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1.
Diagn Microbiol Infect Dis ; 110(1): 116432, 2024 Sep.
Article in English | MEDLINE | ID: mdl-39024932

ABSTRACT

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 (R2> 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.


Subject(s)
Brucella Vaccine , Brucella , Brucellosis , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Brucella/genetics , Brucella/classification , Brucella/isolation & purification , Humans , Brucella Vaccine/genetics , Brucella Vaccine/immunology , Brucellosis/diagnosis , Brucellosis/prevention & control , Brucellosis/microbiology , Polymerase Chain Reaction/methods , Sensitivity and Specificity , China , Vaccines, Attenuated/genetics , Real-Time Polymerase Chain Reaction/methods , DNA, Bacterial/genetics
2.
Nanotechnology ; 35(39)2024 Jul 12.
Article in English | MEDLINE | ID: mdl-38917779

ABSTRACT

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 theirin vitro/in vivoimmunostimulatory 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 thein vitrocytotoxicity and immunostimulatory effects of both proteins and nanoparticles on the J774 murine macrophage cells,in vivoimmunization experiments were conducted using concentrations of 16µg ml-1for each protein. Both free antigens and antigen-containing nanoparticles excessively induced humoral immunity by increasing producedBrucella-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 triggerBrucella-specific humoral and cellular immune response.


Subject(s)
Brucellosis , Nanoparticles , Polylactic Acid-Polyglycolic Acid Copolymer , Recombinant Proteins , Animals , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Brucellosis/prevention & control , Brucellosis/immunology , Mice , Nanoparticles/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/chemistry , Mice, Inbred BALB C , Female , Brucella Vaccine/immunology , Brucella Vaccine/genetics , Brucella Vaccine/administration & dosage , Brucella abortus/immunology , Brucella abortus/genetics , Drug Carriers/chemistry , Nanovaccines
3.
Int Immunopharmacol ; 134: 112204, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38703567

ABSTRACT

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.


Subject(s)
Brucella Vaccine , Brucella abortus , Brucellosis , Lactococcus lactis , Mice, Inbred BALB C , Animals , Brucella abortus/immunology , Brucellosis/prevention & control , Brucellosis/immunology , Lactococcus lactis/genetics , Lactococcus lactis/immunology , Brucella Vaccine/immunology , Brucella Vaccine/administration & dosage , Brucella Vaccine/genetics , Mice , Female , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/genetics , Epitopes/immunology , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Spleen/immunology , Genetic Vectors , Immunoglobulin G/blood , Immunoglobulin G/immunology , Cytokines/metabolism
4.
Vaccine ; 42(17): 3710-3720, 2024 Jun 20.
Article in English | MEDLINE | ID: mdl-38755066

ABSTRACT

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.


Subject(s)
Brucella melitensis , Brucellosis , Placenta , Animals , Brucella melitensis/pathogenicity , Brucella melitensis/immunology , Brucella melitensis/genetics , Female , Sheep , Brucellosis/prevention & control , Brucellosis/immunology , Brucellosis/veterinary , Pregnancy , Placenta/microbiology , Mice , Sheep Diseases/prevention & control , Sheep Diseases/immunology , Sheep Diseases/microbiology , Trophoblasts/immunology , Trophoblasts/microbiology , Brucella Vaccine/immunology , Brucella Vaccine/administration & dosage , Brucella Vaccine/genetics , Humans , Vaccines, Attenuated/immunology , Vaccines, Attenuated/administration & dosage
5.
J Mol Evol ; 92(3): 338-357, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38809331

ABSTRACT

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.


Subject(s)
Brucella abortus , Brucella melitensis , Genome, Bacterial , Genomics , Phylogeny , Brucella melitensis/genetics , Brucella abortus/genetics , Egypt , Genomics/methods , Animals , Brucellosis/microbiology , Brucella Vaccine/genetics , Bacterial Vaccines
6.
Microb Pathog ; 185: 106417, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37866552

ABSTRACT

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.


Subject(s)
Brucella Vaccine , Brucellosis , Lactococcus lactis , Cattle , Mice , Animals , Lactococcus lactis/genetics , Mice, Inbred BALB C , Brucella Vaccine/genetics , Brucellosis/prevention & control , Vaccination/methods , Immunization/methods , Brucella abortus/genetics , Recombinant Proteins/genetics , Immunoglobulin G , Antibodies, Bacterial
7.
Vet Med Sci ; 9(4): 1908-1922, 2023 07.
Article in English | MEDLINE | ID: mdl-37276346

ABSTRACT

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.


