Anti-rabies virus immunization in dogs and evaluation of canine serum antibody titers via the immunoperoxidase monolayer assay (IPMA)On day 0, all the serum samples presented the highest antibody titer at a ratio of 1:10, whereas, samples taken on the same day (no. 3, 6, 7, 10, and 12) presented mild antibody responses. Three weeks after the first dose, all the sera except those from groups 3, 6, 10, and 12 presented higher antibody titers than did those from the first immunization at a ratio of 1:100. Three weeks after the second vaccination (day 42), all the samples exhibited a mild antibody response at a ratio of 1:1000 (Fig. S1). Accordingly, 13 dogs received a booster three weeks after the third dose, and blood samples were collected two weeks after this booster.Measurements of serum-neutralizing antibody titers against the rabies virus via the rapid fluorescent foci inhibition test (RFFIT)Individual sera were collected for two weeks after the last booster and tested for neutralizing antibody titers against the rabies challenge virus standard (CVS) strain via the RFFIT assay. The levels of neutralizing antibodies ranged from 17 to 60 IU/ml (Fig. 1), which were higher than the protective level of 0.5 IU/ml. Two dogs showed high antibody responses, which were greater than 50 IU/ml.Fig. 1Serum VNA titer against rabies virus in dogs via the RFFIT assay. All 13 serum samples presented neutralizing antibody levels greater than 0.5 IU/ml. The average titers ranged from 20 to 30 IU/ml. Two outliers presented levels greater than 50 IU/ml.Variable heavy chain (VH) and variable light chain (VL) fragments amplificationPBMCs were separated from all blood samples, and total RNA was extracted. The pooled RNA samples were then converted to cDNA. The synthesized cDNA served as PCR templates for VH and VL fragment amplifications using specific primers for the canine pComb3XSS-scFv antibody (detailed in the Materials and Methods). The sizes of the VH and VL PCR products and the purified products were approximately 450 and 350 bp, respectively (Fig. 2).Fig. 2VH, VLλ, and VLκ amplification via conventional PCR. (a) VLκ amplicons were amplified via sets of forward primers (CSCK1-F, 24-F, 34-F, 4-F, 5-F, and 6-F; numbers 1, 24, 34, 4, 5, and 6 in the figure). Four reverse primers were used: CSCJK1-B, CSCJK2-B, CSCJK3-B, and CSCJK4-B. (b) VLλ amplicons were amplified via sets of forward primers (CSCLam1a-F–CSCLam13-F; numbers 1a, 1b, 1c, 1d, and 2–13 in the figure). CSCJLam1-B and CSCJLam2-B served as reverse primers. The size of the VLκ and VLλ PCR products was 350 bp. (c) VH amplicons were amplified via the forward primer (CSCVH1-F–CSCVH9-F; numbers 1–9 in the figure) and the CSCG1234-B reverse primer. The size of the VH PCR products was 450 bp. (d) Purified variable chain fragments. After the antibody fragments were amplified via PCR, the corrected PCR products were purified via gel extraction (Lane 1, purified VH; Lane 2, purified VLλ; and Lane 3, purified VLκ). The PCR products were run on 1.2% agarose gels.Single-chain variable fragment (scFv) library constructionsSixteen clones from each library were randomly selected. The results revealed a cloning efficiency of 100% (16/16) for the VHVLκ library and 75% (12/16) for the VHVLλ library (Fig. 3). The VHVLκ and VHVLλ libraries consisted of 2.4 × 108 and 1.3 × 106 clones, respectively.Fig. 3Colony PCR of 16 randomly selected clones from the (a) VHVLκ scFv library and (b) VHVLλ scFv library. The inserted scFv antibodies were amplified via second-round PCR primers (RSC-F and RSC-B(AA) primers), and the size of the PCR product was 850–900 bp, as observed on a 1% agarose gel. M refers to the 100 bp DNA ladder.Determination of scFv library diversity via BstNI analysisThe diversity of the constructed libraries was determined via BstNI analysis (Fig. 4). The patterns of the DNA fingerprints of 30 random clones from the VHVLκ and VHVLλ non-panning libraries completely differed across groups.Fig. 4BstNI