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. 2026 Mar 16;11(12):19161–19170. doi: 10.1021/acsomega.5c12008

Innovative Vaccine Composed of the BAFF Protein Containing an Unnatural Amino Acid Triggers the Production of Autoantibodies against Endogenous BAFF and Alleviates Collagen-Induced Arthritis

Jin Hu †, Yeshuang Yuan ‡, Jing Li §, Shanbo Yang §, Jiaqi Sun ∥, Wei Song §, Lulu Liu §, Hua Bai †,*, Bo Zhang §,*
PMCID: PMC13044639  PMID: 41939304

Abstract

B-cell-activating factor (BAFF) plays a fundamental role in B lymphocyte survival and homeostasis, and aberrant BAFF signaling is closely related to numerous autoimmune disorders. Although passive immunization strategies using monoclonal antibodies and engineered receptors have achieved success in treating autoimmune diseases, disadvantages, including the cost of continuous administration and potential immunogenicity, still exist. Here, we report an innovative active anti-BAFF vaccine that was developed through the site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO2-Phe) in soluble murine BAFF (DmBAFF). It triggered a humoral immune response against endogenous BAFF and successfully alleviated collagen-induced arthritis in a murine model. An analysis of the immune response to the pNO2-Phe-containing DmBAFF mutants in both DBA/1­(H2q) and C57BL/6 (H2b) mice indicated that the mutants disrupted both T cell and B-cell tolerance toward the endogenous molecule and resulted in the production of strong and distinct antibodies against the corresponding modified epitopes of WT-mBAFF. Preventive vaccination with DmBAFF containing pNO2-Phe at Y91 or Y139 significantly reduced the arthritis incidence and joint inflammation compared with DmBAFF but did not obviously affect T and B-cell subpopulations. Based on these data collectively, preventive vaccination with BAFF containing the site-specific incorporation of pNO2-Phe is an effective prophylactic intervention option for autoimmune arthritis. The therapeutic potential of this vaccine after disease onset remains to be evaluated.

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1. Introduction

The tumor necrosis factor family member, B-cell-activating factor (BLyS), also known as BAFF, TALL-1, and TNFSF13B, is a pivotal homeostatic cytokine in B cells that helps regulate both innate and adaptive immune responses. Like most other type II transmembrane proteins, BAFF is expressed as a membrane-bound form and can be cleaved at a furin consensus site in the Golgi apparatus to generate a soluble active cytokine. Soluble BAFF exerts its biological functions by binding to three cell surface receptors: BAFF-R, TACI, and BCMA. The essential role of BAFF in B-cell functions suggests that its deregulation may lead to autoimmune diseases. Indeed, patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren’s syndrome (SS) exhibit elevated levels of soluble BAFF in their sera that correlate with disease activity. −

The critical role of BAFF as a regulator of peripheral B-cell survival suggests that its blockade may be a useful therapeutic option for autoimmune diseases. Belimumab is the first anti-BAFF monoclonal antibody to be approved for the treatment of active, autoantibody-positive SLE in the USA and Europe. − A recent phase III study conducted in 49 centers across China, Japan, and South Korea also confirmed that belimumab significantly improves disease symptoms while reducing prednisone use, with no new safety issues. Atacicept is a soluble glycoprotein composed of human IgG1-Fc fused with the extracellular domain of the human TACI receptor (TACI-Fc). Neutralization of BAFF and its homologue APRIL (a proliferation-inducing ligand) in a murine model with TACI-Fc significantly inhibits antibody production, affinity maturation, and germinal center formation. , Clinical studies are currently ongoing to assess the safety and efficacy of atacicept as a treatment for SLE, RA, and other autoimmune diseases. , Despite the importance of developing an antagonist-targeting BAFF signaling, monoclonal antibodies and engineered receptor fusion proteins are impractical to manufacture in large quantities. In addition, the potential immunogenicity that may elicit antibody responses after long-term use is also of concern. , Thus, the development of alternative strategies that target BAFF and BAFF-related molecules is critical for the widespread adoption of this treatment modality for autoimmune diseases.

Active immunotherapy using anti-BAFF vaccination is an innovative targeted strategy with the potential to treat autoimmune diseases, , but a comprehensive evaluation of the efficacy and safety of this methodology in a murine model has not been conducted. In the present study, a subset of BAFF vaccines that were generated through the site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO2-Phe) into a soluble deletion mutant murine BAFF (DmBAFF) was evaluated. After replacing the endogenous amino acid with pNO2-Phe at different sites, preventive vaccination with DmBAFF containing pNO2-Phe at Y91 or Y139 significantly increased the immune response against endogenous WT-mBAFF, reduced the incidence and onset of arthritis, and alleviated arthritis in vivo. Moreover, the long-term administration of BAFF vaccines did not affect the populations of T cells and B cells, offering preliminary safety observations that warrant further investigation. It should be noted that this study evaluates the vaccine in a preventive setting prior to disease induction. This establishes a proof-of-principle for breaking BAFF tolerance, which is a critical first step. The applicability of this strategy as a prophylactic intervention after the onset of clinical symptoms represents a distinct and important question for future investigation.

