Bioinformatics approach for structure modeling, vaccine design, and molecular docking of Brucella candidate proteins BvrR, OMP25, and OMP31.

Brucellosis is a zoonotic disease with significant economic and healthcare costs. Despite the eradication efforts, the disease persists. Vaccines prevent disease in animals while antibiotics cure humans with limitations. This study aims to design vaccines and drugs for brucellosis in animals and humans, using protein modeling, epitope prediction, and molecular docking of the target proteins (BvrR, OMP25, and OMP31). Tertiary structure models of three target proteins were constructed and assessed using RMSD, TM-score, C-score, Z-score, and ERRAT. The best models selected from AlphaFold and I-TASSER due to their superior performance according to CASP 12 - CASP 15 were chosen for further analysis. The motif analysis of best models using MotifFinder revealed two, five, and five protein binding motifs, however, the Motif Scan identified seven, six, and eight Post-Translational Modification sites (PTMs) in the BvrR, OMP25, and OMP31 proteins, respectively. Dominant B cell epitopes were predicted at (44-63, 85-93, 126-137, 193-205, and 208-237), (26-46, 52-71, 98-114, 142-155, and 183-200), and (29-45, 58-82, 119-142, 177-198, and 222-251) for the three target proteins. Additionally, cytotoxic T lymphocyte epitopes were detected at (173-181, 189-197, and 202-210), (61-69, 91-99, 159-167, and 181-189), and (3-11, 24-32, 167-175, and 216-224), while T helper lymphocyte epitopes were displayed at (39-53, 57-65, 150-158, 163-171), (79-87, 95-108, 115-123, 128-142, and 189-197), and (39-47, 109-123, 216-224, and 245-253), for the respective target protein. Furthermore, structure-based virtual screening of the ZINC and DrugBank databases using the docking MOE program was followed by ADMET analysis. The best five compounds of the ZINC database revealed docking scores ranged from (- 16.8744 to - 15.1922), (- 16.0424 to - 14.1645), and (- 14.7566 to - 13.3222) for the BvrR, OMP25, and OMP31, respectively. These compounds had good ADMET parameters and no cytotoxicity, while DrugBank compounds didn't meet Lipinski's rule criteria. Therefore, the five selected compounds from the ZINC20 databases may fulfill the pharmacokinetics and could be considered lead molecules for potentially inhibiting Brucella's proteins.

A novel vaccine strategy against Brucellosis using Brucella abortus multi-epitope OMPs vaccine based on Lactococcus lactis live bacterial vectors.

Brucella infections typically occur in mucosal membranes, emphasizing the need for mucosal vaccinations. This study evaluated the effectiveness of orally administering Lactococcus lactis (L. lactis) for producing the Brucella abortus multi-epitope OMPs peptide. A multi-epitope plasmid was generated through a reverse vaccinology method, and mice were administered the genetically modified L. lactis orally as a vaccine. The plasmid underwent digestion, synthesizing a 39 kDa-sized protein known as OMPs by the target group. The sera of mice that were administered the pNZ8124-OMPs-L. lactis vaccine exhibited a notable presence of IgG1 antibodies specific to outer membrane proteins (OMPs), heightened levels of interferon (IFN-λ) and tumor necrosis factor alpha (TNF-α), and enhanced transcription rates of interleukin 4 (IL-4) and interleukin 10 (IL-10). The spleen sections from the pNZ8124-OMPs-L. lactis and IRIBA group had less morphological damage associated with inflammation, infiltration of lymphocytes, and lesions to the spleen. The findings present a novel approach to utilizing the food-grade, non-pathogenic L. lactis as a protein cell factory to synthesize innovative immunological candidate OMPs. This approach offers a distinctive way to evaluate experimental medicinal items' practicality, safety, affordability, and long-term sustainability.

Brucella melitensis Rev1Δwzm: Placental pathogenesis studies and safety in pregnant ewes.

