Co-formulation of the rF1V plague vaccine with depot-formulated cytokines enhances immunogenicity and efficacy to elicit protective responses against aerosol challenge in mice.

This study evaluated a depot-formulated cytokine-based adjuvant to improve the efficacy of the recombinant F1V (rF1V) plague vaccine and examined the protective response following aerosol challenge in a murine model. The results of this study showed that co-formulation of the Alhydrogel-adsorbed rF1V plague fusion vaccine with the depot-formulated cytokines recombinant human interleukin 2 (rhuIL-2) and/or recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) significantly enhances immunogenicity and significant protection at lower antigen doses against a lethal aerosol challenge. These results provide additional support for the co-application of the depot-formulated IL-2 and/or GM-CSF cytokines to enhance vaccine efficacy.

SPR and Double Resonance LPG Biosensors for Helicobacter pylori BabA Antigen Detection.

Given the medical and social significance of Helicobacter pylori infection, timely and reliable diagnosis of the disease is required. The traditional invasive and non-invasive conventional diagnostic techniques have several limitations. Recently, opportunities for new diagnostic methods have appeared based on the recent advance in the study of H. pylori outer membrane proteins and their identified receptors. In the present study we assess the way in which outer membrane protein-cell receptor reactions are applicable in establishing a reliable diagnosis. Herein, as well as in other previous studies of ours, we explore the reliability of the binding reaction between the best characterized H. pylori adhesin BabA and its receptor, the blood antigen Leb. For the purpose we developed surface plasmon resonance (SPR) and double resonance long period grating (DR LPG) biosensors based on the BabA-Leb binding reaction for diagnosing H. pylori infection. In SPR detection, the sensitivity was estimated at 3000 CFU/mL-a much higher sensitivity than that of the RUT test. The DR LPG biosensor proved to be superior in terms of accuracy and sensitivity-concentrations as low as 102 CFU/mL were detected.

Diagnosing adult and pediatric extrapulmonary tuberculosis by MPT64 antigen detection with immunohistochemistry and immunocytochemistry using reproduced polyclonal antibodies.

Diagnosing extrapulmonary tuberculosis (EPTB) is challenging. Immunohistochemistry or immunocytochemistry has been used to diagnose tuberculosis (TB) by detection of MPT64 antigen from various extrapulmonary specimens and has shown good diagnostic performance in our previous studies. The test can distinguish between disease caused by Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria and can be applied on formalin-fixed paraffin-embedded tissue. As the antibodies previously used were in limited supply, a new batch of polyclonal antibodies was developed for scale-up and evaluated for the first time in this study. Our aim was to assess the diagnostic accuracy of the MPT64 test with reproduced antibodies in the high burden settings of Pakistan and India. Patients were enrolled prospectively. Samples from suspected sites of infection were collected and subjected to histopathologic and/or cytologic evaluation, routine TB diagnostics, GeneXpert MTB/RIF (Xpert), and the MPT64 antigen detection test. Patients were followed until the end of treatment. Based on a composite reference standard (CRS), 556 patients were categorized as TB cases and 175 as non-TB cases. The MPT64 test performed well on biopsies with a sensitivity and specificity of 94% and 75%, respectively, against a CRS. For cytology samples, the sensitivity was low (36%), whereas the specificity was 81%. Overall, the MPT64 test showed higher sensitivity (73%) than Xpert (38%) and Mtb culture (33%). The test performed equally well in adults and children. We found an additive diagnostic value of the MPT64 test in conjunction with histology and molecular tests, increasing the yield for EPTB. In conclusion, immunochemical staining with MPT64 antibodies improves the diagnosis of EPTB in high burden settings and could be a valuable addition to routine diagnostics.

Production and Evaluation of Ag85B:HspX:hFcγ1 Immunogenicity as an Fc Fusion Recombinant Multi-Stage Vaccine Candidate Against Mycobacterium tuberculosis.