2. Materials and Methods

2.1. General Materials

The TOP10 strain was used to clone and propagate plasmid DNA. Miniprep and maxiprep kits (Qiagen) were used to harvest and purify plasmid DNA. Go-TagGreenMaster Mix (Promega) and the PCR cleanup system (Promega) were used to perform PCR and purify DNA fragments, respectively. The QuikChange Lightning site-directed mutagenesis kit (Agilent) was used to generate site-directed mutations. The phenylalanine derivative p-nitrophenylalanine (pNO2-Phe) was purchased from Aladdin (Beijing, China). Female DBA/1 and C57BL/6 mice aged 6 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China) and maintained under pathogen-free conditions in-house on a 12-h light cycle with access to food and water provided ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at Calibr.

2.2. Incorporation of pNO2-Phe into Murine BAFF (mBAFF)

The original gene encoding DmBAFF (mBAFF in which amino acids 115–122 are replaced by two glycine residues) was synthesized by The Beijing Genomics Institute (BGI, Beijing, China) and cloned into vector pET21-a­(+) at the NdeI and XhoI sites to generate pET21-DmBAFF-WT (opt) and pET21-DmBAFF-WT (nonopt). A 6× His tag was added to the C-terminus for affinity purification. The proteins were expressed and purified as described in a previous report. Briefly, the OrigamiB (DE3) strain hosting the vector(s) was inoculated into 2 mL of Luria–Bertani (LB) medium containing 100 μg/mL ampicillin and grown at 37 °C while being shaken at 220 rpm. Then, the overnight Escherichia coli cultures were added to 2× YT medium and incubated at 37 °C. When optical densities of the cultures reached 0.5 (OD600), IPTG was added to a final concentration of 0.5 mM. After an overnight induction at 25 °C, cells were harvested by centrifugation and resuspended in His-Bind buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, and 5 mM imidazole). Protein extraction was performed by passing cells through a microfluidizer twice at 1200 bar with cooling. The supernatant was collected by centrifugation, and the samples were analyzed by separation on 12% SDS-PAGE gel under reducing conditions, followed by Coomassie blue staining.

A pEVOL-based plasmid containing a Methanococcus jannaschii-derived aminoacyl tRNA synthetase/tRNA pair specific for pNO2-Phe (pEVOL-pNO2-Phe) was first constructed using previously reported methods to generate DmBAFF variants containing pNO2-Phe at specific sites. , The plasmids encoding pNO2-Phe-DmBAFF variants were constructed by site-directed PCR mutagenesis to insert pNO2-Phe at each corresponding position of pET21-DmBAFF-WT (opt). The obtained mutant plasmid, pET-DmBAFF-TAG, was transformed into OrigamiB (DE3) cell line along with pEVOL-pNO2-Phe. The expression of pNO2-Phe-bearing DmBAFF was similar to the expression of wild-type DmBAFF, except that GMML minimal medium was used for culture. When the OD600 reached 0.5, pNO2-Phe was added to a final concentration of 1 mM, and then protein expression was induced 0.5 h later by adding IPTG and l-arabinose at final concentrations of 0.5 mM and 0.1%, respectively.

2.3. Protein Purification

The general procedure for the purification of His-tag DmBAFF is described below. The bacterial lysate, which was resuspended in a His-Bind buffer, was passed through a microfluidizer twice. After centrifugation, the DmBAFF protein in the supernatant was mixed with Ni-NTA His-Bind Resin (Thermo) for 2 h. Then, the unbound proteins were removed by washes with 10 volumes of washing buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 20 mM imidazole). The DmBAFF protein was eluted with an eluent buffer (20 mM Tris-HCl, pH 7.6, 250 mM NaCl, and 300 mM imidazole). The eluted pool was diluted 20-fold with anion exchange binding buffer (20 mM Tris-HCl, pH 8.0) and then loaded onto a 1 mL SOURCE 15Q (GE Healthcare) column equilibrated with the same buffer. DmBAFF was eluted with a 0–30% linear gradient in 15 column volumes. The elution buffer contained 20 mM Tris-HCl, pH 8.0, and 2 M NaCl. The main elution peak was pooled, concentrated, and buffer exchanged with PBS using an ultrafiltration centrifuge tube (Merck Millipore). The purity of DmBAFF proteins obtained with this procedure was usually greater than 95%.

The purified DmBAFF proteins were buffer-exchanged into endotoxin-free PBS and stored at −80 °C. While endotoxin levels were not quantified in this study, the purification process involving Ni-NTA affinity chromatography followed by anion-exchange chromatography (SOURCE 15Q) is an established method for significantly reducing endotoxin contamination from E. coli-expressed proteins.

2.4. Affinity Analysis of WT-mBAFF, DmBAFF, and Different Variants of DmBAFF

The affinities of WT-mBAFF, DmBAFF, and different variants of DmBAFF for BAFF-R were determined with biolayer interferometry (BLI) by using an Octet RED 96 system (ForteBio, PALL). Biotinylated BAFF-R (Biolegend Inc.) was captured on streptavidin-coated biosensor tips according to the manufacturer’s directions. No regeneration was needed between measurements because the dissociation was completed within 10 min. Data were analyzed using the Octet RED 96 evaluation system, and a 1:1 Langmuir binding model was used to fit all binding curves.

