Lipidation of pneumococcal proteins enables activation of human antigen-presenting cells and initiation of an adaptive immune response.

Streptococcus pneumoniae remains a significant global threat, with existing vaccines having important limitations such as restricted serotype coverage and high manufacturing costs. Pneumococcal lipoproteins are emerging as promising vaccine candidates due to their surface exposure and conservation across various serotypes. While prior studies have explored their potential in mice, data in a human context and insights into the impact of the lipid moiety remain limited. In the present study, we examined the immunogenicity of two pneumococcal lipoproteins, DacB and MetQ, both in lipidated and non-lipidated versions, by stimulation of primary human immune cells. Immune responses were assessed by the expression of common surface markers for activation and maturation as well as cytokines released into the supernatant. Our findings indicate that in the case of MetQ lipidation was crucial for activation of human antigen-presenting cells such as dendritic cells and macrophages, while non-lipidated DacB demonstrated an intrinsic potential to induce an innate immune response. Nevertheless, immune responses to both proteins were enhanced by lipidation. Interestingly, following stimulation of dendritic cells with DacB, LipDacB and LipMetQ, cytokine levels of IL-6 and IL-23 were significantly increased, which are implicated in triggering potentially important Th17 cell responses. Furthermore, LipDacB and LipMetQ were able to induce proliferation of CD4+ T cells indicating their potential to induce an adaptive immune response. These findings contribute valuable insights into the immunogenic properties of pneumococcal lipoproteins, emphasizing their potential role in vaccine development against pneumococcal infections.

Type VII secretion system extracellular protein B targets STING to evade host anti-Staphylococcus aureus immunity.

Staphylococcus aureus (S. aureus) can evade antibiotics and host immune defenses by persisting within infected cells. Here, we demonstrate that in infected host cells, S. aureus type VII secretion system (T7SS) extracellular protein B (EsxB) interacts with the stimulator of interferon genes (STING) protein and suppresses the inflammatory defense mechanism of macrophages during early infection. The binding of EsxB with STING disrupts the K48-linked ubiquitination of EsxB at lysine 33, thereby preventing EsxB degradation. Furthermore, EsxB-STING binding appears to interrupt the interaction of 2 vital regulatory proteins with STING: aspartate-histidine-histidine-cysteine domain-containing protein 3 (DHHC3) and TNF receptor-associated factor 6. This persistent dual suppression of STING interactions deregulates intracellular proinflammatory pathways in macrophages, inhibiting STING's palmitoylation at cysteine 91 and its K63-linked ubiquitination at lysine 83. These findings uncover an immune-evasion mechanism by S. aureus T7SS during intracellular macrophage infection, which has implications for developing effective immunomodulators to combat S. aureus infections.

Analysis of the components of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) and its regulation of γδ T-cell function.

BACKGROUND: Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown. METHODS: Mtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni-NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs. RESULTS: In this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αß T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb. CONCLUSION: Activated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.

Streptococcus pneumoniae endopeptidase O induces trained immunity and confers protection against various pathogenic infections.

Antibiotic resistance and the surge of infectious diseases during the pandemic present significant threats to human health. Trained immunity emerges as a promising and innovative approach to address these infections. Synthetic or natural fungal, parasitic and viral components have been reported to induce trained immunity. However, it is not clear whether bacterial virulence proteins can induce protective trained immunity. Our research demonstrates Streptococcus pneumoniae virulence protein PepO, is a highly potent trained immunity inducer for combating broad-spectrum infection. Our findings showcase that rPepO training confers robust protection to mice against various pathogenic infections by enhancing macrophage functionality. rPepO effectively re-programs macrophages, re-configures their epigenetic modifications and bolsters their immunological responses, which is independent of T or B lymphocytes. In vivo and in vitro experiments confirm that trained macrophage-secreted complement C3 activates peritoneal B lymphocyte and enhances its bactericidal capacity. In addition, we provide the first evidence that granulocyte colony-stimulating factor (G-CSF) derived from trained macrophages plays a pivotal role in shaping central-trained immunity. In summation, our research demonstrates the capability of rPepO to induce both peripheral and central trained immunity in mice, underscoring its potential application in broad-spectrum anti-infection therapy. Our research provides a new molecule and some new target options for infectious disease prevention.

