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Brucellosis is a bacterial zoonosis caused by the genus Brucella , which mainly affects domestic animals. In these natural hosts, brucellae display a tropism towards the reproductive organs, such as the placenta, replicating in high numbers and leading to placentitis and abortion, an ability also exerted by the B. melitensis live-attenuated Rev1 strain, the only vaccine available for ovine brucellosis. It is broadly accepted that this tropism is mediated, at least in part, by the presence of certain preferred nutrients in the placenta, particularly erythritol, a polyol that is ultimately incorporated into the Brucella central carbon metabolism via two reactions dependent on transaldolase (Tal) or fructose-bisphosphate aldolase (Fba). In the light of these remarks, we propose that blocking the incorporation of erythritol into the central carbon metabolism of Rev1 by deleting the genes encoding Tal and Fba may impair the ability of the vaccine to proliferate massively in the placenta. Therefore, a Rev1Δ fba Δ tal double mutant was generated and confirmed to be unable to use erythritol. This mutant exhibited a reduced intracellular fitness both in BeWo trophoblasts and THP-1 macrophages. In the murine model, Rev1Δ fba Δ tal provided comparable protection to the Rev1 reference vaccine while inducing fewer adverse reproductive events in pregnant animals. Altogether, these results postulate the Rev1Δ fba Δ tal mutant as a reproductively safer Rev1-derived vaccine candidate to be studied in the natural host.

In recent years, immunization with the S2 live-attenuated vaccine has been recognized as the most economical and effective strategy for preventing brucellosis in Inner Mongolia, China. However, there are still challenges related to vaccine toxicity and the inability to distinguish between vaccine immunization and natural infection. Therefore, in this study, we developed a digital droplet polymerase chain reaction (ddPCR) assay based on single-nucleotide polymorphism (SNP) loci to identify wild Brucella strains and S2 vaccine strains. The assay demonstrated excellent linearity (R 2 > 0.99) with a lower detection limit of 10 copies/µL for both wild and vaccine strains. Additionally, the ddPCR assay outperformed the real-time fluorescent quantitative PCR (qPCR) assay in screening 50 clinical samples. We have established an effective and highly sensitive ddPCR assay for Brucella, providing an efficient method for detecting and differentiating wild strains of Brucella from the S2 vaccine strain., Competing Interests: 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., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Pantanal region, the largest floodplain in the world, has a huge biodiversity and is an important livestock center. Bovine brucellosis has been reported in the region over the last three decades, posing implications for cattle industry as well as for the maintenance of biodiversity. We aimed to investigate the presence of B. abortus S19 vaccine strain DNA in unvaccinated domestic and wild ungulates from the Brazilian Pantanal. Fifty-two heifers, 63 ovine, 24 domestic pigs, 28 feral pigs, and three Pampas deer were sampled. Brucella spp. was detected through bcsp31 PCR of blood samples in 45.3% (77/170) of the sampled animals, of which 36.4% (28/77) showed positivity in ery PCR corresponding to B. abortus S19 strain. Feral pigs presented the highest occurrence of positive samples in bcsp31 PCR (75%), followed by ovine (47.6%), domestic pigs (41.7%), and unvaccinated heifers (30.8%). We did not observe positivity in Pampas deer. Our results strongly suggest that vaccination against bovine brucellosis may promote spill-over of B. abortus S19 strain in the Pantanal region. Moreover, our data indicate that wild strains of Brucella circulates in the Pantanal Biome., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Live attenuated vaccines (LAVs) whose virulence would be controlled at the tissue level could be a crucial tool to effectively fight intracellular bacterial pathogens, because they would optimize the induction of protective immune memory while avoiding the long-term persistence of vaccine strains in the host. Rational development of these new LAVs implies developing an exhaustive map of the bacterial virulence genes according to the host organs implicated. We report here the use of transposon sequencing to compare the bacterial genes involved in the multiplication of Brucella melitensis, a major causative agent of brucellosis, in the lungs and spleens of C57BL/6 infected mice. We found 257 and 135 genes predicted to be essential for B. melitensis multiplication in the spleen and lung, respectively, with 87 genes common to both organs. We selected genes whose deletion is predicted to produce moderate or severe attenuation in the spleen, the main known reservoir of Brucella, and compared deletion mutants for these genes for their ability to protect mice against challenge with a virulent strain of B. melitensis. The protective efficacy of a deletion mutant for the plsC gene, implicated in phospholipid biosynthesis, is similar to that of the reference Rev.1 vaccine but with a shorter persistence in the spleen. Our results demonstrate that B. melitensis faces different selective pressures depending on the organ and underscore the effectiveness of functional genome mapping for the design of new safer LAV candidates., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Barbieux et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Safe and effective vaccine candidates are needed to address the limitations of existing vaccines against Brucellosis, a disease responsible for substantial economic losses in livestock. The present study aimed to encapsulate recombinant Omp25 and EipB proteins, knowledged antigen properties, into PLGA nanoparticles, characterize synthesized nanoparticles with different methods, and assessed their in vitro / in vivo immunostimulatory activities to develop new vaccine candidates. The recombinant Omp25 and EipB proteins produced with recombinant DNA technology were encapsulated into PLGA nanoparticles by double emulsion solvent evaporation technique. The nanoparticles were characterized using FE-SEM, Zeta-sizer, and FT-IR instruments to determine size, morphology, zeta potentials, and polydispersity index values, as well as to analyze functional groups chemically. Additionally, the release profiles and encapsulation efficiencies were assessed using UV-Vis spectroscopy. After loading with recombinant proteins, O-NPs reached sizes of 221.2 ± 5.21 nm, while E-NPs reached sizes of 274.4 ± 9.51 nm. The cumulative release rates of the antigens, monitored until the end of day 14, were determined to be 90.39% for O-NPs and 56.1% for E-NPs. Following the assessment of the in vitro cytotoxicity and immunostimulatory effects of both proteins and nanoparticles on the J774 murine macrophage cells, in vivo immunization experiments were conducted using concentrations of 16 µ g ml -1 for each protein. Both free antigens and antigen-containing nanoparticles excessively induced humoral immunity by increasing produced Brucella -specific IgG antibody levels for 3 times in contrast to control. Furthermore, it was also demonstrated that vaccine candidates stimulated Th1-mediated cellular immunity as well since they significantly raised IFN-gamma and IL-12 cytokine levels in murine splenocytes rather than IL-4 following to immunization. Additionally, the vaccine candidates conferred higher than 90% protection from the infection according to challenge results. Our findings reveal that PLGA nanoparticles constructed with the encapsulation of recombinant Omp25 or EipB proteins possess great potential to trigger Brucella -specific humoral and cellular immune response., (Creative Commons Attribution license.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rev1Δwzm is protected under a CSIC-UPNA patent WO/2019/101993 (PCT/EP2018/082539) and both are public institutions without any conflict of interests with this publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: 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., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Brucellosis is a notifiable disease induced by a facultative intracellular Brucella pathogen. In this study, eight Brucella abortus and eighteen Brucella melitensis strains from Egypt were annotated and compared with RB51 and REV1 vaccines respectively. RAST toolkit in the BV-BRC server was used for annotation, revealing genome length of 3,250,377 bp and 3,285,803 bp, 3289 and 3323 CDS, 48 and 49 tRNA genes, the same number of rRNA (3) genes, 583 and 586 hypothetical proteins, 2697 and 2726 functional proteins for B. abortus and B. melitensis respectively. B. abortus strains exhibit a similar number of candidate genes, while B. melitensis strains showed some differences, especially in the SRR19520422 Faiyum strain. Also, B. melitensis clarified differences in antimicrobial resistance genes (KatG, FabL, MtrA, MtrB, OxyR, and VanO-type) in SRR19520319 Faiyum and (Erm C and Tet K) in SRR19520422 Faiyum strain. Additionally, the whole genome phylogeny analysis proved that all B. abortus strains were related to vaccinated animals and all B. melitensis strains of Menoufia clustered together and closely related to Gharbia, Dameitta, and Kafr Elshiek. The Bowtie2 tool identified 338 (eight B. abortus) and 4271 (eighteen B. melitensis) single nucleotide polymorphisms (SNPs) along the genomes. These variants had been annotated according to type and impact. Moreover, thirty candidate genes were predicted and submitted at GenBank (24 in B. abortus) and (6 in B. melitensis). This study contributes significant insights into genetic variation, virulence factors, and vaccine-related associations of Brucella pathogens, enhancing our knowledge of brucellosis epidemiology and evolution in Egypt., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (Published by Elsevier Ltd.)