2.5. Detection of Anti-BAFF Antibodies in Mouse Serum and T Cell Restimulation Assay

Mice were subcutaneously injected with PBS or 5 μg of DmBAFF or pNO2-Phe-bearing DmBAFF mutants emulsified 1:1 in Freund’s complete adjuvant (CFA) in a final volume of 200 μL once a week for 3 weeks. Serum was obtained through a retro-orbital eye bleed on days 7, 14, and 21 after the initial injection and stored at −80 °C until analysis. For the detection of anti-BAFF antibodies in mouse serum, enzyme-linked immunosorbent assay (ELISA) plates were coated with 100 ng/well of recombinant WT-mBAFF (Biolegend Inc.). Serum samples were diluted 100-fold and incubated for 1 h at room temperature. After washing, bound IgG was detected by adding a horseradish peroxidase-conjugated goat antimouse IgG antibody (Thermo) and TMB substrate, and the absorbance was read at 450 nm on a microplate reader (TIANGEN Inc.).

For T cell restimulation assays, splenocytes collected on day 28 after injection were seeded in microtiter plates at a density of 1 × 106 cells in 100 μL per well in triplicate and treated with the respective antigens at concentrations of 10 μg/mL. All cells were cultured in RPMI 1640 medium supplemented with 10% calf serum and penicillin/streptomycin. For the detection of interferon-γ (IFN-γ), 50 μL of culture supernatant was removed after 96 h and analyzed with a commercial IFN-γ ELISA kit (Dakewe Biotech Co., Ltd.), and cytokine concentrations were calculated based on the respective standard curves. Viable cell densities were determined using the CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer’s instructions.

2.6. Construction of the Collagen-Induced Arthritis (CIA) Model and Preventive DmBAFF Vaccination

DBA/1 mice (6 weeks of age) were vaccinated with 5 μg of DmBAFF, pNO2-Phe91-DmBAFF, or pNO2-Phe139-DmBAFF emulsified 1:1 in CFA through a subcutaneous injection on day 0. On days 12 and 25, the mice received booster doses (5 μg per mouse) of each sample without adjuvant. Arthritis was induced on day 15 by immunization with bovine type II collagen (COII, 100 μg/mouse, Chondrex Inc.) emulsified 1:1 in CFA. On day 36, the mice received a booster dose of COII (100 μg/mouse) emulsified 1:1 in IFA. The development of arthritis was monitored in a blinded manner by using a visual scoring system. The clinical arthritis scoring and the evaluation of radiographic images (Figure D) were performed by two independent investigators who were blinded to the treatment group assignments of the mice.

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Effects of DmBAFF vaccination in CIA mice. (A) Flowchart of the in vivo analysis in DBA/1 mice. (B) Arthritis scores (left) and incidence of CIA (right) in DBA/1 mice. The mice were vaccinated with 5 μg DmBAFF, pNO2-Phe91-DmBAFF, or pNO2-Phe139-DmBAFF on day 0, followed by booster immunizations on days 12 and 25. CIA was induced on day 15, followed by a COII booster on day 36. Vaccine booster doses were administered on days 12 and 25. (C) Anti-WT-mBAFF IgG titers (left) and anti-COII IgG titers (right) in the serum of mice treated with DmBAFF, pNO2-Phe91-, and 139-DmBAFF on day 43. The titers of IgG were quantified by ELISA assay. Values are the means ± SEM (n = 8 mice per group). (D) Radiographs and bone mineral density (BMD) analysis of paws in CIA mice. On day 65, both fore- and hind-paws were radiographed, and BMD was analyzed. A radiograph from a control DmBAFF-treated mouse shows signs of arthritis with disfiguration, osteolysis, and osteophyte production; a radiograph from a pNO2-Phe-DmBAFF-treated mouse shows no lesions or evidence of joint damage, bone loss, or disfiguration. All clinical scoring and radiographic assessments were performed in a blinded manner. Values are the means ± SEM (n = 8 mice per group for (B, C); n = 5–6 mice per group for radiographic analysis in (D). The P values shown were determined by a two-tailed unpaired Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

The titers of total anti-COII or anti-mBAFF IgG antibodies were determined on day 43 using quantitative ELISAs, as described above. Briefly, microtiter plates were coated with native bovine COII or WT-mBAFF (100 ng/well), blocked, and incubated with serially diluted test sera. Bound IgG was detected by incubating the protein with HRP-conjugated goat antimouse IgG, followed by the substrate (TMB). Optical densities were measured at 450 nm with an ELISA plate reader. For the radiological analysis, on day 65, both fore- and hind-paws were radiographed with an X-ray Faxitron Imaging System (Faxitron X-ray Inc., Wheeling, IL). Data were digitized, and the images of radiographs were prepared. On day 70, mice were euthanized with CO2, and peripheral blood mononuclear cells (PBMC) and splenocytes were collected. The cells were stained with both antimouse-thy (1.2)-FITC and antimouse-B220-APC antibodies (Biolegend Inc.), and stained single-cell suspensions were analyzed using a CytoFLEX flow cytometer (Beckman Inc.).