Subject(s)
Brucella Vaccine , Brucellosis , Lactococcus lactis , Animals , Mice , Antigens, Bacterial , Bacterial Vaccines , Brucella abortus , Brucella Vaccine/genetics , Brucellosis/microbiology , Brucellosis/veterinary , Immunoglobulin G/metabolism , Lactococcus lactis/genetics , Lactococcus lactis/metabolism , Mice, Inbred BALB C
8.
Arch Microbiol ; 205(4): 122, 2023 Mar 20.
Article in English | MEDLINE | ID: mdl-36939918

ABSTRACT

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.


Subject(s)
Brucella Vaccine , Lactococcus lactis , Mice , Animals , Brucella abortus/genetics , Lactococcus lactis/genetics , Vaccination , Brucella Vaccine/genetics , Mice, Inbred BALB C
9.
Immunol Res ; 71(2): 247-266, 2023 04.
Article in English | MEDLINE | ID: mdl-36459272

ABSTRACT

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.


Subject(s)
Brucella Vaccine , Brucella suis , Brucellosis , Animals , Humans , Brucella suis/genetics , Brucella suis/immunology , Brucellosis/genetics , Brucellosis/immunology , Brucellosis/prevention & control , Computational Biology , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte , Escherichia coli , Molecular Docking Simulation , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccines, Subunit/therapeutic use , Drug Resistance, Bacterial/genetics , Drug Resistance, Bacterial/immunology , Proteome/genetics , Proteome/immunology , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Brucella Vaccine/genetics , Brucella Vaccine/immunology , Brucella Vaccine/therapeutic use , Epitopes/genetics , Epitopes/immunology , Vaccine Development , Drug Design
10.
Front Cell Infect Microbiol ; 12: 1023243, 2022.
Article in English | MEDLINE | ID: mdl-36530431

ABSTRACT

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_0002189, was selected as a marker to differentiate the BrucellaS2vaccine strain from B. abortus and B. melitensis. A loop-mediated isothermal amplification (LAMP) assay was developed, based on the GL_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.


Subject(s)
Brucella Vaccine , Brucella melitensis , Brucella suis , Brucellosis , Animals , Sheep/genetics , Brucella Vaccine/genetics , Nucleic Acid Amplification Techniques/methods , Brucellosis/diagnosis , Brucellosis/prevention & control , Brucellosis/veterinary , Brucella suis/genetics , Brucella melitensis/genetics , Brucella abortus/genetics
11.
Gene ; 830: 146521, 2022 Jul 01.
Article in English | MEDLINE | ID: mdl-35447245

ABSTRACT

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.


Subject(s)
Brucella Vaccine , Brucellosis , ATP-Binding Cassette Transporters , Animals , Brucella Vaccine/genetics , Brucella abortus/genetics , Brucellosis/prevention & control , Vaccination , Virulence/genetics
12.
Microb Biotechnol ; 15(6): 1811-1823, 2022 06.
Article in English | MEDLINE | ID: mdl-35166028

ABSTRACT

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.


Subject(s)
Brucella Vaccine , Brucellosis , Animals , Antibodies, Bacterial , Brucella Vaccine/genetics , Brucella abortus/genetics , Brucellosis/prevention & control , Immunity, Humoral , Immunization , Immunoglobulin G , Mice , Mice, Inbred BALB C , Recombinant Proteins/genetics , Vaccines, Subunit
13.
Front Immunol ; 12: 679560, 2021.
Article in English | MEDLINE | ID: mdl-34163479

ABSTRACT

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.


Subject(s)
Brucella Vaccine/genetics , Brucella Vaccine/immunology , Brucellosis, Bovine/diagnosis , Brucellosis, Bovine/prevention & control , Genetic Markers , Vaccines, Synthetic , Animals , Brucella Vaccine/administration & dosage , Brucellosis, Bovine/immunology , Brucellosis, Bovine/metabolism , Cattle , Chromatography, High Pressure Liquid , Cytokines/metabolism , Diagnosis, Differential , Disease Models, Animal , Genetic Engineering , Immunization , Immunogenicity, Vaccine , Mice , Outcome Assessment, Health Care , Proteomics/methods , Tandem Mass Spectrometry , Virulence
14.
Infect Dis Poverty ; 10(1): 13, 2021 Feb 16.
Article in English | MEDLINE | ID: mdl-33593447

