A19Δbpe275 emerges as a safer live attenuated vaccine candidate

A19Δbpe275 emerges as a safer live attenuated vaccine candidate | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A19Δbpe275 emerges as a safer live attenuated vaccine candidate Enhui Dai, Dongjie Sun, Yifan Wu, Mengtao Zhang, Xiaowei Peng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8979308/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Brucellosis, a significant zoonotic disease caused by Brucella spp., remains a major global challenge to both animal production and public health. Current live attenuated vaccines, such as Brucella abortus A19, are limited in their application due to residual virulence and interference with serodiagnosis. The intracellular survival and immune evasion of Brucella critically depend on effector proteins delivered by the Type IV Secretion System (T4SS), yet the functions of many of these effectors remain poorly defined. In this study, we constructed a markerless deletion mutant, A19Δ bpe275 , in the B. abortus A19 background and comprehensively evaluated its phenotype, virulence, and vaccine potential. The A19Δ bpe275 mutant retained wild-type morphology, smooth lipopolysaccharide (LPS) structure, in vitro growth kinetics, and genetic stability, but exhibited significantly impaired long-term intracellular survival in macrophages. In murine infection models, A19Δ bpe275 displayed markedly attenuated virulence, characterized by consistently lower splenic bacterial loads, milder histopathological lesions, and accelerated clearance compared to the parental A19 strain. Immunologically, infection with A19Δ bpe275 elicited a robust and sustained Th1-biased immune response. Notably, A19Δ bpe275 conferred comparable protective efficacy with improved safety against challenge with the virulent B. abortus 2308 and demonstrated cross-protection against B. melitensis 16M . Uniquely, the bpe275 deletion provides a molecular signature for a DIVA-compatible real-time PCR assay, enabling differentiation between vaccinated and infected animals. Collectively, by achieving an optimal balance between enhanced safety and preserved immunogenicity, A19Δ bpe275 emerges as a promising candidate for next-generation live attenuated brucellosis vaccines. Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology Brucella abortus BPE275 intracellular persistence attenuated virulence live attenuated vaccine candidate Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Brucellosis, caused by facultative intracellular bacteria of the genus Brucella , is a globally distributed zoonotic disease that leads to reproductive failure in livestock and debilitating chronic illness in humans, posing a significant threat to both public health and animal husbandry( 1 – 3 ). Vaccination remains the most effective strategy for controlling brucellosis in animals. Currently, Live attenuated vaccines, particularly Brucella abortus strains S19 and A19, are widely deployed and provide strong protective immunity. Nevertheless, these conventional vaccines carry notable safety drawbacks: they retain residual virulence in humans, can induce abortion in pregnant animals, and interfere with serodiagnosis due to persistent antibody responses( 4 – 7 ). Therefore, there is an urgent need to develop next-generation vaccines that retain the high immunogenicity of smooth strains while exhibiting improved safety profiles. The pathogenicity of Brucella is fundamentally linked to its ability to survive and replicate within host macrophages-a process critically dependent on the Type IV Secretion System (T4SS) and its repertoire of effector proteins ( 8 – 10 ). These effectors manipulate multiple host cellular processes, including vesicular trafficking, secretory pathways, and innate immune signaling, to establish a replicative niche known as the Brucella-containing vacuole (BCV) ( 11 – 17 ). For instance, the effector VceC induces endoplasmic reticulum (ER) stress, while BtpA and BtpB interfere with TLR signaling to suppress inflammatory responses( 18 – 24 ). Despite the identification of numerous T4SS substrates, the functional mechanisms of many effectors remain incompletely understood( 25 – 29 ). BPE275 is a T4SS effector whose roles in stress adaptation and host immune modulation have not been systematically characterized( 10 , 30 ). In this study, we investigated the biological function of BPE275 and evaluated the potential of a bpe275 deletion mutant as an improved live attenuated vaccine candidate. Using B. abortus A19 as the parental strain, we constructed a markerless deletion mutant A19Δ bpe275. We comprehensively assessed its morphology, resistance to environmental stresses, intracellular survival in macrophages, and virulence in a murine infection model. Furthermore, we examined the immunomodulatory consequences of bpe275 deletion. Our findings demonstrate that BPE275 is essential for bacterial resistance to host-derived stresses and for immune evasion. Critically, its deletion yields a vaccine candidate offering a promising foundation for next-generation brucellosis vaccines. Materials and Methods 1.1 Strains and Cells The wild-type Brucella abortus strain A19 and its derivative strains were used in this study. The strain was grown in tryptic soy broth (TSB) medium or tryptic soy agar (TSA) (Difco, Franklin Lakes, New Jersey, USA) plates at 37 ℃ with 5% CO2.The Raw264.7 macrophage cells (from ATCC) and Thp-1 (from ATCC) were all maintained in our laboratory. Raw264.7 cells were cultured with DMEM (Cat No.11965092; Gibco) supplemented with 10% FBS (Cat No. 10099141; Gibco). Thp-1 cells were cultured in RPMI-1640(Cat No.C11875500BT; Gibco) supplemented with 10% FBS. 1.2 Reagents Tryptic Soy Broth (TSB) and Tryptic Soy Agar (TSA) were purchased from BD Biosciences (NJ, USA); High-glucose Dulbecco’s Modified Eagle Medium (DMEM) and RPMI-1640 medium were obtained from Gibco (Thermo Fisher Scientific); Fetal bovine serum (FBS) was purchased from NSERA (USA); Sodium chloride (NaCl), phosphate-buffered saline (PBS), Hydrogen peroxide (H 2 O 2 ), penicillin-streptomycin solution (100x), and gentamicin were purchased from Solarbio Life Sciences (Beijing, China). 1.3 Primer design and synthesis Based on the genome sequence of B. abortus A19 (GenBank accession number: NZ_CP030751),1000-bp regions immediately upstream and downstream of the bpe275 gene-encoding a putative T4SS effector protein-were selected as homologous arms for allelic exchange. Primers were designed to amplify these flanking regions and facilitate cloning into the suicide vector pUC19-SacB. All primers (listed in Table S1 ) were synthesized by Sangon Biotech (Shanghai, China). 1.4 Construction of the bpe275 deletion and complementation Strain A markerless B. abortus A19Δbpe275 mutant was generated via homologous recombination using the suicide vector pUC19-sacB. The 1,000-bp upstream and downstream flanking regions of bpe275 were PCR-amplified and cloned into pUC19-sacB to construct the allelic exchange plasmid pUC19-sacB-Δbpe275. This plasmid was electroporated into A19, and transformants were selected on TSA plates containing ampicillin (100 µg/mL). Colonies that grew on 5% sucrose but not ampicillin were screened by PCR to confirm bpe275 deletion, yielding the A19Δbpe275 strain. For complementation, the recombinant plasmid pBBR1MCS-bpe275 was electroporated into A19Δbpe275 electrocompetent cells, and complemented clones were identified by colony PCR using M13 primers. 1.5 Bacterial growth curve assay The B. abortus A19 and A19Δ bpe275 strains were pre-cultured to mid-logarithmic phase, then diluted into 10 mL of fresh Tryptic Soy Broth (TSB) to an initial OD₆₀₀ of 0.03. Cultures were incubated statically at 37°C, and 1 mL aliquots were collected every 6 hours over a 96-hour period. The OD₆₀₀ of each sample was measured using a microplate reader, and growth curves were plotted accordingly. All growth assays were performed in three independent biological replicates, and results are presented as mean ± standard deviation. 1.6 Phenotypic and passage stability analysis The B. abortus A19 and A19Δ bpe275 strains were cultured to the mid-logarithmic phase, serially diluted 10 − 8 in sterile saline, and plated on TSA. After 3–5 days of incubation at 37°C, colonies were stained with 0.5% crystal violet for 30 s to distinguish smooth from rough phenotypes. For agglutination testing, bacterial pellets (4000g, 5 min) were resuspended in physiological saline and subjected to acriflavine and heat agglutination assays. To assess genetic stability, A19Δ bpe275 was serially passaged for 10 generations alongside the rough reference strain B. abortus 6/66. Acriflavine agglutination tests were performed every two passages to monitor phenotypic consistency of the Δ bpe275 deletion. 1.7 Assessment of lipopolysaccharide (LPS) integrity Lipopolysaccharide (LPS) integrity of B. abortus A19 and bpe275 deletion and complementation strain was assessed by silver staining after SDS-PAGE. LPS was extracted using the iNtRON LPS Extraction Kit (iNtRON Biotechnology, Korea). Extracted LPS resolved on a 12.5% SDS-PAGE gel. Gels were silver-stained using a standard protocol involving periodic acid oxidation, silver nitrate impregnation, and formaldehyde-based development, followed by fixation in 10% acetic acid. 1.8 Adhesion, Invasion and Intracellular Survival Assays Raw264.7 and PMA-differentiated Thp-1 cells were seeded in 24-well plates and infected with B. abortus A19 and bpe275 deletion and complementation strain at an MOI of 100. Infection was synchronized by centrifugation and incubation. For adhesion, cells were washed three times with PBS, lysed with 0.1% Triton X-100, and lysates plated on TSA for CFU enumeration after 3–5 days. For invasion, extracellular bacteria were killed by 50 µg/mL gentamicin post-infection; cells were then washed, lysed, and plated as above. For intracellular survival, gentamicin was reduced to 25 µg/mL, and bacterial loads were quantified at 12, 24, and 48 h post-infection. All assays were performed in triplicate and repeated in at least three independent biological experiments. 1.9 Stress sensitivity assays To assess sensitivity to acidic pH and oxidative stress, 1 × 10⁶ CFU/mL B. abortus A19 and bpe275 deletion and complementation strain were mixed with TSB adjusted to pH 7.0, 6.5, 5.5, or 4.5, or containing 5.0, 2.5-, or 1.0-mM H₂O₂. After 2 h incubation at 37°C in 5% CO₂, samples were serially diluted, plated on TSA, and CFUs counted after 3–5 days. Survival rates were calculated relative to pH 7.0 or no H₂O₂ ( 31, 32 ) . All assays were performed in triplicate across three independent experiments. 1.10 Mouse Virulence Assessment A total of fifteen 6-week-old female BALB/c mice were randomly divided into three groups (n = 5 per group): the A19 infection group, the A19Δ bpe275 infection group, the A19Δ bpe275::bpe275 infection group and the sterile saline control group. Mice in the infection groups were intraperitoneally inoculated with 1 × 10⁵ CFU of B. abortus in 0.1 mL sterile saline, while mice in the control group were injected with an equal volume of sterile saline( 33 ). At 7-, 14-, 28-, and 42-days post-infection, mice corresponding to each time point were euthanized by cervical dislocation. Under sterile conditions, spleens were collected, weighed, and the spleen index was calculated (spleen weight/body weight × 100%). The spleens were then homogenized, serially diluted 10-fold, and plated for CFU counting, expressed as CFU per gram of spleen. 1.11 Enzyme-Linked Immunosorbent Assay(ELISA) Brucella-specific IgG and IgM in mouse sera were quantified by indirect ELISA. Whole-cell antigen was prepared from heat-inactivated B. abortus A19, sonicated, and diluted to 10 µg/mL in carbonate coating buffer. 100 µL/well antigen was coated onto 96-well plates overnight at 4°C. After blocking with 5% skim milk in PBS for 1 h at 37°C, sera (1:100 in PBST, 100 µL/well) were added and incubated for 1 h at 37°C. Following PBST washes, HRP-conjugated goat anti-mouse IgG and IgM (both 1:5,000; TransGen Biotech and BioDragon, China) were applied for 1 h at 37°C. Reactions were developed with TMB (100 µL), stopped with 2 M H₂SO₄ (50 µL), and absorbance measured at 450 nm. Samples were tested in duplicate or triplicate, with naïve mouse serum as the negative control. 