2.7. Analysis of pNO2-Phe-Bearing DmBAFF Mutant Epitopes

Peptides containing biotin were immobilized on ELISA plates coated with avidin at a concentration of 100 ng/well. The serum samples collected on day 70 were applied at 1:100 dilution, and bound IgG was detected with HRP-antimouse-IgG as described above. The sequences of peptides used in these experiments are P1: CLQLIADSDTPTC, P2: CIRKGTYTFVPWLLSFKRGNALC, P3: CKENKIVVRQTGYFFIYSQVLYTDPIFC, P4: CAMGHVIQRKKVHVFGDELSC, P5: CLVTLFRCIQNMPKTLPNC, and P6: CYSAGIARLEEGDEIQLAIPRENAQISRNGDDTC. The disulfide linkage was constructed by adding two additional cysteine residues at the terminus of each peptide. P3 and P6 contain amino acids Y91 and Y139, respectively, and P4 contains the deleted fragment (115–122) and was used as a negative control.

2.8. Statistical Analysis

Data were analyzed using GraphPad Prism 6 software. For comparisons between two groups, the two-tailed unpaired Student’s t test was used. For comparisons involving more than two groups (e.g., vaccination groups in immunogenicity and CIA studies), differences were assessed using one-way ANOVA, followed by Student’s t test for specific pairwise comparisons as indicated in the figure legends. It is noted that such multiple comparisons without formal correction (e.g., Tukey, Bonferroni) may increase the risk of Type I error. Therefore, P values should be interpreted with caution, and the reported differences are considered exploratory. In all cases, P values <0.05 were considered statistically significant.

3. Results

3.1. Expression of Murine BAFF and Its Mutants

Both human and murine BAFF are expressed either as a membrane-bound molecule or cleaved into soluble cytokines. For human BAFF, the soluble form consists of a 152 amino acid (residues 134–285) extracellular fragment of the membrane-bound molecule that is released by proteolysis and is the principal active domain of BAFF. As reported in a previous study, a soluble human BAFF mutant (hmBAFF) in which amino acids 217–224 are replaced by two glycine residues retains the binding activity of BAFF with its receptors but does not activate B-lymphocyte proliferation. This inactivated form of BAFF may represent an ideal vaccine candidate for prevention. Therefore, we replaced the corresponding region of murine BAFF (AA 115–122) with two glycine residues by aligning the sequences of human and murine BAFF. This gene encoding the mouse BAFF mutant (DmBAFF) was synthesized and cloned into the pET21-(a+) vector for expression in E. coli (Figure A). Surprisingly, the gene encoding soluble DmBAFF expressed ∼20-fold more protein than the WT-mBAFF gene (Figure A), and approximately 40% of the protein was soluble.

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Construction and expression of DmBAFF and pNO2-Phe-DmBAFF. (A) Construction of plasmids encoding WT-mBAFF and DmBAFF. The genes encoding mBAFF and DmBAFF (amino acids 115–122 of WT-mBAFF were replaced by two glycine residues) were cloned into pET21-(a+) vector for E. coli expression (Top) and SDS-PAGE analysis of Ni-NTA-purified WT-mBAFF and DmBAFF (bottom). (B) Site-specific incorporation of pNO2-Phe in DmBAFF. A model of murine BAFF based on the crystal structure of human BAFF (PBD: 1OQE) was referred to for the selection of incorporation sites, and four sites (Y91, Y100, Y139, and R158) within the three regions of the highest scores of epitope prediction according to Immune Epitope Database (IEDB) were selected. (C) SDS-PAGE analysis of pNO2-Phe-incorporated DmBAFF expressed in the presence (+) or absence (−) of pNO2-Phe. Different variants of DmBAFF were expressed in the presence of the orthogonal tRNA/tRNA synthetase pair specific for pNO2-Phe. For SDS-PAGE analysis in (A) and (C), equal volumes of culture supernatant after induction were loaded per lane. Total protein expression was compared under identical culture and induction conditions but not normalized to cell density or culture volume.

3.2. Production of Site-Specific pNO2-Phe-Substituted DmBAFF

Nitroaryl groups have historically been used as highly immunogenic haptens, most likely because of the propensity of the electron-deficient pi system to bind to germline antibody-combining sites. The phenylalanine derivative pNO2-Phe was incorporated into DmBAFF at different sites in bacteria in response to the amber nonsense codon to disrupt immunological tolerance toward self-WT-BAFF and induce the production of a specific anti-WT-BAFF antibody. Several criteria were considered to determine which DmBAFF residues would be appropriate to substitute with pNO2-Phe and trigger the production of a high titer of antibodies that cross-react with self-WT-BAFF. First, the residue should be exposed on the surface of the protein. Second, the residue should be within a region of the protein that is potentially present by class II MHC molecules. We analyzed the immune epitopes of DmBAFF using the Immune Epitope Database (IEDB), and the top 15 percentile ranks of the predictions were selected. Additionally, the p-phenyl-containing tyrosine was given priority due to its structural similarity with pNO2-Phe.

