Development and Epitope Characterization of Monoclonal Antibodies Targeting the Rabies Virus P Protein

ABSTRACT

Rabies is a fatal zoonotic disease caused by rabies virus (RABV), resulting in approximately 59,000 deaths annually worldwide and posing a serious threat to public health. The RABV phosphoprotein (P protein) plays crucial roles in viral replication, transcription, and immune antagonism; however, its immunogenic properties have not been fully characterized. In this study, the RABV P protein was expressed in an Escherichia coli expression system and used to immunize mice, resulting in the generation of six P protein‐specific monoclonal antibodies (mAbs). Using an overlapping peptide‐based truncation strategy, two linear B‐cell epitopes were identified: ⁵²DMKRLHLDDEKSSNL⁶⁶ and ¹⁷⁷VAPGPPALEWSATNE¹⁹¹. Alanine‐scanning mutagenesis revealed that residues D52, M53, R55, L56, and L58 were critical for the recognition of epitope ⁵²DMKRLHLDDEKSSNL⁶⁶ by mAbs 2D2 and 18G7. Residues G180, P181, and W186 were essential for recognition of epitope ¹⁷⁷VAPGPPALEWSATNE¹⁹¹ by mAbs 3D7, 15D8, 16D3, and 16C6. Notably, although some amino acid residues within epitopes P1‐5 and P4‐2 exhibited high variability among representative RABV strains, the critical residues recognized by these monoclonal antibodies were highly conserved. These findings provide new insights into the antigenic structure of the RABV P protein and may contribute to future studies on its functional characterization, as well as the development of P protein‐based diagnostic reagents and subunit vaccines.

1. Introduction

Rabies is a fatal zoonotic disease caused by infection with rabies virus (RABV), which primarily affects the central nervous system [1, 2]. Clinically, rabies is characterized by agitation, hydrophobia, aerophobia, and progressive paralysis, and the case fatality rate approaches 100% following the onset of encephalitic symptoms [3, 4]. Although rabies is vaccine‐preventable and inactivated rabies vaccines are widely used worldwide, several limitations remain, including suboptimal cell‐mediated immune responses and the requirement for multiple‐dose administration [5]. Despite the availability of effective vaccines, rabies remains endemic in more than 150 countries and territories, resulting in approximately 59,000 deaths annually and posing a substantial global public health burden [6]. Furthermore, the clinical diagnosis of rabies remains a major challenge, particularly in the absence of a clear history of animal exposure or characteristic clinical manifestations. To date, no diagnostic tool has been approved by the World Health Organization for the detection of rabies infection prior to the onset of clinical symptoms. Therefore, continued efforts to better understand RABV antigenicity and host immune recognition are essential for the development of improved diagnostic tools and next‐generation vaccines.

RABV, a member of the genus Lyssavirus within the family Rhabdoviridae, is an enveloped, bullet‐shaped virion [7]. The RABV genome comprises a non‐segmented, single‐stranded, negative‐sense RNA of approximately 11.9 kb [8], encoding five viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and the large RNA‐dependent RNA polymerase (L) [9]. Among these viral proteins, the G protein has been extensively investigated because it is the primary target of virus‐neutralizing antibodies and serves as the major antigen in rabies vaccine development. The N protein, owing to its high conservation and abundant expression, has been widely used in diagnostic assays. In contrast, the P protein has received less attention despite its essential roles in viral replication [10], transcription [11], and immune evasion [12, 13]. For example, the P protein serves as a co‐factor of the viral polymerase complex [14], and suppresses host innate immune signaling pathways [15, 16]. Despite its critical biological roles and confirmed necessity for viral virulence, the antigenic structure of the P protein remains insufficiently characterized. Specifically, detailed mapping of B‐cell epitopes on the P protein is crucial for understanding host humoral immune responses and developing precision diagnostic tools.