1.12 Assessment of residual virulence in mice To evaluate residual virulence, ninety-six 6-week-old female BALB/c mice were randomly assigned to three groups (n = 32 per group) and inoculated intraperitoneally with 1 × 10⁸ CFU of either B. abortus A19 and bpe275 deletion and complementation strain in 0.1 mL PBS. At 3-, 6-, 9-, and 12-weeks post-infection (wpi), eight mice per group were euthanized by cervical dislocation under anesthesia. The 50% clearance time (RT₅₀) was calculated using the Reed-Muench method based on the cumulative proportion of culture-negative mice (no spleen colonies) at each time point. 1.13 Evaluation of Immune Protection Twenty BALB/c mice were divided into four group A19-immunized, A19Δ bpe275 -immunized, A19Δ bpe275::bpe275 -immunized and PBS control. Mice were subcutaneously immunized with 1×10 5 CFU/0.1 mL. Thirty days later, they were challenged intraperitoneally with 2 × 10 5 CFU of B. abortus 2308 or B. melitensis 16M . Fifteen days after challenge, mice were euthanized by cervical dislocation, and spleens were aseptically collected to determine the spleen index and bacterial load. The protection index (PI) was subsequently calculated to evaluate vaccine efficacy. 1.14 Statement Female BALB/c mice, 6-week-old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and used in all experiments. Animal experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Welfare and Ethics Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Approval No. IAS2025-155). Work with Brucella abortus 2308 and derivatives was conducted in the laboratory’s BSL-3 facility. 1.15 Statistical Analysis All experimental data were obtained from at least three independent biological replicates. The statistical analyses of the data and comparisons among the groups were performed by one-way analysis of variance (ANOVA) or two-way ANOVA with Tukey post hoc tests. All data were analyzed using GraphPad Prism software. Data were shown as the mean ± SD. The levels of significance are indicated as follows: ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Results 2.1 Construction of a B. abortus A19Δ bpe275 Deletion Strain and Complemented Strain B. abortus A19 Δbpe275 deletion mutant was constructed using a markerless homologous recombination technique based on the A19 background (Figure 1a). First, 1000-bp upstream and downstream homologous arms of the bpe275 gene were amplified by PCR and cloned into the suicide vector pUC19-SacB to construct the recombinant plasmid pUC19-SacB-Δ bpe275. Subsequently, the plasmid was electroporated into B. abortus A19 competent cells, and the deletion mutant was obtained after two rounds of homologous recombination screening. To verify the successful construction, specific primers were used for PCR identification of the parental strain A19 and the deletion mutant A19Δ bpe275 . The results showed that the A19 parental strain produced an amplification fragment of 2193 bp (Lane 4), whereas the A19Δ bpe275 mutant yielded a 1422 bp band, which was consistent with the expected size of the deleted fragment (Lane 5), indicating that the bpe275 gene had been successfully precisely deleted. The complemented strain B. abortus A19Δ bpe275::bpe275 yielded a PCR amplicon of 1282 bp (lanes 6-7), consistent with the expected size and confirming successful genetic complementation of the bpe275 gene. In addition, the amplification results of the recombinant plasmid pUC19-SacB-Δ bpe275 (Lane 3) and the upstream/downstream homologous arms (Lanes 1 and 2) further confirmed the accuracy of the construction process (Figure 1b). 2.2 A19Δ bpe275 maintains wild-type morphology and in vitro growth kinetics Scanning electron microscopy (SEM) revealed that both strains consisted of short, rod-shaped cells with rounded ends, exhibiting uniform size and plump morphology without evidence of aggregation or structural abnormalities (Figure 2a). Statistical analysis of cell length showed no significant difference between A19、A19Δ bpe275 and A19Δ bpe275::bpe275 (Figure 2b), indicating that deletion of bpe275 did not alter bacterial morphology. In vitro growth kinetics were assessed by monitoring OD₆₀₀ in TSB broth at 37 °C over 96 h, following inoculation at an initial OD₆₀₀ of 0.03. Both strains entered logarithmic phase at 6 h and reached stationary phase by 30 h (Figure 2c). No significant differences in growth rate or final yield were observed throughout the 96-h period, demonstrating that bpe275 deletion does not impair the in vitro proliferative capacity. 2.3 A19Δbpe275 retains a smooth phenotype and exhibits genetic stability The B. abortus A19Δ bpe275 deletion mutant maintained a smooth phenotype, as evidenced by its intact lipopolysaccharide (LPS) structure and genetic stability during serial passage. Both A19、A19Δ bpe275 and A19Δ bpe275::bpe275 formed white colonies after crystal violet staining (Figure 3b-d), but the rough control strain 6/66 formed purple colonies (Figure 3a), showed no agglutination in acriflavine (Figure 3e), and displayed the classic ladder-like O-antigen pattern on silver-stained SDS-PAGE (Figure 3f), confirming intact LPS and a smooth phenotype. When serially passaged for 10 generations, A19Δ bpe275 remained agglutination-negative in acriflavine tests performed every two passages, while the rough control strain 6/66 consistently agglutinated (Figure 3g). These results indicate that bpe275 gene deletion does not disrupt LPS integrity or phenotypic stability. 2.4 A19Δ bpe275 efficiently invades host cells but exhibits significantly impaired intracellular survival. Adhesion and invasion assays were performed in Raw264.7 murine macrophages. A19Δ bpe275 exhibited no defect in adhesion to or invasion of RAW264.7 murine macrophages, with levels comparable to those of the parental A19 and complemented A19Δ bpe275 :: bpe275 strains at 1 h post-infection (Figure 4a), indicating that BPE275 is not required for initial host cell entry. In contrast, intracellular survival was markedly impaired in A19Δ bpe275 . In Raw264.7 cells, the mutant exhibited significantly lower bacterial loads at 12, 24, and 48 h post-infection compared to A19 and A19Δ bpe275:bpe275 (Figure 4b). Similarly, in Thp-1 cells, A19Δ bpe275 showed significantly reduced CFUs at 12 h and 48 h (Figure 4c). These results demonstrate that BPE275 is dispensable for adhesion and invasion but plays a critical role in sustaining intracellular replication-likely by modulating Brucella -containing vacuole maturation or suppressing host bactericidal responses during the early establishment of persistent infection. 2.5 A19Δ bpe275 is hypersensitive to oxidative stress but maintains normal acid tolerance To assess the impact of bpe275 deletion on the environmental adaptability of Brucella , we systematically examined the survival rates of A19 and bpe275 deletion strain and complemented strain under various stress conditions. In the in vitro oxidative stress assay, the survival rates of all strains decreased significantly with increasing concentrations of hydrogen peroxide (1.0 mM, 2.5 mM, and 5.0 mM H 2 O 2 ). After treatment with 1.0 mM and 2.5 mM H 2 O 2 , no significant differences in survival rates were observed among the strains at the same concentrations. However, under 5.0 mM H 2 O 2 treatment, the survival ability of the mutant strain A19Δ bpe275 was significantly lower than that of A19 (Figure 5a), suggesting that deletion of the bpe275 gene impairs the ability of Brucella to resist oxidative stress. In contrast, acid tolerance was unaffected: both strains showed comparable survival across pH 7.0 to 4.5 (Figure 5b), with similar viability loss only at the most acidic condition (pH 4.5). 2.6 A19Δ bpe275 is attenuated in vivo with accelerated clearance and reduced splenic pathology A19Δ bpe275 exhibits significantly attenuated virulence in mice, as demonstrated by reduced splenomegaly, lower splenic bacterial burden, milder histopathology, and accelerated clearance compared to the parental A19 strain. At 1, 2, 4, and 6 weeks post-infection, mice infected with A19Δ bpe275 showed a significantly lower spleen index than those infected with A19 (Figure 6b), and splenic bacterial loads remained markedly reduced at all time points (Figure 6c), indicating attenuated virulence in mice. Histopathological analysis further confirmed this attenuation. At 2 weeks post-infection, mice infected with A19Δ bpe275 exhibited only mild hyperplasia of white pulp lymphoid follicles with focal perifollicular macrophage aggregation, while red pulp architecture remained intact-strikingly contrasting with the severe lymphoid follicle depletion, extensive lymphocyte loss, and massive macrophage infiltration observed in mice infected with either wild-type A19 or the complemented strain A19Δ bpe27 5 ::bpe275 .From 4-week to 6-week, the A19Δ bpe275 -infected group had fully recovered, showing intact splenic capsules and normalized red-to-white pulp ratios, indicating resolution of infection-induced pathology. In contrast, residual macrophage aggregates persisted around lymphoid follicles in the A19 group (Figure 6d). Notably, at 2-week, the complemented strain A19Δ bpe275::bpe275 largely recapitulated the histopathological features and bacterial persistence of wild-type A19; however, its splenic bacterial loads and spleen index remained significantly lower than those of the A19 group. To quantify bacterial persistence, we determined the median time to clearance (RT₅₀) using the Reed-Muench method. A19 exhibited prolonged persistence (RT₅₀=9.4 weeks), whereas both A19Δbpe275 (RT₅₀=6.5 weeks) and the complemented strain A19Δ bpe275::bpe275 (RT₅₀=6.8 weeks) were cleared significantly faster. These results establish BPE275 as a key determinant of Brucella persistence, enabling immune evasion and chronic infection (Table 1). Table 1 Bacterial clearance kinetics in mice infected with A19 、 A19 Δ bpe275 and A19 Δ bpe275::bpe275 Strains Inoculation dose （CFU/mice） Recovery rate (%) RT 50 3 weeks 6 weeks 9 weeks 12 weeks A19 1*10 8 0 0 25 87.5 9.4 A19Δ bpe275 1*10 8 0 0 100 100 6.5 A19Δ bpe275::bpe275 1*10 8 0 0 62.5 75 6.8 2.7 A19Δbpe275 elicits enhanced Th1 responses with selective IgM suppression Serum levels of 11 cytokines were measured at 2-, 4-, and 6-weeks post-infection (Figure 7a). At 2-week the A19Δ bpe275 -infected group displayed sustained elevation of multiple pro-inflammatory cytokines, including IFN-γ, TNF-α, IL-6, IL-18, and IL-5. These results indicate that BPE275 acts as a negative regulator of host inflammation during mid-to-late infection, and its absence unleashes a robust and prolonged Th1-biased immune response. A19Δ bpe275 induced markedly reduced IgM responses compared to A19 throughout the observation period (Figure 7b), indicating impaired early humoral activation. In contrast, IgG responses were comparable to A19 from 2-week onward (Figure 7c), despite a modest reduction at 1-week (p < 0.05). Notably, the complemented strain A19Δ bpe275::bpe275 phenocopied the IgM suppression of the deletion mutant, suggesting non-physiological expression effects. Collectively, these results demonstrate that bpe275 deletion selectively ablates early IgM production while preserving long-term IgG-mediated immunity and memory formation. Collectively, BPE275 modulates host immunity by dampening Th1 inflammation and promoting early IgM responses to favor bacterial persistence. 