analysis of the scFv libraries. (a) Fingerprint BstNI digestion of 15 randomly selected clones from the VHVLκ library. (b) Fifteen randomly selected clones from the VHVLλ library.Affinity selection (Biopanning)Purified inactivated rabies virus (RABV) and recombinant glycoprotein of rabies virus (rG) were used as target antigens to screen and obtain scFv clones with binding activity from either the VHVLҡ or VHVLλ library. Figure 5 shows the results of the affinity binding selection for both anti-rG and anti-RABV. The overall input titers of the VHVLҡ and VHVLλ libraries were approximately 1010 – 1011 CFU/ml. Target-bound phages were enriched in the output titer. Titers slightly increased in successive rounds (2nd–4th rounds) of the panning selection procedure for all experiments except for the anti-rG binding outputs of the VHVLκ library.Fig. 5Input and output phage titers (CFU/ml) of the (a) VHVLҡ and (b) VHVLλ libraries. Four selection rounds were performed per target antigen. The results of the affinity selection process are shown in the scatter plots. (a1) (b1) rG binding selection. (a2) (b2) RABV binding selection. The Y-axis shows the power of ten logarithmic scales, the left side indicates the input titers, and the right side indicates the output titers. The gray and black dots indicate the input and output phage titers of each round, respectively.For the VHVLκ anti-rG binding tests, the outputs of the second round were slightly higher than those of the first round. Compared with that of the second round, the third-round output slightly decreased, and the fourth-round output phage titer was 10 times higher than that of the third round. Compared with that in the first round, the target-bound phage titer in the VHVLκ library in the anti-RABV binding tests slightly decreased. The titers tended to increase in the third panning, and the titer increased 10-fold in the fourth output.In the anti-rG binding test of the VHVLλ library, the third output titer was 10 times higher than the second, and the fourth output titer was 10 times higher than the third titer. In the anti-RABV binding test of the VHVLλ library, the titer of the third output phage was 10 times higher than that of the second round, but the titer of the fourth output was similar to that of the third.ELISA screening of individual canine scFv clonesPooled phage clones (TG1) with RABV binding affinity were collected during biopanning; individual clones were subsequently obtained via ELISA. Fifty-eight phage clones from the fourth-round output plates were randomly chosen. Clone numbers 1–19, 20–37, and 38–58 are shown in Fig. 6a–c, respectively. The inactivated RABV binding signals of 33 out of 58 clones (clones no. 1–6, 10–18, 36, 37, 39, 41–46, 49, and 51–58; *) were significantly greater than those of the negative control. Twenty-five unselected clones were excluded from further characterization. The negative control binding signals of 19 clones (clones no. 7, 8, and 19–35; #) were greater than the target binding signals, and the signal levels of the other six clones (9, 38, 40, 47, 48, and 50; ‘NS’ symbol) were not significantly different from those of the negative control.Fig. 6ELISA absorbance signal at 450 nm from soluble fractions of all 37 clones. Each sample consisted of four wells: expression control, RABV, rG, and negative control (SM). (a) Clone numbers 1–19. (b) Clone numbers 20–37. (c) Clone numbers 38–58. Differences between the absorbance levels of the target antigens and SM binding were assessed via independent t tests. The asterisks indicate significant differences as follows: P value ≤ 0.05 (*), ≤ 0.01 (**), and ≤ 0.001 (***). The symbol “#” refers to an excluded sample whose signal from the negative control was greater than or equal to that of the scFv samples. A secondary anti-HA-conjugated HRP antibody bound to the scFv was detected using a TMB substrate. The error bars indicate the SDs of two replicates.Thirty-six soluble scFv antibodies from 58 