In this study, we generated a panel of monoclonal antibodies (mAbs) against the rabies virus phosphoprotein and systematically characterized its antigenic properties. Using truncated protein constructs and alanine‐scanning mutagenesis, we identified two novel linear B‐cell epitopes and defined their critical residues. These findings provide new insights into the antigenic architecture of the RABV P protein and a basis for the development of diagnostic tools and subunit vaccines targeting the P protein.

2. Materials and Methods

2.1. Genes, Vectors, and Cells

The encoding sequence of the P gene from the rabies virus CVS‐11 strain (GenBank: GQ918139.1) was codon‐optimized, synthesized, and inserted into the pET‐28a expression vector by Tsingke Biological Co. Ltd. The pET‐32a(+) and pEGFP‐C1 vectors, DH5α and BL21(DE3) competent cells, as well as HEK 293 T and SP2/0 cells were maintained in our laboratory.

2.2. Expression, Purification, and Identification of the P Protein

The pET‐28a‐P plasmid was transformed into E. coli BL21(DE3) competent cells. Positive colonies were selected and cultured in LB medium at 37°C until reaching the mid‐logarithmic growth phase (OD600 = 0.6‐0.8). Protein expression was induced by adding 0.2 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG), followed by incubation at 16°C for 16 h. The bacterial cells were subjected to ultrasonic disruption on ice and centrifugation, and the supernatant was collected. Protein expression was analyzed by SDS‐PAGE and Western blot.

The supernatant was loaded onto a pre‐equilibrated Ni Sepharose Excel resin (Cytiva, USA). The column was washed with binding buffer to remove non‐specifically bound proteins, and the target protein was eluted with elution buffer (50 mM Tris‐HCl, 150 mM NaCl, 250 mM imidazole, pH 7.0). The eluted fractions were further purified by anion‐exchange chromatography (Cytiva, USA). SDS‐PAGE and Western blot analysis confirmed that the target protein was obtained with high purity in the elution fractions. Protein concentration was determined using a BCA Protein Assay Kit (Solarbio, China) with bovine serum albumin as the standard.

2.3. Immunization of Mice

For immunization, purified P protein was employed as the antigen. A total of 20 μg of P protein emulsified with an equal volume of complete Freund's adjuvant (Sigma, USA) was administered subcutaneously to each BALB/c mouse. Three weeks after the primary immunization, a second immunization was performed using incomplete Freund's adjuvant (Sigma, USA), followed by a third immunization with the same adjuvant 2 weeks later. One week after the final immunization, blood was collected from the tail vein to evaluate antibody production, and antibody titers were measured by indirect ELISA. To enhance the immune response, a booster immunization with 40 μg of the antigen was administered intraperitoneally 3 days prior to cell fusion.

2.4. Monoclonal Antibody Production and Characterization

Splenocytes from immunized mice were mixed with myeloma cells in the logarithmic growth phase at an 8:1 ratio, and cell fusion was induced using PEG‐1500 (Roche, Germany). The fused cells were cultured in 96‐well plates (Corning, NY, USA) pre‐seeded with feeder layers and maintained under the conditions of 37°C and 5% CO₂ for 8 days. Hybridomas stably secreting specific antibodies were obtained after three rounds of limiting‐dilution subcloning and repeated screening by indirect ELISA.

Monoclonal antibodies were produced using the ascites method. Briefly, multiparous female BALB/c mice were intraperitoneally injected with 500 μL of sterile incomplete Freund's adjuvant (Sigma, USA). Seven days later, hybridoma cells exhibiting high antibody titers and good viability were harvested, resuspended in sterile PBS, and adjusted to 1 × 10⁶ cells/500 μL. The cell suspension was then injected intraperitoneally into the mice. Ascitic fluid was collected 7‐10 days after inoculation when obvious abdominal distension was observed. Following centrifugation, the supernatant was collected and purified using a Protein A affinity chromatography column (Yeasen, China). Bound antibodies were eluted with glycine buffer (pH 3.0; Solarbio, China). To neutralize immediately the eluate, 33 μL of Tris‐HCl buffer (pH 8.5) was pre‐added to each collection tube. The purified monoclonal antibodies were subsequently characterized using an isotyping kit, and their titers and affinity were determined by indirect ELISA [17, 18].