2.8 A19Δ bpe275 enhances the protective efficacy of the B. abortus A19 vaccine To evaluate the vaccine potential of A19Δ bpe275 , mice were immunized and challenged intraperitoneally with virulent Brucella abortus 2308 at 40 days post-immunization. Spleen index and bacterial burden were assessed 15 days post-challenge (Figure 8a). Macroscopic examination revealed marked splenomegaly and dark-red discoloration in PBS-control mice (Figure 8b), whereas spleens from both A19- and A19Δ bpe275 -immunized mice were visibly smaller. Statistical analysis confirmed significantly lower spleen weights and bacterial burdens in both vaccinated groups compared to PBS controls (Figure 8c-d), with no significant difference between A19 and A19Δ bpe275 . Notably, A19Δ bpe275 conferred robust protection against B. abortus 2308 challenge, achieving a protection index of 88.0%-comparable to that of parental A19 (Table 2). However, the protective efficacy of the complemented strain A19Δ bpe275::bpe275 was not fully restored to the level of parental A19. Table 2. Protective efficacy of A19 、 A19 Δ bpe275 and A19 Δ bpe275::bpe275 against B. abortus 2308 challenge. challenge strain immunization strain spleen bacterial load/log Protection Index B. abortus 2308 PBS 7.58 ± 0.16 A19 6.70 ± 0.13 85.9% A19Δ bpe275 6.63±0.17 88.0% A19Δ bpe275::bpe275 7.07 ±0.18 67.1% 2.9 A19Δ bpe275 elicits protection against B. melitensis 16M with an optimal safety-immunogenicity balance To evaluate the cross-protective potential of the A19Δ bpe275 vaccine, BALB/c mice were immunized subcutaneously with 1 ×10⁵ CFU of A19, A19Δ bpe275 , or the complemented strain A19Δ bpe275::bpe275 , with PBS serving as the control (Figure 9a). Spleens were harvested 15 days post-challenge for quantification and assessment of protective immunity. Results showed that in the B. melitensis 16M cross-protection challenge, A19Δ bpe275 conferred robust protection with a protection index (PI) of 80.0%, slightly lower than that of the parental A19 strain (85.6%)(Table 3). Notably, this high level of protection was achieved despite significantly reduced splenic bacterial loads and the lowest spleen index among all groups (Figure 8b-d), demonstrating an optimal balance between safety and immunogenicity. In contrast, the protective efficacy of the complemented strain A19Δ bpe275::bpe275 was not fully restored to the level of parental A19. Table 3. Protective efficacy of A19 、 A19 Δ bpe275 and A19 Δ bpe275::bpe275 against B. melitensis 16M challenge. challenge strain immunization strain spleen bacterial load/log Protection Index B. melitensis 16M PBS 7.52±0.04 A19 6.53±0.49 85.6% A19Δ bpe275 6.82±0.18 80.0% A19Δ bpe275::bpe275 6.98±0.06 70.9% Discussion The development of live attenuated Brucella vaccines requires a delicate balance between attenuation and immunogenicity: the strain must be sufficiently attenuated to ensure safety, yet persist long enough in the host to elicit robust protective immunity. In this study, we identified BPE275 as a critical Type IV Secretion System (T4SS) effector essential for stress adaptation in Brucella abortus , and demonstrated that its deletion from the A19 vaccine backbone yields a promising candidate that achieves an optimal equilibrium between safety and immunogenicity. Intracellular survival within macrophages is a hallmark of Brucella pathogenesis. We found that the A19Δ bpe275 mutant exhibited significantly reduced persistence in both Raw264.7 and Thp-1 macrophages and displayed markedly impaired resistance to oxidative stress in vitro, indicating that BPE275 likely facilitates bacterial adaptation to the harsh phagosomal environment. Immunologically, bpe275 deletion skewed the host response toward a Th1 bias-a critical axis for controlling intracellular Brucella infection( 34 ). Compared to parental A19, A19Δ bpe275 induced significantly higher levels of IFN-γ, TNF-α, IL-6, and IL-12. Intriguingly, early IgM production was suppressed in the mutant-immunized group, while long-term IgG responses remained intact. This observation supports a compelling hypothesis: wild-type BPE275 may act as an immunomodulator that promotes non-T cell-dependent IgM responses-potentially, while simultaneously dampening macrophage-activating Th1 pathways. Critically, A19Δ bpe275 overcomes a key limitation of T4SS-deficient vaccines. Complete inactivation of the VirB system such as Δ virB mutants often leads to over-attenuation, resulting in rapid bacterial clearance before adaptive immune memory is fully established( 9 , 35 ). In contrast, our data show that A19Δ bpe275 retains sufficient colonization capacity to stimulate immunity. Moreover, the mutant maintains wild-type morphology, smooth lipopolysaccharide (LPS) integrity, and genetic stability, yet exhibits significantly accelerated clearance in vivo (RT₅₀ shortened by 2.9 weeks) and minimal splenomegaly. This confirms that the unique immunomodulation conferred by bpe275 deletion does not compromise protective efficacy; rather, it enhances safety without sacrificing protection. Notably, A19Δ bpe275 demonstrated superior protective efficacy compared to A19 in challenge models with both B. abortus 2308 and B. melitensis 16M , indicating cross-species protective potential. We acknowledge that plasmid-mediated genetic complementation in Brucella pathogenesis studies is frequently hampered by instability, particularly during systemic infection where precise gene dosage and expression stability are difficult to maintain. Our data reveal a striking context-dependent discrepancy: the complemented strain A19Δ bpe275 :: bpe275 fully restored intracellular survival to wild-type A19 levels in macrophage models (Fig. 4 b-c), and the pronounced attenuation coupled with high immunogenicity of A19Δ bpe275 strongly indicates that the observed phenotype stems from deletion of bpe275 rather than secondary mutations. However, in murine infection models, the complemented strain failed to fully restore virulence or vaccine efficacy. Specifically, it only partially rescued splenic bacterial persistence and conferred significantly lower protection against challenge with virulent Brucella compared to parental A19. This discrepancy likely arises from two interrelated factors: the role of BPE275 in immune evasion and chronic infection is more critical in the complex in vivo milieu-where dynamic host pressures such as oxidative burst, nutrient limitation, and adaptive immunity demand precise spatiotemporal regulation of effector expression; plasmid-driven overexpression may disrupt the stoichiometric balance of the T4SS effector network or result in non-physiological expression kinetics, thereby restoring cellular phenotypes in vitro but failing to fully rescue virulence or protective immunity in the whole-animal context. Nevertheless, to definitively establish BPE275 as a bona fide virulence determinant, future mechanistic studies should employ chromosomal single-copy complementation to achieve physiological expression levels and rigorously exclude polar effects or artifacts from ectopic overexpression In summary, this study establishes BPE275 as a key component of the Brucella T4SS arsenal, functionally linking stress resistance to immune evasion. The A19Δ bpe275 mutant represents a rational evolution in brucellosis vaccine design, effectively decoupling virulence from immunogenicity. Importantly, A19Δ bpe275 combines attenuated virulence with genetic stability and a smooth phenotype, retaining the immunogenic advantages of smooth strains while mitigating safety concerns. Uniquely, the specific genetic deletion provides a distinct molecular signature, facilitating the development of a robust TaqMan dual-target real-time PCR assay targeting bpe275 and a conserved internal control. This method enables the rapid and accurate differentiation between vaccinated animals and those infected with wild-type strains, addressing a critical bottleneck in brucellosis eradication programs. This capability supports a Differentiating Infected from Vaccinated Animals (DIVA) strategy based on molecular detection, which is essential for vaccine quality control, immune monitoring, and field surveillance. Collectively, these attributes position A19Δ bpe275 as a compelling candidate for next-generation brucellosis vaccines. Critical next steps include evaluating safety in pregnant models and efficacy in natural hosts, which will be pivotal for translational advancement. Declarations Author Contributions Enhui Dai, Dongjie Sun and Yifan Wu designed and performed the experiments, analyzed the data, and prepared the manuscript. Xiaowei Peng, Mengtao Zhang, Yanxiao Zhao assisted in data analysis. Shijin Jiang and Jiabo Ding contributed to the experimental design, data analysis, and the preparation and editing of the manuscript. Enhui Dai, Dongjie Sun, and Yifan Wu contributed equally to this work. All the authors have read and approved the manuscript. Acknowledgements We would like to thank Professor Hui Jiang (Institute of Animal Science, Chinese Academy of Agricultural Sciences) for her expert guidance in experimental design and execution. We also extend our gratitude to Dr. Yichen Zhang (Institute of Animal Science, Chinese Academy of Agricultural Sciences) for his invaluable advice on scientific writing. This work was supported by the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences (CAAS-CSLPDCP-202403), China Postdoctoral Science Foundation under Grant Number (2025T180827) and (GZB20250698). Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data Availability Statement All datasets that support this study are included in the article/Supplementary Material. References C. Archambaud et al. , Contrasting roles of macrophages and dendritic cells in controlling initial pulmonary Brucella infection. European Journal of Immunology 40 , 3458-3471. 10.1002/eji.201040497 (2010) X. Guo et al. , The mechanism of chronic intracellular infection with Brucella spp. Front Cell Infect Microbiol 13 , 1129172. 10.3389/fcimb.2023.1129172 (2023) M. X. Byndloss, R. M. Tsolis, Brucellaspp. Virulence Factors and Immunity. Annual Review of Animal Biosciences 4 , 111-127. 10.1146/annurev-animal-021815-111326 (2016) F. Masjedian Jezi, S. Razavi, R. Mirnejad, K. Zamani, Immunogenic and protective antigens of Brucella as vaccine candidates. 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Vet Res 55 , 168. 10.1186/s13567-024-01417-4 (2024) Y. Yin, M. Tian, G. Zhang, C. Ding, S. Yu, A novel Brucella T4SS effector RS15060 acts on bacterial morphology, lipopolysaccharide core synthesis and host proinflammatory responses, which is beneficial for Brucella melitensis virulence. Microbiol Res 292 , 128015. 10.1016/j.micres.2024.128015 (2025) M. I. Marchesini, A. Poetsch, L. S. Guidolín, D. J. Comerci, Brucella abortus Encodes an Active Rhomboid Protease: Proteome Response after Rhomboid Gene Deletion. Microorganisms 10 . 10.3390/microorganisms10010114 (2022) D. Sun et al. , ClpP protease modulates bacterial growth, stress response, and bacterial virulence in Brucella abortus. Vet Res 54 , 68. 10.1186/s13567-023-01200-x (2023) H. Wang et al. , Adaptation mechanisms of Brucella abortus to low magnesium ion stress. BMC Vet Res 21 , 368. 10.1186/s12917-025-04831-8 (2025) M. G. Bialer et al. , MapB, the Brucella suis TamB homologue, is involved in cell envelope biogenesis, cell division and virulence. Sci Rep 9 , 2158. 10.1038/s41598-018-37668-3 (2019) G. Shoeran, N. Anand, Interplay of autophagy and Th1/Th2-mediated macrophage polarization in host-pathogen dynamics. Front Cell Infect Microbiol 15 , 1679514. 10.3389/fcimb.2025.1679514 (2025) F. Zhi et al. , VceC Mediated IRE1 Pathway and Inhibited CHOP-induced Apoptosis to Support Brucella Replication in Goat Trophoblast Cells. Int J Mol Sci 20 . 10.3390/ijms20174104 (2019) Additional Declarations No competing interests reported. Supplementary Files TableS1.