Immune profiles of MCP-1 with M tb antigens and recombinant cytokines stimulation in tuberculosis.

Tuberculosis is caused by Mycobacterium tuberculosis (M tb), which is recognized by macrophages and produces inflammatory cytokines, and chemokines at the site of infection. The present study was proposed to understand the interaction of M tb antigens, cytokines, and chemokines. We have evaluated the chemokine MCP-1 levels and its expression in PBMCs stimulated with M tb antigens Ag85A, ESAT6 and recombinant cytokines rhTNF-α, rhIFN-γ, rhTGF-ß, and rhIL-10 in active pulmonary TB (APTB) patients, household contacts (HHC) at 0 months, 6 months and healthy controls (HC). We have observed low levels of MCP-1 with Ag85A, ESAT6, and rhTNF-α stimulations in APTB 0M compared to HHC and HC (p < 0.0067, p < 0.0001, p < 0.01, p < 0.005, p < 0.0065, p < 0.0001) and significantly increased after treatment with rhTNF-α. The MCP-1 levels with rhIFN-γ were high in APTB, HHC at 0 M and significant between APTB 0 M vs. 6 M, HHC vs. HC, and HHC 0M vs. 6M (p < 0.0352, p < 0.0252, p < 0.00062). The rhTGF-ß, rhIL-10 induced high MCP-1 levels in APTB, HHC compared to HC (p < 0.0414, p < 0.0312, p < 0.004, p < 0.0001) and significantly decreased after treatment with rhIL-10 (p < 0.0001). The MCP-1 expression was low with all the stimulations in APTB 0M when compared to HC and after treatment. Whereas, HHC shown low MCP-1 expression with rhTNF-α, rhIFN-γ and Ag85A and high with rhTGF-ß, rhIL-10 and ESAT6. In conclusion, the study determined the differential expression and production of MCP-1 with M tb antigens and recombinant cytokines. Further, cohort studies are required to study these interaction to identify the high risk individuals, which might help for TB control.

Evaluation of cytokine profiles related to Mycobacterium tuberculosis latent antigens using a whole-blood assay in the Philippines.

Introduction: There is no useful method to discriminate between latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (PTB). This study aimed to investigate the potential of cytokine profiles to discriminate between LTBI and active PTB using whole-blood stimulation with Mycobacterium tuberculosis (MTB) antigens, including latency-associated antigens. Materials and methods: Patients with active PTB, household contacts of active PTB patients and community exposure subjects were recruited in Manila, the Philippines. Peripheral blood was collected from the participants and used for whole-blood stimulation (WBS) with either the early secretory antigenic target and the 10-kDa culture filtrate protein (ESAT-6/CFP-10), Rv3879c or latency-associated MTB antigens, including mycobacterial DNA-binding protein 1 (MDP-1), α-crystallin (Acr) and heparin-binding hemagglutinin (HBHA). Multiple cytokine concentrations were analyzed using the Bio-Plex™ multiplex cytokine assay. Results: A total of 78 participants consisting of 15 active PTB patients, 48 household contacts and 15 community exposure subjects were eligible. The MDP-1-specific IFN-γ level in the active PTB group was significantly lower than that in the household contact group (p < 0.001) and the community exposure group (p < 0.001). The Acr-specific TNF-α and IL-10 levels in the active PTB group were significantly higher than those in the household contact (TNF-α; p = 0.001, IL-10; p = 0.001) and community exposure (TNF-α; p < 0.001, IL-10; p = 0.01) groups. However, there was no significant difference in the ESAT-6/CFP-10-specific IFN-γ levels among the groups. Conclusion: The patterns of cytokine profiles induced by latency-associated MTB antigens using WBS have the potential to discriminate between LTBI and active PTB. In particular, combinations of IFN-γ and MDP-1, TNF-α and Acr, and IL-10 and Acr are promising. This study provides the first demonstration of the utility of MDP-1-specific cytokine responses in WBS.