An urgent need is to introduce an effective vaccine against Mycobacterium tuberculosis (M.tb) infection. In the present study, a multi-stage M.tb immunodominant Fcγ1 fusion protein (Ag85B:HspX:hFcγ1) was designed and produced, and the immunogenicity of purified protein was evaluated. This recombinant fusion protein was produced in the Pichia pastoris expression system. The HiTrap-rPA column affinity chromatography purified and confirmed the fusion protein using ELISA and Western blotting methods. The co-localisation assay was used to confirm its proper folding and function. IFN-γ, IL-12, IL-4, and TGF-ß expression in C57BL/6 mice then evaluated the immunogenicity of the construct in the presence and absence of BCG. After expression optimisation, medium-scale production and the Western blotting test confirmed suitable production of Ag85B:HspX:hFcγ1. The co-localisation results on antigen-presenting cells (APCs) showed that Ag85B:HspX:hFcγ1 properly folded and bound to hFcγRI. This strong co-localisation with its receptor can confirm inducing proper Th1 responses. The in vivo immunisation assay showed no difference in the expression of IL-4 but a substantial increase in the expression of IFN-γ and IL-12 (P ≤ 0.02) and a moderate increase in TGF-ß (P = 0.05). In vivo immunisation assay revealed that Th1-inducing pathways have been stimulated, as IFN-γ and IL-12 strongly, and TGF-ß expression moderately increased in Ag85B:HspX:hFcγ1 group and Ag85B:HspX:hFcγ1+BCG. Furthermore, the production of IFN-γ from splenocytes in the Ag85B:HspX:hFcγ1 group was enormously higher than in other treatments. Therefore, this Fc fusion protein can make a selective multi-stage delivery system for inducing appropriate Th1 responses and is used as a subunit vaccine alone or in combination with others.

Effect of concurrent infection of Helicobacter pylori with Toxoplasma gondii infection on gastric pathology.

BACKGROUND: Toxoplasma gondii (T. gondii) and Helicobacter pylori (H. pylori) are among the most prevalent foodborne parasitic and bacterial infections worldwide. However, the concurrent impact of coinfection on gastric pathology has yet to be studied in depth. The effect of coinfection generally either adds a synergetic or antagonistic impact; we aimed in the current work to assess the impact of T. gondii coinfection on the progression of H. pylori-associated gastric pathology and reporting H. pylori virulent strains. The study was conducted on 82 patients complaining of persistent gastrointestinal symptoms with failed treatment response and prone to endoscopy. They were subjected to stool examination to detect H. pylori antigen, serological screening for latent toxoplasmosis, endoscopy, histopathological examination, and molecular detection of H. pylori virulence strains in gastric biopsies. Out of the 82 patients, 62 patients were positive for H. pylori antigen in stool and 55 patients confirmed positivity by histopathology; out of them, 37 patients had isolated Vac As1 variants, 11 patients had combined Vac As1 and Cag A variants, and 7 patients had combined Vac As1, Cag A and VacAs2 variants. Patients with the combined two or three variances showed significantly deteriorated histopathological features than patients with a single Vac As1 variant (P < 0.05). Latent toxoplasmosis was positive among 35/82 patients. Combined H. pylori and Toxoplasma gondii infection had significantly marked inflammation than patients with isolated infection (P < 0.05). CONCLUSION: Screening for toxoplasmosis among H. pylori-infected patients is recommended as it is considered a potential risk factor for gastric inflammation severity. H. pylori gastric inflammation may be heightened by Toxoplasma coinfection.

The role of ESAT-6 in tuberculosis immunopathology.

Despite major global efforts to eliminate tuberculosis, which is caused by Mycobacterium tuberculosis (Mtb), this disease remains as a major plague of humanity. Several factors associated with the host and Mtb interaction favor the infection establishment and/or determine disease progression. The Early Secreted Antigenic Target 6 kDa (ESAT-6) is one of the most important and well-studied mycobacterial virulence factors. This molecule has been described to play an important role in the development of tuberculosis-associated pathology by subverting crucial components of the host immune responses. This review highlights the main effector mechanisms by which ESAT-6 modulates the immune system, directly impacting cell fate and disease progression.

Antagonizing roles of SHP1 in the pathogenesis of Helicobacter pylori infection.