Background: Most Brucella infections take place on mucosal membranes. Therefore, creating vaccinations delivered through the mucosa may be crucial for managing brucellosis. Consequently, we assessed the efficacy of a recombinant oral antigen delivery system based on Lactococcus lactis for Brucella abortus omp25 antigen., Method: Oral vaccinations with L. lactis transformed with pNZ8148 variants encoding for omp25 (pNZ8148:omp25) and free-pNZ8148 were administered to mice. On day 30, following immunization in animal groups, anti-omp25-specific IgG1 antibodies were assessed by the ELISA test. Additionally, nasal and bronchoalveolar lavages containing omp25-specific secretory IgA (sIgA) were analysed by ELISA. ELISA test and real-time PCR were also used to analyse cytokine responses up to 28 days following the last boost. In addition, the protective potential of L. lactis pNZ8148:omp25 vaccines was assessed in BALB/c mice by exposing them to the B. abortus strain., Results: Based on the initial screening results, the omp25 protein was identified for immunogenicity because it had the maximum solubility and flexibility and antigenic values of 0.75. The produced plasmid was digested using KpnI and XbaI. By electrophoretic isolation of the digestion fragments at 786 bp, the omp25 gene, the successful production of the recombinant plasmid, was confirmed. Antigen expression at the protein level revealed that the target group generated the 25 kDa-sized omp25 protein, but there was no protein expression in the control group. Fourteen days after priming, there was a considerable amount of omp25-specific IgG1 in the sera of mice vaccinated with pNZ8148-Usp45-omp25-L. lactis (p < 0.001 in target groups compared to the phosphate-buffered saline control group). IFN-γ and TNF-α levels were more significant in samples from mice that had been given the pNZ8148-Usp45-omp25-L. lactis and IRBA vaccinations, in samples taken on days 14 and 28, respectively (p < 0.001). The pNZ8148-Usp45-omp25-L. lactis and IRBA immunization groups had significantly greater IL-4 and IL-10 transcription levels than the other groups. The spleen portions from the pNZ8148-Usp45-omp25-L. lactis and IRIBA vac group had less extensive spleen injuries, alveolar oedema, lymphocyte infiltration and morphological damage due to the inflammatory process., Conclusion: Our study offers a novel method for using the food-grade, non-pathogenic and noncommercial bacterium L. lactis as a protein cell factory to produce the novel immunogenic fusion candidate romp25. This method offers an appealing new approach to assessing the cost-effective, safe, sustainable, simple pilot development of pharmaceutical products., (© 2023 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Brucella suis mediates the transmission of brucellosis in humans and animals and a significant facultative zoonotic pathogen found in livestock. It has the capacity to survive and multiply in a phagocytic environment and to acquire resistance under hostile conditions thus becoming a threat globally. Antibiotic resistance is posing a substantial public health threat, hence there is an unmet and urgent clinical need for immune-based non-antibiotic methods to treat brucellosis. Hence, we aimed to explore the whole proteome of Brucella suis to predict antigenic proteins as a vaccine target and designed a novel chimeric vaccine (multi-epitope vaccine) through subtractive genomics-based reverse vaccinology approaches. The applied subsequent hierarchical shortlisting resulted in the identification of Multidrug efflux Resistance-nodulation-division (RND) transporter outer membrane subunit (gene BepC) that may act as a potential vaccine target. T-cell and B-cell epitopes have been predicted from target proteins using a number of immunoinformatic methods. Six MHC I, ten MHC II, and four B-cell epitopes were used to create a 324-amino-acid MEV construct, which was coupled with appropriate linkers and adjuvant. To boost the immunological response to the vaccine, the vaccine was combined with the TLR4 agonist HBHA protein. The MEV structure predicted was found to be highly antigenic, non-toxic, non-allergenic, flexible, stable, and soluble. To confirm the interactions with the receptors, a molecular docking simulation of the MEV was done using the human TLR4 (toll-like receptor 4) and HLAs. The stability and binding of the MEV-docked complexes with TLR4 were assessed using molecular dynamics (MD) simulation. Finally, MEV was reverse translated, its cDNA structure was evaluated, and then, in silico cloning into an E. coli expression host was conducted to promote maximum vaccine protein production with appropriate post-translational modifications. These comprehensive computer calculations backed up the efficacy of the suggested MEV in protecting against B. suis infections. However, more experimental validations are needed to adequately assess the vaccine candidate's potential. HIGHLIGHTS: • Subtractive genomic analysis and reverse vaccinology for the prioritization of novel vaccine target • Examination of chimeric vaccine in terms of allergenicity, antigenicity, MHC I, II binding efficacy, and structural-based studies • Molecular docking simulation method to rank based vaccine candidate and understand their binding modes., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This work aimed to provide recombinant Lactococcus lactis as a potential live vector for the manufacture of recombinant Brucella abortus (rBLS-Usp45). The sequences of the genes were collected from the GenBank database. Using Vaxijen and ccSOL, the proteins' immunogenicity and solubility were evaluated. Mice were given oral vaccinations with recombinant L. lactis. Anti-BLS-specific IgG antibodies were measured by ELISA assay. Cytokine reactions were examined using real-time PCR and the ELISA technique. The BLS protein was chosen for immunogenicity based on the vaccinology screening findings since it had maximum solubility and antigenic values ​​of 99% and 0.75, respectively. The BLS gene, digested at 477 bp, was electrophoretically isolated to demonstrate that the recombinant plasmid was successfully produced. Protein-level antigen expression showed that the target group produced the 18 kDa-sized BLS protein, whereas the control group did not express any proteins. In the sera of mice given the L. lactis-pNZ8148-BLS-Usp45 vaccine 14 days after priming, there was a significant level of BLS-specific IgG1, IgG2a (P < 0.001) compared to the PBS control group. Vaccinated mice showed higher levels of IFN-γ, TNFα, IL-4, and IL-10 in samples obtained on days 14 and 28, after receiving the L. lactis-pNZ8148-BLS-Usp45 and IRBA vaccines (P < 0.001). The inflammatory reaction caused less severe spleen injuries, alveolar edema, lymphocyte infiltration, and morphological damage in the target group's spleen sections. Based on our findings, an oral or subunit-based vaccine against brucellosis might be developed using L. lactis-pNZ8148-BLS-Usp45 as a novel, promising, and safe alternative to the live attenuated vaccines now available., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Brucellosis is a highly prevalent zoonotic disease caused by Brucella spp. Brucella suis S2 vaccination is an effective strategy to prevent animal brucellosis. However, S2 induces antibodies against the smooth lipopolysaccharide,making it challenging to distinguish field infected from vaccinated livestock. Early and accurate diagnosis is essential for infection control and prevention. In this study, we aimed to develop a quick and accurate assay to distinguish the BrucellaS2 vaccine strain from closely related B. abortus and B. melitensis ., Methods: Whole-genome sequencing of B. suis S2 was performed, and the sequence was compared with that of the genomes of B. abortus and B. melitensis . One specific gene, GL&#95;0002189 , was selected as a marker to differentiate the Brucella S2vaccine strain from B. abortus and B. melitensis . A loop-mediated isothermal amplification (LAMP) assay was developed, based on the GL&#95;0002189 gene, and then assessed for target specificity, lower limit of detection, and repeatability., Results: Our results revealed that there was no cross-reaction with other strains, and the LAMP assay displayed high sensitivity for detecting S2 with a minimum detection limit of 18.9×103 copies/µL DNA input, it is nearly 100 times higher than conventional PCR technology. Concordance between the LAMP assay and a conventional polymerase chain reaction method was assessed using 54 blood samples collected from sheep with suspected brucellosis. Total concordance between the two assays was 92.6%, without a significant difference (p > 0.05) in the test results., Conclusion: This is the first report of a LAMP assay for the detection of the B. suis S2vaccine strain. Our approach can be helpful for the control and eradication of brucellosis, and its simplicity in requiring no specialized equipment or personnel makes it useful for implementation in resource-limited settings as well as for field use., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mu, Li, Yan, Mao, Shi, Qin, Liu and Wang.)