Structures and activation mechanism of the Gabija anti-phage system.

Prokaryotes have evolved intricate innate immune systems against phage infection1-7. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB8. GajA functions as a DNA endonuclease that is inactive in the presence of ATP9. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg2+. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.

Association of Helicobacter pylori CagA seropositivity with gastric precancerous lesions: a systematic review and meta-analysis.

The objective of this meta-analysis is to delineate the association between H. pylori CagA serological status and the prevalence of gastric precancerous lesions (GPL). We searched peer-reviewed articles up to October 2023. The extraction of data from the included studies was carried out as well as the quality assessment. Pooled effect sizes were calculated using a random effect model. Thirteen studies met the inclusion criteria, comprising 2728 patients with GPL and 17 612 controls. The aggregate odds ratio (OR) for the association between serum CagA and GPL was 2.74 (95% CI = 2.25-3.32; P  = 0.00; I 2  = 60.4%), irrespective of H. pylori infection status. Within the H. pylori -infected cohort, the OR was 2.25 (95% CI = 1.99-2.56; P  = 0.00; I 2  = 0.0%). Conversely, among the non-infected individuals, the OR was 1.63 (95% CI = 1.04-2.54; P  = 0.038; I 2  = 0.0%). Heterogeneity was explored using subgroup and meta-regression analyses, indicating that the variability between studies likely stemmed from differences in disease classification. Our results demonstrated robustness and negligible publication bias. The meta-analysis underscores a more pronounced association between H. pylori CagA seropositivity and the risk of developing GPL than between seronegativity and the same risk, irrespective of H. pylori infection status at the time. Additionally, the strength of the association was heightened in the presence of an active H. pylori infection. The implications of these findings advocate for the utility of CagA serostatus as a potential biomarker for screening GPL.

Microbial peptides activate tumour-infiltrating lymphocytes in glioblastoma.

Microbial organisms have key roles in numerous physiological processes in the human body and have recently been shown to modify the response to immune checkpoint inhibitors1,2. Here we aim to address the role of microbial organisms and their potential role in immune reactivity against glioblastoma. We demonstrate that HLA molecules of both glioblastoma tissues and tumour cell lines present bacteria-specific peptides. This finding prompted us to examine whether tumour-infiltrating lymphocytes (TILs) recognize tumour-derived bacterial peptides. Bacterial peptides eluted from HLA class II molecules are recognized by TILs, albeit very weakly. Using an unbiased antigen discovery approach to probe the specificity of a TIL CD4+ T cell clone, we show that it recognizes a broad spectrum of peptides from pathogenic bacteria, commensal gut microbiota and also glioblastoma-related tumour antigens. These peptides were also strongly stimulatory for bulk TILs and peripheral blood memory cells, which then respond to tumour-derived target peptides. Our data hint at how bacterial pathogens and bacterial gut microbiota can be involved in specific immune recognition of tumour antigens. The unbiased identification of microbial target antigens for TILs holds promise for future personalized tumour vaccination approaches.

New Rapid Helicobacter Pylori Blood Test Based on Dual Detection of FliD and CagA Antibodies for On-Site Testing.

