Current Understanding of Bacillus Calmette-Guérin-Mediated Trained Immunity and Its Perspectives for Controlling Intracellular Infections.

The bacillus Calmette-Guérin (BCG) is an attenuated bacterium derived from virulent Mycobacterium bovis. It is the only licensed vaccine used for preventing severe forms of tuberculosis in children. Besides its specific effects against tuberculosis, BCG administration is also associated with beneficial non-specific effects (NSEs) following heterologous stimuli in humans and mice. The NSEs from BCG could be related to both adaptive and innate immune responses. The latter is also known as trained immunity (TI), a recently described biological feature of innate cells that enables functional improvement based on metabolic and epigenetic reprogramming. Currently, the mechanisms related to BCG-mediated TI are the focus of intense research, but many gaps are still in need of elucidation. This review discusses the present understanding of TI induced by BCG, exploring signaling pathways that are crucial to a trained phenotype in hematopoietic stem cells and monocytes/macrophages lineage. It focuses on BCG-mediated TI mechanisms, including the metabolic-epigenetic axis and the inflammasome pathway in these cells against intracellular pathogens. Moreover, this study explores the TI in different immune cell types, its ability to protect against various intracellular infections, and the integration of trained innate memory with adaptive memory to shape next-generation vaccines.

Recombinant Bacillus Calmette-Guérin Expressing SARS-CoV-2 Chimeric Protein Protects K18-hACE2 Mice against Viral Challenge.

COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette-Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.

Neutrophils and schistosomiasis: a missing piece in pathology.

Schistosomiasis is a chronic human parasitic disease that causes serious health problems worldwide. The disease-associated liver pathology is one of the hallmarks of infections by Schistosoma mansoni and Schistosoma japonicum, and is accountable for the debilitating condition found in infected patients. In the past few years, investigative studies have highlighted the key role played by neutrophils and the influence of inflammasome signalling pathway in different pathological conditions. However, it is noteworthy that the study of inflammasome activation in neutrophils has been overlooked by reports concerning macrophages and monocytes. This interplay between neutrophils and inflammasomes is much more poorly investigated during schistosomiasis. Herein, we reviewed the role of neutrophils during schistosomiasis and addressed the potential connection between these cells and inflammasome activation in this context.

S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity.

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.

The role of the adaptor molecule STING during Schistosoma mansoni infection.

Schistosomiasis is a human parasitic disease responsible for serious consequences for public health, as well as severe socioeconomic impacts in developing countries. Here, we provide evidence that the adaptor molecule STING plays an important role in Schistosoma mansoni infection. S. mansoni DNA is sensed by cGAS leading to STING activation in murine embryonic fibroblasts (MEFs). Sting-/- and C57BL/6 (WT) mice were infected with schistosome cercariae in order to assess parasite burden and liver pathology. Sting-/- mice showed worm burden reduction but no change in the number of eggs or granuloma numbers and area when compared to WT animals. Immunologically, a significant increase in IFN-γ production by the spleen cells was observed in Sting-/- animals. Surprisingly, Sting-/- mice presented an elevated percentage of neutrophils in lungs, bronchoalveolar lavage, and spleens. Moreover, Sting-/- neutrophils exhibited increased survival rate, but similar ability to kill schistosomula in vitro when stimulated with IFN-γ when compared to WT cells. Finally, microbiota composition was altered in Sting-/- mice, revealing a more inflammatory profile when compared to WT animals. In conclusion, this study demonstrates that STING signaling pathway is important for S. mansoni DNA sensing and the lack of this adaptor molecule leads to enhanced resistance to infection.

NLRP6 Plays an Important Role in Early Hepatic Immunopathology Caused by Schistosoma mansoni Infection.

Schistosomiasis is a debilitating parasitic disease that affects more than 200 million people worldwide and causes approximately 280,000 deaths per year. Inside the definitive host, eggs released by Schistosoma mansoni lodge in the intestine and especially in the liver where they induce a granulomatous inflammatory process, which can lead to fibrosis. The molecular mechanisms initiating or promoting hepatic granuloma formation remain poorly understood. Inflammasome activation has been described as an important pathway to induce pathology mediated by NLRP3 receptor. Recently, other components of the inflammasome pathway, such as NLRP6, have been related to liver diseases and fibrotic processes. Nevertheless, the contribution of these components in schistosomiasis-associated pathology is still unknown. In the present study, using dendritic cells, we demonstrated that NLRP6 sensor is important for IL-1ß production and caspase-1 activation in response to soluble egg antigens (SEA). Furthermore, the lack of NLRP6 has been shown to significantly reduce periovular inflammation, collagen deposition in hepatic granulomas and mRNA levels of α-SMA and IL-13. Livers of Nlrp6-/- mice showed reduced levels of CXCL1/KC, CCL2, CCL3, IL-5, and IL-10 as well as Myeloperoxidase (MPO) and Eosinophilic Peroxidase (EPO) enzymatic activity. Consistently, the frequency of macrophage and neutrophil populations were lower in the liver of NLRP6 knockout mice, after 6 weeks of infection. Finally, it was further demonstrated that the onset of hepatic granuloma and collagen deposition were also compromised in Caspase-1-/- , IL-1R-/- and Gsdmd-/- mice. Our findings suggest that the NLRP6 inflammasome is an important component for schistosomiasis-associated pathology.