One of the main causes of human brucellosis is Brucella melitensis infecting small ruminants. To date, Rev1 is the only vaccine successfully used to control ovine and caprine brucellosis. However, it is pathogenic for pregnant animals, resulting in abortions and vaginal and milk shedding, as well as being infectious for humans. Therefore, there is an urgent need to develop an effective vaccine that is safer than Rev1. In efforts to further attenuate Rev1, we recently used wzm inactivation to generate a rough mutant (Rev1Δwzm) that retains a complete antigenic O-polysaccharide in the bacterial cytoplasm. The aim of the present study was to evaluate the placental pathogenicity of Rev1Δwzm in trophoblastic cells, throughout pregnancy in mice, and in ewes inoculated in different trimesters of pregnancy. This mutant was evaluated in comparison with the homologous 16MΔwzm derived from a virulent strain of B. melitensis and the naturally rough sheep pathogen B. ovis. Our results show that both wzm mutants triggered reduced cytotoxic, pro-apoptotic, and pro-inflammatory signaling in Bewo trophoblasts, as well as reduced relative expression of apoptosis genes. In mice, both wzm mutants produced infection but were rapidly cleared from the placenta, in which only Rev1Δwzm induced a low relative expression of pro-apoptotic and pro-inflammatory genes. In the 66 inoculated ewes, Rev1Δwzm was safe and immunogenic, displaying a transient serological interference in standard RBT but not CFT S-LPS tests; this serological response was minimized by conjunctival administration. In conclusion, these results support that B. melitensis Rev1Δwzm is a promising vaccine candidate for use in pregnant ewes and its efficacy against B. melitensis and B. ovis infections in sheep warrants further study.

[Analysis on personal protection in occupational population at high risk for brucellosis and influencing factor in China].

Objective: To understand the current status of personal protection in occupational population at high risk for brucellosis in China and provide evidence for the evaluation of implementation of National Brucellosis Prevention and Control Plan (2016-2020). Methods: Four counties in Shanxi Province and Xinjiang Uygur Autonomous Region were selected to conduct a questionnaire survey in occupational population at high risk for brucellosis from December 2019 to July 2020 by using cross-sectional survey methods. Results: A total of 2 384 persons at high risk for brucellosis were surveyed, and the standardized utilization rate of personal protective equipment (PPE) was 20.13% (480/2 384). The utilization rate of glove, mask, rubber shoe, and work cloth were 38.26% (912/2 384), 31.80% (758/2 384), 32.01% (763/2 384) and 30.87% (736/2 384),respectively. There were significant differences in the utilization rate and standardized utilization rate of the four types of PPE among populations in different age, occupation, educational level and area groups (all P<0.001). The utilization rate and standardized utilization rate of PPE were lower in people over 60 years old, women, farmers, and those with lower educational level. The results of multivariate analysis showed that occupation and area were the influencing factors for the standardized utilization of PPE, the standardized utilization rates of PPE were higher in herdsmen and veterinarians. The standardized utilization rate of PPE in Yanggao County and Huocheng County was significantly higher than that in Zuoyun County and Hunyuan County. Conclusions: The utilization rate of the four types of PPE in occupational population at high risk for brucellosis was not high in China, and the standardized utilization rate was low, lower than the requirement in National Brucellosis Prevention and Control Plan, and there were significant differences among different areas. It is urgent to distribute PPE to occupational population at high risk for brucellosis and carry out health education about PPE utilization. Meanwhile, it is necessary to strengthen information exchange or sharing among different areas.

Knowledge-attitude and practice of Anthrax and brucellosis: Implications for zoonotic disease surveillance and control in pastoral communities of Afar and Somali region, Ethiopia.