As a result, four sites (Y91, Y100, Y139, and R158) within the three sequences with the highest scores from the epitope prediction were selected for unnatural amino acid incorporation (Figure B). As shown in Figure C, pNO2-Phe-substituted DmBAFF displayed a mobility on SDS-PAGE gels similar to that of DmBAFF, and no expression of full-length DmBAFF was detected in the absence of pNO2-Phe in the culture medium, which confirmed our genetic code expansion for engineering pNO2-Phe insertion into DmBAFF at specific sites. The incorporation of pNO2-Phe at site R158 was confirmed by peptide sequencing (Figure S1), and a mass shift of approximately 85 Da (±1 Da), which is equal to the mass difference between an arginine residue and pNO2-Phe, was detected at this specific position. We measured the binding affinity of these DmBAFF mutants for BAFF-R and compared them with the non-pNO2-Phe-substituted and wild-type proteins to detect their biological activities. As shown in Figure S2, the K D value for the standard wild-type BAFF (WT-BAFF) was 1.41 nM, consistent with previously reported values. The affinities of DmBAFF and its pNO2-Phe mutants were equivalent, whereas a substantial reduction (an approximately 100-fold decrease) was observed for these mutants compared with wild-type BAFF. Clearly, the biological activity of DmBAFF mutants was much lower than that of WT-BAFF, confirming that DmBAFF mutants may represent ideal candidates for vaccine design.

3.3. Analysis of the Effects of Immunization with DmBAFF and pNO2-Phe-Substituted DmBAFF

The ability of pNO2-Phe to mediate the induction of anti-BAFF antibody production and prevent the development of arthritis depends on class II MHC molecules. , Therefore, we used arthritis-susceptible mouse strains and detected their ability to produce IgG that cross-reacts with WT-mBAFF. Mice with different MHC molecular backgrounds (DBA/1 H2q and C57BL/6 H2b) were vaccinated with PBS, DmBAFF, or pNO2-Phe-91-, 100-, 139-, or 158-DmBAFF mutants and administered a CFA adjuvant once a week for 3 weeks, following the same immunization schedule described in Section . Plasma samples were collected 1 week after each immunization, and the production of autoantibodies against WT-mBAFF was determined using an ELISA. Based on our results, mice immunized with pNO2-Phe91-DmBAFF displayed markedly higher titers of specific anti-BAFF antibodies at week 3 than mice treated with DmBAFF in both the C57BL/6 and DBA/1 strains, and the mice treated with pNO2-Phe-100 and 139-DmBAFF exhibited an intermediate capacity to produce cross-reacting antibodies (Figure A,B). In contrast, the titers of cross-reacting IgG from the pNO2-Phe158-DmBAFF-immunized group were comparable to the DmBAFF group for up to 3 weeks, which was nonsignificant for DBA/1 mice and slightly higher than C57BL/6 mice (P = 0.034).

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Immunogenicity of pNO2-Phe-containing DmBAFF in CIA-susceptible mouse strains. (A) DBA/1 (H2q) and (B) C57BL/6 (H2b) mice were immunized with 5 μg WT-mBAFF, DmBAFF, and pNO2-Phe-bearing DmBAFF variants with CFA adjuvant once a week for 3 weeks. Sera were collected 1 week after each immunization and tested for IgG against WT-mBAFF by ELISA at a 1:100 dilution. Results are presented as optical density (OD) values. IgG antibodies against WT-mBAFF were quantified by ELISA at a single serum dilution of 1:100, and results are presented as optical density (OD) values. Values are the mean ± SEM (n = 6–8 mice per group). The P values shown were determined by one-way ANOVA with Tukey’s multiple comparisons test. *P ≤ 0.05, **P ≤ 0.01.

Splenocytes from DBA/1 mice immunized with DmBAFF and pNO2-Phe-containing DmBAFF were prepared at the fourth week (7 days after the last injection) and restimulated in vitro with either DmBAFF or the respective antigens at a concentration of 10 μg/mL to determine whether the disruption of tolerance toward mBAFF by pNO2-Phe-incorporated DmBAFF involves T cells. The proliferation of T cells and the secretion of IFN-γ were measured 96 h after restimulation. In the IFN-γ analysis, mice treated with pNO2-Phe-incorporated DmBAFF generally produced higher levels of IFN-γ than mice treated with DmBAFF, and splenocytes that were restimulated with the corresponding pNO2-Phe-incorporated DmBAFF secreted higher levels of IFN-γ than cells restimulated with DmBAFF (Figure A). Consistent with the antibody production, splenocytes from pNO2-Phe91-DmBAFF-treated mice displayed the highest DmBAFF and pNO2-Phe-DmBAFF responses compared to the other mutants, and the mice treated with pNO2-Phe100- or pNO2-Phe139-DmBAFF secreted intermediate levels of IFN-γ. Similar results were obtained from cell proliferation analysis, except that splenocytes from the pNO2-Phe100-DmBAFF group showed a comparable proliferation response to the DmBAFF group (Figure B). It should be noted that while IFN-γ is a marker of Th1-type T-cell responses, the cytokine profiles of other T helper subsets (e.g., Th2, Th17) were not assessed in this study. Thus, the insertion of pNO2-Phe disrupts both T-cell and B-cell tolerance toward the endogenous molecule and results in the production of cross-reacting antibodies against WT-mBAFF. The immunization analysis revealed different levels of immunogenicity of pNO2-Phe positional isomers of DmBAFF, with a general order of Y91 ∼ Y100 > Y139 > R158. Because Y91 and Y100 generally belong to the same fragment of the immune epitope according to the prediction (Figure B), we selected pNO2-Phe91- and pNO2-Phe139-DmBAFF for studies of the effects of preventive vaccination using the classic CIA model.