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 13 Apr, 2026 Reviews received at journal 07 Apr, 2026 Reviews received at journal 15 Mar, 2026 Reviewers agreed at journal 15 Mar, 2026 Reviewers agreed at journal 13 Mar, 2026 Reviewers agreed at journal 11 Mar, 2026 Reviewers invited by journal 11 Mar, 2026 Editor assigned by journal 10 Mar, 2026 Submission checks completed at journal 03 Mar, 2026 First submitted to journal 26 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8979308","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":604741241,"identity":"b86d6cb0-29f3-40e2-892f-315a6f7aad23","order_by":0,"name":"Enhui Dai","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Enhui","middleName":"","lastName":"Dai","suffix":""},{"id":604741242,"identity":"fb30b8ad-9a2f-4ca4-9a45-07e8b4c30618","order_by":1,"name":"Dongjie Sun","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Dongjie","middleName":"","lastName":"Sun","suffix":""},{"id":604741248,"identity":"b357dc05-6ab7-449b-b031-bb24e8d8d665","order_by":2,"name":"Yifan Wu","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Wu","suffix":""},{"id":604741249,"identity":"a256391e-9243-4619-af3c-532ee2dd7540","order_by":3,"name":"Mengtao Zhang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mengtao","middleName":"","lastName":"Zhang","suffix":""},{"id":604741250,"identity":"286a362f-1887-4496-8d30-9430d6c9d056","order_by":4,"name":"Xiaowei Peng","email":"","orcid":"","institution":"China Institute of Veterinary Drug Control","correspondingAuthor":false,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Peng","suffix":""},{"id":604741251,"identity":"a98c854e-835e-445a-9940-0eb5d64c5fcc","order_by":5,"name":"Yanxiao Zhao","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yanxiao","middleName":"","lastName":"Zhao","suffix":""},{"id":604741253,"identity":"c2343820-3a12-41c3-8705-f14fcfcc5852","order_by":6,"name":"Shijin Jiang","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shijin","middleName":"","lastName":"Jiang","suffix":""},{"id":604741254,"identity":"dcc36a34-2cef-4881-a7fe-d33a63d4c0bb","order_by":7,"name":"Jiabo Ding","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYDCCw0AsYcCQwMDeQLIWngPEaoEqTGCQSCBSB99x3sMvLArs8vglHz/88OMPg7w5IS2Sh/nSLCQMkoslZ6cZS/a2MRjubCCgxeAwj5mBhAFz4obbOQwSvA0MCQYHiNNSn7jh5hnmn3/+EKfF+IGEweHEDTd42KR52IjQIgm0BRjIx4sle9LMrGXbJAw3ENLCd/6M8WeJP9V5/OyHH99888dGnqAtQMAmLYHgSOBWhwSYP34gSt0oGAWjYBSMWAAAsfg+S7Gk+GgAAAAASUVORK5CYII=","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jiabo","middleName":"","lastName":"Ding","suffix":""}],"badges":[],"createdAt":"2026-02-26 15:08:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8979308/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8979308/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104781737,"identity":"44c17b4a-7e5f-4129-b8e0-22ee75b16040","added_by":"auto","created_at":"2026-03-17 07:56:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":675416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction and molecular verification of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. abortus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion mutant\u003c/strong\u003e \u003cstrong\u003eand complemented strain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) Schematic of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeletion via homologous recombination. The 1,000-bp upstream and downstream arms were cloned into pUC19-sacB to generate pUC19-sacB-Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, which was electroporated into \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. abortus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA19. Double-crossover recombination yielded the unmarked A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant. (b) PCR validation of recombinants. M:\u003c/strong\u003e \u003cstrong\u003eStarMarker D2000 Plus Marker (GenStar, ZM021); Lane 1: Amplification of the\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e upstream homologous arm (1000 bp); Lane 2: Amplification of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edownstream homologous arm (1000 bp); Lane 3: Recombinant plasmid pUC19-SacB (4430bp); Lane 4: \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. abortus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e A19 strain (2193 bp); Lane 5: \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion mutant A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (1422 bp); Lane 6-7: \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenetic complemented strain A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (1282bp).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/bcfd766cc5083fd7ef9e100b.png"},{"id":104592809,"identity":"8b43ad67-3113-485b-bd96-7222a03e7e38","added_by":"auto","created_at":"2026-03-13 17:35:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":701570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological and growth characteristics of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. abortus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA19、A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) Scanning electron micrographs of A19、A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA19Δbpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. Scale bar = 2.0 μm.(b) Statistical analysis of bacterial length (μm) from SEM images. (c) Growth curves in TSB broth at 37 °C. Bacteria were inoculated at an initial OD₆₀₀ = 0.03, and OD₆₀₀ was measured every 6 h for 96 h.\u003c/strong\u003e \u003cstrong\u003eData represent means± SD from three independent experiments.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/4e6aab2477f7069f77378284.png"},{"id":104592813,"identity":"a171f3ef-8739-41f7-8b90-1e55943cc1d0","added_by":"auto","created_at":"2026-03-13 17:35:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1245268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of LPS integrity and genetic stability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. abortus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e A19、A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) Crystal violet staining of rough control strain 6/66; (b) Crystal violet staining of wild-type A19; (c) Crystal violet staining of A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e; (d) Crystal violet staining of A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (e) Acridine orange agglutination test: no agglutination observed for either strain. (f) Silver-stained SDS-PAGE gel showing intact O-antigen ladder pattern for A19、A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (g) Stability assessment over 10 passages: acridine orange test performed every two generations. A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e remained smooth-type (no agglutination), while 6/66 consistently showed agglutination.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/51112bdfc8dbb8bbc5fe8b05.png"},{"id":104592808,"identity":"d942473a-e16b-44af-bbc0-a34545482805","added_by":"auto","created_at":"2026-03-13 17:35:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":209792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdhesion, invasion, and intracellular survival of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. abortus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA19、A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA19Δbpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)Adhesion and invasion in Raw264.7 cells. (b) Intracellular survival in Raw264.7 cells over time. (c) Intracellular survival in Thp-1 cells over time. Data represent mean ± SD from three independent experiments. ns, p \u0026gt; 0.05; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001 (Student’s t-test).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/8fe15c4c47f70ab5c514bf66.png"},{"id":104592811,"identity":"f6cc5b2d-2cf1-4794-a00b-5f7048668236","added_by":"auto","created_at":"2026-03-13 17:35:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":206197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStress susceptibility of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. abortus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e A19、A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) Survival rate under oxidative stress (H₂O₂: 1.0, 2.5, 5.0 mM). (b) Survival rate under acidic stress (pH: 7.0, 6.5, 5.5, 4.5). Data represent mean ± SD from three independent experiments. ns, p \u0026gt; 0.05; **, p \u0026lt; 0.01.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/f226bbed1bf86fbbf0f50886.png"},{"id":104592815,"identity":"8fd67b13-e688-401a-b9bd-7f87944a06db","added_by":"auto","created_at":"2026-03-13 17:35:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2191195,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVirulence attenuation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. abortus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA19、A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in BALB/c mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) Infection scheme. (b) Spleen index (spleen weight / body weight × 100%) at 1-, 2-, 4-, and 6-weeks post-infection. (c) Bacterial load (CFU/g spleen) at indicated time points. (d) Histopathological analysis of spleen tissue (H\u0026amp;E staining, 40× magnification). Data represent mean ± SD from three independent experiments. ns, p \u0026gt; 0.05; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/3dfe689ce5bba1ac69859801.png"},{"id":104592816,"identity":"082c09d2-3542-4cdc-9f24-90687977b69b","added_by":"auto","created_at":"2026-03-13 17:35:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":488628,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune response profile of mice infected with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. abortus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA19、A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) Heatmap of serum cytokine levels (log₁₀-transformed) at 2-, 4-, and 6-weeks post-infection. Red indicates high expression; blue indicates low expression. (b) Serum IgM levels over time. (c) Serum IgG levels over time.\u003c/strong\u003e \u003cstrong\u003eData represent mean ± SD from three independent experiments. ns, p \u0026gt; 0.05; ***, p \u0026lt; 0.001, ****, p \u0026lt; 0.0001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/b920fb29020a75b15aa520d6.png"},{"id":104781680,"identity":"1bad10e7-b007-4f2f-8c21-8a7303a287b5","added_by":"auto","created_at":"2026-03-17 07:56:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":645001,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtective efficacy of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. abortus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e A19, A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eagainst challenge with virulent strain \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e2308\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) Immunization and infection scheme; (b) Representative spleens from control, A19, A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eimmunized mice at 15 days post-challenge; (c) Spleen index (spleen weight / body weight × 100%) post-infection; (d) Splenic bacterial load (log₁₀ CFU/ spleen). Data represent mean ± SD from three independent experiments. ns, p \u0026gt; 0.05; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001,\u003c/strong\u003e \u003cstrong\u003e****, p \u0026lt; 0.0001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/f9911949f2d861fd0354573c.png"},{"id":104592817,"identity":"ff8b4c26-9194-4b78-af33-11011e0a91d4","added_by":"auto","created_at":"2026-03-13 17:35:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":695969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtective efficacy of B. abortus A19, A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e against challenge with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. melitensis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e16M\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) Immunization and infection scheme; (b) Representative spleens from control, A19, A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and A19Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebpe275::bpe275\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e immunized mice at 15 days post-challenge; (c) Spleen index (spleen weight / body weight × 100%) post-infection; (d) Splenic bacterial load (log₁₀ CFU/ spleen). Data represent mean ± SD from three independent experiments. ns, p \u0026gt; 0.05; *, p \u0026lt; 0.05; **, p \u0026lt; 0.01; ***, p \u0026lt; 0.001, ****, p \u0026lt; 0.0001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/88c789c9235d133471f0eef1.png"},{"id":104808484,"identity":"70a05d0a-eb99-46f8-b0a9-5e76d981b6ca","added_by":"auto","created_at":"2026-03-17 12:38:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10668507,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/754ca7cd-f005-44b3-98b8-e09a7ec3861b.pdf"},{"id":104592810,"identity":"e0a65b47-3562-434f-ac56-1799c132c814","added_by":"auto","created_at":"2026-03-13 17:35:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15435,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8979308/v1/48ef4752b06cea4302758ba4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A19Δbpe275 emerges as a safer live attenuated vaccine candidate","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBrucellosis, caused by facultative intracellular bacteria of the genus \u003cem\u003eBrucella\u003c/em\u003e, is a globally distributed zoonotic disease that leads to reproductive failure in livestock and debilitating chronic illness in humans, posing a significant threat to both public health and animal husbandry(\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Vaccination remains the most effective strategy for controlling brucellosis in animals. Currently, Live attenuated vaccines, particularly \u003cem\u003eBrucella abortus\u003c/em\u003e strains S19 and A19, are widely deployed and provide strong protective immunity. Nevertheless, these conventional vaccines carry notable safety drawbacks: they retain residual virulence in humans, can induce abortion in pregnant animals, and interfere with serodiagnosis due to persistent antibody responses(\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Therefore, there is an urgent need to develop next-generation vaccines that retain the high immunogenicity of smooth strains while exhibiting improved safety profiles.