Background: Brucellosis is a widespread disease that affects animals and humans. The live attenuated Brucella abortus A19 strain is used for vaccination against brucellosis in China. In addition, the main mechanisms supporting the residual toxicity of A19 have not been elucidated. Here, we performed a comprehensive comparative analysis of the genome-wide sequence of A19 against the whole genome sequences of the published virulent reference strain 9-941. The primary objective of this study was to identify candidate virulence genes by systematically comparing the genomic sequences between the two genomes., Results: This analysis revealed two deletion regions in the A19 genome, in which all included large fragments of 63 bp, and one of their gene function is related to ABC transporter permease protein. In addition, we have identified minor mutations in important virulence-related genes that can be used to determine the underlying mechanisms of virulence attenuation. The function of its virulence gene covers LysR family transcriptional regulator, outer membrane, MFS transporter and oxidoreductase etc. At the same time, a PCR differential diagnosis method was constructed, which can distinguish A19, S19 and most other commonly used Brucella viruent strains and vaccine strains., Conclusion: The data may help to provide resources for further detailed analysis of mechanisms for other Brucella vaccines. It laid the foundation for further distinguishing between vaccine immunity and virulent strains infection., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely, ribosomal protein L7/L12, outer membrane protein (OMP) 22, OMP25 and OMP31, was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model. Four proteins were cloned, expressed and purified, and their immunocompetence was analysed. BALB/c mice were immunized subcutaneously with single subunit vaccines (SSVs) or CSV. Cellular and humoral immune responses were determined by ELISA. Results of immunoreactivity showed that these four recombinant proteins reacted with Brucella-positive serum individually but not with Brucella-negative serum. A massive production of IFN-γ and IL-2 but low degree of IL-10 was observed in mice immunized with SSVs or CSV. In addition, the titres of IgG2a were heightened compared with IgG1 in SSV- or CSV-immunized mice, which indicated that SSVs and CSV induced a typical T-helper-1-dominated immune response in vivo. Further investigation of the CSV showed a superior protective effect in mice against brucellosis. The protection level induced by CSV was significantly higher than that induced by SSVs, which was not significantly different compared with a group immunized with RB51. Collectively, these antigens of Brucella could be potential candidates to develop subunit vaccines, and the CSV used in this study could be a potential candidate therapy for the prevention of brucellosis., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and animals. Currently available live attenuated vaccines against brucellosis still have drawbacks. Therefore, subunit vaccines, produced using epitope-based antigens, have the advantage of being safe, cost-effective and efficacious. Here, we identified B. abortus small RNAs expressed during early infection with bone marrow-derived macrophages (BMDMs) and an apolipoprotein N-acyltransferase (Int) was identified as the putative target of the greatest expressed small RNA. Decreased expression of Int was observed during BMDM infection and the protein sequence was evaluated to rationally select a putative immunogenic epitope by immunoinformatic, which was explored as a vaccinal candidate. C57BL/6 mice were immunized and challenged with B. abortus , showing lower recovery in the number of viable bacteria in the liver, spleen, and axillary lymph node and greater production of IgG and fractions when compared to non-vaccinated mice. The vaccinated and infected mice showed the increased expression of TNF-α , IFN-γ , and IL-6 following expression of the anti-inflammatory genes IL-10 and TGF-β in the liver, justifying the reduction in the number and size of the observed granulomas. BMDMs stimulated with splenocyte supernatants from vaccinated and infected mice increase the CD86+ marker, as well as expressing greater amounts of iNOS and the consequent increase in NO production, suggesting an increase in the phagocytic and microbicidal capacity of these cells to eliminate the bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Oliveira, Brancaglion, Santos, Araújo, Novaes, Santos, Oliveira, Corsetti and de Almeida.)

Brucella abortus is an important zoonotic pathogen that causes severe economic loss to husbandry and poses a threat to human health. The B. abortus A19 live vaccine has been extensively used to prevent bovine brucellosis in China. However, it is difficult to distinguish the serological response induced by A19 from that induced by natural infection. In this study, a novel genetically marked vaccine, A19ΔvirB12, was generated and evaluated. The results indicated that A19ΔvirB12 was able to provide effective protection against B. abortus 2308 (S2308) challenge in mice. Furthermore, the safety and protective efficacy of A19ΔvirB12 have been confirmed in natural host cattle. Additionally, the VirB12 protein allowed for serological differentiation between the S2308 challenge/natural infection and A19ΔvirB12 vaccination. However, previous studies have found that the accuracy of the serological detection based on VirB12 needs to be improved. Therefore, we attempted to identify potential supplementary antigens with differential diagnostic functions by combining label-free quantitative proteomics and protein chip technology. Twenty-six proteins identified only in S2308 were screened; among them, five proteins were considered as potential supplementary antigens. Thus, the accuracy of the differential diagnosis between A19ΔvirB12 immunization and field infection may be improved through multi-antigen detection. In addition, we explored the possible attenuation factors of Brucella vaccine strain. Nine virulence factors were downregulated in A19ΔvirB12. The downregulation pathways of A19ΔvirB12 were significantly enriched in quorum sensing, ATP-binding cassette transporter, and metabolism. Several proteins related to cell division were significantly downregulated, while some proteins involved in transcription were upregulated in S2308. In conclusion, our results contribute to the control and eradication of brucellosis and provide insights into the mechanisms underlying the attenuation of A19ΔvirB12., Competing Interests: Authors CH, ML, HZ, LR, DW, and SH were employed by Tecon Biological Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, He, Zhang, Liu, Zhao, Ren, Wu, Du, Liu, Han, He and Chen.)

Background: A new candidate vector vaccine against human brucellosis based on recombinant influenza viral vectors (rIVV) subtypes H5N1 expressing Brucella outer membrane protein (Omp) 16, L7/L12, Omp19 or Cu-Zn SOD proteins has been developed. This paper presents the results of the study of protection of the vaccine using on guinea pigs, including various options of administering, dose and frequency. Provided data of the novel vaccine candidate will contribute to its further movement into the preclinical stage study., Methods: General states of guinea pigs was assessed based on behavior and dynamics of a guinea pig weight-gain test. The effectiveness of the new anti-brucellosis vector vaccine was determined by studying its protective effect after conjunctival, intranasal and sublingual administration in doses 10 5 EID 50 , 10 6 EID 50 and 10 7 EID 50 during prime and boost vaccinations of animals, followed by challenge with a virulent strain of B. melitensis 16 M infection. For sake of comparison, the commercial B. melitensis Rev.1 vaccine was used as a control. The protective properties of vaccines were assessed by quantitation of Brucella colonization in organs and tissues of infected animals and compared to the control groups., Results: It was observed a gradual increase in body weight of guinea pigs after prime and booster immunization with the vaccine using conjunctival, intranasal and sublingual routes of administration, as well as after using various doses of vaccine. The most optimal way of using the vaccine has been established: double intranasal immunization of guinea pigs at a dose of 10 6 EID 50 , which provides 80% protection of guinea pigs from B. melitensis 16 M infection (P < 0.05), which is comparable to the results of the effectiveness of the commercial B. melitensis Rev.1 vaccine., Conclusions: We developed effective human vaccine candidate against brucellosis and developed its immunization protocol in guinea pig model. We believe that because of these studies, the proposed vaccine has achieved the best level of protection, which in turn provides a basis for its further promotion.