clones (numbers 1–6, 9–18, 36–46, 49, and 51–58; *) significantly bound to rG. Nineteen clones were unselected (7, 8, and 19–35; #) because the rG binding signals were lower than the SM binding signals, or the inactivated RABV binding signals of some positive rG binding clones were lower than those of the negative control. The antigen binding signals of the other three unselected clones (47, 48, and 50; “NS” symbol) were not significantly different from those of the SM control.Finally, only 20 scFv clones (numbers 1–6, 10–17, 36, 37, 39, 44, 54, and 58) presented either inactivated RABV or recombinant RABV-G binding signals, which were significantly different, with P values ≤ 0.01 (**) or ≤ 0.001 (***), from those of the SM control. ELISA clones no. 1–6, 12, 16, 36, 39, 44, and 54 were from the VH-VLҡ output, whereas clones no. 15, 37, and 58 were from the VH-VLλ output library. All these antibodies were determined, and miniprep plasmid purifications were performed for further DNA sequencing characterizations.DNA sequencing and amino acid translationDNA from 20 ELISA-specific binding clones was sequenced; corrected full-length DNA sequences of canine scFv antibodies (approximately 800–850 bp) were observed for all antibodies except for two clones (nos. 36 and 37) whose 550-bp DNA sequences of VH fragments and linkers were incomplete. The full scFv DNA sequences were further analyzed via IMGT data (IMGT/V-QUEST; https://www.imgt.org/IMGT_vquest/analysis) for Canis lupus familiaris antibodies. Among the 18 sequences, only nine amino acid sequence patterns (named K9RABVscFv1, 2, 12, 15, 16, 39, 44, 54, and 58) were categorized on the basis of combinations of different amino acid sequences in complementarity-determining regions (CDRs)-1, -2, and − 3 of the VL and VH fragments. ELISA clones no. 1, 3, 4, 5, 10, 11, and 13 contained the same CDR amino acid sequences and were named K9RABVscFv1. ELISA clones no. 2, 6, and 17 were K9RABVscFv2. ELISAs revealed that clones no. 12 and 14 were K9RABVscFv12, whereas clones no. 15, 16, 39, 44, and 58 were K9RABVscFv15, K9RABVscFv16, K9RABVscFv39, K9RABVscFv44, and K9RABVscFv58, respectively. We subsequently performed a pilot RFFIT neutralization assay of nine unpurified soluble scFvs in PBS (TG1 E. coli) before performing large-scale protein expression and purification for neutralization testing of the purified scFv (Table S1). Five clones (K9RABVscFv1, K9RABVscFv2, K9RABVscFv12, K9RABVscFv15, and K9RABVscFv16) with superior neutralization ability were selected because their neutralization ability tended to be high. The amino acid sequences and alignments are shown in Table S2 and Fig. 7, respectively. The complementarity-determining regions (CDRs) and frameshift region amino acid sequences of the variable chains are also shown in Fig. 7. IMGT database analysis revealed that the variable light chains of K9RABVscFv clone numbers 1, 2, 12, and 16 were the kappa subtype, whereas only clone number 15 was the lambda subtype. Because of randomized combinations of VH and VL fragments, the CDRs of individual antibodies mostly manifested with different amino acid sequences. However, some CDRs containing patterns similar to those of clones K9RABVscFv1, 2, 15, and 16 shared the same CDR1 amino acid sequences on VL chains (QSLLHSNGNTY). Only one residue on CDR1-VL of K9RABVscFv12 was dissimilar to that of the other clones (QSLLHSDGNTY). CDR2-VL of K9RABVscFv2 and 15 had the same sequence, “QVS,” whereas the “KVS” sequence was observed in K9RABVscFv12 and 16. A unique “AVS” sequence was found in the CDR2-VL of K9RABVscFv1.Fig. 7Amino acid sequence alignments for the five selected K9RABVscFv clones. The complementarity-determining regions (CDRs) of the VL (top) and VH (below) fragments are in black-bordered rectangles.Germline immunoglobulin repertoire characterizationThe immunoglobulin germline repertoires of the five K9RABVscFv clones were investigated via IMGT reference allele databases. Table 1 shows the