BACKGROUND: SHP1 has been documented as a tumor suppressor and it was thought to play an antagonistic role in the pathogenesis of Helicobacter pylori infection. In this study, the exact mechanism of this antagonistic action was studied. MATERIALS AND METHODS: AGS, MGC803, and GES-1 cells were infected with H. pylori, intracellular distribution changes of SHP1 were first detected by immunofluorescence. SHP1 overexpression and knockdown were then constructed in these cells to investigate its antagonistic roles in H. pylori infection. Migration and invasion of infected cells were detected by transwell assay, secretion of IL-8 was examined via ELISA, the cells with hummingbird-like alteration were determined by microexamination, and activation of JAK2/STAT3, PI3K/Akt, and ERK pathways were detected by immunoblotting. Mice infection model was established and gastric pathological changes were evaluated. Finally, the SHP1 activator sorafenib was used to analyze the attenuating effect of SHP1 activation on H. pylori pathogenesis in vitro and in vivo. RESULTS: The sub-localization of SHP1 changed after H. pylori infection, specifically that the majority of the cytoplasmic SHP1 was transferred to the cell membrane. SHP1 inhibited H. pylori-induced activation of JAK2/STAT3 pathway, PI3K/Akt pathway, nuclear translocation of NF-κB, and then reduced EMT, migration, invasion, and IL-8 secretion. In addition, SHP1 inhibited the formation of CagA-SHP2 complex by dephosphorylating phosphorylated CagA, reduced ERK phosphorylation and the formation of CagA-dependent hummingbird-like cells. In the mice infection model, gastric pathological changes were observed and increased IL-8 secretion, indicators of cell proliferation and EMT progression were also detected. By activating SHP1 with sorafenib, a significant curative effect against H. pylori infection was obtained in vitro and in vivo. CONCLUSIONS: SHP1 plays an antagonistic role in H. pylori pathogenesis by inhibiting JAK2/STAT3 and PI3K/Akt pathways, NF-κB nuclear translocation, and CagA phosphorylation, thereby reducing cell EMT, migration, invasion, IL-8 secretion, and hummingbird-like changes.

Dual RNA sequencing of Helicobacter pylori and host cell transcriptomes reveals ontologically distinct host-pathogen interaction.

Helicobacter pylori is a highly successful pathogen that poses a substantial threat to human health. However, the dynamic interaction between H. pylori and the human gastric epithelium has not been fully investigated. In this study, using dual RNA sequencing technology, we characterized a cytotoxin-associated gene A (cagA)-modulated bacterial adaption strategy by enhancing the expression of ATP-binding cassette transporter-related genes, metQ and HP_0888, upon coculturing with human gastric epithelial cells. We observed a general repression of electron transport-associated genes by cagA, leading to the activation of oxidative phosphorylation. Temporal profiling of host mRNA signatures revealed the downregulation of multiple splicing regulators due to bacterial infection, resulting in aberrant pre-mRNA splicing of functional genes involved in the cell cycle process in response to H. pylori infection. Moreover, we demonstrated a protective effect of gastric H. pylori colonization against chronic dextran sulfate sodium (DSS)-induced colitis. Mechanistically, we identified a cluster of propionic and butyric acid-producing bacteria, Muribaculaceae, selectively enriched in the colons of H. pylori-pre-colonized mice, which may contribute to the restoration of intestinal barrier function damaged by DSS treatment. Collectively, this study presents the first dual-transcriptome analysis of H. pylori during its dynamic interaction with gastric epithelial cells and provides new insights into strategies through which H. pylori promotes infection and pathogenesis in the human gastric epithelium. IMPORTANCE: Simultaneous profiling of the dynamic interaction between Helicobacter pylori and the human gastric epithelium represents a novel strategy for identifying regulatory responses that drive pathogenesis. This study presents the first dual-transcriptome analysis of H. pylori when cocultured with gastric epithelial cells, revealing a bacterial adaptation strategy and a general repression of electron transportation-associated genes, both of which were modulated by cytotoxin-associated gene A (cagA). Temporal profiling of host mRNA signatures dissected the aberrant pre-mRNA splicing of functional genes involved in the cell cycle process in response to H. pylori infection. We demonstrated a protective effect of gastric H. pylori colonization against chronic DSS-induced colitis through both in vitro and in vivo experiments. These findings significantly enhance our understanding of how H. pylori promotes infection and pathogenesis in the human gastric epithelium and provide evidence to identify targets for antimicrobial therapies.