BACKGROUND: Anthrax and brucellosis are endemic national priority zoonotic diseases in Ethiopia. This study assess the possible factors explaining the current limited information available on animal and human cases in pastoral communities. METHODS: Two questionnaire surveys gathered data from 509 pastoralists and 51 healthcare providers between February and April 2019 in five districts of Afar and the Somali region (SRS). RESULTS: Among the 51 healthcare providers, 25 (49%) and 38 (74.5%) had heard of brucellosis, and anthrax, respectively. Of those, only 3 (12%) and 14 (36.8%) knew the symptoms of brucellosis and Anthrax. None of the Health Extension Workers knew any disease symptoms. Healthcare providers recalled two human cases of brucellosis and 39 cases of Anthrax in the last 12 months, based on symptom-based diagnosis. Pastoralists had a moderate level of knowledge about diseases in their animals, with over half (52.4%; n = 267/509) understanding that animals can transmit diseases to people. Overall, 280 out of 508 (55.1%) and 333 out of 507 (65.7%) pastoralists had heard of brucellosis and Anthrax, respectively. Among the latter, 282 (51.3%) knew at least one preventive measure for Anthrax. However, disease knowledge among women was poor. Despite their knowledge, pastoralists engaged in risky unprotected animal handling, animal product consumption/usage as well as husbandry behaviors exposing them to pathogens and favoring the spread of diseases. They identified Anthrax as the most important zoonosis (47.6%) and as one of top three diseases suspected to cause mortality in their livestock. Pastoralists highlighted lack of vaccine coverage, availability and their timely administration. Both, pastoralists and healthcare providers stated the lack of disease awareness and the unavailability of drugs in the market as important challenges. Health facilities lacked protocols and standard operating procedures for managing zoonotic diseases, and did not have access to laboratory confirmation of pathogens. CONCLUSION: Our study revealed significant under-reporting of Anthrax and brucellosis, and weak prevention and response in humans, mostly associated with poor disease knowledge of healthcare providers. Ability to respond to animal outbreaks was limited by vaccine and drugs availability, timely vaccine administration and the mobility of pastoralists.

The immunostimulatory roles of gold nanoparticles in immunization and vaccination against Brucella abortus antigens.

One effective antigen carrier proposed for use in immunization and vaccination is gold nanoparticles. Prior work has shown that gold nanoparticles themselves have adjuvant properties. Currently, gold nanoparticles are used to design new diagnostic tests and vaccines against viral, bacterial, and parasitic infections. We investigated the use of gold nanoparticles as immunomodulators in immunization and vaccination with an antigen isolated from Brucella abortus. Gold nanoparticles with a diameter of 15 nm were synthesized for immunization of animals and were then conjugated to the isolated antigen. The conjugates were used to immunize white BALB/c mice. As a result, high-titer (1:10240) antibodies were produced. The respiratory and proliferative activities of immune cells were increased, as were the serum interleukin concentrations. The minimum antigen amount detected with the produced antibodies was âˆ¼ 0.5 pg. The mice immunized with gold nanoparticles complexed with the B. abortus antigen were more resistant to B. abortus strain 82 than were the mice immunized through other schemes. This fact indicates that animal immunization with this conjugate enhances the effectiveness of the immune response. The results of this study are expected to be used in further work to examine the protective effect of gold nanoparticles complexed with the B. abortus antigen on immunized animals and to develop test systems for diagnosing brucellosis in the laboratory and in the field.

Efficacy of antibiotic prophylaxis to preventing brucellosis in accidental exposure: A systematic review.

BACKGROUND: Antibiotic prophylaxis to prevent brucellosis after accidental exposure to Brucella is an important topic in public health. This study aimed to systematically review the efficacy of antibiotic prophylaxis following accidental exposure to Brucella in preventing human brucellosis disease. METHODS: The study protocol was registered in PROSPERO (CRD42023456812). The outcomes included the incidence of brucellosis disease, adverse events rate, and antibiotic prophylaxis adherence. A comprehensive literature search, conducted until 20 November, 2023, involved Medline, Embase, Cochrane Library, and LILACS databases. Descriptive analysis and meta-analysis using R software were performed, risk of bias was assessed using JBI Critical appraisal tools, and certainty of evidence was assessed using the GRADE tool. RESULTS: Among 3102 initially identified records, eight studies involving 97 individuals accidentally exposed, all focused on high-risk accidental exposure to Brucella in laboratory settings, were included in the review. All studies reported the prophylactic treatment comprising doxycycline at a dosage of 100 mg twice daily, combined with rifampicin at 600 mg, both administered over 21 days. Prophylaxis adherence was reported in 86% of cases, and incidence of brucellosis post-treatment was 0.01. Adverse events, mainly gastrointestinal, occurred in 26% of cases. Critical appraisal revealed limitations in reporting demographics and clinical information. The certainty of evidence was rated as 'very low,' emphasising the need for caution in interpreting the observed outcomes due to study design constraints and the absence of comparative groups. CONCLUSIONS: PEP is an alternative practice reported in the literature, used in accidents with high-risk exposure to Brucella. The currently available evidence of the efficacy of antibiotic prophylaxis is insufficient to support a recommendation for or against the widespread use of antibiotic prophylaxis, so caution is needed in interpreting results due to the very low certainty of evidence, primarily stemming from case series and lack of comparative groups.