results for the germline genes and families in the V region of CDR3. VL-VH scFv revealed that pairing of the IGKV2-IGHV3 canine antibody germline family was predominant (4/5 clones; K9RABVscFvs no. 1, 2, 12, and 16), whereas VL-VH pairing of the IGLV2-IGHV3 family was discovered in K9RABVscFv15. Analysis of the V-alleles of VH revealed a predominance of the IGHV3-5*01 allele (clone numbers 1, 2, and 12), followed by IGHV3-6*01 (clone no. 15) and IGHV3-41*01 (clone no. 16). All the scFv clones used the same IGHJ4*01 (VH) and IGKJ1*01 (VL) alleles, except for the IGLJ2*01 allele for K9RABVscFv15.Table 1 Sequence diversity of the five selected K9RABVscFv fragments compared with the ig germline IMGT database.Soluble K9RABVscFv production, purification, and concentrationThe results of the western blot analyses of the concentrated K9RABVscFv clones are presented in Fig. 8. The total protein concentrations of the purified K9RABVscFv clones were measured with a Pierce BCA Protein Assay Kit (Thermo Scientific), and the absorbance at 560 nm was as follows: K9RABVscFv1 (5.35 and 7.35 mg/ml), K9RABVscFv2 (6.12 mg/ml), K9RABVscFv12 (4.08 mg/ml), K9RABVscFv15 (4.30 mg/ml), and K9RABVscFv16 (5.88 and 7.11 mg/ml).Fig. 8SDS‒PAGE and Western blot analysis of the five purified, concentrated, and soluble K9RABVscFv clones. The actual scFv molecular weight was approximately 30–37 kDa (black arrow), and a 60-kDa (white arrow) dimeric form of scFv was observed in four clones. (a) SDS‒PAGE analysis of 5 K9RABVscFv (b) Anti-6XHIS tag-conjugated HRP secondary monoclonal antibody (clone HIS-1, Sigma‒Aldrich, UK) at a concentration of 1:10,000 bound to the scFv antibody in the WB assay and detected by ECL reagents (Cytiva Amersham). (c) All five scFv antibodies incorporated with primary antibody 1:5,000 dilution of unconjugated goat anti-human IgG [F(ab’)2] (Invitrogen; Thermo Fisher Scientific) and a 1:5,000 dilution of HRP-conjugated rabbit anti-goat IgG (H + L) (Invitrogen; Thermo Fisher Scientific) as a secondary antibody. The secondary antibodies were detected via enhanced chemiluminescence (ECL) reagents (Cytiva Amersham). The original Western blot images are shown in Fig. S3. A Spectra Multicolor Broad Range Protein Ladder (Thermo Scientific) was used in panels (a) and (c).The binding ability of these five purified K9RABVscFv clones was assessed via ELISA. Five hundred nanograms of inactivated RABV were coated on the bottom ofeach well, and 100 µl of individually purified and concentrated scFv antibodies (0.6 µg/µl) were added to each well. ELISA signals were measured by absorbance at 450 nm (Fig. 9). K9RABVscFv1 presented the highest binding signal, followed by K9RABVscFv15, K9RABVscFv2, K9RABVscFv16, and K9RABVscFv12. All the clones were significantly different from the BSA-negative control.Fig. 9ELISA binding absorbance signal at 450 nm of five purified soluble K9RABVscFv clones (0.6 µg/µl; total 60 µg/well). BSA was used as a negative control. Asterisks indicate significant differences at P values ≤ 0.05 (*), ≤ 0.01 (**), and ≤ 0.001 (***). A TMB substrate was used for detection. The error bars indicate the SDs of duplicate samples.RFFIT neutralization activity of the purified K9RABVscFv clonesSeven purified K9RABVscFv samples consisting of K9RABVscFv1_1 (5.35 mg/ml), K9RABVscFv1_2 (7.35 mg/ml), K9RABVscFv2 (6.12 mg/ml), K9RABVscFv12 (4.08 mg/ml), K9RABVscFv15 (4.30 mg/ml), K9RABVscFv16_1 (5.88 mg/ml), and K9RABVscFv16_2 (7.11 mg/ml) were tested for rabies virus neutralizing antibody activity (RVNA) via the RFFIT assay. K9RABVscFv1 and K9RABVscFv16 showed neutralization activity, whereas K9RABVscFv2, 12, and 15 did not inhibit CVS-11 (Table 2). The neutralizing titers of K9RABVscFv1_1 and K9RABVscFv1_2 were 3.51 and 4.96 IU/ml, respectively, whereas those of K9RABVscFv16_1 and K9RABVscFv16_2 were 2.37 and 5.65 IU/ml, respectively. Moreover, these 4 purified scFv samples presented neutralizing titers greater than the protective level of 0.5 