Helicobacter pylori is the most prevalent bacterial infection, affecting half of the world's population, with a high morbidity and mortality rate.1,2 Several invasive and noninvasive testing procedures are available, and their selective use serves the specific needs of diverse clinical scenarios. For gastric cancer prevention, mass screening is necessary and requires a noninvasive, rapid, accurate and cost-effective test. For this purpose H pylori serology currently seems to be the preferred noninvasive diagnostic method. Population-based testing and treatment for H pylori is cost effective in high-risk countries, but less effective in low- and medium-risk countries.3,4 Many serologic tests are available on the market, with inconsistent performance often being observed. Therefore, international guidelines recommend considering only serologic tests with high accuracy that have been validated in the respective local populations. To date, no rapid point-of-care test (POCT) has reached a sufficient degree of accuracy.

Cryo-EM structure of an active bacterial TIR-STING filament complex.

Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes1-4. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide4-13, but the molecular basis of STING filament assembly and extension remains unknown. Here we use cryogenic electron microscopy to determine the structure of the active Toll/interleukin-1 receptor (TIR)-STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR-STING filament formation is driven by STING interfaces that become exposed on high-affinity recognition of the cognate cyclic dinucleotide signal c-di-GMP. Repeating dimeric STING units stack laterally head-to-head through surface interfaces, which are also essential for human STING tetramer formation and downstream immune signalling in mammals5. The active bacterial TIR-STING structure reveals further cross-filament contacts that brace the assembly and coordinate packing of the associated TIR NADase effector domains at the base of the filament to drive NAD+ hydrolysis. STING interface and cross-filament contacts are essential for cell growth arrest in vivo and reveal a stepwise mechanism of activation whereby STING filament assembly is required for subsequent effector activation. Our results define the structural basis of STING filament formation in prokaryotic antiviral signalling.

Mycobacterium tuberculosis PPE51 Inhibits Autophagy by Suppressing Toll-Like Receptor 2-Dependent Signaling.

Autophagy is an ubiquitous homeostatic pathway in mammalian cells and plays a significant role in host immunity. Substantial evidence indicates that the ability of Mycobacterium tuberculosis (Mtb) to successfully evade immune responses is partially due to inhibition of autophagic pathways. Our previous screening of Mtb transposon mutants identified the PPE51 protein as an important autophagy-inhibiting effector. We found that expression of PPE51, either by infecting bacteria or by direct expression in host cells, suppressed responses to potent autophagy-inducing stimuli and interfered with bacterial phagocytosis. This phenotype was associated with reduced activation of extracellular signal-regulated kinase 1/2 (ERK1/2), a key component of signaling pathways that stimulate autophagy. Multiple lines of evidence demonstrated that the effects of PPE51 are attributable to signal blocking by Toll-like receptor 2 (TLR2), a receptor with known involvement of activation of ERK1/2 and autophagy. Consistent with these results, mice with intact TLR2 signaling showed striking virulence attenuation for an Mtb ppe51 deletion mutant (Δ51) compared to wild-type Mtb, whereas infection of TLR2-deficient mice showed no such attenuation. Mice infected with Δ51 also displayed increased T cell responses to Mtb antigens and increased autophagy in infected lung tissues. Together, these results suggest that TLR2 activates relevant host immune functions during mycobacterial infection, which Mtb then evades through suppression of TLR2 signaling by PPE51. In addition to its previously identified function transporting substrates across the bacterial cell wall, our results demonstrate a direct role of PPE51 for evasion of both innate and adaptive immunity to Mtb. IMPORTANCE Tuberculosis is a significant global infectious disease caused by infection of the lungs with Mycobacterium tuberculosis, which resides and replicates mainly within host phagocytic cells. During coevolution with humans, Mtb has acquired various mechanisms to inhibit host cellular processes, including autophagy. Autophagy is a complex host cellular process that helps control intracellular infections by enhancing innate and adaptive immune responses. We identified the Mtb protein PPE51 as a mycobacterial effector that inhibits autophagy. We discovered TLR2 and mitogen-activated protein kinase signaling as the axis by which PPE51 mediates this effect. Autophagy regulation by PPE51, along with suppression of other TLR2-activated host cell functions, leads to increased bacterial survival in phagocytic cells and tissues of infected mice. A better understanding of how Mtb regulates autophagy and other host immune effectors could facilitate the design of new therapeutics or vaccines against tuberculosis.