Contrasting roles for IgM and B-cell MHCII expression in Brucella abortus S19 vaccine-mediated efficacy against B. melitensis infection.

Brucellosis, caused by the bacterium Brucella, poses a significant global threat to both animal and human health. Although commercial live Brucella vaccines including S19, RB51, and Rev1 are available for animals, their unsuitability for human use and incomplete efficacy in animals necessitate the further study of vaccine-mediated immunity to Brucella. In this study, we employed in vivo B-cell depletion, as well as immunodeficient and transgenic mouse models, to comprehensively investigate the roles of B cells, antigen uptake and presentation, antibody production, and class switching in the context of S19-mediated immunity against brucellosis. We found that antibody production, and in particular secretory IgM plays a protective role in S19-mediated immunity against virulent Brucella melitensis early after the challenge in a manner associated with complement activation. While T follicular helper cell deficiency dampened IgG production and vaccine efficacy at later stages of the challenge, this effect appeared to be independent of antibody production and rather was associated with altered T-cell function. By contrast, B-cell MHCII expression negatively impacted vaccine efficacy at later timepoints after the challenge. In addition, B-cell depletion after vaccination, but before the challenge, enhanced S19-mediated protection against brucellosis, suggesting a deleterious role of B cells during the challenge phase. Collectively, our findings indicate antibody production is protective, while B-cell MHCII expression is deleterious, to live vaccine-mediated immunity against brucellosis. IMPORTANCE: Brucella is a neglected zoonotic pathogen with a worldwide distribution. Our study delves into B-cell effector functions in live vaccine-mediated immunity against brucellosis. Notably, we found antibody production, particularly secretory IgM, confers protection against virulent Brucella melitensis in vaccinated mice, which was associated with complement activation. By contrast, B-cell MHCII expression negatively impacted vaccine efficacy. In addition, B-cell depletion after vaccination, but before the B. melitensis challenge, enhanced protection against infection, suggesting a detrimental B-cell role during the challenge phase. Interestingly, deficiency of T follicular helper cells, which are crucial for aiding germinal center B cells, dampened vaccine efficacy at later stages of challenge independent of antibody production. This study underscores contrasting and phase-dependent roles of B-cell effector functions in vaccine-mediated immunity against Brucella.

Effects of Pregnancy Prevention on Brucella abortus Shedding in American bison (Bison bison).

Products of parturition are the predominant source of Brucella abortus for transmission in bison (Bison bison). Our objective was to assess whether preventing pregnancy in Brucella-seropositive bison reduced B. abortus shedding. Brucella-seropositive and -seronegative bison from Yellowstone National Park, Wyoming, USA were used in a replicated experiment. Each of two replicates (rep1, rep2) included a group of seropositive females treated with a single dose of gonadotropin-releasing hormone-based immunocontraceptive (Treatment rep1, n=15; Treatment rep2, n=20) and an untreated group (Control rep1, n=14; Control rep2, n=16) housed separately. Seronegative sentinel females were placed in each group to monitor horizontal transmission. Seronegative males were co-mingled for breeding each year. Pregnant females were removed from treatment groups in the first year, but not thereafter. Each January-June we monitored for B. abortus shedding events-any parturition associated with culture-positive fluids or tissues. We analyzed probability of shedding events using a negative binomial generalized linear mixed model fit by maximum likelihood using Laplace approximation. Over 5 yr, we observed zero shedding events in Treatment rep1 vs. 12 in Control rep1. All five Control rep1 sentinels but zero (0/5) Treatment rep1 sentinels seroconverted. In the second replicate, Treatment rep2 had two shedding events over 3 yr and Control rep2 had five events over 2 yr. Sentinels in both Control rep2 (3/6) and Treatment rep2 (5/6) seroconverted by trial endpoint. Treatment rep1 showed a reduced shedding probability relative to Control rep1, Treatment rep2, and Control rep2 (log odds value -25.36 vs. -1.71, -1.39, and -0.23, respectively). Fixed effect predictor covariates, year and age, had no explanatory value. These data suggest that successful contraception of brucellosis-seropositive female bison prevents shedding of B. abortus by individual animals. However, contraceptive treatment may or may not sufficiently reduce disease transmission to reduce brucellosis prevalence in an affected herd.