IU/ml.Table 2 The RFFIT neutralizing activity of seven purified K9RABVscFv samples.Notably, both neutralizing scFv concentrations were approximately 5 mg/ml, and the RVNA titers of K9RABVscFv1 and K9RABVscFv16 were 3.51 IU/ml and 2.37 IU/ml, respectively. This means that the neutralization ability of K9RABVscFv1 is slightly better than that of K9RABVscFv16, as described in Fig. 10. Notably, dose-dependent effects on RVNA titers were detected when the concentrations of both antibodies were increased to approximately 7 mg/ml. However, the neutralizing potencies of these two clones at a concentration of 7 mg/ml were contrary to the results at a concentration of 5 mg/ml.Fig. 10Percent neutralization of K9RABVscFv1 (5.35 mg/ml) and K9RABVscFv16 (5.88 mg/ml) interpreted from the RFFIT results. Twofold dilutions of both antibodies were conducted from 1:2 to 1:256. A total of 8 fields per well were observed. Percent neutralization was calculated from the number of negative fields.Bioinformatics: K9RABVscFv1 and K9RABVscFv16 homology modeling and molecular docking analysisHomology model constructions of K9RABVscFv1, K9RABVscFv16, and RABV-GTo predict the interaction sites between neutralizing scFv antibodies (K9RABVscFv1 and K9RABVscFv16, the full amino acid sequences are shown in Fig. S2) and the rabies virus glycoprotein (RABV-G; SQ382, Thai-street rabies virus, Fig. S2), bioinformatics was implemented before in vitro epitope mapping. The analysis parameter scores of the homology models are presented in Table S3. The crystal structure of the human stapled scFv (spFv) GLK1 clone (PDBID: 8dy2.1. A, X-ray, 1.65 Å resolution)24 was selected as an appropriate template for homology modeling and 3D-structural model generation of the K9RABVscFv1 antibody, whereas the crystal structure of the 4M5.3 anti-fluorescein human scFv (PDBID: 1 × 9q, X-ray, 1.5 Å resolution)25 was used as a template for K9RABVscFv16. In the case of RABV-G (SQ382, 524 amino acids) homology modeling, the monomer form of the prefusion RABV-G trimers PDBID no. 7u9g (electron microscope, 3.39 Å resolution)4 was the best template.The tertiary structural graphics of K9RABVscFv1, K9RABVscFv16, and RABV-G (SQ382) are shown in Fig. 11. Notably, all the predicted CDRs were located on the outer loop of the VL and VH chains. Previously reported antigenic sites on RABV-G26,27 were indicated as colorful clusters on the ectodomain of RABV-G.Fig. 11Three-dimensional (3D) tertiary structure prediction of the K9RABVscFv1 and K9RABVscFv16 antibodies and the rabies virus glycoprotein (RABV-G). PDBID no. 1 × 9q, 8dy2.1. A and the monomer form of PDBID no. 7u9g were used as templates for K9RABVscFv1, K9RABVscFv16, and RABV-G protein structure simulations. The light purple and pink clusters represent the VL and VH chains, respectively. The orange fragment was a glycine/serine linker (G/S linker). CDR1–3 on the VH and VL chains are shown in several colors. Surface display and line-ribbon style of (a) K9RABVscFv1, (b) K9RABVscFv16, and (c) RABV-G (SQ382). The SWISS model online server (https://swissmodel.expasy.org/) was used to construct protein structure homology models, which were decorated with BIOVIA Discovery Studio Visualizer 2021.In silico molecular docking of neutralizing scFv clones and RABV-GThe best prediction models for the K9RABVscFv1 vs. RABV-G (SQ382) and K9RABVscFv16 vs. RABV-G (SQ382) complexes were selected on the basis of the smallest HADDOCK scores, which were − 100.8 ± 6.4 and − 79.5 ± 3.9, respectively. The root mean square deviations of K9RABV scFv1 and K9RABVscFv16 were 0.5 ± 0.3 °A and 0.4 ± 0.4 °A, respectively. The most likely binding conformation between the CDR1-3 area on K9RABVscFv1 and the antigenic sites on RABV-G is shown in Fig. 12, and the K9RABVscFv16-bound RABV-G complex is shown in Fig. 13.Fig. 12The most likely binding conformation between the CDR1–3 area on K9RABVscFv1 and the antigenic sites on RABV-G was predicted by the HADDOCK 2.4 web server. (a) Surface