Immuno-profiling of Brucella proteins for developing improved vaccines and DIVA capable serodiagnostic assays for brucellosis.

Brucellosis remains a worldwide zoonotic disease with a serious impact on public health and livestock productivity. Controlling brucellosis in livestock is crucial for limiting human infections in the absence of effective human vaccines. Brucellosis control measures are majorly dependent on rigorous monitoring of disease outbreaks and mass vaccination of livestock. Live attenuated vaccines are available for livestock vaccination that play a vital role in brucellosis control programs in many countries. Even though the existing animal vaccines confer protection against brucellosis, they carry some drawbacks, including their infectivity to humans and interference with sero-monitoring. The available serodiagnostic assays for brucellosis depend on detecting anti-LPS antibodies in the serum. Since diagnosis plays a vital role in controlling brucellosis, developing improved serodiagnostic assays with enhanced specificity, sensitivity and DIVA capability is required. Therefore, it is essential to identify novel antigens for developing improved vaccines and serodiagnostic assays for brucellosis. In the present study, we performed a high throughput immunoprofiling of B. melitensis protein microarray using brucellosis-positive human and animal serum samples. The screening identified several serodominant proteins of Brucella that exhibited common or differential reactivity with sera from animals and humans. Subsequently, we cloned, expressed, and purified ten serodominant proteins, followed by analyzing their potential to develop next-generation vaccines and improved serodiagnostic assays for brucellosis. Further, we demonstrated the protective efficacy of one of the serodominant proteins against the B. melitensis challenge in mice. We found that the seroreactive protein, Dps (BMEI1980), strongly reacted with brucellosis-positive serum samples, but it did not react with sera from B. abortus S19-vaccinated cattle, indicating DIVA capability. A prototype lateral flow assay and indirect ELISA based on Dps protein exhibited high sensitivity, specificity, and DIVA capability. Thus, the present study identified promising candidates for developing improved vaccines and affordable, DIVA-capable serodiagnostic assays for animal and human brucellosis.

Outer membrane protein 25 of Brucella suppresses TLR-mediated expression of proinflammatory cytokines through degradation of TLRs and adaptor proteins.

Toll-like receptors (TLRs) are essential components of innate immunity that serves as the first line of defense against the invaded microorganisms. However, successful infectious pathogens subvert TLR signaling to suppress the activation of innate and adaptive responses. Brucella species are infectious intracellular bacterial pathogens causing the worldwide zoonotic disease, brucellosis, that impacts economic growth of many countries. Brucella species are considered as stealthy bacterial pathogens as they efficiently evade or suppress host innate and adaptive immune responses for their chronic persistence. However, the bacterial effectors and their host targets for modulating the immune responses remain obscure. Brucella encodes various outer membrane proteins (Omps) that facilitate their invasion, intracellular replication, and immunomodulation. Outer membrane protein 25 (Omp25) of Brucella plays an important role in the immune modulation through suppression of proinflammatory cytokines. However, the mechanism and the signaling pathways that are targeted by Omp25 to attenuate the production of proinflammatory cytokines remain obscure. Here, we report that Omp25 and its variants, viz. Omp25b, Omp25c, and Omp25d, suppress production of proinflammatory cytokines that are mediated by various TLRs. Furthermore, we demonstrate that Omp25 and its variants promote enhanced ubiquitination and degradation of TLRs and their adaptor proteins to attenuate the expression of proinflammatory cytokines. Targeting multiple TLRs and adaptor proteins enables Omp25 to effectively suppress the expression of proinflammatory cytokines that are induced by diverse pathogen-associated molecular patterns. This can contribute to the defective adaptive immune response and the chronic persistence of Brucella in the host.

The neurosteroid pregnenolone promotes degradation of key proteins in the innate immune signaling to suppress inflammation.

Pregnenolone is a steroid hormone precursor that is synthesized in various steroidogenic tissues, in the brain, and in lymphocytes. In addition to serving as the precursor for other steroid hormones, pregnenolone exerts its own effect as an anti-inflammatory molecule to maintain immune homeostasis in various inflammatory conditions. Pregnenolone and its metabolic derivatives have been shown to have beneficial effects in the brain, including enhancing memory and learning, reversing depressive disorders, and modulating cognitive functions. A decreased level of pregnenolone has been observed in neuroinflammatory diseases, which emphasizes its role in neuroprotection and neuroregeneration. Although the anti-inflammatory property of pregnenolone was recognized several decades ago, its mechanism of action remains unknown. Here we report that pregnenolone promotes ubiquitination and degradation of the TLR2/4 adaptor protein TIRAP and TLR2 in macrophages and microglial cells. Pregnenolone and its metabolites suppressed the secretion of tumor necrosis factor α and interleukin-6 mediated through TLR2 and TLR4 signaling. Pregnenolone has been reported to induce activation of cytoplasmic linker protein 170, and this protein has recently been shown to promote targeted degradation of TIRAP. We observed enhanced degradation of TIRAP and TLR4 suppression by cytoplasmic linker protein 170 in the presence of pregnenolone. Our experimental data reveal novel nongenomic targets of pregnenolone and provide important leads to understand its role in restoring immune homeostasis in various inflammatory conditions.