What risk do Brucella vaccines pose to humans? A systematic review of the scientific literature on occupational exposure.

BACKGROUND: Currently, vaccination of livestock with attenuated strains of Brucella remains an essential measure for controlling brucellosis, although these vaccines may be dangerous to humans. The aim of this study was to review the risk posed to humans by occupational exposure to vaccine strains and the measures that should be implemented to minimize this risk. METHODS: This article reviewed the scientific literature indexed in PubMed up to September 30, 2023, following "the PRISMA guidelines". Special emphasis was placed on the vaccine strain used and the route of exposure. Non-occupational exposure to vaccine strains, intentional human inoculation, publications on exposure to wild strains, and secondary scientific sources were excluded from the study. RESULTS: Nineteen primary reports were found and classified in three subgroups: safety accidents in vaccine factories that led to an outbreak (n = 2), survellaince studies on vaccine manufacturing workers with a serologic diagnosis of Brucella infection (n = 3), and publications of infection by vaccine strains during their administration, including case reports, records of occupational accidents and investigations of outbreaks in vaccination campaigns (n = 14). Although accidental exposure during vaccine manufacturing were uncommon, they could provoke large outbreaks through airborne spread with risk of spread to the neighboring population. Besides, despite strict protection measures, a percentage of vaccine manufacturing workers developed positive Brucella serology without clinical infection. The most frequent type of exposure with symptomatic infection was needle injury during vaccine administration. Prolonged contact with the pathogen, lack of information and a low adherence to personal protective equipment (PPE) use in the work environment were commonly associated with infection. CONCLUSIONS: Brucella vaccines pose occupational risk of contagion to humans from their production to their administration to livestock, although morbidity is low and deaths were not reported. Recommended protective measures and active surveillance of exposed workers appeared to reduce this risk. It would be advisable to carry out observational studies and/or systematic registries using solid diagnostic criteria.

A multi-epitope subunit vaccine based on CU/ZN-SOD, OMP31 and BP26 against Brucella melitensis infection in BALB/C mice.

Brucellosis, a zoonosis caused by Brucella, is highly detrimental to both humans and animals. Most existing vaccines are live attenuated vaccines with safety flaws for people and animals. Therefore, it is advantageous to design a multi-epitope subunit vaccine (MEV) to prevent Brucella infection. To this end, we applied a reverse vaccinology approach. Six cytotoxic T cell (CTL) epitopes, seven T helper cell (HTL) epitopes, and four linear B cell epitopes from CU/ZN-SOD, Omp31, and BP26 were obtained. We linked the CTL, HTL, B-cell epitopes, the appropriate CTB molecular adjuvant, and the universal T helper lymphocyte epitope, PADRE, with linkers AAY, GPPGG, and KK, respectively. This yielded a 412-amino acid MEV construct, which we named MEVcob. The immunogenicity, stability, safety, and feasibility of the construct were evaluated by bioinformatics tools (including the AlphaFold2 prediction tool, the AlphaFold2 tool, NetMHC-I pan 4.0 server, IEDB MHC-I server, ABCpred service, and C-ImmSim server); the physicochemical properties, secondary and tertiary structures, and binding ability of MEVocb to toll-like receptor 4 (TLR4) was analyzed. Then, codon adaptation and computer cloning studies were performed. MEVocb is highly immunogenic in immunostimulation experiments, The proteins translated by these sequences were relatively stable, exhibiting a high antigenic index. Furthermore, mouse experiments confirmed that the MEVocb construct could raise IFN-γ, IgG, IgG2a, IgG1, IL-2, TNF-α levels in mice, indicating that induced a specific humoral and cellular immune response in BALB/c mice. This vaccine induced a statistically significant level of protection in BALB/c mice when challenged with Brucella melitensis 043 in Xinjiang. Briefly, we utilized immunoinformatic tools to design a novel multi-epitope subunit candidate vaccine against Brucella. This vaccine aims to induce host immune responses and confer specific protective effects. The study results offer a theoretical foundation for the development of a novel Brucella subunit vaccine.