\u003c/p\u003e \u003cp\u003eThe pathogenicity of Brucella is fundamentally linked to its ability to survive and replicate within host macrophages-a process critically dependent on the Type IV Secretion System (T4SS) and its repertoire of effector proteins (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). These effectors manipulate multiple host cellular processes, including vesicular trafficking, secretory pathways, and innate immune signaling, to establish a replicative niche known as the Brucella-containing vacuole (BCV) (\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). For instance, the effector VceC induces endoplasmic reticulum (ER) stress, while BtpA and BtpB interfere with TLR signaling to suppress inflammatory responses(\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Despite the identification of numerous T4SS substrates, the functional mechanisms of many effectors remain incompletely understood(\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). BPE275 is a T4SS effector whose roles in stress adaptation and host immune modulation have not been systematically characterized(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we investigated the biological function of BPE275 and evaluated the potential of a \u003cem\u003ebpe275\u003c/em\u003e deletion mutant as an improved live attenuated vaccine candidate. Using \u003cem\u003eB. abortus\u003c/em\u003e A19 as the parental strain, we constructed a markerless deletion mutant A19Δ\u003cem\u003ebpe275.\u003c/em\u003e We comprehensively assessed its morphology, resistance to environmental stresses, intracellular survival in macrophages, and virulence in a murine infection model. Furthermore, we examined the immunomodulatory consequences of \u003cem\u003ebpe275\u003c/em\u003e deletion. Our findings demonstrate that BPE275 is essential for bacterial resistance to host-derived stresses and for immune evasion. Critically, its deletion yields a vaccine candidate offering a promising foundation for next-generation brucellosis vaccines.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Strains and Cells\u003c/h2\u003e \u003cp\u003eThe wild-type Brucella abortus strain A19 and its derivative strains were used in this study. The strain was grown in tryptic soy broth (TSB) medium or tryptic soy agar (TSA) (Difco, Franklin Lakes, New Jersey, USA) plates at 37 ℃ with 5% CO2.The Raw264.7 macrophage cells (from ATCC) and Thp-1 (from ATCC) were all maintained in our laboratory. Raw264.7 cells were cultured with DMEM (Cat No.11965092; Gibco) supplemented with 10% FBS (Cat No. 10099141; Gibco). Thp-1 cells were cultured in RPMI-1640(Cat No.C11875500BT; Gibco) supplemented with 10% FBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Reagents\u003c/h2\u003e \u003cp\u003eTryptic Soy Broth (TSB) and Tryptic Soy Agar (TSA) were purchased from BD Biosciences (NJ, USA); High-glucose Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) and RPMI-1640 medium were obtained from Gibco (Thermo Fisher Scientific); Fetal bovine serum (FBS) was purchased from NSERA (USA); Sodium chloride (NaCl), phosphate-buffered saline (PBS), Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), penicillin-streptomycin solution (100x), and gentamicin were purchased from Solarbio Life Sciences (Beijing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1.3 Primer design and synthesis\u003c/h2\u003e \u003cp\u003eBased on the genome sequence of \u003cem\u003eB. abortus\u003c/em\u003e A19 (GenBank accession number: NZ_CP030751),1000-bp regions immediately upstream and downstream of the \u003cem\u003ebpe275\u003c/em\u003e gene-encoding a putative T4SS effector protein-were selected as homologous arms for allelic exchange. Primers were designed to amplify these flanking regions and facilitate cloning into the suicide vector pUC19-SacB. All primers (listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were synthesized by Sangon Biotech (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e1.4 Construction of the \u003cem\u003ebpe275\u003c/em\u003e deletion and complementation Strain\u003c/h2\u003e \u003cp\u003eA markerless B. abortus A19Δbpe275 mutant was generated via homologous recombination using the suicide vector pUC19-sacB. The 1,000-bp upstream and downstream flanking regions of bpe275 were PCR-amplified and cloned into pUC19-sacB to construct the allelic exchange plasmid pUC19-sacB-Δbpe275. This plasmid was electroporated into A19, and transformants were selected on TSA plates containing ampicillin (100 \u0026micro;g/mL). Colonies that grew on 5% sucrose but not ampicillin were screened by PCR to confirm bpe275 deletion, yielding the A19Δbpe275 strain. For complementation, the recombinant plasmid pBBR1MCS-bpe275 was electroporated into A19Δbpe275 electrocompetent cells, and complemented clones were identified by colony PCR using M13 primers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e1.5 Bacterial growth curve assay\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eB. abortus\u003c/em\u003e A19 and A19Δ\u003cem\u003ebpe275\u003c/em\u003e strains were pre-cultured to mid-logarithmic phase, then diluted into 10 mL of fresh Tryptic Soy Broth (TSB) to an initial OD₆₀₀ of 0.03. Cultures were incubated statically at 37\u0026deg;C, and 1 mL aliquots were collected every 6 hours over a 96-hour period. The OD₆₀₀ of each sample was measured using a microplate reader, and growth curves were plotted accordingly. All growth assays were performed in three independent biological replicates, and results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e1.6 Phenotypic and passage stability analysis\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eB. abortus\u003c/em\u003e A19 and A19Δ\u003cem\u003ebpe275\u003c/em\u003e strains were cultured to the mid-logarithmic phase, serially diluted 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e in sterile saline, and plated on TSA. After 3\u0026ndash;5 days of incubation at 37\u0026deg;C, colonies were stained with 0.5% crystal violet for 30 s to distinguish smooth from rough phenotypes. For agglutination testing, bacterial pellets (4000g, 5 min) were resuspended in physiological saline and subjected to acriflavine and heat agglutination assays. To assess genetic stability, A19Δ\u003cem\u003ebpe275\u003c/em\u003e was serially passaged for 10 generations alongside the rough reference strain \u003cem\u003eB. abortus\u003c/em\u003e 6/66. Acriflavine agglutination tests were performed every two passages to monitor phenotypic consistency of the Δ\u003cem\u003ebpe275\u003c/em\u003e deletion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e1.7 Assessment of lipopolysaccharide (LPS) integrity\u003c/h2\u003e \u003cp\u003eLipopolysaccharide (LPS) integrity of \u003cem\u003eB. abortus\u003c/em\u003e A19 and \u003cem\u003ebpe275\u003c/em\u003e deletion and complementation strain was assessed by silver staining after SDS-PAGE. LPS was extracted using the iNtRON LPS Extraction Kit (iNtRON Biotechnology, Korea). Extracted LPS resolved on a 12.5% SDS-PAGE gel. Gels were silver-stained using a standard protocol involving periodic acid oxidation, silver nitrate impregnation, and formaldehyde-based development, followed by fixation in 10% acetic acid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e1.8 Adhesion, Invasion and Intracellular Survival Assays\u003c/h2\u003e \u003cp\u003eRaw264.7 and PMA-differentiated Thp-1 cells were seeded in 24-well plates and infected with \u003cem\u003eB. abortus\u003c/em\u003e A19 and \u003cem\u003ebpe275\u003c/em\u003e deletion and complementation strain at an MOI of 100. Infection was synchronized by centrifugation and incubation. For adhesion, cells were washed three times with PBS, lysed with 0.1% Triton X-100, and lysates plated on TSA for CFU enumeration after 3\u0026ndash;5 days. For invasion, extracellular bacteria were killed by 50 \u0026micro;g/mL gentamicin post-infection; cells were then washed, lysed, and plated as above. For intracellular survival, gentamicin was reduced to 25 \u0026micro;g/mL, and bacterial loads were quantified at 12, 24, and 48 h post-infection.\u003c/p\u003e \u003cp\u003eAll assays were performed in triplicate and repeated in at least three independent biological experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e1.9 Stress sensitivity assays\u003c/h2\u003e \u003cp\u003eTo assess sensitivity to acidic pH and oxidative stress, 1 \u0026times; 10⁶ CFU/mL \u003cem\u003eB. abortus\u003c/em\u003e A19 and \u003cem\u003ebpe275\u003c/em\u003e deletion and complementation strain were mixed with TSB adjusted to pH 7.0, 6.5, 5.5, or 4.5, or containing 5.0, 2.5-, or 1.0-mM H₂O₂. After 2 h incubation at 37\u0026deg;C in 5% CO₂, samples were serially diluted, plated on TSA, and CFUs counted after 3\u0026ndash;5 days. Survival rates were calculated relative to pH 7.0 or no H₂O₂ \u003csup\u003e(\u003cem\u003e31, 32\u003c/em\u003e)\u003c/sup\u003e. All assays were performed in triplicate across three independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e1.10 Mouse Virulence Assessment\u003c/h2\u003e \u003cp\u003eA total of fifteen 6-week-old female BALB/c mice were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;5 per group): the A19 infection group, the A19Δ\u003cem\u003ebpe275\u003c/em\u003e infection group, the A19Δ\u003cem\u003ebpe275::bpe275\u003c/em\u003e infection group and the sterile saline control group. Mice in the infection groups were intraperitoneally inoculated with 1 \u0026times; 10⁵ CFU of \u003cem\u003eB. abortus\u003c/em\u003e in 0.1 mL sterile saline, while mice in the control group were injected with an equal volume of sterile saline(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). At 7-, 14-, 28-, and 42-days post-infection, mice corresponding to each time point were euthanized by cervical dislocation. Under sterile conditions, spleens were collected, weighed, and the spleen index was calculated (spleen weight/body weight \u0026times; 100%). The spleens were then homogenized, serially diluted 10-fold, and plated for CFU counting, expressed as CFU per gram of spleen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e1.11 Enzyme-Linked Immunosorbent Assay(ELISA)\u003c/h2\u003e \u003cp\u003eBrucella-specific IgG and IgM in mouse sera were quantified by indirect ELISA. Whole-cell antigen was prepared from heat-inactivated \u003cem\u003eB. abortus\u003c/em\u003e A19, sonicated, and diluted to 10 \u0026micro;g/mL in carbonate coating buffer. 