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Restimulation of splenic T cells from DBA/1 mice. The (A) IFN-γ production and (B) cell viability of spleen cells obtained from mice injected with DmBAFF and 91-, 100-, 139-, and 158-pNO2-Phe-DmBAFF were determined by ELISA and ATP-dependent method, respectively. Cells were restimulated with 10 μg/mL of the respective antigens as well as DmBAFF for 96 h; pNO2-Phe-100 DmBAFF was used for the restimulation of cells from the DmBAFF-treated group as a representative. Values are the means ± SEM (n = 3 mice per group). IFN-γ secretion is indicative of a Th1-type response, but the cytokine profiles of other T helper subsets were not characterized.

3.4. Vaccination Protects a Murine CIA Model

DBA/1 mice were vaccinated with DmBAFF, pNO2-Phe91-DmBAFF, or pNO2-Phe139-DmBAFF on day 0, followed by booster immunizations on days 12 and 25. Arthritis was induced on day 15 by immunization with type II collagenas previously reported. Booster doses of vaccines were also administered at an intermediate time during the COII injections (day 25). Starting on day 36, animals were monitored and scored for arthritis every other day, and plasma was collected on day 43 to detect antibodies against both WT-mBAFF and COII (Figure A).

As shown in Figure B, mice in the control group developed typical clinical symptoms of severe arthritis, which began on approximately the fifth day after the booster injection of COII and progressed rapidly to a mean clinical score of ∼7 in the next 2 weeks. In contrast, in mice treated with pNO2-Phe91-DmBAFF and pNO2-Phe139-DmBAFF, the progression of arthritis was substantially inhibited, reaching mean clinical scores of ∼1.5 and ∼2, respectively, at the end of the detection period (day 57). Consistent with the arthritis scores, the incidence of arthritis in mice treated with pNO2-Phe91-DmBAFF and pNO2-Phe139-DmBAFF was significantly reduced compared with DmBAFF-treated mice; paw swelling was observed among all control mice just 1 week after arthritis onset, but in only half of mutant DmBAFF-treated mice (Figure B). Because antibodies against collagen participate in the development of arthritis and their production is related to the function of BAFF, we then investigated whether vaccination with DmBAFF mutants affected the B-cell activities and inhibited the anticollagen immune response. As anticipated, mice immunized with pNO2-Phe91- and pNO2-Phe139- DmBAFF produced higher antibody titers that cross-reacted with WT-mBAFF compared to animals treated with DmBAFF, which was negatively correlated with anti-COII IgG titers (Figure C). Nevertheless, although vaccination with mutant DmBAFF significantly ameliorated arthritis in mice, it had minimal effect on the proportions of T cells or B cells in spleens and peripheral blood lymphocytes (Figure S3). However, future studies incorporating detailed immunophenotyping (e.g., transitional, follicular, marginal zone, and plasma cell subsets), total immunoglobulin quantification, and assessment of immune competence will be necessary to comprehensively evaluate the immunological safety of this vaccine. In addition, radiographs and BMD analysis of the paws revealed that the joints in the control group mice displayed obvious osteolysis and disfigurement, while these signs were absent from most of the pNO2-Phe-DmBAFF-treated mice, confirming that vaccination prevented CIA-associated damage to bone and cartilage (Figure D).