2.5. Western Blot Analysis

The reactivity of hybridoma supernatants against E. coli‐expressed P protein was evaluated by Western blot. Protein samples were mixed with loading buffer, denatured at 100°C for 10 min, and separated on a 12% SDS‐PAGE gel. Proteins were then transferred onto a PVDF membrane (Millipore, USA), blocked with 5% skim milk in PBST at 4°C overnight, and incubated with hybridoma supernatant as the primary antibody, followed by HRP‐conjugated Goat Anti‐Mouse IgG (1: 5000, Abbkine, China). Target bands were visualized using a chemiluminescence kit (CWBIO, China).

2.6. Determination of Monoclonal Antibody Affinity

Indirect ELISA was used to determine the affinity of the monoclonal antibodies. Recombinant P protein was diluted to 1 μg/mL and 2 μg/mL in carbonate buffer (CBS), and 100 μL per well was added for coating 96‐well plates at 37°C for 2 h. The plates were washed three times with PBST and blocked with 5% skim milk at 37°C for 2 h. Purified ascites‐derived antibodies were adjusted to a concentration of 1 mg/mL and serially two‐fold diluted starting at 1: 1000. The diluted antibodies were added to the wells as primary antibodies, followed by incubation with HRP‐conjugated goat anti‐mouse IgG as the secondary antibody. After washing, TMB substrate was added for color development for 5 min in the dark. The reaction was terminated with stop solution, and the absorbance was measured at 450 nm using a microplate reader.

The reciprocal of OD450 (1/OD450) was plotted against the reciprocal of the monoclonal antibody concentration (1/$[\text{Ab}]$) to generate double‐reciprocal plots, and linear regression analysis was performed. The antibody molar concentrations corresponding to 50% maximal binding under the two antigen coating concentrations were determined from the regression equations. The affinity constant (K _aff) was then calculated according to the following formula:$K\text{aff}=(\mathrm{n}-1)/2(\mathrm{n}[\text{Ab}\prime ]\mathrm{t}-[\text{Ab}]\mathrm{t})$, where n is the ratio of the two antigen coating concentrations, and $[\text{Ab}\prime ]\mathrm{t}$ and $[\text{Ab}]\mathrm{t}$ represent the antibody molar concentrations corresponding to 50% maximal binding under the respective antigen coating conditions [17, 18].

2.7. Indirect Immunofluorescence Assay (IFA)

IFA was performed to evaluate mAb reactivity. HEK 293 T cells were transfected with pEGFP‐C1‐P or empty pEGFP‐C1 using PEI (Yeasen, China). At 48 h post‐transfection, cells were fixed with 4% paraformaldehyde (Beyotime, China) for 15 min, permeabilized with 0.1% Triton X‐100 (Beyotime, China) for 15 min, and blocked with 5% skim milk in PBST at 37°C for 2 h. Cells were incubated with hybridoma supernatants for 1 h, washed, and then incubated with Dylight 594‐conjugated goat anti‐mouse IgG (1: 200, Abbkine, China) for 1 h. Fluorescence was observed with a Nikon Ti2‐U microscope.

2.8. Expression and Characterization of P Protein Fragments

The P protein was truncated into five overlapping fragments, each of which was cloned into the prokaryotic expression vector pET‐32a(+) for expression. After induction, the supernatant was collected to determine the solubility of each truncated fusion protein. Subsequently, indirect ELISA and Dot blot were performed to screen for linear epitope segments that exhibited specific binding to the monoclonal antibodies. Based on the preliminary screening results, The positive reactive regions were subjected to further fine truncation until the precise epitope regions recognized by the monoclonal antibodies were accurately identified.