Group A streptococcal collagen-like protein 1 restricts tumor growth in murine pancreatic adenocarcinoma and inhibits cancer-promoting neutrophil extracellular traps.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer associated with an immunosuppressive environment. Neutrophil extracellular traps (NETs) were initially described in the context of infection but have more recently been implicated in contributing to the tolerogenic immune response in PDAC. Thus, NETs are an attractive target for new therapeutic strategies. Group A Streptococcus (GAS) has developed defensive strategies to inhibit NETs. Methods: In the present work, we propose utilizing intra-tumoral GAS injection to stimulate anti-tumor activity by inhibiting cancer-promoting NETs. Mice harboring Panc02 or KPC subcutaneous tumors injected with three different M-type GAS strains. Tumors and spleens were harvested at the endpoint of the experiments to assess bacterial colonization and systemic spread, while sera were analyzed for humoral responses toward the streptococcal antigens, especially the M1 and Scl1 proteins. Role of the streptococcal collagen-like protein 1 (Scl1) in anti-PDAC activity was assessed in vivo after intratumoral injection with M1 GAS wild-type, an isogenic mutant strain devoid of Scl1, or a complemented mutant strain with restored scl1 expression. In addition, recombinant Scl1 proteins were tested for NET inhibition using in vitro and ex vivo assays assessing NET production and myeloperoxidase activity. Results: Injection of three different M-type GAS strains reduced subcutaneous pancreatic tumor volume compared to control in two different murine PDAC models. Limitation of tumor growth was dependent on Scl1, as isogenic mutant strain devoid of Scl1 did not reduce tumor size. We further show that Scl1 plays a role in localizing GAS to the tumor site, thereby limiting the systemic spread of bacteria and off-target effects. While mice did elicit a humoral immune response to GAS antigens, tested sera were weakly immunogenic toward Scl1 antigen following intra-tumoral treatment with Scl1-expressing GAS. M1 GAS inhibited NET formation when co-cultured with neutrophils while Scl1-devoid mutant strain did not. Recombinant Scl1 protein inhibited NETs ex vivo in a dose-dependent manner by suppressing myeloperoxidase activity. Discussion: Altogether, we demonstrate that intra-tumoral GAS injections reduce PDAC growth, which is facilitated by Scl1, in part through inhibition of cancer promoting NETs. This work offers a novel strategy by which NETs can be targeted through Scl1 protein and potentiates its use as a cancer therapeutic.

Dachshund Homolog 1: Unveiling Its Potential Role in Megakaryopoiesis and Bacillus anthracis Lethal Toxin-Induced Thrombocytopenia.

Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34-megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis.

Diagnostic value of IgG antibody and stool antigen tests for chronic Helicobacter pylori infections in Ibb Governorate, Yemen.

The stool antigen test (SAT) and the serum Helicobacter pylori (H. pylori) IgG antibody assays exhibit significant utility in the clinical diagnosis of H. pylori infection and in distinguishing between acute and chronic infections. The main objective of the current study was to identify the diagnostic value of serum H. pylori IgG antibody and SAT in the detection of H. pylori infections among chronic H. pylori-infected patients residing in Ibb Governorate, Yemen. 200 patients with H. pylori infection, confirmed through positive results in the serum immunochromatographic antibody test, were selected for H. pylori infection confirmation using serum H. pylori IgG antibodies and SAT across diverse hospitals, gastroenterology, and Hepatology clinics in Ibb Governorate. After the selection of patients, blood and stool specimens were obtained from all participants and underwent analysis via the Statistical Package for the Social Sciences (SPSS). The prevalence of H. pylori infection demonstrated variability based on the confirmatory tests, with rates of 54% for SAT and 78.5% for serum H. pylori IgG antibody, contrasting with a 100% prevalence observed in the screening serum immunochromatographic antibody test. Clinically, the study categorized H. pylori infections into four stages, whereby a significant proportion of patients (40.5%) exhibited positivity for both serum H. pylori IgG antibody and SAT, indicative of active chronic infections. The majority of positive cases only manifested serum H. pylori IgG antibody presence (chronic infections) at 38%, whereas 13.5% exclusively tested positive for SAT, corresponding to acute infections. Moreover, 88% of patients did not have either serum H. pylori IgG antibody or SAT (absence of infections) during confirmatory tests. Noteworthy is the study's approach employing multiple tests for H. pylori infection detection, focusing predominantly on chronic infections-prevailing types caused by H. pylori. The results revealed a significant association between serum levels of H. pylori IgG antibody and SAT results with the presence of diverse gastrointestinal symptoms among patients, which increased with long H. pylori infection durations.