Cytoplasmic Linker Protein CLIP170 Negatively Regulates TLR4 Signaling by Targeting the TLR Adaptor Protein TIRAP.

Cytoplasmic linker protein 170 (CLIP170) is a CAP-Gly domain-containing protein that is associated with the plus end of growing microtubules and implicated in various cellular processes, including the regulation of microtubule dynamics, cell migration, and intracellular transport. Our studies revealed a previously unrecognized property and role of CLIP170. We identified CLIP170 as one of the interacting partners of Brucella effector protein TcpB that negatively regulates TLR2 and TLR4 signaling. In this study, we demonstrate that CLIP170 interacts with the TLR2 and TLR4 adaptor protein TIRAP. Furthermore, our studies revealed that CLIP170 induces ubiquitination and subsequent degradation of TIRAP to negatively regulate TLR4-mediated proinflammatory responses. Overexpression of CLIP170 in mouse macrophages suppressed the LPS-induced expression of IL-6 and TNF-α whereas silencing of endogenous CLIP170 potentiated the levels of proinflammatory cytokines. In vivo silencing of CLIP170 in C57BL/6 mice by CLIP170-specific small interfering RNA enhanced LPS-induced IL-6 and TNF-α expression. Furthermore, we found that LPS modulates the expression of CLIP170 in mouse macrophages. Overall, our experimental data suggest that CLIP170 serves as an intrinsic negative regulator of TLR4 signaling that targets TIRAP.

The Brucella effector protein TcpB induces degradation of inflammatory caspases and thereby subverts non-canonical inflammasome activation in macrophages.

The inflammasome contains intracellular receptors that recognize various pathogen-associated molecular patterns and play crucial roles in innate immune responses to invading pathogens. Non-canonical inflammasome activation is mediated by caspase-4/11, which recognizes intracellular LPS and promotes pyroptosis and secretion of proinflammatory cytokines. Brucella species are infectious intracellular pathogens that replicate in professional and non-professional phagocytic cells and subvert immune responses for chronic persistence in the host. The Brucella effector protein TcpB suppresses Toll-like receptor 2 (TLR2)- and TLR4-mediated innate immune responses by targeted degradation of the Toll/interleukin-1 receptor (TIR) domain-containing adaptor protein. TcpB is a cell-permeable protein with multiple functions, and its intracellular targets other than TIR domain-containing adaptor protein remain unclear. Here, we report that TcpB induces ubiquitination and degradation of the inflammatory caspases 1, 4, and 11. Furthermore, in both mouse and human macrophages, TcpB attenuated LPS-induced non-canonical inflammasome activation and suppressed pyroptosis and secretion of IL-1α and IL-1ß induced by intracellular LPS delivery. The intact TIR domain was essential for TcpB to subvert the non-canonical inflammasome activation as a TcpB(G158A) mutant failed to suppress pyroptotic cell death and inflammatory responses. Brucella-infected macrophages exhibited minimal pyroptosis but secreted IL-1ß, which was suppressed by TcpB. We also demonstrated that TcpB protein can efficiently attenuate Salmonella enterica serovar Typhimurium-induced pyroptosis and proinflammatory cytokine secretion in macrophages. Because TcpB suppresses both TLR4- and caspase-4/11-mediated inflammation, TcpB might be a candidate target for developing drugs against LPS-induced septicemia.

Recombinant haemagglutinin protein of highly pathogenic avian influenza A (H5N1) virus expressed in Pichia pastoris elicits a neutralizing antibody response in mice.

Recombinant avian influenza vaccines offer several advantages over the conventional vaccines. In this study, the haemagglutinin (HA) gene of highly pathogenic avian influenza H5N1 was cloned and expressed as His tagged protein in methylotropic yeast Pichia pastoris. The expression of recombinant HA (rHA) protein was confirmed by SDS-PAGE and western blot analysis. The rHA protein was purified using Ni-NTA affinity chromatography under denaturing conditions and the functions of the protein was assessed by the haemagglutinin assay after refolding. The immunogenicity of the rHA was evaluated by immunizing four groups of mice with different payloads (2.5, 5.0, 10 and 25µg) of purified rHA and the production of rHA specific antibodies were analysed by haemagglutinin inhibition assay (HI) and enzyme-linked immunosorbent assay (ELISA). An antigen specific immune response was observed against rHA indicating that the rHA antigen could be used as a vaccine candidate against avian influenza. These results suggest that this strategy would pave the way for the development of rapid and cost effective method for the production of an avian influenza vaccine.