2.9. Alanine Scanning

Alanine‐scanning mutagenesis was performed to evaluate the functional contribution of individual residues within the identified epitopes (Table 1). Target residues were substituted with alanine, whereas native alanine residues were replaced with glycine [19]. Mutations were introduced by incorporating the desired nucleotide substitutions into primer sequences. The annealed primers were ligated into restriction enzyme‐digested pEGFP‐C1 vectors using T4 DNA ligase at 16°C overnight to generate mutant plasmids.

Table 1.

Alanine scanning mutagenesis scheme for epitope peptides P1‐5 and P4‐2.

P1‐5	Sequence	P4‐2	Sequence
D52A	AMKRLHLDDEKSSNL	V177A	AAPGPPALEWSATNE
M53A	DAKRLHLDDEKSSNL	A178G	VGPGPPALEWSATNE
K54A	DMARLHLDDEKSSNL	P179A	VAAGPPALEWSATNE
R55A	DMKALHLDDEKSSNL	G180A	VAPAPPALEWSATNE
L56A	DMKRAHLDDEKSSNL	P181A	VAPGAPALEWSATNE
H57A	DMKRLALDDEKSSNL	P182A	VAPGPAALEWSATNE
L58A	DMKRLHADDEKSSNL	A183G	VAPGPPGLEWSATNE
D59A	DMKRLHLADEKSSNL	L184A	VAPGPPAAEWSATNE
D60A	DMKRLHLDAEKSSNL	E185A	VAPGPPALAWSATNE
E61A	DMKRLHLDDAKSSNL	W186A	VAPGPPALEASATNE
K62A	DMKRLHLDDEASSNL	S187A	VAPGPPALEWAATNE
S63A	DMKRLHLDDEKASNL	A188G	VAPGPPALEWSGTNE
S64A	DMKRLHLDDEKSANL	T189A	VAPGPPALEWSAANE
N65A	DMKRLHLDDEKSSAL	N190A	VAPGPPALEWSATAE
L66A	DMKRLHLDDEKSSNA	E191A	VAPGPPALEWSATNA

2.10. Conservation Analysis and Spatial Localization Visualization of the Epitopes

Amino acid sequences of the P protein from various RABV strains were retrieved from GenBank to assess B‐cell epitope conservation. Multiple sequence alignment was performed using ClustalW (https://www.genome.jp/tools-bin/clustalw), and conservation profiles were visualized with Jalview. The 3D structure of the P protein was predicted with AlphaFold3, and the identified epitopes were mapped onto the model using PyMOL to evaluate their spatial localization and surface accessibility.

3. Results

3.1. Expression and Identification of the Recombinant P Protein

The recombinant P protein was expressed in a soluble form in E. coli and purified. SDS‐PAGE revealed a protein band at approximately 40 kDa, consistent with the predicted size (Figure 1A), and Western blot confirmed its reactivity with an anti‐His monoclonal antibody (Figure 1B). Mice were immunized according to the schedule shown in Figure 1C, and serum antibody titers reached 1: 64,000 (Figure 1D), demonstrating strong immunogenicity.

Figure 1.

Expression, purification, and immunogenicity evaluation of recombinant P protein. (A) SDS‐PAGE analysis of the purified recombinant P protein. (B) Western blot using anti‐His monoclonal antibody. Lane M, marker (10–180 kDa). (C) Immunization schedule of BALB/c mice. (D) Serum antibody titers measured by indirect ELISA. NC, non‐immunized mouse.

3.2. Generation and Characterization of mAbs against the RABV P Protein

Six mAbs specific to the RABV P protein (2D2, 3D7, 15D8, 16D3, 16C6, and 18G7) were obtained by indirect ELISA screening. All mAbs showed specific reactivity with the recombinant P protein, as confirmed by Western blot (Figure 2A) and IFA (Figure 2B). Notably, IFA demonstrated that the P protein expressed in 293 T cells was effectively recognized by mAbs generated against the prokaryotically expressed antigen.

Figure 2.

