at a 1:2000 dilution. After incubation with the antibody and washing, TMB-ELISA substrate was added to the wells, allowed to develop, and then stopped with 2 M H2SO4 following the manufacturer’s instructions. The plate was then read at 450 nm. All assays were performed in at least two rounds of biological triplicate.

Protein Purification

Protein purification was carried out using Shuffle T7 Express E. coli (NEB) to allow for disulfide formation. We used the pET28a expression vector. 400 mL cultures were incubated at 30 °C with shaking until an approximate OD600 of 0.5 and induced with 0.1 mM IPTG for 6 h. After 6 h, the culture was pelleted and the bacteria were frozen at −80 °C. After freezing, the pellet was suspended on ice in phosphate buffer (20 mM NaH2PO4, 500 mM NaCl, 25 mM imidazole, EDTA free protease inhibitor, pH 7.4). The cells were lysed by sonication and the cellular debris was pelleted. The nanobodies were purified from the supernatant by Ni-NTA resin. The eluted samples containing the nanobodies were then passaged on a size exclusion column (Superdex 75 Increase 10/300 gl) using an Akta Pure FPLC and fractionated to obtain pure nanobody.

Thermal Shift Assay

For the thermal shift assay we used the Applied Biosciences Protein Thermal Shift Dye Kit (catalog no. 4461146) according to manufacturer guidelines at a concentration of 25 μM.

Acknowledgments

B.W.D. is supported by the NIH (R01 AI148419, R21 AI159203), DTRA (HDTRA1-17-C0008), and Tito’s Handmade Vodka. Figure 1 and the TOC graphic were generated using BioRender.com.

Data Availability Statement

All nanobody sequencing data has been deposited at the NCBI under the GEO accession GSE202972.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.3c00024.

  • Table S1, sequences of nanobody CDRs; Table S2, bacteria strains used in this study and their characteristics; Figure S1, R3_3 thermal denaturation curve (PDF)

Author Contributions

# G.A.K. and K.E.G. contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

id3c00024_si_001.pdf (186.7KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

id3c00024_si_001.pdf (186.7KB, pdf)

Data Availability Statement

All nanobody sequencing data has been deposited at the NCBI under the GEO accession GSE202972.

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