Investigating Associations between HLA-DR Genotype, H. pylori Infection, and Anti-CagA IgA Seropositivity in a Turkish Gastritis Cohort.

Helicobacter pylori (H. pylori) is associated with gastric inflammation and mucosal antibodies against its cytotoxin-associated gene A (CagA) are protective. Vaccine-elicited immunity against H. pylori requires MHC class II expression, indicating that CD4+ T cells are protective. We hypothesized that the HLA-DR genotypes in human populations include protective alleles that more effectively bind immunogenic CagA peptide fragments and susceptible alleles with an impaired capacity to present CagA peptides. We recruited patients (n = 170) admitted for gastroendoscopy procedures and performed high-resolution HLA-DRB1 typing. Serum anti-CagA IgA levels were analyzed by ELISA (23.2% positive) and H. pylori classified as positive or negative in gastric mucosal tissue slides (72.9% positive). Pearson Chi-square analysis revealed that H. pylori infection was significantly increased in DRB1*11:04-positive individuals (p = 0.027). Anti-CagA IgA was significantly decreased in DRB1*11:04 positive individuals (p = 0.041). In contrast, anti-CagA IgA was significantly increased in DRB1*03:01 positive individuals (p = 0.030). For these HLA-DRB1 alleles of interest, we utilized two in silico prediction methods to compare their capacity to present CagA peptides. Both methods predicted increased numbers of peptides for DRB1*03:01 than DRB1*11:04. In addition, both alleles preferred distinctively different CagA 15mer peptide sequences for high affinity binding. These observations suggest that DRB1*11:04 is a susceptible genotype with impaired CagA immunity, whereas DRB1*03:01 is a protective genotype that promotes enhanced CagA immunity.

Combined Use of Helicobacter pylori Genotyping and CDX2 Expression as a Predictor of Malignant Potential in Gastric Intestinal Metaplasia.

OBJECTIVE: Gastrointestinal metaplasia (GIM) has a close relationship with gastric cancer (GC), but it is unclear how to judge which GIM could develop into GC. This study aimed to assess the role of CDX2 and its association with Helicobacter pylori (H.pylori) genotypes in GIM. METHODS: CagA and vacA genes were identified via PCR in 466 H. pylori-positive gastric tissues, including gastritis (n=104), GIM diagnosed endoscopically (GIM-1; n=82), gastric cancer (GC; n=173), and paired adjacent GIM tumors resected surgically (GIM-2; n=107). GIM was subclassified per the HID- AB pH2.5-PAS as follows: type I (n=23), type II (n=43), and type III (n=16) in GIM-1; type I (n=8), type II (n=40), and type III (n=59) in GIM-2. CDX2 expression was evaluated immunohistochemically. RESULTS: In GIM-1, the infection rate of vacAm2 (55.8%) and vacAs1m2 (53.5%) was higher in subtype II than in others (P<0.05), while that of vacAm1 (49.2%) and vacAs1m1 (33.9%) was higher in subtype III than in others. The cagA+ rate was higher in subtypes I (75.0%) and III (64.4%) than in subtype II (40.0%; P<0.05) respectively. CDX2 was upregulated in subtype I than in subtypes II and III in GIM-1 and GIM-2. In GIM-2 and GC, CDX2 was downregulated in vacAm1, vacAs1m1, and cagA+ (P<0.05). The predominant genotype was vacAs1m2 in subtype II of GIM-1, CDX2 expression remaining unaltered; however, the predominant genotype was cagA+ vacAs1m1 in subtypes II and III of GIM-2, negatively correlated with CDX2 expression. CONCLUSION: These GIM subtypes (cagA+ vacAs1m1 H. pylori-positive GIM with negative CDX2 expression) resemble GC and should be evaluated similar to cancerous GIM.