ABSTRACT

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 105 EID50, 106 EID50 and 107 EID50 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 106 EID50, 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.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Brucella Vaccine/administration & dosage , Brucella Vaccine/genetics , Brucella melitensis/immunology , Brucellosis/prevention & control , Influenza A Virus, H5N1 Subtype/genetics , Administration, Intranasal , Administration, Ophthalmic , Administration, Sublingual , Animals , Bacterial Outer Membrane Proteins/immunology , Body Weight , Brucella Vaccine/immunology , Brucella melitensis/genetics , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Genetic Vectors/immunology , Guinea Pigs , Humans , Immunization, Secondary
15.
Front Immunol ; 12: 778475, 2021.
Article in English | MEDLINE | ID: mdl-34992597

ABSTRACT

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-ß 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.


Subject(s)
Bacterial Zoonoses/prevention & control , Brucella Vaccine/immunology , Brucella abortus/immunology , Brucellosis/prevention & control , Acyltransferases/genetics , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Bacterial Zoonoses/immunology , Bacterial Zoonoses/microbiology , Brucella Vaccine/administration & dosage , Brucella Vaccine/genetics , Brucella abortus/genetics , Brucellosis/immunology , Brucellosis/microbiology , Computer Simulation , Disease Models, Animal , Epitope Mapping/methods , Humans , Immunogenicity, Vaccine , Macrophages/immunology , Macrophages/microbiology , Mice , Primary Cell Culture , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology
16.
Probiotics Antimicrob Proteins ; 13(1): 80-89, 2021 02.
Article in English | MEDLINE | ID: mdl-32661939

ABSTRACT

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.


Subject(s)
Bacterial Outer Membrane Proteins , Brucella Vaccine , Brucella melitensis/genetics , Brucellosis , Lactococcus lactis , Microorganisms, Genetically-Modified , Animals , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Brucella Vaccine/genetics , Brucella Vaccine/immunology , Brucella melitensis/immunology , Brucellosis/immunology , Brucellosis/prevention & control , Female , Lactococcus lactis/genetics , Lactococcus lactis/immunology , Mice , Mice, Inbred BALB C , Microorganisms, Genetically-Modified/genetics , Microorganisms, Genetically-Modified/immunology
17.
J Microbiol Biotechnol ; 30(4): 497-504, 2020 Apr 28.
Article in English | MEDLINE | ID: mdl-31986561

ABSTRACT

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 omp31 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.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Brucella Vaccine/administration & dosage , Brucella melitensis/immunology , Brucellosis/prevention & control , Animals , Bacterial Outer Membrane Proteins/immunology , Brucella Vaccine/genetics , Brucella melitensis/genetics , Brucella melitensis/pathogenicity , Disease Models, Animal , Female , Immunization , Male , Mice , Mice, Inbred BALB C , Mutation , Spleen/drug effects , Spleen/pathology , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/genetics , Virulence/genetics
18.
Genomics ; 112(2): 1444-1453, 2020 03.
Article in English | MEDLINE | ID: mdl-31454518

ABSTRACT

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.


Subject(s)
Brucella Vaccine/genetics , Brucella abortus/genetics , Genes, Bacterial , Immunogenicity, Vaccine/genetics , ATP-Binding Cassette Transporters/genetics , Bacterial Outer Membrane Proteins/genetics , Brucella Vaccine/immunology , Brucella abortus/immunology , Cytochrome P-450 Enzyme System/genetics , Sequence Homology
19.
Biologicals ; 63: 62-67, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31843357

ABSTRACT

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.


Subject(s)
Brucella Vaccine , Brucella abortus , Brucellosis/prevention & control , Mutation , Animals , Brucella Vaccine/genetics , Brucella Vaccine/immunology , Brucella abortus/genetics , Brucella abortus/immunology , Brucella abortus/pathogenicity , Brucellosis/genetics , Brucellosis/immunology , Mice , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology
20.
Can J Microbiol ; 66(1): 39-45, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31574230

ABSTRACT

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.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Brucella Vaccine/genetics , Brucella Vaccine/immunology , Brucella melitensis/immunology , Interleukin-2/genetics , Lactococcus lactis/genetics , Brucella melitensis/genetics , Brucellosis/prevention & control , Cloning, Molecular , Humans , Lactococcus lactis/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccines, Synthetic/metabolism
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