Insights for brucellosis eradication in Italy through a model-based spread evaluation in grazing livestock - Sicily case study.

Brucellosis is one of the world's major zoonotic pathogens and is responsible for enormous economic losses as well as considerable human morbidity in endemic areas. Definitive control of human brucellosis requires control of brucellosis in livestock through practical solutions that can be easily applied to the field. In Italy, brucellosis remains endemic in several southern provinces, particularly in Sicily Region. The purpose of this paper is to describe the developed brucellosis model and its applications, trying to reproduce as faithfully as possible the complex transmission process of brucellosis accounting for the mixing of grazing animals. The model focuses on the contaminated environment rather than on the infected animal, uses real data from the main grazing areas of the Sicily Region, and aims to identify the best control options for minimizing the spread (and the prevalence) and to reach the eradication within the concerned areas. Simulation results confirmed the efficacy of an earlier application of the controls, showed the control should take place 30 days after going to pasture, and the culling time being negligible. Moreover, results highlighted the importance of the timing of both births and grazing pastures (and their interaction) more than other factors. As these factors are region­specific, the study encourages the adoption of different and new eradication tools, tuned on the grazing and commercial behavior of each region. This study will be further extended to improve the model's adaptability to the real world, with the purpose of making the model an operational tool able to help decision makers in accelerating brucellosis eradication in Italy.

The Control Program of Brucellosis by the Iranian Veterinary Organization in Industrial Dairy Cattle Farms.

Brucellosis is a zoonotic infection in livestock that induces a major public health concern in developing countries, including Iran. Despite the efforts of the Iranian veterinary organization (IVO) to control brucellosis, it is still prevalent in domestic animals. In this regard, the present study aimed to evaluate the efficiency of the control strategy used by the IVO in infected herds on serological, cultural, and molecular methods. For this purpose, blood specimens were sampled from a total of 8750 vaccinated dairy cattle in two Brucella-infected farms. These farms were recognized as positive for Brucella by a screening program. Sera were evaluated by the Rose Bengal Plate Test and Wright test analysis. Positive dairy cattle were slaughtered under IVO supervision. The remaining cattle were evaluated every 3 weeks and positive animals were slaughtered. This procedure continued until the remaining animals revealed three successive negative responses in serological tests. Several lymph nodes and milk samples were collected from 164 seropositive cattle and subjected to bacterial isolation and confirmation by Bruceladder-polymerase chain reaction. Brucella melitensis biovar 1 and RB51 vaccine strains were recovered from milk and lymph node samples, respectively. Shedding of B. melitensis in the milk of vaccinated cows is a serious problem resulting in the further spread of brucellosis. The policy of "test and slaughter" performed on infected dairy cattle farms showed their usefulness for the control of brucellosis outbreaks. For the uncontrolled spread of brucellosis in Iran, effective control of bovine brucellosis required several serological surveillances to identify infected herds, eradication of the reservoirs, and vaccination of young heifers with RB51.

One-step preparation of a self-assembled bioconjugate nanovaccine against Brucella.