LVS ΔcapB-vectored multiantigenic melioidosis vaccines protect against lethal respiratory Burkholderia pseudomallei challenge in highly sensitive BALB/c mice.

Melioidosis, caused by the intracellular bacterial pathogen and Tier 1 select agent Burkholderia pseudomallei (Bp), is a highly fatal disease endemic in tropical areas. No licensed vaccine against melioidosis exists. In preclinical vaccine studies, demonstrating protection against respiratory infection in the highly sensitive BALB/c mouse has been especially challenging. To address this challenge, we have used a safe yet potent live attenuated platform vector, LVS ΔcapB, previously used successfully to develop vaccines against the Tier 1 select agents of tularemia, anthrax, and plague, to develop a melioidosis vaccine. We have engineered melioidosis vaccines (rLVS ΔcapB/Bp) expressing multiple immunoprotective Bp antigens among type VI secretion system proteins Hcp1, Hcp2, and Hcp6, and membrane protein LolC. Administered intradermally, rLVS ΔcapB/Bp vaccines strongly protect highly sensitive BALB/c mice against lethal respiratory Bp challenge, but protection is overwhelmed at very high challenge doses. In contrast, administered intranasally, rLVS ΔcapB/Bp vaccines remain strongly protective against even very high challenge doses. Under some conditions, the LVS ΔcapB vector itself provides significant protection against Bp challenge, and consistent with this, both the vector and vaccines induce humoral immune responses to Bp antigens. Three-antigen vaccines expressing Hcp6-Hcp1-Hcp2 or Hcp6-Hcp1-LolC are among the most potent and provide long-term protection and protection even with a single intranasal immunization. Protection via the intranasal route was either comparable to or statistically significantly better than the single-deletional Bp mutant Bp82, which served as a positive control. Thus, rLVS ΔcapB/Bp vaccines are exceptionally promising safe and potent melioidosis vaccines. IMPORTANCE: Melioidosis, a major neglected disease caused by the intracellular bacterial pathogen Burkholderia pseudomallei, is endemic in many tropical areas of the world and causes an estimated 165,000 cases and 89,000 deaths in humans annually. Moreover, B. pseudomallei is categorized as a Tier 1 select agent of bioterrorism, largely because inhalation of low doses can cause rapidly fatal pneumonia. No licensed vaccine is available to prevent melioidosis. Here, we describe a safe and potent melioidosis vaccine that protects against lethal respiratory challenge with B. pseudomallei in a highly sensitive small animal model-even a single immunization is highly protective, and the vaccine gives long-term protection. The vaccine utilizes a highly attenuated replicating intracellular bacterium as a vector to express multiple key proteins of B. pseudomallei; this vector platform has previously been used successfully to develop potent vaccines against other Tier 1 select agent diseases including tularemia, anthrax, and plague.

Systems and Computational Screening identifies SRC and NKIRAS2 as Baseline Correlates of Risk (CoR) for Live Attenuated Oral Typhoid Vaccine (TY21a) associated Protection.

AIM: We investigated the molecular underpinnings of variation in immune responses to the live attenuated typhoid vaccine (Ty21a) by analyzing the baseline immunological profile. We utilized gene expression datasets obtained from the Gene Expression Omnibus (GEO) database (accession number: GSE100665) before and after immunization. We then employed two distinct computational approaches to identify potential baseline biomarkers associated with responsiveness to the Ty21a vaccine. MAIN METHODS: The first pipeline (knowledge-based) involved the retrieval of differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction network construction, and topological network analysis of post-immunization datasets before gauging their pre-vaccination expression levels. The second pipeline utilized an unsupervised machine learning algorithm for data-driven feature selection on pre-immunization datasets. Supervised machine-learning classifiers were employed to computationally validate the identified biomarkers. KEY FINDINGS: Baseline activation of NKIRAS2 (a negative regulator of NF-kB signalling) and SRC (an adaptor for immune receptor activation) was negatively associated with Ty21a vaccine responsiveness, whereas LOC100134365 exhibited a positive association. The Stochastic Gradient Descent (SGD) algorithm accurately distinguished vaccine responders and non-responders, with 88.8%, 70.3%, and 85.1% accuracy for the three identified genes, respectively. SIGNIFICANCE: This dual-pronged novel analytical approach provides a comprehensive comparison between knowledge-based and data-driven methods for the prediction of baseline biomarkers associated with Ty21a vaccine responsiveness. The identified genes shed light on the intricate molecular mechanisms that influence vaccine efficacy from the host perspective while pushing the needle further towards the need for development of precise enteric vaccines and on the importance of pre-immunization screening.