Molecular Identification of Nocardia seriolae and Comparative Analysis of Spleen Transcriptomes of Hybrid Snakehead (Channa maculata Female × Channa argus Male) With Nocardiosis Disease.

Hybrid snakehead (Channa maculata female × Channa argus male) is a new freshwater aquaculture fish species in southern China. During intensive aquaculture, hybrid snakeheads are often infected by Nocardia seriolae. In this study, hybrid snakehead infected suspiciously by N. seriolae in an artificial breeding pond were examined. Diseased hybrid snakeheads swam slowly without food intake, and the clinical symptoms included skin wound, anal swelling and ascites, and white granulomatous in liver, spleen, and kidney of fish. Through bacterial isolation, 16S rDNA sequencing, fluorescence in situ hybridization (FISH) and artificial infection experiment, the pathogen was identified as N. seriolae. Furthermore, the spleen samples from diseased and healthy male hybrid snakeheads in the same pond were used for RNA-Seq analysis. A total of 3,512 unique transcripts (unigenes) were identified as differentially expressed genes (DEGs), and 1,886 of them were up-regulated in diseased fish. The expression patterns of 20 DEGs were verified by quantitative polymerase chain reaction (qPCR). Several immune-related pathways and many immune-related genes were identified. qPCR results showed that the expression patterns of immune-related genes in the liver and kidney of diseased fish were comparable to that in the spleen. This study provides deep-sequencing data of hybrid snakehead spleen and will help understand the immune response of hybrid snakehead to N. seriolae. It is also helpful for the biomarker screening of fish-borne Nocardia spp. and the breeding of nocardiosis-resistant fish species.

Peptide-Based Vaccines for Tuberculosis.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. As a result of the coronavirus disease 2019 (COVID-19) pandemic, the global TB mortality rate in 2020 is rising, making TB prevention and control more challenging. Vaccination has been considered the best approach to reduce the TB burden. Unfortunately, BCG, the only TB vaccine currently approved for use, offers some protection against childhood TB but is less effective in adults. Therefore, it is urgent to develop new TB vaccines that are more effective than BCG. Accumulating data indicated that peptides or epitopes play essential roles in bridging innate and adaptive immunity and triggering adaptive immunity. Furthermore, innovations in bioinformatics, immunoinformatics, synthetic technologies, new materials, and transgenic animal models have put wings on the research of peptide-based vaccines for TB. Hence, this review seeks to give an overview of current tools that can be used to design a peptide-based vaccine, the research status of peptide-based vaccines for TB, protein-based bacterial vaccine delivery systems, and animal models for the peptide-based vaccines. These explorations will provide approaches and strategies for developing safer and more effective peptide-based vaccines and contribute to achieving the WHO's End TB Strategy.

Macrophage-Mediated Melanoma Reduction after HP-NAP Treatment in a Zebrafish Xenograft Model.

The Helicobacter pylori Neutrophil Activating Protein (HP-NAP) is endowed with immunomodulatory properties that make it a potential candidate for anticancer therapeutic applications. By activating cytotoxic Th1 responses, HP-NAP inhibits the growth of bladder cancer and enhances the anti-tumor activity of oncolytic viruses in the treatment of metastatic breast cancer and neuroendocrine tumors. The possibility that HP-NAP exerts its anti-tumor effect also by modulating the activity of innate immune cells has not yet been explored. Taking advantage of the zebrafish model, we examined the therapeutic efficacy of HP-NAP against metastatic human melanoma, limiting the observational window to 9 days post-fertilization, well before the maturation of the adaptive immunity. Human melanoma cells were xenotransplanted into zebrafish embryos and tracked in the presence or absence of HP-NAP. The behavior and phenotype of macrophages and the impact of their drug-induced depletion were analyzed exploiting macrophage-expressed transgenes. HP-NAP administration efficiently inhibited tumor growth and metastasis and this was accompanied by strong recruitment of macrophages with a pro-inflammatory profile at the tumor site. The depletion of macrophages almost completely abrogated the ability of HP-NAP to counteract tumor growth. Our findings highlight the pivotal role of activated macrophages in counteracting melanoma growth and support the notion that HP-NAP might become a new biological therapeutic agent for the treatment of metastatic melanomas.