Since Brucella infection mostly occurs through the mucosal surfaces, immune response induced by vaccine that is delivered by a way of mucosal route can be drastically enhanced to control the brucellosis. Omp31is the major outer membrane protein of Brucella, and is considered as a protective antigen against Brucella infection. Accordingly, Lactococcus lactis has been used as an antigen-delivering vector to develop a vaccine-induced mucosal response for having a safer vaccination against brucellosis. A designed omp31 gene fused to the usp45 signal peptide and M6 cell wall anchor was sub cloned in the pNZ7021 expression vector, and a recombinant L. lactis displaying Omp31 was constructed. Omp31 protein expression was confirmed using Western blotting and immunofluorescence analysis. Animals were orally and intraperitoneally immunized with live or killed L. lactis expressing Omp31, respectively. The humoral and cellular immune responses were evaluated by measuring the specific cytokines and antibodies. sIgA, serum IgA, IgM, and total IgG antibodies significantly increased in the mice immunized with live recombinant L. lactis expressing Omp31 and also serum IgM, and total IgG antibodies significantly increased in mice immunized with killed recombinant L. lactis expressing Omp31. Among IgG subtypes, IgG2a response was significantly higher in both groups compared to IgG1. In mice groups immunized with recombinant L. lactis, the IFN-γ and IL-10 level elevated; however, there was no change in the level of IL-4. These results indicated that recombinants L. lactis induce both humoral and cellular immune responses in mice, and also vaccines based on L. lactis-derived live carriers are promising interventions against Brucella melitensis infections.

For control of brucellosis in small ruminants, attenuated B. melitensis Rev1 is used but it can be virulent for animals and human. Based on these aspects, it is essential to identify potential immunogens to avoid these problems in prevention of brucellosis. The majority of OMPs in the Omp25/31 family have been studied because these proteins are relevant in maintaining the integrity of the outer membrane but their implication in the virulence of the different species of this genus is not clearly described. Therefore, in this work we studied the role of Omp31 on virulence by determining the residual virulence and detecting lesions in spleen and testis of mice inoculated with the B. melitensis LVM31 mutant strain. In addition, we evaluated the conferred protection in mice immunized with the mutant strain against the challenge with the B. melitensis Bm133 virulent strain. Our results showed that the mutation of omp 31 caused a decrease in splenic colonization without generating apparent lesions or histopathological changes apparent in both organs in comparison with the control strains and that the mutant strain conferred similar protection as the B. melitensis Rev1 vaccine strain against the challenge with B. melitensis Bm133 virulent strain. These results allow us to conclude that Omp31 plays an important role on the virulence of B. melitensis in the murine model, and due to the attenuation shown by the strain, it could be considered a vaccine candidate for the prevention of goat brucellosis.

The Brucella melitensis chronic infection and drug resistance emerged as a severe health problem in humans and domestic cattle. The pathogens fast genome sequences availability fetched the possibility to address novel therapeutics targets in a rationale way. We acquired the core genes set from 56 B. melitensis publically available complete genome sequences. A stringent bioinformatics layout of comparative genomics and reverse vaccinology was followed to identify potential druggable proteins and multi-epitope vaccine constructs from core genes. The 23 proteins were shortlisted as novel druggable targets based on their role in pathogen-specific metabolic pathways, non-homologous to human and human gut microbiome proteins and their druggability potential. Furthermore, potential chimeric vaccine constructs were generated from lead T and B-cell overlapped epitopes in combination with immune enhancer adjuvants and linkers sequences. The molecular docking and MD simulation analyses ensured stable molecular interaction of a finally prioritized vaccine construct with human immune cells receptors., Competing Interests: Declaration of Competing Interest The authors declare that no conflict of interest exists., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Brucellosis is a bacterial disease caused by Brucella infection. Brucella abortus strain A19 is a spontaneously attenuated vaccine strain that has been used in vaccination of cattle against brucellosis. Until now, the physiological and molecular mechanisms of A19 are still unknown., Results: In this paper, the whole-genome sequence of B. abortus A19 was performed using Illumina Hiseq 4000 and PacBio sequencing technology and comparative genomics analysis were carried out with the whole genome sequences of B. abortus strains S19. This analysis indicated that the two vaccine strains have a high degree of similarity in genomic structure. We further analysis of the difference in genomic structure between A19 and S19. And found some differential genes such as eryC, eryD and eryF. Of the other different proteins between A19 and S19, such as outer membrane protein, 2-isopropylmalate synthase, citramalate synthase, GntR family transcriptional regulator and ABC transporters, no clear effects related to bacterial virulence were found, pending further investigation., Conclusion: The data presented here provide a reasonable basis for designing Brucella vaccines that can be used in other strains., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2019. Published by Elsevier Inc.)

Brucella abortus S19 is an important tool for controlling bovine brucellosis across the globe. However, vaccination with S19 suffers critical shortcomings such as, presence of residual virulence, induction of abortion and sero-diagnostic interference. In this study, rfbD gene deleted mutant S19 was developed. The mutant strain designated S19ΔR displayed rough LPS phenotype, which was confirmed by acriflavine dye-agglutination and LPS-SDS-PAGE analysis. The virulence was amply reduced as suggested by increased sensitivity to complement killing; reduction in splenic-bacterial load and the recovery time RT50 as validated in mice model. Anti-brucella humoral response was significantly lower as compared to S19 immunization. The minimal induction of Brucella specific IgG1, IgG2a & IgG2b, and IgG3 resulted in no apparent reactivity to RBPT antigen. S19ΔR showed protective index of 1.90 against virulent challenge. S19ΔR being highly attenuated and DIVA compatible may facilitate a platform for developing a safer bovine adulthood vaccine., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus -based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

Brucella abortus is the causative agent of brucellosis, a neglected endemic zoonotic disease. It causes devastating economic losses in low income and developing countries. Clinical symptoms of infected cows include abortion, poor weight, reduced fertility gain and reduction in milk production. Transmission of the zoonotic disease from cattle to human can occur through direct contact with infected cows, their tissues (e.g. placenta or aborted tissues), or their products (e.g. dairy) whereas human-to-human transmission can occur transplacentally or via breastfeeding. Malaise, fatigue, fever, arthritis are some clinical symptom of the disease in humans. Recent studies have revealed that Brucella abortus show resistance to several antibiotics. There are worldwide concerns about rising levels of antibiotic resistance resulting in the treatment failure as well as the reduced usefulness of older broad-spectrum antibiotics. Hence, a rather novel method has been in use to combat resistant pathogens since the last decade. To overcome this challenge, subtractive genomic analysis has been successfully carried out with the whole proteome of Brucella abortus strain 2308 using various bioinformatic tools and servers. Proteins nonhomologous to cattle and human were selected for metabolic analysis. Only three membrane proteins (ABC transporter permease, acriflavine resistance protein B, penicillin-binding protein 2) were found to be potential novel vaccine candidates with cattle as the host whereas one membrane protein (ABC transporter permease) was selected as novel drug target with human as the host. Development of novel vaccines and therapeutics through targeting inhibition of the functions of any of these essential proteins can lead to disruption of pathogen-specific metabolic pathways and thereby to the destruction and the eradication of this pathogen from respective hosts. The results of this study could facilitate the discovery and release of new and effective drugs and help in designing and producing potent vaccines against Brucella abortus strain 2308., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Designing a potent recombinant vaccine, using the appropriate subunits with the greatest effect on stimulating the immune system, especially in the case of intracellular pathogens such as gram negative Brucella Melitensis bacteria, is of great importance. In this study, three repeats of 27 amino acids of the immunogenic epitope derived from OMP31 antigen (3E) from the Brucella melitensis, in a protective manner against Brucellosis have been used. To fortify the delivery system of recombinant antigens, IL-2 cytokine as a molecular adjuvant was fused to recombinant constructs. Recombinant proteins were evaluated for immunological studies in a mouse model (BALB/c)., Results: The results showed that all recombinant proteins could stimulate the immune system to produce Th1 cytokines and antibodies in compare to the negative control treatments. 3E-IL2 and then OMP31-IL2 proteins stimulated higher levels of IFN-γ and IL-2 compared to the other treatments (p < 0.05). Also, the results indicated that experimental treatments produced a higher level of IgG2a isotype than IgG1 isotype. In addition, the findings of the experiment showed that the presence of chemical adjuvant (IFA) along with molecular adjuvant can play a significant role in stimulating the immune system. After determining the potency of recombinant structures, their efficacy in stimulating the immune system were also evaluated. B. melitensis M16 strain was used to challenge 30 days after last immunization. The microbial load of the splenocyte in the treatments receiving chimeric proteins were significantly lower. Also, Wright serological test confirmed that these treatments had the lowest agglutination rate, as well as the positive treatment, while in the negative treatments in excess of blood serum dilutions, agglutination rate were more than 2 + ., Conclusions: 3E-IL2 treatment showed the best performance compared to other recombinant proteins and could be considered as the suitable candidate for further research on the production of recombinant vaccine against Brucella.