100 \u0026micro;L/well antigen was coated onto 96-well plates overnight at 4\u0026deg;C. After blocking with 5% skim milk in PBS for 1 h at 37\u0026deg;C, sera (1:100 in PBST, 100 \u0026micro;L/well) were added and incubated for 1 h at 37\u0026deg;C. Following PBST washes, HRP-conjugated goat anti-mouse IgG and IgM (both 1:5,000; TransGen Biotech and BioDragon, China) were applied for 1 h at 37\u0026deg;C. Reactions were developed with TMB (100 \u0026micro;L), stopped with 2 M H₂SO₄ (50 \u0026micro;L), and absorbance measured at 450 nm. Samples were tested in duplicate or triplicate, with na\u0026iuml;ve mouse serum as the negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e1.12 Assessment of residual virulence in mice\u003c/h2\u003e \u003cp\u003eTo evaluate residual virulence, ninety-six 6-week-old female BALB/c mice were randomly assigned to three groups (n\u0026thinsp;=\u0026thinsp;32 per group) and inoculated intraperitoneally with 1 \u0026times; 10⁸ CFU of either \u003cem\u003eB. abortus\u003c/em\u003e A19 and \u003cem\u003ebpe275\u003c/em\u003e deletion and complementation strain in 0.1 mL PBS. At 3-, 6-, 9-, and 12-weeks post-infection (wpi), eight mice per group were euthanized by cervical dislocation under anesthesia. The 50% clearance time (RT₅₀) was calculated using the Reed-Muench method based on the cumulative proportion of culture-negative mice (no spleen colonies) at each time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e1.13 Evaluation of Immune Protection\u003c/h2\u003e \u003cp\u003eTwenty BALB/c mice were divided into four group A19-immunized, A19Δ\u003cem\u003ebpe275\u003c/em\u003e-immunized, A19Δ\u003cem\u003ebpe275::bpe275\u003c/em\u003e-immunized and PBS control. Mice were subcutaneously immunized with 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU/0.1 mL. Thirty days later, they were challenged intraperitoneally with 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU of \u003cem\u003eB. abortus 2308\u003c/em\u003e or \u003cem\u003eB. melitensis 16M\u003c/em\u003e. Fifteen days after challenge, mice were euthanized by cervical dislocation, and spleens were aseptically collected to determine the spleen index and bacterial load. The protection index (PI) was subsequently calculated to evaluate vaccine efficacy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e1.14 Statement\u003c/h2\u003e \u003cp\u003eFemale BALB/c mice, 6-week-old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and used in all experiments. Animal experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Welfare and Ethics Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Approval No. IAS2025-155). Work with \u003cem\u003eBrucella abortus 2308\u003c/em\u003e and derivatives was conducted in the laboratory\u0026rsquo;s BSL-3 facility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e1.15 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were obtained from at least three independent biological replicates. The statistical analyses of the data and comparisons among the groups were performed by one-way analysis of variance (ANOVA) or two-way ANOVA with Tukey post hoc tests. All data were analyzed using GraphPad Prism software. Data were shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The levels of significance are indicated as follows: ns, no significance; *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003ch2\u003e\u003cstrong\u003e2.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConstruction of a \u003cem\u003eB. abortus\u003c/em\u003e A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e Deletion Strain and Complemented Strain\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eB. abortus\u003c/em\u003e A19\u003cem\u003e\u0026Delta;bpe275\u003c/em\u003e deletion mutant was constructed using a markerless homologous recombination technique based on the A19 background (Figure 1a). First, 1000-bp upstream and downstream homologous arms of the \u003cem\u003ebpe275\u003c/em\u003e gene were amplified by PCR and cloned into the suicide vector pUC19-SacB to construct the recombinant plasmid pUC19-SacB-\u0026Delta;\u003cem\u003ebpe275.\u003c/em\u003e Subsequently, the plasmid was electroporated into \u003cem\u003eB. abortus\u003c/em\u003e A19 competent cells, and the deletion mutant was obtained after two rounds of homologous recombination screening. To verify the successful construction, specific primers were used for PCR identification of the parental strain A19 and the deletion mutant A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e. The results showed that the A19 parental strain produced an amplification fragment of 2193 bp (Lane 4), whereas the A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e mutant yielded a 1422 bp band, which was consistent with the expected size of the deleted fragment (Lane 5), indicating that the \u003cem\u003ebpe275\u003c/em\u003e gene had been successfully precisely deleted. The complemented strain B. abortus A19\u0026Delta;\u003cem\u003ebpe275::bpe275\u003c/em\u003e yielded a PCR amplicon of 1282 bp (lanes 6-7), consistent with the expected size and confirming successful genetic complementation of the\u003cem\u003e\u0026nbsp;bpe275\u003c/em\u003e gene. In addition, the amplification results of the recombinant plasmid pUC19-SacB-\u0026Delta;\u003cem\u003ebpe275\u0026nbsp;\u003c/em\u003e(Lane 3) and the upstream/downstream homologous arms (Lanes 1 and 2) further confirmed the accuracy of the construction process (Figure 1b).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.2 A19\u0026Delta;\u003cem\u003ebpe275\u0026nbsp;\u003c/em\u003emaintains wild-type morphology and in vitro growth kinetics\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eScanning electron microscopy (SEM) revealed that both strains consisted of short, rod-shaped cells with rounded ends, exhibiting uniform size and plump morphology without evidence of aggregation or structural abnormalities (Figure 2a). Statistical analysis of cell length showed no significant difference between A19、A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e and A19\u0026Delta;\u003cem\u003ebpe275::bpe275\u003c/em\u003e (Figure 2b), indicating that deletion of \u003cem\u003ebpe275\u0026nbsp;\u003c/em\u003edid not alter bacterial morphology.\u003c/p\u003e\n\u003cp\u003eIn vitro growth kinetics were assessed by monitoring OD₆₀₀ in TSB broth at 37 \u0026deg;C over 96 h, following inoculation at an initial OD₆₀₀ of 0.03. Both strains entered logarithmic phase at 6 h and reached stationary phase by 30 h (Figure 2c). No significant differences in growth rate or final yield were observed throughout the 96-h period, demonstrating that \u003cem\u003ebpe275\u003c/em\u003e deletion does not impair the in vitro proliferative capacity.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.3 A19\u0026Delta;bpe275 retains a smooth phenotype and exhibits genetic stability\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe \u003cem\u003eB. abortus\u003c/em\u003e A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e deletion mutant maintained a smooth phenotype, as evidenced by its intact lipopolysaccharide (LPS) structure and genetic stability during serial passage. Both A19、A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e and A19\u0026Delta;\u003cem\u003ebpe275::bpe275\u0026nbsp;\u003c/em\u003eformed white colonies after crystal violet staining (Figure 3b-d), but the rough control strain 6/66 formed purple colonies (Figure 3a), showed no agglutination in acriflavine (Figure 3e), and displayed the classic ladder-like O-antigen pattern on silver-stained SDS-PAGE (Figure 3f), confirming intact LPS and a smooth phenotype.\u003c/p\u003e\n\u003cp\u003eWhen serially passaged for 10 generations, A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e remained agglutination-negative in acriflavine tests performed every two passages, while the rough control strain 6/66 consistently agglutinated (Figure 3g). These results indicate that\u003cem\u003e\u0026nbsp;bpe275\u0026nbsp;\u003c/em\u003egene deletion does not disrupt LPS integrity or phenotypic stability.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.4 A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003eefficiently invades host cells but exhibits significantly impaired intracellular survival.\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAdhesion and invasion assays were performed in Raw264.7 murine macrophages. A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e exhibited no defect in adhesion to or invasion of RAW264.7 murine macrophages, with levels comparable to those of the parental A19 and complemented A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e::\u003cem\u003ebpe275\u003c/em\u003e strains at 1 h post-infection (Figure 4a), indicating that BPE275 is not required for initial host cell entry.\u003c/p\u003e\n\u003cp\u003eIn contrast, intracellular survival was markedly impaired in A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e. In Raw264.7 cells, the mutant exhibited significantly lower bacterial loads at 12, 24, and 48 h post-infection compared to A19 and A19\u0026Delta;\u003cem\u003ebpe275:bpe275\u003c/em\u003e (Figure 4b). Similarly, in Thp-1 cells, A19\u0026Delta;\u003cem\u003ebpe275\u0026nbsp;\u003c/em\u003eshowed significantly reduced CFUs at 12 h and 48 h (Figure 4c).\u003c/p\u003e\n\u003cp\u003eThese results demonstrate that BPE275 is dispensable for adhesion and invasion but plays a critical role in sustaining intracellular replication-likely by modulating \u003cem\u003eBrucella\u003c/em\u003e-containing vacuole maturation or suppressing host bactericidal responses during the early establishment of persistent infection.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.5 A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e is hypersensitive to oxidative stress but maintains normal acid tolerance\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo assess the impact of \u003cem\u003ebpe275\u003c/em\u003e deletion on the environmental adaptability of \u003cem\u003eBrucella\u003c/em\u003e, we systematically examined the survival rates of A19 and \u003cem\u003ebpe275\u003c/em\u003e deletion strain and complemented strain under various stress conditions. In the in vitro oxidative stress assay, the survival rates of all strains decreased significantly with increasing concentrations of hydrogen peroxide (1.0 mM, 2.5 mM, and 5.0 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). After treatment with 1.0 mM and 2.5 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, no significant differences in survival rates were observed among the strains at the same concentrations. However, under 5.0 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, the survival ability of the mutant strain A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e was significantly lower than that of A19 (Figure 5a), suggesting that deletion of the \u003cem\u003ebpe275\u0026nbsp;\u003c/em\u003egene impairs the ability of \u003cem\u003eBrucella\u003c/em\u003e to resist oxidative stress. In contrast, acid tolerance was unaffected: both strains showed comparable survival across pH 7.0 to 4.5 (Figure 5b), with similar viability loss only at the most acidic condition (pH 4.5).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.6\u003c/strong\u003e \u003cstrong\u003eA19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e is attenuated in vivo with accelerated clearance and reduced splenic pathology\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eA19\u0026Delta;\u003cem\u003ebpe275\u0026nbsp;\u003c/em\u003eexhibits significantly attenuated virulence in mice, as demonstrated by reduced splenomegaly, lower splenic bacterial burden, milder histopathology, and accelerated clearance compared to the parental A19 strain. At 1, 2, 4, and 6 weeks post-infection, mice infected with A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e showed a significantly lower spleen index than those infected with A19 (Figure 6b), and splenic bacterial loads remained markedly reduced at all time points (Figure 6c), indicating attenuated virulence in mice.