display structures of RABV-G with residues on antigenic sites that interact with (b) residues on the CDR of K9RABVscFv1. The numbers in brackets “()” in the RABV-G labels refer to the positions without 19 amino acids in the signal sequence. (c) Interactions between the residues on the CDR of K9RABVscFv1 and the antigenic sites of RABV-G on the basis of the results in Table 3. The sticks display the amino acid residues on the CDR (chain A) that interact with the residues on the antigenic sites of RABV-G (chain B). Significant interactions are shown in panels (c1), (c2), and (c3). The VH chain, VL chain, G/S linker, CDR1–3 VH, CDR1–3 VL, antigenic major sites I, II, III, IV, and G5, and minor site G1 (or “a”) are shown in different colors.Fig. 13The most likely binding conformation between the CDR1–3 area on K9RABVscFv16 and the antigenic sites on RABV-G was predicted by the HADDOCK 2.4 web server. (a) Surface display structures of RABV-G with residues on antigenic sites that interact with (b) residues on the CDR of K9RABVscFv1. The numbers in brackets “()” in the RABV-G labels refer to the positions without 19 amino acids in the signal sequence. (c) Interactions between the residues on the CDR of K9RABVscFv1 and the antigenic sites of RABV-G on the basis of the results in Table 3. The sticks display the amino acid residues on the CDR (A chain) that interact with the residues on the antigenic sites of RABV-G (B chain). Significant interactions are shown in panels (c1) and (c2). The VH chain, VL chain, G/S linker, CDR1–3 VH, CDR1–3 VL, antigenic major sites I, II, III, IV, and G5, and minor site G1 (or “a”) are shown in different colors.The analysis also revealed the binding sites on scFv (CDR1, 2, 3 and FR1, 2, 3 of VL and VH) and RABV-G (antigenic sites I, IIa, IIb, III, IV, and a), which were determined from the positions of amino acid residues. The interactions between the residues on the CDR of the scFv and the antigenic sites on RABV-G are shown in Table 3.Computational predictions of binding between K9RABVscFv1 and the RABV-G complex revealed that residues on antigenic sites IIa, IIb, and III together with CDR-1 and CDR-3 of the VL chain and CDR-1, CDR-2, and CDR-3 of the VH chain are involved in these interactions. The 18 binding sites showed interactions between residues on CDRs and antigenic sites, but two residues on scFv (Q24 and E226) recognized the same residues on RABV-G (R352 and K217, respectively), with different types of hydrogen bonds and ion pairs. Overall, 100% of the residues on epitope III significantly interacted with residues on the CDR of the VL chain, especially R352 on epitope III of RABV-G, which could bind to several sites on the CDR of VL. All residues on antigenic site IIa interact with residues on the CDR of the VH chain. Residues on epitope IIb interact with residues on the CDR of the VL or VH chain. The 11 residues on the CDRs of VL, CDR1-VL (6/18), and CDR3-VL (5/18) recognized residues on RABV-G epitopes IIb (4/18) and III (7/18), whereas the seven residues on the CDRs of VH, CDR1-VH (2/18), CDR2-VH (1/18), and CDR3-VH (4/18) interacted with epitopes IIa (3/18) and IIb (4/18).K9RABVscFv16 also interacted with RABV-G, particularly residues at antigenic sites IIa, IIb, and III, together with the CDR-1 of VL and CDR-1, -2, and − 3 of the VH chain. Twelve binding sites showed interactions between residues on the CDR and antigenic sites; the binding residues on the CDR were predominantly located on the CDRs of VH (9/12), consisting of CDR1-VH (4/12), CDR2-VH (1/12), and CDR3-VH (4/12). Almost all of them possibly bind to epitope II, which is divided into IIb (7/12) and IIa (2/12), whereas only three residues of CDR1-VL may bind to antigenic sites on RABV-G, especially epitope III (3/12). Docking prediction indicated that S158 on scFv could bind to two residues of epitope II, L57 (IIb) and K217 (IIa).Table 3 Summary of the docking results for the CDRs of K9RABVscFv and the antigenic sites on RABV-G.