Osteoarticular brucellosis is the most common complication in Brucella -infected humans regardless of age, sex, or immune status. The mechanism of bone destruction caused by Brucella species remained partially unknown due to the lack of a suitable animal model. Here, to study this complication, we explored the suitability of the use of the NOD- scid IL2rγ null mouse to study osteoarticular brucellosis and examined the potential use of this strain to evaluate the safety of live attenuated vaccine candidates. Mice were inoculated intraperitoneally with a single dose of 1 × 10 4 , 1 × 10 5 , or 1 × 10 6 CFU of B. abortus S19 or the vaccine candidate B. abortus S19 ΔvjbR and monitored for the development of side effects, including osteoarticular disease, for 13 weeks. Decreased body temperature, weight loss, splenomegaly, and deformation of the tails were observed in mice inoculated with B. abortus S19 but not in those inoculated with S19 ΔvjbR Histologically, all S19-inoculated mice had a severe dose-dependent inflammatory response in multiple organs. The inflammatory response at the tail was characterized by the recruitment of large numbers of neutrophils, macrophages, and osteoclasts with marked bone destruction. These lesions histologically resembled what is typically observed in Brucella -infected patients. In contrast, mice inoculated with B. abortus S19 ΔvjbR did not show significant bone changes. Immunofluorescence, in situ hybridization, and confocal imaging demonstrated the presence of Brucella at the sites of inflammation, both intra- and extracellularly, and large numbers of bacteria were observed within mature osteoclasts. These results demonstrate the potential use of the NOD- scid IL2rγ null mouse model to evaluate vaccine safety and further study osteoarticular brucellosis., (Copyright © 2019 American Society for Microbiology.)

Brucella abortus RB51 is an attenuated, stable, spontaneous rough mutant derived in the laboratory from the virulent strain B. abortus 2308. Previous studies discovered that the wboA gene, which encodes a glycosyltransferase required for synthesis of the O-polysaccharide, is disrupted in strain RB51 by an IS711 element. However, complementation of strain RB51 with a functional wboA gene (strain RB51WboA) does not confer it a smooth phenotype but results in low levels of cytoplasmic O-polysaccharide synthesis. In this study, we asked if increasing the potential availability of bactoprenol priming precursors in strain RB51WboA would increase the levels of O-polysaccharide synthesis and enhance the protective efficacy against virulent Brucella challenge. To achieve this, we overexpressed the wbkF gene, which encodes a putative undecaprenyl-glycosyltransferase involved in bactoprenol priming for O-polysaccharide polymerization, in strain RB51WboA to generate strain RB51WboAKF. In comparison to strain RB51WboA, strain RB51WboAKF expressed higher levels of O-polysaccharide, but was still attenuated and remained phenotypically rough. Mice immunized with strain RB51WboAKF developed increased levels of smooth LPS-specific serum antibodies, primarily of IgG2a and IgG3 isotype. Splenocytes from mice vaccinated with strain RB51WboAKF secreted higher levels of antigen-specific IFN-γ and TNF-α and contained more numbers of antigen-specific IFN-γ secreting CD4+ and CD8+ T lymphocytes when compared to those of the RB51 or RB51WboA vaccinated groups. Immunization with strain RB51WboAKF conferred enhanced protection against virulent B. abortus 2308, B. melitensis 16M and B. suis 1330 challenge when compared to the currently used vaccine strains. Our results suggest that strain RB51WboAKF has the potential to be a more efficacious vaccine than its parent strain in natural hosts., Competing Interests: The authors have declared that no competing interests exist.

The Capripoxvirus (CaPV) has a large double‑stranded DNA genome and a restricted host‑range. At present, it is being investigated as an ideal vaccine vector. In the present study, a novel recombinant goat pox virus (rGTPV) was constructed to express Brucella outer membrane protein (OMP)25, and was validated by in vitro and in vivo immunization assays. A novel rGTPV vector was created, in which the thymidine kinase gene was used as a flanking sequence, I1L was inserted as a promoter element to enhance Brucella OMP25 expression, and p7.5 as another promoter element was used to regulate guanine phosphoribosyl‑transferase as a selection maker. The rGTPV vector was transfected into sheep fetal fibroblast/lamb testis cells pre‑infected with GTPV G14‑STV44‑55 to recombine. Brucella OMP25 protein was expressed in cells by rGTPV, and activated immune reactivity to Brucella OMP25 protein, as detected by western blotting. Furthermore, rGTPV elicited, anti‑Brucella‑specific immunoglobulin G responses, as determined by ELISA. Mice vaccinated with rGTPV did not exhibit pathology alterations in the kidney and liver. These results suggested that the novel rGTPV was able to efficiently drive Brucella OMP25 protein expression and activate immune reactivity, and may have applications in CaPV live vector vaccines and associated research.

Feral swine cost around $1.5 billion each year in agricultural, environmental, and personal property damages. They are also the most widespread carriers of the zoonotic disease brucellosis, which threatens both livestock bio-security and public health. Currently, there is no approved vaccine against brucellosis in pigs. This is a preliminary report on the development of a live-attenuated B. suis vaccine that could be employed to deliver heterologous antigens to control swine populations. An attenuated vaccine strain provided significant protection against B. suis challenge in mice. Leucine auxotrophy in the vaccine strain allowed the over-expression of heterologous antigens without the use of antibiotic resistant markers. Vaccinated mice showed the development of antibodies against expressed antigen. Further evaluation is required to assess its ability to cause infertility using the mouse model prior to further testing for use as a tool for feral swine population and disease control., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Brucellosis is an infectious disease that brings enormous economic burdens for developing countries. The Brucella melitensis (B. melitensis) M5-90 vaccine strain (M5-90) has been used on a large scale in China, but may cause abortions if given to pregnant goats or sheep subcutaneously during the late stages of gestation. Moreover, the vaccine M5-90 cannot differentiate natural from vaccinated infection. Therefore, a safer and more potent M5-90 vaccine is required. In this study, a vjbR mutant of M5-90 (M5-90ΔvjbR) was constructed and overcame these drawbacks. M5-90ΔvjbR strain showed reduced survival capability in murine macrophages (RAW 264.7) and BALB/c mice and induced high protective immunity in mice. In addition, M5-90ΔvjbR induced an anti-Brucella-specific immunoglobulin G (IgG) response and stimulated the expression of gamma interferon (INF-γ) and interleukin-4 (IL-4) in vaccinated mice. Furthermore, M5-90ΔvjbR induced IgG response and stimulated the secretion of IFN-γ and IL-4 in immunized sheep. Moreover, the VjbR antigen allowed serological differentiation between infected and vaccinated animals. These results suggest that M5-90ΔvjbR is an ideal live attenuated and efficacious live vaccine candidate against B. melitensis 16 M infection., (Copyright © 2018. Published by Elsevier Ltd.)

Brucella as intracellular pathogen requires a coordinate interaction between Th1 subset of gamma interferon-secreting CD4 T cells and CD8 T cells for optimal protective immunity. It was previously recognized that L7/L12 as T cell-reactive antigen from the pathogen. On other hand, Omp25 was found as another antigen to provide protection against the Brucella infection by eliciting both Th1 and Th2 type of immune responses in mice. Here, we analyzed the prophylactic and therapeutic efficacy of a divalent fusion protein (rL7/L12-Omp25) comprising these two promising immunogens of Brucella in the presence of murine IFN-gamma in mice against B. abortus 544 challenge. rIFN-gamma with rL7/L12-Omp25 resulted in superior immune response when compared to the animal vaccine strain B. abortus S19. The vaccine candidate caused dominance of IgG1 over IgG2a and upregulated cytokine secretion (IFN-gamma, TNF-α, and IL-10) among immunized mice. Moreover, the antigen in combination with murine IFN-gamma elicited stronger cell-mediated immune response among the immunized animals when compared to standard vaccine (S19). The registered log protection unit among challenged mice with B. abortus 544 pathogen was 2.16, p = 0.0001 when rL7/L12-Omp25 was administered alone and 2.4, p = 0.0001 when it was administered along with rIFN-gamma. However, the molecule upon administration with murine IFN-gamma imparted very minimal or no therapeutic effect against brucellosis. To conclude, our study demonstrates the potential of rL7/L12-Omp25 as an immunogen of prospective and efficient prophylaxis as it is capable of eliciting both cell-mediated and humoral immune responses against brucellosis.

