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

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

Crosstalk of Brucella abortus nucleomodulin BspG and host DNA replication process/mitochondrial respiratory pathway promote anti-apoptosis and infection.

The secretory proteins of Brucella mediate the expression of the bacterium in the host, thereby facilitating intracellular parasitism. With the exception of the recently reported BspJ, the Brucella nucleomodulin has not yet been characterized. We defined the Brucella nucleomodulin BspG and verified six proteins (PCBP1, KMT5C, NDUFS6, PCNA, CIAO2B, and SDHB) that interacted with BspG using a yeast two-hybrid assay and co-immunoprecipitation (CO-IP) screening. The deletion of BspG decreased the intracellular proliferation of B. abortus in both in vivo and in vitro experiments. The analysis found that these interacting proteins were related to energy generation, gene expression, and apoptosis of host cells. The crosstalk between B. abortus nucleomodulin BspG and host DNA replication/mitochondrial respiratory pathways promotes anti-apoptosis and infection, but the mechanism needs additional study.

Hypervirulent FAdV-4 infection induces activation of the NLRP3 inflammasome in chicken macrophages.

Fowl adenovirus serotype 4 (FAdV-4) is the primary causative agent of hepatitis-hydropericardium syndrome (HHS) causing great economic losses to the world poultry industry. The exact factors responsible for the pathogenesis of hypervirulent FAdV-4 have not been completely elucidated. Hypervirulent FAdV-4 infection induces inflammatory damages in accompany with a high level of proinflammatory interleukin-1 beta (IL-1ß) secretion in a variety of organs. Investigation of the mechanisms underlying hypervirulent FAdV-4-induced IL-1ß secretion would contribute to understanding of the pathogenesis of FAdV-4. Here, we investigated whether FAdV-4 infection activates NLRP3 inflammasome in chicken macrophage cell line HD11. The results showed that stimulation of HD11 with hypervirulent FAdV-4 induced NLRP3- and Caspase-1-dependent secretion of IL-1ß. Genetic knockdown of NLRP3 or Caspase-1 expression, a critical component of inflammasome, significantly downregulated IL-1ß expression, indicating that activation of the NLRP3 inflammasome contributed to the FAdV-4-induced IL-1ß secretion. Moreover, ATP signaling and potassium efflux were involved in the process of NLRP3 inflammasome activation. Our data indicated that hypervirulent FAdV-4 infection induces the activation of NLRP3 inflammasome and followed by massive secretion of IL-1ß of macrophages, which thereby contribute to the inflamed lesion of tissues.

Deletion of the type IV secretion system promoter VirB in Brucella abortus A19 strain attenuated the virulence of the bacteria and promotes autophagy.

Brucella abortus is a gram-negative intracellular parasite bacteria that causes serious health hazards in humans and animals. The type IV secretion system (T4SS), encoded by the virB promoter, has been identified as an important virulence factor for Brucella abortus, but its impact on Brucella abortus A19 remains unclear. In this study, the T4SS of Brucella abortus A19 was inactivated by deletion of the virB promoter, resulting in a mutant strain A19ΔvirB. Real-time PCR and western blotting analysis demonstrated that T4SS-related proteins were not expressed after virB promoter deletion. Moreover, the survival rate of A19 in high-salt and strong acidic environments decreased after virB promoter deletion. Compared to the parental strain A19, the A19ΔvirB mutant strain showed reduced growth rate in TSB, decreased invasion ability to macrophages and dendritic cells, and reduced virulence of the mutant strain in macrophages, dendritic cells, and mice. In addition, the A19ΔvirB mutant strain showed enhanced autophagy in macrophages and dendritic cells compared with A19, and the A19ΔvirB mutant strain was able to upregulate IL-6 and downregulate IL-10 in macrophages. These data help us to better understand the T4SS of the A19 vaccine strain and contribute to our efforts to improve Brucella vaccines.

Nucleomodulin BspJ as an effector promotes the colonization of Brucella abortus in the host.

BACKGROUND: Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified. OBJECTIVES: To verify the functions of nucleomodulin BspJ in Brucella's intracellular infection cycles. METHODS: Constructed Brucella abortus BspJ gene deletion strain (B. abortus ΔBspJ) and complement strain (B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro. RESULTS: BspJ gene deletion reduced the survival and intracellular proliferation of Brucella at the replicating Brucella-containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1ß, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella. CONCLUSIONS: BspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella's intracellular survival.