The Mycobacterium tuberculosis PE_PGRS Protein Family Acts as an Immunological Decoy to Subvert Host Immune Response.

Mycobacterium tuberculosis (M.tb) is a successful pathogen that can reside within the alveolar macrophages of the host and can survive in a latent stage. The pathogen has evolved and developed multiple strategies to resist the host immune responses. M.tb escapes from host macrophage through evasion or subversion of immune effector functions. M.tb genome codes for PE/PPE/PE_PGRS proteins, which are intrinsically disordered, redundant and antigenic in nature. These proteins perform multiple functions that intensify the virulence competence of M.tb majorly by modulating immune responses, thereby affecting immune mediated clearance of the pathogen. The highly repetitive, redundant and antigenic nature of PE/PPE/PE_PGRS proteins provide a critical edge over other M.tb proteins in terms of imparting a higher level of virulence and also as a decoy molecule that masks the effect of effector molecules, thereby modulating immuno-surveillance. An understanding of how these proteins subvert the host immunological machinery may add to the current knowledge about M.tb virulence and pathogenesis. This can help in redirecting our strategies for tackling M.tb infections.

Identification of Leptospiral Protein Antigens Recognized by WC1+ γδ T Cell Subsets as Target for Development of Recombinant Vaccines.

Pathogenic Leptospira species cause leptospirosis, a neglected zoonotic disease recognized as a global public health problem. It is also the cause of the most common cattle infection that results in major economic losses due to reproductive problems. γδ T cells play a role in the protective immune response in livestock species against Leptospira, while human γδ T cells also respond to Leptospira. Thus, activation of γδ T cells has emerged as a potential component in the optimization of vaccine strategies. Bovine γδ T cells proliferate and produce gamma interferon (IFN-γ) in response to vaccination with inactivated leptospires, and this response is mediated by a specific subpopulation of the WC1-bearing γδ T cells. WC1 molecules are members of the group B scavenger receptor cysteine-rich (SRCR) superfamily and are composed of multiple SRCR domains, of which particular extracellular domains act as ligands for Leptospira. Since WC1 molecules function as both pattern recognition receptors and γδ TCR coreceptors, the WC1 system has been proposed as a novel target to engage γδ T cells. Here, we demonstrate the involvement of leptospiral protein antigens in the activation of WC1+ γδ T cells and identify two leptospiral outer membrane proteins able to interact directly with them. Interestingly, we show that the protein-specific γδ T cell response is composed of WC1.1+ and WC1.2+ subsets, although a greater number of WC1.1+ γδ T cells respond. Identification of protein antigens will enhance our understanding of the role γδ T cells play in the leptospiral immune response and in recombinant vaccine development.

Enhanced High Mutation Rate and Natural Selection to Produce Attenuated Viral Vaccine with CRISPR Toolkit in RNA Viruses especially SARS-CoV-2.

The best and most effective way to combat pandemics is to use effective vaccines and live attenuated vaccines are among the most effective vaccines. However, one of the major problems is the length of time it takes to get the attenuated vaccines. Today, the CRISPR toolkit (Clustered Regularly Inerspaced Short Palindromic Repeats) has made it possible to make changes with high efficiency and speed. Using this toolkit to make point mutations on the RNA virus's genome in a coculture of permissive and nonpermissive cells and under controlled conditions can accelerate changes in the genome and accelerate natural selection to obtain live attenuated vaccines.

EASINESS: E. coli Assisted Speedy affINity-maturation Evolution SyStem.