In this study, we assessed the protective efficacy of single subunit vaccines, encoded by the B. abortus 544 genes aspC, dps, yaeC and inpB, against B. abortus infection in mice. First, immunization with these antigens, with the exception of the YaeC protein, was found to elicit both humoral and cellular immune responses with IgG2a being dominant over IgG1. In addition, a massive production of IFN-γ but lower degree of IL-10 was observed, suggesting that all three antigens were able to induce predominantly cell-mediated immunity in response to B. abortus infection. Further investigation of a combined subunit vaccine (CSV) consisting of purified AspC, Dps, InpB and Ndk proteins showed a superior protective effect in mice against brucellosis. The intraperitoneal injection of this combination was shown to induce a remarkable production of IFN-γ and IL-2, which occurred in conjunction with an increase of blood CD4 + and CD8 + T cell proportions. In addition, the higher titer of IgG2a compared to IgG1 elicited by this CSV was obtained, suggesting that this CSV induced a typical T-helper-1-dominated immune response in vivo. Furthermore, the protection level induced by this combination was significantly higher than that induced by single antigens and was not significantly different compared to a group immunized with a live attenuated vaccine (RB51). Altogether, our findings suggest that the combination of different immunogenic antigens could be a useful approach for the development of a new, effective and safe brucellosis vaccine that can replace current vaccine strains., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Brucellosis is an important zoonotic disease causing huge economic losses worldwide. Currently no effective immunotherapy for Brucellosis or any biomarker to monitor the efficacy of therapy is available. Treatment is ineffective and animals remain carrier lifelong. S19 and RB51 are live attenuated vaccine strains of Brucella abortus. However, S19 induces only antibody, ineffective for intracellular pathogen. RB51 induces cell mediated immunity (CMI) but it is Rifampicin resistant. Both organisms are secreted in milk and can infect humans and cause abortions in animals. Phage lysed bacteria (lysates) retain maximum immunogenicity as opposed to killing by heat or chemicals. We report here the successful immunotherapy of bovine Brucellosis by phage lysates of RB51 (RL) and S19 (SL). The SL induced strong antibody response and RL stimulated CMI. In vitro restimulation of leukocytes from RL immunized cattle induced interferon gamma production. A single subcutaneous dose of 2 ml of cocktail lysate (both RL and SL), eliminated live virulent Brucella from Brucellosis affected cattle with plasma level of Brucella specific 223 bp amplicon undetectable by RT-PCR and blood negative for live Brucella by culture in 3 months post-immunization. This is the first report on minimally invasive monitoring of the efficacy of antibacterial therapy employing plasma RNA specific for live bacteria as a biomarker as well as on the use of RB51 phage lysate for successful immunotherapy of Brucellosis in cattle.

An anti-Brucella vaccine candidate comprising rough Salmonella vector delivering Brucella antigens was developed. This system provides a platform for live Brucella-free vaccine development as it can mimic active-intracellular infection of Brucella organism. Exploiting this phenomenon thus provides significant protection at a single dose and also re-assured the safety. To date, no human anti-Brucella vaccines are available, owing to the lack of safe and effective formulation. This study investigated the safety of the vaccine formulation in mice model and in vitro human cell cultures. The experiment was designed to determine the LD50 of the vaccine formulation. The vaccine formulation did not induce any mortality even when mice were administered at 8 × 10 9 CFU per oral or per subcutaneous (SC), which was 100-times more than the actual vaccine dose intended for mice model. In contrast, wild-type (WT) Salmonella positive control strain induced 100% mortality at 8 × 10 7 CFU per mice via oral or SC routes. Interaction of the vaccine with phagocytic (THP-1 derived macrophage) and non-phagocytic (Caco-2) human cell lines as well as human PBMC was investigated. In in vitro experiments, inflammatory and pyretic cytokines TNF-α, and IL-1β inductions were significantly lower in vaccine group as compared to WT group. Further, apoptosis, nitric oxide synthase and cytotoxicity inductions were comparable and not exacerbated, given that the strain is based on a rough bacterial vector that may have endotoxic lipid-A more readily exposed. These findings corroborated that the vaccine formulation is highly safe in mice model and is relatively mild in the induction of inflammatory cytokines and cellular changes in human cell lines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Brucellosis is one the serious infectious diseases caused deleterious health and economic losses. Vaccination with subunit vaccines is the efficient alternative way than live attenuated vaccines against infectious diseases. Herein a new chimeric OMP25-BLS antigen emulsified in Chitosan Nanoparticles was designed and its immune responses were compared with control groups. Also, the role of heat shock protein 60 kDa in combination with OMP25-BLS antigen was assessed. Structural and antigenic features of chimeric antigen were predicted using bioinformatics tools. Moreover, the humoral and cellular immune responses were measured by ELISA in seven different groups. Observations showed rOMP25-BLS structure was highly stable and antigenic. Cytokines analysis showed rOMP25 and rOMP25-BLS + rHSP60 induced higher titer of INF-γ than rHSP60 and rOMP25-BLS. There was no statistically significant difference between positive control group and rOMP25-BLS + rHSP60 in inducing TNF-α (p < .05). Additionally, the highest titer of IL-4 was dedicated to rOMP25 among other immunized treatments, while there were no significant differences between positive control group and other immunized groups with recombinant proteins (p < .05). In addition, rOMP25-BLS and rHSP60 induced higher titer of total antibody compared to other groups. Also, rHSP60 could improve IgG2a to IgG1 ratio when it used in combination with chimeric antigen. Moreover, the lymphocyte proliferation index was higher in chimeric rOMP25-BLS + HSP60 antigen. In conclusion, while rOMP25-BLS chimeric antigen unable to induce efficient cellular response than individual injection of rOMP25, its injection in combination with rHSP60 could improve cellular immunity., (Copyright © 2017. Published by Elsevier Ltd.)

Background: Brucellosis is a widespread zoonotic disease caused by Gram-negative Brucella bacteria. Immunisation with attenuated vaccine is an effective method of prevention, but it can interfere with diagnosis. Live, attenuated Brucella abortus strain 104M has been used for the prevention of human brucellosis in China since 1965. However, at present, no fast and reliable method exists that can distinguish this strain from field strains. Single nucleotide polymorphism (SNP)-based assays offer a new approach for such discrimination. SNP-based minor groove binder (MGB) and Cycleave assays have been used for rapid identification of four Brucella vaccine strains (B. abortus strains S19, A19 and RB51, and B. melitensis Rev1). The main objective of this study was to develop a PCR assay for rapid and specific detection of strain 104M., Results: We developed a SNP-based MGB PCR assay that could successfully distinguish strain 104M from 18 representative strains of Brucella (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae, and B. ovis), four Brucella vaccine strains (A19, S19, S2, M5), and 55 Brucella clinical field strains. The assay gave a negative reaction with four non-Brucella species (Escherichia coli, Pasteurella multocida, Streptococcus suis and Pseudomonas aeruginosa). The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 220 fg for the 104M strain and 76 fg for the single non-104M Brucella strain tested (B. abortus A19). The assay was also reproducible (intra- and inter-assay coefficients of variation = 0.006-0.022 and 0.012-0.044, respectively)., Conclusions: A SNP-based MGB PCR assay was developed that could straightforwardly and unambiguously distinguish B. abortus vaccine strain 104M from non-104M Brucella strains. Compared to the classical isolation and identification approaches of bacteriology, this real-time PCR assay has substantial advantages in terms of simplicity and speed, and also reduces potential exposure to live Brucella. The assay developed is therefore a simple, rapid, sensitive, and specific tool for brucellosis diagnosis and control.