3.5. Recognition of Immune Epitopes of Mutant DmBAFF

Vaccination with both pNO2-Phe-DmBAFF and DmBAFF induced the production of antibodies against WT-mBAFF. However, only mice vaccinated with site-specific pNO2-Phe-substituted DmBAFF (91 or 139) exhibited significant relief of clinical symptoms of the disease. Because the incorporation of pNO2-Phe might create new T-cell epitopes on the protein and thus enhance the ability of helper T cells to trigger an effective immune response, , we performed an epitope mapping analysis to investigate whether the differences between DmBAFF- and mutant DmBAFF-induced antibodies contributed to the efficacy of treatment. Six cyclic peptides were designed based on the structure of the analogous hBAFF protein using internal cysteine residues along with two additionally introduced cysteine residues at the N-terminal and C-terminal sites (Figure A). Biotinylated peptides were immobilized on avidin-coated ELISA plates, and antibodies in plasma obtained from mice treated with DmBAFF, pNO2-Phe91-DmBAFF, pNO2-Phe139-DmBAFF, or PBS were analyzed using serum samples collected on day 70. As anticipated, mice vaccinated with pNO2-Phe91-DmBAFF or pNO2-Phe139-DmBAFF exclusively produced high titers of antibodies against P3 and P6, which correspond to the sequences of WT-mBAFF ranging from 79 to 104 amino acids (for P3) and 138 to 169 amino acids (for P6) (Figure B). In contrast, IgG in plasma from DmBAFF-immunized mice recognized only P2, and no significant differences were observed between mutant DmBAFF- and DmBAFF-immunized mice. No antibody binding above the PBS-treated background was observed for P4, which was synthesized as a negative control corresponding to the absent and substituted sequence of WT-mBAFF. Based on these results, the replacement of Y91 or Y139 by pNO2-Phe in DmBAFF associated with the disruption of immune self-tolerance toward WT-mBAFF and the production of antibodies that preferentially recognize the corresponding epitopes. However, a direct functional link between the recognition of these specific epitopes and the therapeutic efficacy has not been established.

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Epitope mapping of DmBAFF, pNO2-Phe91-, and 139-DmBAFF-induced IgG. (A) Six biotinylated-cyclic peptides (P1 to P6) were designed and synthesized based on the human BAFF structure. The disulfide linkage was constructed by adding two additional cysteine residues at the terminus of each peptide. P3 and P6 contain amino acids Y91 and Y139, respectively, and P4 contains the deleted fragment (115–122) used as a negative control. (B) DmBAFF, pNO2-Phe91-, and 139-DmBAFF-induced IgG to P1 to P6. Serum obtained on day 70 from mice treated with DmBAFF, pNO2-Phe91-, and 139-DmBAFF (n = 8 per group) was tested for their reactivities with the individual peptides. Serum obtained from PBS-treated mice (shown in Figure , n = 3) was used as a negative control. Values are the means ± SEM. The P values shown were determined by Student’s unpaired t test, *P ≤ 0.05.

4. Discussion

BAFF is a ligand that plays an important role in B lymphocyte maturation and survival. , Overexpression of BAFF is closely associated with the pathogenesis and progression of autoimmune diseases, including SLE, RA, and SS. Therefore, BAFF is considered an important therapeutic target for the treatment of autoimmune diseases. Indeed, BAFF antagonists based on antibodies and soluble receptors have been used successfully to treat both murine and human autoimmune diseases, and belimumab has now been approved as treatment for SLE, and Atacicept remains investigational and is currently undergoing clinical evaluation for SLE and other autoimmune conditions. , Despite these recent advances, therapy with passive immunization targeting BAFF has numerous disadvantages, including the requirement for the repeated administration of an exogenous protein, which may inevitably elicit unwanted antibody responses and limit long-term treatment efficacy. − In addition, based on current serological and genetic analyses and known environmental factors, the development of some autoimmune diseases, such as SLE and RA, is likely predictable to some extent, particularly in preclinical individuals who are seropositive but latent and asymptomatic. ,, Therefore, we believe that the development of active immunotherapy for the preventive treatment of autoimmune diseases is promising.

Here, we report a novel BAFF-based vaccine generated by incorporating the unnatural amino acid pNO2-Phe into specific sites of DmBAFF. A soluble murine BAFF mutant (DmBAFF), in which amino acids 115–122 were replaced by two glycine residues, was chosen for the incorporation of pNO2-Phe due to its high yield and solubility compared with that of WT-mBAFF (Figure A). Based on our experiments, higher titers of autoantibodies cross-reacting with WT-mBAFF were produced upon vaccination with pNO2-Phe-modified DmBAFF than DmBAFF. According to our results (Figure ), the incorporation of pNO2-Phe at position 91 or 100 resulted in strong immunization against WT-mBAFF in mice with either the H-2q or H-2b haplotype, whereas relatively lower immunogenicity was observed for DmBAFF incorporating pNO2-Phe at position 139 or 158. In restimulation assays, splenocytes obtained from pNO2–Phe-DmBAFF-treated mice showed significantly increased cell proliferation and IFN-γ production upon restimulation with DmBAFF (Figure ). Thus, a T cell neoepitope is generated when pNO2-Phe is incorporated into DmBAFF and results in the incorporated site- and class II MHC-dependent production of antibodies against WT-mBAFF.