Brucellosis, caused by Brucella, is a severe zoonosis, and the current Brucella live attenuated vaccine cannot be used in humans due to major safety risks. Although polysaccharide antigens can be used to prepare the Brucella vaccine, their lower immunogenicity limits them from producing efficient and broad protection. In this study, we produced a high-performance bioconjugate nanovaccine against different species of Brucella by introducing a self-assembly nanoparticle platform and an O-linked glycosylation system into Yersinia enterocolitica serotype O:9, which has an O-polysaccharide composed of the same unit as Brucella. After successfully preparing the vaccine and confirming its stability, we subsequently demonstrated the safety of the vaccine in mice by high-dose immunization. Then, by a series of mouse experiments, we found that the nanovaccine greatly promoted antibody responses. In particular, the increase of IgG2a was more obvious than that of IgG1. Most importantly, this nanovaccine could provide cross-protection against B. abortus, B. melitensis, and B. suis strains by lethal dose challenged models, and could improve the clearance of B. melitensis, the most common pathogenic species in human brucellosis, by non-lethal dose infection. Overall, for the first time, we biocoupled polysaccharide antigens with nano carriers to prepare a Brucella vaccine, which showed pronounced and extensive protective effects in mice. Thus, we provided a potential candidate vaccine and a new direction for Brucella vaccine design.

Nano and microparticle drug delivery systems for the treatment of Brucella infections.

Nano-based drug delivery systems are increasingly used for diagnosis, prevention and treatment of several diseases, thanks to several beneficial properties, including the ability to target specific cells or organs, allowing to reduce treatment costs and side effects frequently associated with chemotherapeutic medications, thereby improving treatment compliance of patients. In the field of communicable diseases, especially those caused by intracellular bacteria, the delivery of antibiotics targeting specific cells is of critical importance to maximize their treatment efficacy. Brucella melitensis, an intracellular obligate bacterium surviving and replicating inside macrophages is hard to be eradicated, mainly because of the low ability of antibiotics to enter these phagocityc cells . Although different antibiotics regimens including gentamicin, doxycycline and rifampicin are in fact used against the Brucellosis, no efficient treatment has been attained yet, due to the intracellular life of the respective pathogen. Nano-medicines responding to environmental stimuli allow to maximize drug delivery targeting macropages, thereby boosting treatment efficacy. Several drug delivery nano-technologies, including solid lipid nanoparticles, liposomes, chitosan, niosomes, and their combinations with chitosan sodium alginate can be employed in combination of antibiotics to successfully eradicate Brucellosis infection from patients.

Immunization of BALB/c mice with BAB1-0278: An initial investigation of a novel potential vaccine for brucellosis based on Lactococcus Lactis vector.

The gram-negative intracellular bacterium Brucella abortus causes bovine brucellosis, a zoonotic disease that costs a lot of money. This work developed a vector vaccine against brucellosis utilizing recombinant L. lactis expressing Brucella outer membrane protein BAB1-0278. Gene sequences were obtained from GenBank. The proteins' immunogenicity was tested with Vaxijen. The target vector was converted into L. lactis after enzymatic digestion and PCR validated the BAB1-0278 gene cloning in the pNZ8148 vector. The target protein was extracted using a Ni-NTA column and confirmed using SDS-PAGE and western blot. After vaccination with the target vaccine, the expression of IgG subclasses was evaluated by the ELISA method. Cytokine production was also measured by the qPCR method in the small intestine and spleen. Lymphocyte proliferation and innate immune response (NLR, CRP, and PLR) were also assessed. Finally, after the challenge test, the spleen tissue was examined by H&E staining. BAB1-0278 was chosen because of its antigenicity score of 0.5614. A 237-bp gene fragment was discovered using enzymatic digestion and PCR. The presence of a 13 kDa protein band was confirmed by SDS-PAGE and western blot. In comparison to the PBS group, mice given the L. lactis-pNZ8148-BAB1-0278-Usp45 vaccine 14 days after priming had substantially greater levels of total IgG, IgG1, and IgG2a (P < 0.001). Also, the production of cytokines (IFN-γ, TNFα, IL-4, and IL-10) indicating cellular immunity increased compared to the control group (P < 0.001). The target group had a lower inflammatory response, morphological impairment, alveolar edema, and lymphocyte infiltration. An efficient probiotic-based oral brucellosis vaccination was created. These studies have proven that the recommended immunization gives the best protection, which supports its promotion.