The live attenuated Brucella melitensis Rev.1 Elberg-originated vaccine strain has been widely used to control brucellosis in small ruminants. However, despite extensive research, the molecular mechanisms underlying the attenuation of this strain are still unknown. In the current study, we conducted a comprehensive comparative analysis of the whole-genome sequence of Rev.1 against that of the virulent reference strain, B. melitensis 16M. This analysis revealed five regions of insertion and three regions of deletion within the Rev.1 genome, among which, one large region of insertion, comprising 3,951 bp, was detected in the Rev.1 genome. In addition, we found several missense mutations within important virulence-related genes, which may be used to determine the mechanism underlying virulence attenuation. Collectively, our findings provide new insights into the Brucella virulence mechanisms and, therefore, may serve as a basis for the rational design of new Brucella vaccines.

Recombinant engineering of immunologically active chimeric protein consisting of Omp19 and P39 domains of B. abortus (rOP), was purified under denaturing conditions upon expression in E. coli BL21 (DE3) and refolded to dynamic form. The immuno-protective efficacy of rOP was evaluated by challenging the BALB/c mice intraperitoneally (I.P) with the infective species of Brucella in the absence or presence of adjuvants, such as Aluminum hydroxide gel (Al), or Freund's Complete Adjuvant (FCA)/Incomplete Freund's Adjuvant (IFA). Surprisingly, after second boosting, mice received rOP per se were found to be immunogenic in terms of IgG response with the dominant expression of IgG2a and significant IFN-γ by splenic T cells, suggesting that rOP is a strong inducer of anti-Brucella immunity. The resulted anti-rOP antibodies recognized native Omp19 and P39 among species of Brucella with distinct double bands and single band against chimera in immunoblotting. An enhanced and comparable antibody response with varied IgG isotype combinations were noticed in the mice primed and boosted with rOP in adjuvants. However, rOP+FCA/IFA formulation was found to be the most effective in lymphocyte recall assays at inducing significant (P<0.001) proliferation index (P.I.) as well as increased Th1-coupled cytokines (IFN-γ, IL-2 and IL-12p70) than rOP+Al in response to rOP re-stimulation. Furthermore, in vitro defensive assay revealed that compared to anti-rOP antisera, the polyclonal anti-sera from rOP+adjuvants exhibited enhanced protection of RAW264.7 cells against virulent challenge by B. melitensis 16M and B. abortus 544. In addition, compared to sham group, enumeration of Brucella CFU after challenge with the above species showed a significant (P<0.01) reduction of bacteria (log CFU) in the macrophage cell lines and organs of vaccinated mice. On the whole, a relatively higher and faster reduction was noticed in the mice vaccinated with similar amount of purified antigen in Freund's adjuvant. Ability of inducing Th1 directed immune protection in the absence of adjuvant support, postulated rOP as a plausible entrant for developing a chimeric based subunit vaccine against Brucella., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Brucellosis, which is caused by Brucella spp., is a zoonotic infectious disease that can cause great hazard to public health and safety. The virulence of Brucella is essential for survive and multiply in host macrophages. GntR is a transcriptional regulator in Brucella that is required for virulence in macrophages and mice, and involved in resistance to stress responses. To determine the expression levels of target genes of GntR, we detected the expression levels of the GntR target genes in Brucella infected BALB/c mice. The results showed that several genes related to virulence, including omp25, virB1, vjbR, dnaK, htrA and hfq, were regulated by GntR during infection in BALB/c mice. Moreover, the 2308ΔgntR mutant induced high protective immunity in BALB/c mice challenge with B. abortus 2308 (S2308), and elicited an anti-Brucella-specific immunoglobulin G (IgG) response and induced the secretion of gamma interferon (IFN-γ) and interleukin-4 (IL-4). All together, these results indicated that gntR promoted the virulence of Brucella. The 2308ΔgntR was significantly attenuated in macrophages and mice and induced protective immune response during infection, suggested that 2308ΔgntR mutant is an attractive candidate for the design of a live attenuated vaccine against Brucella., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. Although effective, the current Brucella vaccines (strain M5-90 or others) have several drawbacks. The first is their residual virulence for animals and humans and the second is their inability to differentiate natural infection from that caused by vaccination. In the present study, Brucella melitensis M5-90 manB mutant (M5-90ΔmanB) was generated to overcome these drawbacks. M5-90ΔmanB showed significantly reduced survival in macrophages and mice, and induced strong protective immunity in BALB/c mice. It elicited anti-Brucella-specific IgG1 and IgG2a subtype responses and induced the secretion of gamma interferon (IFN-γ) and interleukin-4(IL-4). Results of immune assays showed, M5-90ΔmanB immunization induced the secretion of IFN-γ in goats, and serum samples from goats inoculated with M5-90ΔmanB were negative by Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). Further, the ManB antigen also allows serological assays differentiate infections caused by wild strains from infections by vaccination. These results show that M5-90ΔmanB is a suitable attenuated vaccine candidate against virulent Brucella melitensis 16 M (16 M) infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We previously developed a potent candidate vaccine against bovine brucellosis caused by Brucella abortus using the influenza viral vector expressing Brucella Omp16 and L7/L12 proteins (Flu-BA). Our success in the Flu-BA vaccine trial in cattle and results of a pilot study in non-pregnant small ruminants prompted us in the current study to test its efficacy against B. melitensis infection in pregnant sheep and goats. In this study, we improved the Flu-BA vaccine formulation and immunization method to achieve maximum efficacy and safety. The Flu-BA vaccine formulation had two additional proteins Omp19 and SOD, and administered thrice with 20% Montanide Gel01 adjuvant, simultaneously by both subcutaneous and conjunctival routes at 21 days intervals in pregnant sheep and goats. At 42 days post-vaccination (DPV) we detected antigen-specific IgG antibodies predominantly of IgG2a isotype but also IgG1, and also detected a strong lymphocyte recall response with IFN-γ production. Importantly, our candidate vaccine prevented abortion in 66.7% and 77.8% of pregnant sheep and goats, respectively. Furthermore, complete protection (absence of live B. melitensis 16M) was observed in 55.6% and 66.7% of challenged sheep and goats, and 72.7% and 90.0% of their fetuses (lambs/yeanlings), respectively. The severity of B. melitensis 16M infection in vaccinated sheep and goats and their fetuses (index of infection and rates of Brucella colonization in tissues) was significantly lower than in control groups. None of the protection parameters after vaccination with Flu-BA vaccine were statistically inferior to protection seen with the commercial B. melitensis Rev.1 vaccine (protection against abortion and vaccination efficacy, alpha = 0.18-0.34, infection index, P = 0.37-0.77, Brucella colonization, P = 0.16 to P > 0.99). In conclusion, our improved Flu-BA vaccine formulation and delivery method were found safe and effective in protecting pregnant sheep and goats against adverse consequences of B. melitensis infection.

Brucella melitensis (B. melitensis) is a facultative intracellular pathogen, which is the main epidemic strain in China. To overcome disadvantages of traditional live attenuated vaccines, in this study a rough mutant RM57 was induced from a B. melitensis isolate M1981. In order to uncover the reason of changes in the LPS of RM57, the nucleotide sequences and transcription levels of all known genes related to LPS synthesis were detected. As LPS plays an important role in outer membrane integrity, the sensitivities of RM57 to SDS and polymyxin B were examined. The results showed that the expression level of LPS genes of RM57 was not significantly changed, and RM57 was sensitive to polymyxin B, compared to its parent strain. In further study, the virulence and protective efficacy of RM57 in mice and guinea pigs were determined, and our data indicated that RM57 was attenuated and had good protection effects, especially in guinea pigs model. Overall, these results demonstrated that the artificially induced rough mutant strain RM57 was an efficacious vaccine candidate against the challenge of virulent B. melitensis. Our data presented here provided additional insight into the mechanism of LPS synthesis of Brucella spp., (Copyright © 2017. Published by Elsevier Ltd.)