\u003c/p\u003e\n\u003cp\u003eHistopathological analysis further confirmed this attenuation. At 2 weeks post-infection, mice infected with A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e exhibited only mild hyperplasia of white pulp lymphoid follicles with focal perifollicular macrophage aggregation, while red pulp architecture remained intact-strikingly contrasting with the severe lymphoid follicle depletion, extensive lymphocyte loss, and massive macrophage infiltration observed in mice infected with either wild-type A19 or the complemented strain A19\u0026Delta;\u003cem\u003ebpe27\u003c/em\u003e5\u003cem\u003e::bpe275\u003c/em\u003e.From 4-week to 6-week, the A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e-infected group had fully recovered, showing intact splenic capsules and normalized red-to-white pulp ratios, indicating resolution of infection-induced pathology. In contrast, residual macrophage aggregates persisted around lymphoid follicles in the A19 group (Figure 6d).\u003c/p\u003e\n\u003cp\u003eNotably, at 2-week, the complemented strain A19\u0026Delta;\u003cem\u003ebpe275::bpe275\u003c/em\u003e largely recapitulated the histopathological features and bacterial persistence of wild-type A19; however, its splenic bacterial loads and spleen index remained significantly lower than those of the A19 group.\u003c/p\u003e\n\u003cp\u003eTo quantify bacterial persistence, we determined the median time to clearance (RT₅₀) using the Reed-Muench method. A19 exhibited prolonged persistence (RT₅₀=9.4 weeks), whereas both A19\u0026Delta;bpe275 (RT₅₀=6.5 weeks) and the complemented strain A19\u0026Delta;\u003cem\u003ebpe275::bpe275\u003c/em\u003e (RT₅₀=6.8 weeks) were cleared significantly faster. These results establish BPE275 as a key determinant of \u003cem\u003eBrucella\u003c/em\u003e persistence, enabling immune evasion and chronic infection (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1 Bacterial clearance kinetics in mice infected with A19\u003c/strong\u003e\u003cstrong\u003e、\u003c/strong\u003e\u003cstrong\u003eA19\u003c/strong\u003e\u003cstrong\u003e\u0026Delta;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ebpe275\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and A19\u003c/strong\u003e\u003cstrong\u003e\u0026Delta;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ebpe275::bpe275\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"591\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 23.4913%;\"\u003e\n \u003cp\u003eStrains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 20.5019%;\"\u003e\n \u003cp\u003eInoculation dose\u003c/p\u003e\n \u003cp\u003e（CFU/mice）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 312px;\"\u003e\n \u003cp\u003eRecovery rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 62px;\"\u003e\n \u003cp\u003eRT\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e6 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e9 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e12 weeks\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.4913%;\"\u003e\n \u003cp\u003eA19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.5019%;\"\u003e\n \u003cp\u003e1*10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e87.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e9.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.4913%;\"\u003e\n \u003cp\u003eA19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.5019%;\"\u003e\n \u003cp\u003e1*10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.4913%;\"\u003e\n \u003cp\u003eA19\u0026Delta;\u003cem\u003ebpe275::bpe275\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.5019%;\"\u003e\n \u003cp\u003e1*10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e62.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e6.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch2\u003e\u003cstrong\u003e2.7 A19\u0026Delta;bpe275 elicits enhanced Th1 responses with selective IgM suppression\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eSerum levels of 11 cytokines were measured at 2-, 4-, and 6-weeks post-infection (Figure 7a). At 2-week the A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e-infected group displayed sustained elevation of multiple pro-inflammatory cytokines, including IFN-\u0026gamma;, TNF-\u0026alpha;, IL-6, IL-18, and IL-5. These results indicate that BPE275 acts as a negative regulator of host inflammation during mid-to-late infection, and its absence unleashes a robust and prolonged Th1-biased immune response.\u003c/p\u003e\n\u003cp\u003eA19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e induced markedly reduced IgM responses compared to A19 throughout the observation period (Figure 7b), indicating impaired early humoral activation. In contrast, IgG responses were comparable to A19 from 2-week onward (Figure 7c), despite a modest reduction at 1-week (p \u0026lt; 0.05). Notably, the complemented strain A19\u0026Delta;\u003cem\u003ebpe275::bpe275\u003c/em\u003e phenocopied the IgM suppression of the deletion mutant, suggesting non-physiological expression effects. Collectively, these results demonstrate that \u003cem\u003ebpe275\u003c/em\u003e deletion selectively ablates early IgM production while preserving long-term IgG-mediated immunity and memory formation. Collectively, BPE275 modulates host immunity by dampening Th1 inflammation and promoting early IgM responses to favor bacterial persistence.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.8 A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e enhances the protective efficacy of the \u003cem\u003eB. abortus\u003c/em\u003e A19 vaccine\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo evaluate the vaccine potential of A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e mice were immunized and challenged intraperitoneally with virulent \u003cem\u003eBrucella abortus\u003c/em\u003e \u003cem\u003e2308\u003c/em\u003e at 40 days post-immunization. Spleen index and bacterial burden were assessed 15 days post-challenge (Figure 8a). Macroscopic examination revealed marked splenomegaly and dark-red discoloration in PBS-control mice (Figure 8b), whereas spleens from both A19- and A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e-immunized mice were visibly smaller. Statistical analysis confirmed significantly lower spleen weights and bacterial burdens in both vaccinated groups compared to PBS controls (Figure 8c-d), with no significant difference between A19 and A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e. Notably, A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e conferred robust protection against \u003cem\u003eB. abortus 2308\u003c/em\u003e challenge, achieving a protection index of 88.0%-comparable to that of parental A19 (Table 2). However, the protective efficacy of the complemented strain A19\u0026Delta;\u003cem\u003ebpe275::bpe275\u0026nbsp;\u003c/em\u003ewas not fully restored to the level of parental A19.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Protective efficacy of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA19\u003c/strong\u003e\u003cstrong\u003e、\u003c/strong\u003e\u003cstrong\u003eA19\u003c/strong\u003e\u003cstrong\u003e\u0026Delta;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ebpe275\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eand A19\u003c/strong\u003e\u003cstrong\u003e\u0026Delta;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ebpe275::bpe275\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;against \u003cem\u003eB. abortus\u003c/em\u003e \u003cem\u003e2308\u003c/em\u003e challenge.\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"515\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 142px;\"\u003e\n \u003cp\u003echallenge strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003eimmunization strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003espleen bacterial load/log\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eProtection Index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cem\u003eB. abortus 2308\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e7.58 \u0026plusmn; 0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003eA19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e6.70 \u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e85.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003eA19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e6.63\u0026plusmn;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e88.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003eA19\u0026Delta;\u003cem\u003ebpe275::bpe275\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e7.07 \u0026plusmn;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e67.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch2\u003e\u003cstrong\u003e2.9 A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e elicits protection against \u003cem\u003eB. melitensis\u003c/em\u003e \u003cem\u003e16M\u003c/em\u003e with an optimal safety-immunogenicity balance\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo evaluate the cross-protective potential of the A19\u0026Delta;\u003cem\u003ebpe275\u0026nbsp;\u003c/em\u003evaccine, BALB/c mice were immunized subcutaneously with 1 \u0026times;10⁵ CFU of A19, A19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e, or the complemented strain A19\u0026Delta;\u003cem\u003ebpe275::bpe275\u003c/em\u003e, with PBS serving as the control (Figure 9a). Spleens were harvested 15 days post-challenge for quantification and assessment of protective immunity. Results showed that in the \u003cem\u003eB. melitensis\u003c/em\u003e \u003cem\u003e16M\u003c/em\u003e cross-protection challenge, A19\u0026Delta;\u003cem\u003ebpe275\u0026nbsp;\u003c/em\u003econferred robust protection with a protection index (PI) of 80.0%, slightly lower than that of the parental A19 strain (85.6%)(Table 3). Notably, this high level of protection was achieved despite significantly reduced splenic bacterial loads and the lowest spleen index among all groups (Figure 8b-d), demonstrating an optimal balance between safety and immunogenicity. In contrast, the protective efficacy of the complemented strain A19\u0026Delta;\u003cem\u003ebpe275::bpe275\u003c/em\u003e was not fully restored to the level of parental A19.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Protective efficacy of A19\u003c/strong\u003e\u003cstrong\u003e、\u003c/strong\u003e\u003cstrong\u003eA19\u003c/strong\u003e\u003cstrong\u003e\u0026Delta;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ebpe275\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and A19\u003c/strong\u003e\u003cstrong\u003e\u0026Delta;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ebpe275::bpe275\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;against \u003cem\u003eB. melitensis\u0026nbsp;\u003c/em\u003e\u003cem\u003e16M\u003c/em\u003e challenge.\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"505\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 143px;\"\u003e\n \u003cp\u003echallenge strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003eimmunization strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003espleen bacterial load/log\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eProtection Index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 143px;\"\u003e\n \u003cp\u003e\u003cem\u003eB. melitensis 16M\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e7.52\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003eA19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e6.53\u0026plusmn;0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e85.6%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003eA19\u0026Delta;\u003cem\u003ebpe275\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e6.82\u0026plusmn;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e80.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 157px;\"\u003e\n \u003cp\u003eA19\u0026Delta;\u003cem\u003ebpe275::bpe275\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 110px;\"\u003e\n \u003cp\u003e6.98\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e70.