Combining multi-omics analysis to identify host-targeted targets for the control of Brucella infection.

Human infections caused by Brucella (called brucellosis) are among the most common zoonoses worldwide with an estimated 500,000 cases each year. Since chronic Brucella infections are extremely difficult to treat, there is an urgent need for more effective therapeutics. As a facultative intracellular bacterium, Brucella is strictly parasitic in the host cell. Here, we performed proteomic and transcriptomic and metabolomic analyses on Brucella infected patients, mice and cells that provided an extensive "map" of physiological changes in brucellosis patients and characterized the metabolic pathways essential to the response to infection, as well as the associated cellular response and molecular mechanisms. This is the first report utilizing multi-omics analysis to investigate the global response of proteins and metabolites associated with Brucella infection, and the data can provide a comprehensive insight to understand the mechanism of Brucella infection. We demonstrated that Brucella increased nucleotide synthesis in the host, consistent with increased biomass requirement. We also identified IMPDH2, a key regulatory complex that controls nucleotide synthesis during Brucella infection. Pharmacological targeting of IMPDH2, the rate-limiting enzyme in guanine nucleotide biosynthesis, efficiently inhibits B. abortus growth both in vitro and in vivo. Through screening a library of natural products, we identified oxymatrine, an alkaloid obtained primarily from Sophora roots, is a novel and selective IMPDH2 inhibitor. In further in vitro bacterial inhibition assays, oxymatrine effectively inhibited the growth of B. abortus, which was impaired by exogenous supplementation of guanosine, a salvage pathway of purine nucleotides. This moderately potent, structurally novel compound may provide clues for further design and development of efficient IMPDH2 inhibitors and also demonstrates the potential of natural compounds from plants against Brucella.

Design of multi-epitope vaccine candidate against Brucella type IV secretion system (T4SS).

Brucellosis is a common zoonosis, which is caused by Brucella infection, and Brucella often infects livestock, leading to abortion and infertility. At present, human brucellosis remains one of the major public health problems in China. According to previous research, most areas in northwest China, including Xinjiang, Tibet, and other regions, are severely affected by Brucella. Although there are vaccines against animal Brucellosis, the effect is often poor. In addition, there is no corresponding vaccine for human Brucellosis infection. Therefore, a new strategy for early prevention and treatment of Brucella is needed. A multi-epitope vaccine should be developed. In this study, we identified the antigenic epitopes of the Brucella type IV secretion system VirB8 and Virb10 using an immunoinformatics approach, and screened out 2 cytotoxic T lymphocyte (CTL) epitopes, 9 helper T lymphocyte (HTL) epitopes, 6 linear B cell epitopes, and 6 conformational B cell epitopes. These advantageous epitopes are spliced together through different linkers to construct a multi-epitope vaccine. The silico tests showed that the multi-epitope vaccine was non-allergenic and had a strong interaction with TLR4 molecular docking. In immune simulation results, the vaccine construct may be useful in helping brucellosis patients to initiate cellular and humoral immunity. Overall, our findings indicated that the multi-epitope vaccine construct has a high-quality structure and suitable characteristics, which may provide a theoretical basis for the development of a Brucella vaccine.

Bioinformatics-based design of a fusion vaccine with CTLA-4 variable region to combat Brucella.

Brucellosis has become a global zoonotic disease, seriously endangering the health of people all over the world. Vaccination is an effective strategy for protection against Brucella infection in livestock in developed countries. However, current vaccines are pathogenic to humans and pregnant animals, which limits their use. Therefore, it is very important to improve the safety and immune protection of Brucella vaccine. In this study, different bioinformatics approaches were carried out to predict the physicochemical properties, T/B epitope, and tertiary structure of Omp2b and Omp31. Then, these two proteins were sequentially linked, and the Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) variable region was fused to the N-terminal of the epitope sequence. In addition, molecular docking was performed to show that the structure of the fusion protein vaccine had strong affinity with B7 (B7-1, B7-2). This study showed that the designed vaccine containing CTLA-4 had high potency against Brucella, which could provide a reference for the future development of efficient brucellosis vaccines.