Identification of P protein‐specific monoclonal antibodies. (A) Western blot analysis of hybridoma supernatants for reactivity with the P protein. Lane M, protein marker (10–180 kDa); Lane 1, P protein; Lane 2, pET‐32a‐BL21(DE3). (B) IFA to assess the reactivity of monoclonal antibodies. Green fluorescence, EGFP‐P expression; red fluorescence, specific monoclonal antibody binding. NC, serum from non‐immunized mice; PC, serum from immunized mice.

Four mAbs (3D7, 15D8, 16D3, and 18G7) were selected based on growth and titer, then produced by ascites and purified. SDS‐PAGE analysis showed that the purified antibodies displayed distinct heavy and light chain bands at approximately 55 kDa and 25 kDa, respectively, with high purity (Figure 3A). Subtype analysis of the purified antibodies revealed that mAb 18G7 belonged to the IgG1 heavy chain isotype, whereas mAbs 3D7, 15D8, and 16D3 were IgG2a; all of which possessed kappa light chains (Figure 3B). After normalization to 1 mg/mL, indirect ELISA revealed that mAbs 3D7, 15D8, 16D3, and 18G7 exhibited identical titers of 1:2.56 × 10⁵ (Figure 3C). Their affinity constants were determined to be 2.39 × 10⁹, 3.60 × 10⁸, 1.68 × 10⁹, and 4.41 × 10⁸L/mol, respectively (Figure 3D).

Figure 3.

Preparation and characterization of monoclonal antibodies. (A) SDS‐PAGE analysis of purified monoclonal antibodies. Lane M, protein marker (10‐180 kDa); Lanes 1, 3, 5, and 7 show mAbs 3D7, 15D8, 16D3, and 18G7 before purification; lanes 2, 4, 6, and 8 show the corresponding purified antibodies. (B) Isotype determination of six monoclonal antibodies. (C) Antibody titer of mAbs 3D7, 15D8, 16D3, and 18G7 measured by indirect ELISA. (D) Affinity of mAbs 3D7, 15D8, 16D3, and 18G7 estimated by an ELISA‐based assay.

3.3. B‐Cell Epitope Mapping of the P Protein Using Mabs

As shown in Figure 4A, two rounds of truncation were performed to map mAb binding sites. The P protein was first divided into five overlapping fragments (P1‐P5), all of which were successfully expressed (Figure 4B). Indirect ELISA showed that mAbs 2D2 and 18G7 recognized P1, whereas 3D7, 15D8, 16D3, and 16C6 bound to P4 (Figure 4C), which was confirmed by Dot blot (Figure 4D). Based on these results, P1 and P4 were further truncated and cloned into pEGFP‐C1 for fine mapping. IFA results demonstrated that the epitopes recognized by mAbs 2D2 (Figure 5A) and 18G7 (data not shown) were located within the P1‐5 fragment, whereas those epitopes recognized by mAbs 3D7 (Figure 5B), 15D8, 16D3, and 16C6 were mapped to the P4‐2 fragment (data not shown). Due to space limitations, only representative IFA results of mAbs 2D2 and 3D7 are presented.

Figure 4.

Preliminary mapping of the antigenic epitopes recognized by all six P‐specific mAbs. (A) Design of truncations of the P protein for fine mapping of antigenic epitopes; (B) Expression of five truncated P protein fragments in E. coli. Genes encoding P1‐P5 were cloned into pET‐32a(+) and expressed. His‐tagged proteins were detected by Western blot using an anti‐His antibody. Lane M, marker (10–180 kDa); Lanes 1–6, pET‐32a(+) empty vector and P1–P5 fragments. (C) Indirect ELISA; (D) Dot blot. NC, negative control (pET‐32a(+) empty vector); PC, positive control (full‐length P protein).

Figure 5.

Fine mapping of epitope regions within truncated overlapping fragments P1 (P1‐1 to P1‐6) and P4 (P4‐1 to P4‐6) of the P protein by IFA. (A) Reactivity of mAb 2D2 with the truncated overlapping fragment P1 (aa 1–74). (B) Reactivity of mAb 3D7 with the truncated overlapping fragment P4 (aa 167–236). 293 T cells were transfected with EGFP‐fused P fragments (P1‐1 to P1‐6 and P4‐1 to P4‐6). IFA was performed as in Figure 2B. NC, cells transfected with empty pEGFP‐C1; PC, cells transfected with full‐length P plasmid.