Complete Freund's adjuvant as a confounding factor in multiple sclerosis research.

Complete Freund's adjuvant (CFA) is used as a standard adjuvant for the induction of experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model in multiple sclerosis studies. Still, CFA induces glial activation and neuroinflammation on its own and provokes pain. In addition, as CFA contains Mycobacteria, an immune response against bacterial antigens is induced in parallel to the response against central nervous system antigens. Thus, CFA can be considered as a confounding factor in multiple sclerosis-related studies performed on EAE. Here, we discuss the effects of CFA in EAE in detail and present EAE variants induced in experimental animals without the use of CFA. We put forward CFA-free EAE variants as valuable tools for studying multiple sclerosis pathogenesis and therapeutic approaches.

Neisseria gonorrhoeae lipooligosaccharide glycan epitopes recognized by bactericidal IgG antibodies elicited by the meningococcal group B-directed vaccine, MenB-4C.

Introduction: Outer membrane vesicles (OMVs) of Neisseria meningitidis in the group B-directed vaccine MenB-4C (BexseroR) protect against infections with Neisseria gonorrhoeae. The immunological basis for protection remains unclear. N. meningitidis OMV vaccines generate human antibodies to N. meningitidis and N. gonorrhoeae lipooligosaccharide (LOS/endotoxin), but the structural specificity of these LOS antibodies is not defined. Methods: Ten paired human sera obtained pre- and post-MenB-4C immunization were used in Western blots to probe N. meningitidis and N. gonorrhoeae LOS. Post-MenB-4C sera (7v5, 19v5, and 17v5), representing individual human variability in LOS recognition, were then used to interrogate structurally defined LOSs of N. meningitidis and N. gonorrhoeae strains and mutants and studied in bactericidal assays. Results and discussion: Post-MenB-4C sera recognized both N. meningitidis and N. gonorrhoeae LOS species, ~10% of total IgG to gonococcal OMV antigens. N. meningitidis and N. gonorrhoeae LOSs were broadly recognized by post-IgG antibodies, but with individual variability for LOS structures. Deep truncation of LOS, specifically a rfaK mutant without α-, ß-, or γ-chain glycosylation, eliminated LOS recognition by all post-vaccine sera. Serum 7v5 IgG antibodies recognized the unsialyated L1 α-chain, and a 3-PEA-HepII or 6-PEA-HepII was part of the conformational epitope. Replacing the 3-PEA on HepII with a 3-Glc blocked 7v5 IgG antibody recognition of N. meningitidis and N. gonorrhoeae LOSs. Serum 19v5 recognized lactoneotetrose (LNT) or L1 LOS-expressing N. meningitidis or N. gonorrhoeae with a minimal α-chain structure of Gal-Glc-HepI (L8), a 3-PEA-HepII or 6-PEA-HepII was again part of the conformational epitope and a 3-Glc-HepII blocked 19v5 antibody binding. Serum 17v5 LOS antibodies recognized LNT or L1 α-chains with a minimal HepI structure of three sugars and no requirement for HepII modifications. These LOS antibodies contributed to the serum bactericidal activity against N. gonorrhoeae. The MenB-4C vaccination elicits bactericidal IgG antibodies to N. gonorrhoeae conformational epitopes involving HepI and HepII glycosylated LOS structures shared between N. meningitidis and N. gonorrhoeae. LOS structures should be considered in next-generation gonococcal vaccine design.

Mitochondrial variants of complex I genes associated with leprosy clinical subtypes.