Based on accumulating evidence, anticytokine vaccination is a practical strategy that can be applied to autoimmune diseases. , Since cytokines play a major role in homeostasis, the long-term safety of this strategy is a concern. Consistent with the class of B-cell-depleting drugs, BAFF antagonists such as belimumab also impair the survival of immune cells, particularly B cells, resulting in reduced production of autoantibodies and cytokines. Considering the important role of BAFF in maintaining immune homeostasis, the side effects of BAFF-targeting treatments, such as broad-spectrum immunosuppression, remain a challenging safety issue. In the present study, mice treated with pNO2-Phe-BAFF vaccines displayed an amelioration of CIA, with no significant change in total T and B cell numbers compared with DmBAFF-treated mice (Figures B and S3). While this suggests a lack of gross immunosuppression, it should be noted that bulk lymphocyte counts are insufficient to rule out subtler immunomodulatory effects. BAFF primarily influences B cell subset distribution and survival, and our study did not assess transitional, follicular, marginal zone, or plasma cell populations, nor did it evaluate humoral immune competence or BAFF levels postvaccination. Additionally, while radiographic analysis indicated preserved bone architecture, histological assessment of synovitis and cartilage integrity provided more direct evidence of joint protection. Crucially, the long-term immunological impact of sustained anti-BAFF antibody production remains uncharacterized. Future studies must include longitudinal monitoring of memory B cell and plasma cell compartments, germinal center activity, and comprehensive toxicology to rule out unintended consequences for immune memory and overall immune competence. Future studies incorporating these analyses are essential to fully delineate the safety profile of this vaccine strategy. It is important to note that the immunization regimen in this proof-of-concept study employed complete Freund’s adjuvant (CFA), a potent inflammatory stimulant. While effective for breaking immune tolerance in preclinical models, CFA is not clinically translatable. The highly inflammatory milieu induced by CFA (and later IFA during CIA induction) may have contributed to the potency of the anti-BAFF response and influenced disease outcomes. Future development of this vaccine strategy will require evaluation with clinically acceptable adjuvants.

This study demonstrates the efficacy of anti-BAFF vaccination in preventing the development of collagen-induced arthritis. While this preventive approach has clear relevance for individuals at high risk of autoimmune disease (e.g., those with autoantibody positivity but no clinical symptoms), a paramount question for clinical translation is whether such a vaccine can effectively treat established disease. A therapeutic application would involve administering the vaccine in an inflammatory milieu with pre-existing tissue damage and an ongoing autoimmune response, which pose additional challenges. These include potential differences in immune tolerance mechanisms during active disease, the necessity of using clinically acceptable adjuvants, and the critical timing of intervention relative to the disease flare. Future studies are warranted to investigate the prophylactic efficacy of this vaccine strategy in established arthritis models. Such work will be essential to define the full clinical potential of active BAFF vaccination, distinguishing its role as a preventive agent from its utility as a disease-modifying treatment.

In conclusion, in this study, we evaluated the effects of BAFF vaccination on the prevention of autoimmune disease in a mouse model. The incorporation of the unnatural amino acid pNO2-Phe into specific sites of DmBAFF, a biologically inert form of BAFF, disrupts T cell tolerance toward the endogenous molecule and results in the production of strong and distinct antibodies against the corresponding modified epitopes of WT-mBAFF. Our data suggest that the immunogenicity of pNO2-Phe-DmBAFF depends on the incorporation site and MHC although the precise impact on BAFF-R engagement and downstream signaling was not measured here. Moreover, preventive vaccination with pNO2-Phe-Y91- or Y139-DmBAFF significantly reduces the onset and incidence of arthritis and alleviates inflammation and joint destruction in a mouse model of arthritis compared with DmBAFF. It should be emphasized that these effects were demonstrated in a setting using strong, nonclinical adjuvants (CFA/IFA). While initial safety assessments based on total lymphocyte counts are encouraging, comprehensive evaluation, including B cell subset analysis, immunoglobulin profiling, BAFF level monitoring, and histological joint assessment, will be crucial in future preclinical and clinical development. Furthermore, subsequent studies must address the translatability of the approach by employing clinically relevant adjuvants and rigorously controlling for potential confounding factors such as endotoxin. Thus, BAFF-based vaccination represents a novel and promising proof-of-concept strategy for active immunization against a self-cytokine. The findings warrant further development, including evaluation in therapeutic models, mechanistic studies of BAFF neutralization and receptor blockade, and comprehensive long-term safety assessments. This approach may potentially contribute to a new class of immunotherapeutics for autoimmune diseases.

Supplementary Material

ao5c12008_si_001.pdf (337.2KB, pdf)

Acknowledgments

This study was financially supported by grants from the Nonprofit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2024-RC310-06) and the National Natural Science Foundation of China (No. 21805311).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c12008.

  • Validation of site-specific incorporation of pNO2-Phe at R158 by mass spectrometry (Figure S1); binding kinetics and affinities of the indicated variants to BAFF-R measured by biolayer interferometry (BLI) (Figure S2); dynamic changes in splenic and peripheral blood T and B cell proportions in CIA mice (Figure S3) (PDF)

⊥.

J.H. and Y.Y. contributed equally to this work. J.H. and Y.Y. designed the study. J.H., Y.Y., J.L., S.Y., and J.S. performed the experiments, analyzed the data, and wrote the manuscript. J.H. conducted the CIA experiments and acquired the corresponding photographic documentation. W.S. and L.L. helped to polish the language and provided comments. H.B. and B.Z. revised and approved the manuscript. All authors reviewed the manuscript.

The authors declare no competing financial interest.

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