3.4. Identification of Critical Amino Acid Residues Within the Epitopes

Alanine‐scanning mutagenesis was performed to identify key residues within epitopes P1‐5 (aa 52‐66) and P4‐2 (aa 177‐191). All constructs were successfully expressed, as indicated by green fluorescence, mAb 2D2 binding depended on residues D52, M53, R55, L56 and L58, mAb 18G7 showed an identical binding pattern with the same critical residues (data not shown). Alanine substitution abolished red fluorescence, confirming these residues as critical for P1‐5 epitope recognition (Figure 6A). For the P4‐2 epitope, residues 180 G, 181 P, and 186 W were also essential for antibody binding by mAb 3D7 (Figure 6B). Similar reactivity patterns were observed for mAbs 15D8, 16C6, and 16D3, although the data are not shown.

Figure 6.

Identification of critical residues within epitopes P1‐5 (aa 52–66) and P4‐2 (aa 177–191) by alanine‐scanning mutagenesis. (A) Reactivity of mAb 2D2 with epitope P1‐5 (aa 52–66) was assessed by IFA. (B) Reactivity of mAb 3D7 with epitope P4‐2 (aa 177–191) was assessed by IFA as described in Figure 2B. NC and PC represent 293 T cells transfected with pEGFP‐C1 and pEGFP‐C1‐P plasmids, respectively.

3.5. Conservation Analysis and Spatial Visualization of Epitopes

To evaluate the sequence conservation of the identified epitopes in the RABV P protein, laboratory‐adapted and representative street strains were analyzed. The epitopes aa 52‐66 (DMKRLHLDDEKSSNL) and 177‐191 (VAPGPPALEWSATNE) were found to be less conserved across the analyzed strains (Figure 7A). Nevertheless, the key residues (52D, 53 M, 55 R, 56 L, 58 L, 180 G, 181 P, and 186 W) within these epitopes were highly conserved in all strains, except for the HEP‐Flury strain.

Figure 7.

Conservation analysis and structural visualization of the identified B‐cell epitopes on the P protein. (A) Conservation analysis based on multiple sequence alignment of the two epitopes from 17 RABV isolates using ClustalW and visualized with Jalview. (B) Structural mapping of epitopes on the AlphaFold3‐predicted P protein. Green indicates epitope regions (aa 52–66 and aa 177–191), and red indicates key residues.

To determine the spatial localization of the epitopes, the identified linear epitopes were mapped onto the AlphaFold3‐predicted 3D structure of the P protein using PyMOL. Both epitopes were surface‐exposed and exhibited mixed α‐helical and coil conformations (Figure 7B), which may facilitate antibody recognition and binding.

4. Discussion

Although several monoclonal antibodies against the rabies virus P protein have been reported previously, the antigenic structure of the P protein remains incompletely understood. In particular, most studies have localized antibody‐binding regions to relatively broad amino acid intervals, whereas the precise definition of minimal linear B‐cell epitopes and their critical residues has remained limited. Therefore, further characterization of P protein antigenic determinants is important for understanding its immunological properties and facilitating the development of diagnostic reagents.

In this study, BALB/c mice were immunized with recombinant P protein derived from the RABV CVS‐11 strain, resulting in the generation of six mAbs with high specificity and affinity for the P protein. Subsequent analyses using Western blot and IFA demonstrated that all six mAbs (2D2, 3D7, 15D8, 16C6, 16D3, and 18G7) were shown to specifically bind to the P antigen.