Leprosy is a chronic bacterial infection mainly caused by Mycobacterium leprae that primarily affects skin and peripheral nerves. Due to its ability to absorb carbon from the host cell, the bacillus became dependent on energy production, mainly through oxidative phosphorylation. In fact, variations in genes of Complex I of oxidative phosphorylation encoded by mtDNA have been associated with several diseases in humans, including bacterial infections, which are possible influencers in the host response to leprosy. Here, we investigated the presence of variants in the mtDNA genes encoding Complex I regarding leprosy, as well as the analysis of their pathogenicity in the studied cohort. We found an association of 74 mitochondrial variants with either of the polar forms, Pole T (Borderline Tuberculoid) or Pole L (Borderline Lepromatous and Lepromatous) of leprosy. Notably, six variants were exclusively found in both clinical poles of leprosy, including m.4158A>G and m.4248T>C in MT-ND1, m.13650C>A, m.13674T>C, m.12705C>T and m.13263A>G in MT-ND5, of which there are no previous reports in the global literature. Our observations reveal a substantial number of mutations among different groups of leprosy, highlighting a diverse range of consequences associated with mutations in genes across these groups. Furthermore, we suggest that the six specific variants exclusively identified in the case group could potentially play a crucial role in leprosy susceptibility and its clinical differentiation. These variants are believed to contribute to the instability and dysregulation of oxidative phosphorylation during the infection, further emphasizing their significance.

The serodiagnositic value of Chlamydia trachomatis antigens in antibody detection using luciferase immunosorbent assay.

Introduction: Among the different antigens used in the detection of anti-Chlamydia trachomatis antibodies, significant differences in sensitivity and specificity have been observed. Further evaluation of C. trachomatis antigens in antibody detection is urgently needed for the development and application of C. trachomatis serologic assays. Methods: Chlamydia trachomatis antigens Pgp3, TmeA, InaC, and HSP60 were selected and used in luciferase immunosorbent assay (LISA). The detection results obtained from well-defined C. trachomatis positive and negative samples were compared with the commercial C. trachomatis ELISA (Mikrogen) for performance evaluation. Results: Pgp3, TmeA, InaC, and HSP60-based LISA showed sensitivity of 92.8, 88.8, 90.4, and 94.4%, and specificity of 99.2, 99.2, 99.2, and 92%, respectively. ROC analysis indicated that Pgp3-based LISA showed similar performance to Mikrogen ELISA (AUC 0.986 vs. 0.993, p = 0.207). Furthermore, four C. trachomatis antigens achieved strong diagnostic efficiency, i.e., positive likelihood ratios [+LR] ≥ 10 in C. trachomatis-infected women and negative likelihood ratios [-LR] ≤ 0.1 in C. trachomatis negative low exposure risk children, but only Pgp3 and TmeA showed strong diagnostic value in general adults. In addition, Pgp3, TmeA, and InaC, but not HSP60, achieved high performance, i.e., both positive predictive value (PPV) and negative predictive value (NPV) ≥ 90.9%, and showed no significant cross-reactivity with anti-Chlamydiapneumoniae. Conclusion: Three C. trachomatis species-specific antigens Pgp3, TmeA, and InaC show superior performance in the detection of anti-C. trachomatis antibody, indicating the potential to be used in developing C. trachomatis serologic tests.

Viral Immunogenicity Prediction by Machine Learning Methods.

Since viruses are one of the main causes of infectious illnesses, prophylaxis is essential for efficient disease control. Vaccines play a pivotal role in mitigating the transmission of various viral infections and fortifying our defenses against them. The initial step in modern vaccine design and development involves the identification of potential vaccine targets through computational techniques. Here, using datasets of 1588 known viral immunogens and 468 viral non-immunogens, we apply machine learning algorithms to develop models for the prediction of protective immunogens of viral origin. The datasets are split into training and test sets in a 4:1 ratio. The protein structures are encoded by E-descriptors and transformed into uniform vectors by the auto- and cross-covariance methods. The most relevant descriptors are selected by the gain/ratio technique. The models generated by Random Forest, Multilayer Perceptron, and XGBoost algorithms demonstrate superior predictive performance on the test sets, surpassing predictions made by VaxiJen 2.0-an established gold standard in viral immunogenicity prediction. The key attributes determining immunogenicity in viral proteins are specific fingerprints in hydrophobicity and steric properties.