Brucellosis is an important zoonotic disease of worldwide distribution, which causes animal and human disease. However, the current Brucella abortus (B. abortus) vaccines (S19 and RB51) have several drawbacks, including residual virulence for animals and humans. Moreover, S19 cannot allow serological differentiation between infected and vaccinated animals. We constructed double deletion (ΔNodVΔNodW) mutant from virulent B. abortus 2308 (S2308) by deleting the genes encoding two-component regulatory system (TCS) in chromosome II in S2308.2308ΔNodVΔNodW was significantly reduced survival in murine macrophages (RAW 264.7) and BALB/c mice. Moreover, the inoculated mice showed no splenomegaly. The mutant induced high protective immunity in BALB/c mice against challenge with S2308, and elicited an anti-Brucella-specific immunoglobulin G (IgG) response and induced the secretion of gamma interferon (IFN-γ) and interleukin-4 (IL-4). Moreover, NODV and NODW antigens would allow the serological differentiation between infected and vaccinated animals. These results suggest that 2308ΔNodVΔNodW mutant is a potential live attenuated vaccine candidate and can be used effectively against bovine brucellosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

There remains a need for an improved livestock vaccine for brucellosis since conventional vaccines are only ∼70% efficacious, making some vaccinated animals susceptible to Brucella infections. To address this void, a vaccine capable of evoking protective immunity, while still being sufficiently attenuated to produce minimal disease, is sought. In this pursuit, the ΔnorD ΔznuA B. abortus-lacZ (termed as znBAZ) was developed to be devoid of functional norD and znuA B. abortus genes, and to contain the lacZ as a marker gene. The results show that znBAZ is highly attenuated in mouse and human macrophages, and completely cleared from mouse spleens within eight weeks post-vaccination. Producing less splenic inflammation, znBAZ is significantly more protective than the conventional RB51 vaccine by more than four orders of magnitude. Vaccination with znBAZ elicits elevated numbers of IFN-γ + , TNF-α + , and polyfunctional IFN-γ + TNF-α + CD4 + and CD8 + T cells in contrast to RB51-vaccinated mice, which show reduced numbers of proinflammatory cytokine-producing T cells. These results demonstrate that znBAZ is a highly efficacious vaccine candidate capable of eliciting diverse T cell subsets that confer protection against parenteral challenge with virulent, wild-type B. abortus., Competing Interests: The authors declare no financial or commercial conflict of interest., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this study, we explored possibility of increasing the protective efficacy of our novel influenza viral vector based B. abortus vaccine (Flu-BA) in pregnant heifers by adapting an innovative method of vaccine delivery. We administered the vaccine concurrently via the conjunctival and subcutaneous routes to pregnant heifers, and these routes were previously tested individually. The Flu-BA vaccination of pregnant heifers (n=9) against a challenge B. abortus 544 infection provided protection from abortion, infection of heifers and fetuses/calves by 88.8%, 100% and 100%, respectively (alpha=0.004-0.0007 vs. negative control; n=7). Our candidate vaccine using this delivery method provided slightly better protection than the commercial B. abortus S19 vaccine in pregnant heifers (n=8), which provided protection from abortion, infection of heifers and fetuses/calves by 87.5%, 75% and 87.5%, respectively. This improved method of the Flu-BA vaccine administration is highly recommended for the recovery of farms which has high prevalence of brucellosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The Brucella abortus general stress response (GSR) system regulates activity of the alternative sigma factor, σ(E1), which controls transcription of approximately 100 genes and is required for persistence in a BALB/c mouse chronic infection model. We evaluated the host response to infection by a B. abortus strain lacking σ(E1) (ΔrpoE1), and identified pathological and immunological features that distinguish ΔrpoE1-infected mice from wild-type (WT), and that correspond with clearance of ΔrpoE1 from the host. ΔrpoE1 infection was indistinguishable from WT in terms of splenic bacterial burden, inflammation and histopathology up to 6weeks post-infection. However, Brucella-specific serum IgG levels in ΔrpoE1-infected mice were 5 times higher than WT by 4weeks post-infection, and remained significantly higher throughout the course of a 12-week infection. Total IgG and Brucella-specific IgG levels peaked strongly in ΔrpoE1-infected mice at 6weeks, which correlated with reduced splenomegaly and bacterial burden relative to WT-infected mice. Given the difference in immune response to infection with wild-type and ΔrpoE1, we tested whether ΔrpoE1 confers protective immunity to wild-type challenge. Mice immunized with ΔrpoE1 completely resisted WT infection and had significantly higher serum titers of Brucella-specific IgG, IgG2a and IFN-γ after WT challenge relative to age-matched naïve mice. We conclude that immunization of BALB/c mice with the B. abortus GSR pathway mutant, ΔrpoE1, elicits an adaptive immune response that confers significant protective immunity against WT infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Brucella melitensis Rev.1 is an avirulent strain that is widely used as a live vaccine to control brucellosis in small ruminants. Although an assembled draft version of Rev.1 genome has been available since 2009, this genome has not been investigated to characterize this important vaccine. In the present work, we used the draft genome of Rev.1 to perform a thorough genomic comparison and sequence analysis to identify and characterize the panel of its unique genetic markers. The draft genome of Rev.1 was compared with genome sequences of 36 different Brucella melitensis strains from the Brucella project of the Broad Institute of MIT and Harvard. The comparative analyses revealed 32 genetic alterations (30 SNPs, 1 single-bp insertion and 1 single-bp deletion) that are exclusively present in the Rev.1 genome. In silico analyses showed that 9 out of the 17 non-synonymous mutations are deleterious. Three ABC transporters are among the disrupted genes that can be linked to virulence attenuation. Out of the 32 mutations, 11 Rev.1 specific markers were selected to test their potential to discriminate Rev.1 using a bi-directional allele-specific PCR assay. Six markers were able to distinguish between Rev.1 and a set of control strains. We succeeded in identifying a panel of 32 genome-specific markers of the B. melitensis Rev.1 vaccine strain. Extensive in silico analysis showed that a considerable number of these mutations could severely affect the function of the associated genes. In addition, some of the discovered markers were able to discriminate Rev.1 strain from a group of control strains using practical PCR tests that can be applied in resource-limited settings., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Brucellosis is a bacterial disease caused by Brucella infection. In the late fifties, Brucella suis vaccine strain S2 with reduced virulence was obtained by serial transfer of a virulent B. suis biovar 1 strain in China. It has been widely used for vaccination in China since 1971. Until now, the mechanisms underlie virulence attenuation of S2 are still unknown., Results: In this paper, the whole genome sequencing of S2 was carried out by Illumina Hiseq2000 sequencing method. We further performed the comparative genomic analysis to find out the differences between S2 and the virulent Brucella suis strain 1330. We found premature stops in outer membrane autotransporter omaA and eryD genes. Single mutations were found in phosphatidylcholine synthase, phosphorglucosamine mutase, pyruvate kinase and FliF, which have been reported to be related to the virulence of Brucella or other bacteria. Of the other different proteins between S2 and 1330, such as Omp2b, periplasmic sugar-binding protein, and oligopeptide ABC transporter, no definitive implications related to bacterial virulence were found, which await further investigation., Conclusions: The data presented here provided the rational basis for designing Brucella vaccines that could be used in other strains.

Brucellae are Gram-negative intracellular bacterial pathogens that infect humans and animals, bringing great economic burdens to developing countries. Live attenuated Brucella vaccines (strain M5-90 or others) are the most efficient means for prevention and control of animal brucellosis. However, these vaccines have several drawbacks, including residual virulence in animals, and difficulties in differentiating natural infection from vaccine immunization, which limit their application. A vaccine that can differentiate infection from immunization will have extensive applications. A Brucella melitensis (B. melitensis) strain M5-90 pgm mutant (M5-90Δpgm) was constructed to overcome these drawbacks. M5-90Δpgm showed significantly reduced survival in embryonic trophoblast cells and in mice, and induced high protective immunity in BALB/c mice. Moreover, M5-90Δpgm elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon (IFN-γ) and interleukin-2 (IL-2). In addition, M5-90Δpgm induced the secretion of IFN-γ in immunized sheep. Serum samples from sheep inoculated with M5-90Δpgm were negative by the Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). Furthermore, the PGM antigen allowed serological differentiation between infected and vaccinated animals. These results suggest that M5-90Δpgm is an ideal live attenuated vaccine candidate against B. melitensis 16 M and deserves further evaluation for vaccine development.