The P protein was systematically analyzed using an overlapping truncation strategy, leading to the identification of two linear B‐cell epitopes recognized by mAbs. In previous studies, Raux et al. and Nadin‐Davis et al. generated monoclonal antibodies against the P protein using prokaryotic expression systems, and these antibodies exhibited cross‐reactivity among different viral strains [20, 21]. Notably, the epitope regions identified by Raux et al. (aa 20‐82 and aa 177‐297) and those reported by Nadin‐Davis et al. (aa 45‐68 and aa 185‐215) overlapped with the two epitopes characterized in the present study [22]. The consistency between these findings not only validates the reliability of the epitope mapping in this study but also further supports the notion that these regions represent immunodominant sites of the P protein. Unlike previous studies, which often defined epitopes within relatively large amino acid stretches, our study employed a systematic truncation approach to precisely delineate the epitopes to 15‐residue linear sequences, namely ⁵²DMKRLHLDDEKSSNL⁶⁶ and ¹⁷⁷VAPGPPALEWSATNE¹⁹¹.

Sequence alignment results indicated that certain amino acid sites within epitopes P1‐5 and P4‐2 exhibit high variability among representative RABV strains, which we speculate may be related to the role of the P protein as a key factor in viral immune evasion [23]. Specifically, epitope 177‐191 (VAPGPPALEWSATNE) partially overlaps with a previously reported functional region (aa 176‐186), which directly contributes to viral immune evasion by inhibiting IRF3 activation and thereby blocking interferon production [16, 21, 22]. In contrast, the functional significance of the aa 52‐66 region covered by epitope 52‐66 (DMKRLHLDDEKSSNL) remains unclear. Notably, the critical amino acid residues recognized by the monoclonal antibodies generated in this study are highly conserved, suggesting that these residues may play important roles in maintaining the functional integrity of the P protein, particularly in processes related to viral replication and immune evasion. Therefore, designing diagnostic tools or antibodies targeting these conserved residues holds promise for broad‐spectrum detection of different RABV genotypes and provides a theoretical basis for the development of universal diagnostic reagents.

Despite these findings, several limitations should be acknowledged. First, only linear B‐cell epitopes were characterized, and potential conformational epitopes remain unexplored. Second, although key residues were identified, the structural basis of antibody‐epitope interactions was not resolved at high resolution. Future studies integrating structural biology approaches, such as cryo‐EM or X‐ray crystallography, will be essential to further elucidate the molecular mechanisms underlying antibody recognition and to optimize epitope‐based vaccine and diagnostic strategies.

5. Conclusion

In this study, the RABV P protein was successfully expressed, and six specific monoclonal antibodies were generated using hybridoma technology. Two novel linear B‐cell epitopes, aa 52‐66 (DMKRLHLDDEKSSNL) and aa 177‐191 (VAPGPPALEWSATNE), were identified. Alanine‐scanning mutagenesis further revealed key residues critical for antibody binding, including D52, M53, R55, L56, L58, G180, P181, and W186. These findings provide insights into the antigenicity of the RABV P protein and offer a basis for the development of subunit vaccines and serological diagnostics, contributing to efforts toward the elimination of dog‐mediated human rabies.

Author Contributions

Chao Liang: writing original draft, methodology, investigation, formal analysis, data curation. Yanhui Chen: writing original draft, methodology, investigation, formal analysis, data curation. Hongliang Liu: writing review & editing. Jingming Zhou: investigation, formal analysis, data curation., Yumei Chen: investigation, formal analysis, data curation. Yanhua Qi: investigation, formal analysis, data curation. Xifang Zhu: investigation, formal analysis, data curation. Enping Liu: formal analysis, data curation. Sixuan Wu: formal analysis, data curation. Aiping Wang: writing review & editing, supervision, funding acquisition, conceptualization.

Ethics Statement

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with institutional and national guidelines. All efforts were made to minimize animal suffering.

Conflicts of Interest

The authors delcare no conflicts of interest.

Liang C., Chen Y., Liu H., et al., “Development and Epitope Characterization of Monoclonal Antibodies Targeting the Rabies Virus P Protein,” Journal of Medical Virology 98 (2026): e71124. 10.1002/jmv.71124.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.