Antibodies are one of the most important groups of biomolecules for both clinical and basic research and have been developed as potential therapeutics. Affinity is the key feature for biological activity and clinical efficacy of an antibody, especially of therapeutic antibodies, and thus antibody affinity improvement is indispensable and still remains challenging. To address this issue, we developed the E. coli Assisted Speed affINity-maturation Evolution SyStem (EASINESS) for continuous directed evolution of Ag-Ab interactions. Two key components of EASINESS include a mutation system modified from error-prone DNA polymerase I (Pol I) that selectively mutates ColE1 plasmids in E. coli and a protein-protein interaction selection system from mDHFR split fragments. We designed a GCN4 variant which barely forms a homodimer, and during a single round of evolution, we reversed the homodimer formation activity from the GCN4 variant to verify the feasibility of EASINESS. We then selected a potential therapeutic antibody 18A4Hu and improved the affinity of the antibody (18A4Hu) to its target (ARG2) 12-fold in 7 days while requiring very limited hands-on time. Remarkably, these variants of 18A4Hu revealed a significant improved ability to inhibit melanoma pulmonary metastasis in a mouse model. These results indicate EASINESS could be as an attractive choice for antibody affinity maturation.

Neutrophil-Mediated Lung Injury Both via TLR2-Dependent Production of IL-1α and IL-12 p40, and TLR2-Independent CARDS Toxin after Mycoplasma pneumoniae Infection in Mice.

Mycoplasma pneumoniae (Mp) residing extracellularly in the respiratory tract is the primary cause of bacterial community-acquired pneumonia in humans. However, the detailed pathological mechanism of Mp infection, especially inflammation in the lung, remains unclear. This study examined the role of the neutrophils in the inflammation of Mp-induced pneumonia in mice and the mechanism of neutrophil infiltration into the lungs in the Mp-induced pneumonia. We observed massive infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) and lung injury after the Mp challenge. The neutrophils were shown to contribute to lung injury in Mp pneumonia but were not involved in eliminating Mp, suggesting that neutrophils are detrimental to the host in Mp pneumonia. Mp also induced the production of inflammatory cytokines and chemokines in the BALF in a toll-like receptor 2 (TLR2)-dependent manner. Particularly, both interleukin (IL)-1α and IL-12 p40 played a crucial role in neutrophil infiltration into the BALF in a coordinated manner. Both IL-1α and IL-12 p40 were released from the alveolar macrophages depending on the TLR2 and reactive oxygen species. In addition, the community-acquired respiratory distress syndrome (CARDS) toxin from Mp were found to induce neutrophil infiltration into BALF in a TLR2-independent and IL-1α-dependent manner. Collectively, the TLR2-dependent production of both IL-1α and IL-12 p40, and CARDS toxin have been elucidated to play an important role in neutrophil infiltration into the lungs subsequently leading to the lung injury upon Mp infection in mice. These data will aid in the development of therapeutics and vaccines for Mp pneumonia. IMPORTANCE Although Mp-induced pneumonia is usually a self-limiting disease, refractory life-threatening pneumonia is often induced. In addition, the development of alternative therapeutic strategies for Mp is expected because of the emergence of antibiotic-resistant Mp. However, the lack of knowledge regarding the pathogenesis of Mp-induced pneumonia, especially inflammation upon the Mp infection, makes it tedious to design novel therapeutics and vaccines. For example, although neutrophil infiltration is widely recognized as one of the characteristics of Mp-induced pneumonia, the precise role of neutrophils in the aggravation of Mp pneumonia remains unclear. This study showed that the infiltration of neutrophils in the lungs is detrimental to the host in Mp-induced pneumonia in mice. Furthermore, the TLR2-dependent IL-1α and IL-12 p40 production, and CARDS toxin play important roles in neutrophil infiltration into the lung, following lung injury. Our findings apply to the rational design of novel therapeutics and vaccines against Mp.