Schistosoma mansoni SmKI-1 or Its C-Terminal Fragment Induces Partial Protection Against S. mansoni Infection in Mice.

Current schistosomiasis control strategies are mainly based on chemotherapy, but the development of a vaccine against this parasitic disease would contribute to a long-lasting decrease in disease spectrum and transmission. When it comes to vaccine candidates, several genes encoding Schistosoma mansoni proteins expressed at the mammalian host-parasite interface have been tested. Among the most promising molecules are the proteins present on the tegument and digestive tract of the parasite. In this study, we evaluate the potential of SmKI-1, the first Kunitz-type protease inhibitor functionally characterized in S. mansoni, as a vaccine candidate. Bioinformatic analysis points to the C-terminal fragment as the main region of the molecule responsible for the development of a potential protective immune response induced by SmKI-1. Therefore, for the vaccine formulations, we produced the recombinant (r) SmKI-1 and two different fragments, its Kunitz (KI) domain and its C-terminal tail. First, we demonstrate that mice immunized with recombinant SmKI-1 (rSmKI-1) or its fragments, formulated with Freund's adjuvant, induced the production of IgG-specific antibodies. Further, all vaccine formulations tested here also induced a Th1-type of immune response, as suggested by the production of IFN-γ and TNF-α by protein-stimulated cultured splenocytes. However, the protective effect conferred by vaccination was only observed in groups which received rSmKI-1 or C-terminal domain vaccines. Mice administered with rSmKI-1 demonstrated reduction of 47% in worm burden, 36% in egg number in mouse livers, and 33% in area of liver granulomas. Additionally, mice injected with C-terminal domain showed reduction of 28% in worm burden, 38% in egg number in liver, and 25% in area of liver granulomas. In contrast, KI domain immunization was unable to reduce worm burden and ameliorate liver pathology after challenge infection. Taken together, our data demonstrated that SmKI-1 is a potential candidate for use in a vaccine to control schistosomiasis, and its C-terminal tail seems to be the main region of the molecule responsible for protection conferred by this antigen.

The use of gold nanorods as a new vaccine platform against schistosomiasis.

Schistosomiasis is an important parasitic disease affecting >207 million people in 76 countries around the world and causing approximately 250,000 deaths per year. At present, the main strategy adopted for the control of schistosomiasis is the use of safe chemotherapy, such as praziquantel. However, the high rates of reinfection after treatment restrict the use of this treatment approach and assume the need for other forms of control such as vaccination. Sm29 is a protein that is localized in the Schistosoma mansoni tegument of adult worms and schistosomula and is considered a powerful vaccine candidate. Because of the chemical, physical and immunological characteristics of nanoparticles, nanocarriers have received increasing attention. In the field of nanotechnology, gold nanorods are considered potential vaccine carriers. In this study, we bound S. mansoni rSm29 protein to gold nanorods either directly or by cysteamine functionalization. When the worm burden was evaluated, the AuNRs-NH2-rSm29 group of immunized mice showed the best protection level (34%). Following AuNRs-NH2-rSm29 immunization, we observed a Th1 immunological response in mice with higher production of IFN-γ, mainly by CD4+ and CD8+ T cells. Furthermore, AuNRs-NH2-rSm29 could activate dendritic cells in vitro, enhancing MHCII and MHCI expression and the production of IL-1ß in a NLRP3-, ASC- and Caspase-1-dependent manner. In summary, our findings support the use of nanorods as an immunization strategy in vaccine development against infectious diseases.

Serine protease inhibitors containing a Kunitz domain: their role in modulation of host inflammatory responses and parasite survival.

Proteins containing a Kunitz domain have the typical serine protease inhibition function ranging from sea anemone to man. Protease inhibitors play major roles in infection, inflammation disorders and cancer. This review discusses the role of serine proteases containing a Kunitz domain in immunomodulation induced by helminth parasites. Helminth parasites are associated with protection from inflammatory conditions. Therefore, interest has raised whether worm parasites or their products hold potential as drugs for treatment of immunological disorders. Finally, we also propose the use of recombinant SmKI-1 from Schistosoma mansoni as a potential therapeutic molecule to treat inflammatory diseases.