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe development of live attenuated \u003cem\u003eBrucella\u003c/em\u003e vaccines requires a delicate balance between attenuation and immunogenicity: the strain must be sufficiently attenuated to ensure safety, yet persist long enough in the host to elicit robust protective immunity. In this study, we identified BPE275 as a critical Type IV Secretion System (T4SS) effector essential for stress adaptation in \u003cem\u003eBrucella abortus\u003c/em\u003e, and demonstrated that its deletion from the A19 vaccine backbone yields a promising candidate that achieves an optimal equilibrium between safety and immunogenicity.\u003c/p\u003e \u003cp\u003eIntracellular survival within macrophages is a hallmark of \u003cem\u003eBrucella\u003c/em\u003e pathogenesis. We found that the A19Δ\u003cem\u003ebpe275\u003c/em\u003e mutant exhibited significantly reduced persistence in both Raw264.7 and Thp-1 macrophages and displayed markedly impaired resistance to oxidative stress in vitro, indicating that BPE275 likely facilitates bacterial adaptation to the harsh phagosomal environment.\u003c/p\u003e \u003cp\u003eImmunologically, \u003cem\u003ebpe275\u003c/em\u003e deletion skewed the host response toward a Th1 bias-a critical axis for controlling intracellular \u003cem\u003eBrucella\u003c/em\u003e infection(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Compared to parental A19, A19Δ\u003cem\u003ebpe275\u003c/em\u003e induced significantly higher levels of IFN-γ, TNF-α, IL-6, and IL-12. Intriguingly, early IgM production was suppressed in the mutant-immunized group, while long-term IgG responses remained intact. This observation supports a compelling hypothesis: wild-type BPE275 may act as an immunomodulator that promotes non-T cell-dependent IgM responses-potentially, while simultaneously dampening macrophage-activating Th1 pathways.\u003c/p\u003e \u003cp\u003eCritically, A19Δ\u003cem\u003ebpe275\u003c/em\u003e overcomes a key limitation of T4SS-deficient vaccines. Complete inactivation of the VirB system such as Δ\u003cem\u003evirB\u003c/em\u003e mutants often leads to over-attenuation, resulting in rapid bacterial clearance before adaptive immune memory is fully established(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In contrast, our data show that A19Δ\u003cem\u003ebpe275\u003c/em\u003e retains sufficient colonization capacity to stimulate immunity. Moreover, the mutant maintains wild-type morphology, smooth lipopolysaccharide (LPS) integrity, and genetic stability, yet exhibits significantly accelerated clearance \u003cem\u003ein vivo\u003c/em\u003e (RT₅₀ shortened by 2.9 weeks) and minimal splenomegaly. This confirms that the unique immunomodulation conferred by \u003cem\u003ebpe275\u003c/em\u003e deletion does not compromise protective efficacy; rather, it enhances safety without sacrificing protection. Notably, A19Δ\u003cem\u003ebpe275\u003c/em\u003e demonstrated superior protective efficacy compared to A19 in challenge models with both \u003cem\u003eB. abortus 2308\u003c/em\u003e and \u003cem\u003eB. melitensis 16M\u003c/em\u003e, indicating cross-species protective potential.\u003c/p\u003e \u003cp\u003eWe acknowledge that plasmid-mediated genetic complementation in \u003cem\u003eBrucella\u003c/em\u003e pathogenesis studies is frequently hampered by instability, particularly during systemic infection where precise gene dosage and expression stability are difficult to maintain. Our data reveal a striking context-dependent discrepancy: the complemented strain A19Δ\u003cem\u003ebpe275\u003c/em\u003e::\u003cem\u003ebpe275\u003c/em\u003e fully restored intracellular survival to wild-type A19 levels in macrophage models (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-c), and the pronounced attenuation coupled with high immunogenicity of A19Δ\u003cem\u003ebpe275\u003c/em\u003e strongly indicates that the observed phenotype stems from deletion of \u003cem\u003ebpe275\u003c/em\u003e rather than secondary mutations.\u003c/p\u003e \u003cp\u003eHowever, in murine infection models, the complemented strain failed to fully restore virulence or vaccine efficacy. Specifically, it only partially rescued splenic bacterial persistence and conferred significantly lower protection against challenge with virulent \u003cem\u003eBrucella\u003c/em\u003e compared to parental A19. This discrepancy likely arises from two interrelated factors: the role of BPE275 in immune evasion and chronic infection is more critical in the complex in vivo milieu-where dynamic host pressures such as oxidative burst, nutrient limitation, and adaptive immunity demand precise spatiotemporal regulation of effector expression; plasmid-driven overexpression may disrupt the stoichiometric balance of the T4SS effector network or result in non-physiological expression kinetics, thereby restoring cellular phenotypes in vitro but failing to fully rescue virulence or protective immunity in the whole-animal context. Nevertheless, to definitively establish BPE275 as a bona fide virulence determinant, future mechanistic studies should employ chromosomal single-copy complementation to achieve physiological expression levels and rigorously exclude polar effects or artifacts from ectopic overexpression\u003c/p\u003e \u003cp\u003eIn summary, this study establishes BPE275 as a key component of the \u003cem\u003eBrucella\u003c/em\u003e T4SS arsenal, functionally linking stress resistance to immune evasion. The A19Δ\u003cem\u003ebpe275\u003c/em\u003e mutant represents a rational evolution in brucellosis vaccine design, effectively decoupling virulence from immunogenicity. Importantly, A19Δ\u003cem\u003ebpe275\u003c/em\u003e combines attenuated virulence with genetic stability and a smooth phenotype, retaining the immunogenic advantages of smooth strains while mitigating safety concerns. Uniquely, the specific genetic deletion provides a distinct molecular signature, facilitating the development of a robust TaqMan dual-target real-time PCR assay targeting \u003cem\u003ebpe275\u003c/em\u003e and a conserved internal control. This method enables the rapid and accurate differentiation between vaccinated animals and those infected with wild-type strains, addressing a critical bottleneck in brucellosis eradication programs. This capability supports a Differentiating Infected from Vaccinated Animals (DIVA) strategy based on molecular detection, which is essential for vaccine quality control, immune monitoring, and field surveillance. Collectively, these attributes position A19Δ\u003cem\u003ebpe275\u003c/em\u003e as a compelling candidate for next-generation brucellosis vaccines. Critical next steps include evaluating safety in pregnant models and efficacy in natural hosts, which will be pivotal for translational advancement.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnhui Dai, Dongjie Sun and Yifan Wu designed and performed the experiments, analyzed the data, and prepared the manuscript. Xiaowei Peng, Mengtao Zhang, Yanxiao Zhao assisted in data analysis. Shijin Jiang and Jiabo Ding contributed to the experimental design, data analysis, and the preparation and editing of the manuscript. Enhui Dai, Dongjie Sun, and Yifan Wu contributed equally to this work. All the authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Professor Hui Jiang (Institute of Animal Science, Chinese Academy of Agricultural Sciences) for her expert guidance in experimental design and execution. We also extend our gratitude to Dr. Yichen Zhang (Institute of Animal Science, Chinese Academy of Agricultural Sciences) for his invaluable advice on scientific writing.\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences (CAAS-CSLPDCP-202403), China Postdoctoral Science Foundation under Grant Number (2025T180827) and (GZB20250698).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets that support this study are included in the article/Supplementary Material. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eC. 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Zhi\u003cem\u003e et al.\u003c/em\u003e, VceC Mediated IRE1 Pathway and Inhibited CHOP-induced Apoptosis to Support Brucella Replication in Goat Trophoblast Cells. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e. 10.3390/ijms20174104 (2019)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Brucella abortus, BPE275, intracellular persistence, attenuated virulence, live attenuated vaccine candidate","lastPublishedDoi":"10.21203/rs.3.rs-8979308/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8979308/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBrucellosis, a significant zoonotic disease caused by Brucella spp., remains a major global challenge to both animal production and public health. Current live attenuated vaccines, such as Brucella abortus A19, are limited in their application due to residual virulence and interference with serodiagnosis. The intracellular survival and immune evasion of Brucella critically depend on effector proteins delivered by the Type IV Secretion System (T4SS), yet the functions of many of these effectors remain poorly defined. In this study, we constructed a markerless deletion mutant, A19Δ\u003cem\u003ebpe275\u003c/em\u003e, in the \u003cem\u003eB. abortus\u003c/em\u003e A19 background and comprehensively evaluated its phenotype, virulence, and vaccine potential. The A19Δ\u003cem\u003ebpe275\u003c/em\u003e mutant retained wild-type morphology, smooth lipopolysaccharide (LPS) structure, in vitro growth kinetics, and genetic stability, but exhibited significantly impaired long-term intracellular survival in macrophages. In murine infection models, A19Δ\u003cem\u003ebpe275\u003c/em\u003e displayed markedly attenuated virulence, characterized by consistently lower splenic bacterial loads, milder histopathological lesions, and accelerated clearance compared to the parental A19 strain. Immunologically, infection with A19Δ\u003cem\u003ebpe275\u003c/em\u003e elicited a robust and sustained Th1-biased immune response.\u003c/p\u003e \u003cp\u003eNotably, A19Δ\u003cem\u003ebpe275\u003c/em\u003e conferred comparable protective efficacy with improved safety against challenge with the virulent \u003cem\u003eB. abortus 2308\u003c/em\u003e and demonstrated cross-protection against \u003cem\u003eB. melitensis 16M\u003c/em\u003e. Uniquely, the \u003cem\u003ebpe275\u003c/em\u003e deletion provides a molecular signature for a DIVA-compatible real-time PCR assay, enabling differentiation between vaccinated and infected animals. Collectively, by achieving an optimal balance between enhanced safety and preserved immunogenicity, A19Δ\u003cem\u003ebpe275\u003c/em\u003e emerges as a promising candidate for next-generation live attenuated brucellosis vaccines.\u003c/p\u003e","manuscriptTitle":"A19Δbpe275 emerges as a safer live attenuated vaccine candidate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-13 17:35:45","doi":"10.21203/rs.3.rs-8979308/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-13T15:04:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-07T04:23:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-15T13:13:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123855272238203962613819792588170436235","date":"2026-03-15T09:47:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86547253814640357832107942643028204106","date":"2026-03-13T04:57:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27211285057423169951879930466662351732","date":"2026-03-11T12:59:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-11T12:41:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-10T20:05:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-03T08:44:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2026-02-26T14:57:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f3483e94-7b6a-48d8-9c21-cb0763f48063","owner":[],"postedDate":"March 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":64358253,"name":"Health sciences/Diseases"},{"id":64358254,"name":"Biological sciences/Immunology"},{"id":64358256,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-13T15:10:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-13 17:35:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8979308","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8979308","identity":"rs-8979308","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}