Rational Design of a Chimeric Derivative of PcrV as a Subunit Vaccine Against Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is a major cause of nosocomial infections, which remain an unsolved problem in the clinic despite conventional antibiotic treatment. A PA vaccine could be both an effective and economical strategy to address this issue. Many studies have shown that PcrV, a structural protein of the type 3 secretion system (T3SS) from PA, is an ideal target for immune prevention and therapy. However, difficulties in the production of high-quality PcrV likely hinder its further application in the vaccine industry. Thus, we hypothesized that an optimized PcrV derivative with a rational design could be produced. In this study, the full-length PcrV was divided into four domains with the guidance of its structure, and the Nter domain (Met1-Lys127) and H12 domain (Leu251-Ile294) were found to be immunodominant. Subsequently, Nter and H12 were combined with a flexible linker to generate an artificial PcrV derivative (PcrVNH). PcrVNH was successfully produced in E. coli and behaved as a homogenous monomer. Moreover, immunization with PcrVNH elicited a multifactorial immune response and conferred broad protection in an acute PA pneumonia model and was equally effective to full-length PcrV. In addition, passive immunization with anti-PcrVNH antibodies alone also showed significant protection, at least based on inhibition of the T3SS and mediation of opsonophagocytic killing activities. These results provide an additional example for the rational design of antigens and suggest that PcrVNH is a promising vaccine candidate for the control of PA infection

Sex differences in HIV treatment outcomes and adherence by exposure groups among adults in Guangdong, China: A retrospective observational cohort study

Introduction: We aimed to assess sex differences in treatment outcomes and adherence comparing men who have sex with women (MSW), men who have sex with men (MSM), and women who have sex with men (WSM), as well as men and women who inject drugs living with HIV on combination antiretroviral therapy (ART) in Guangdong, China. Methods: We performed a retrospective observational cohort study with data from the National Free Antiretroviral Treatment Program database. We included ART-naive patients aged 18 to 80 years who had contracted HIV through sex or injecting drugs, initiated first-line ART between January 2004 and December 2016, and had at least 60 days of follow-up. Participants were followed for five years. Kaplan-Meier analysis and Cox proportional hazard models were used to evaluate all-cause mortality. Cumulative incidence function and Cox proportional hazards models accounting for competing risks were used to evaluate disease progression to AIDS. Modified Poisson regression models were used to evaluate immunological and virological responses and loss to follow-up. Repeated measures analysis was used to evaluate regular CD4+ cell count, HIV viral load monitoring, ART adherence, side effects, and interruption of ART. Findings: We included 26,409 persons living with HIV. 21,779 (82·5%) people acquired HIV through sex (5118 WSM [23·5%], 8506 MSW [39·0%], 8175 MSM [37·5%]), and 4610 people (17·5%) through injection drug use (249 women [5·4%], 4361 men [94·6%]). Among those infected through sex, MSW had increased risks of all-cause mortality (adjusted hazard ratio [aHR] 1·48, 95% CI 1·20-1·83), progression to AIDS (1·27, 1·09-1·47), virological failure (adjusted incidence rates ratio [aIRR] 1·27, 95% CI 1·09-1·48), and loss to follow-up (1·22, 1·10-1·35) compared to WSM. In contrast, MSM had lower risk of all-cause mortality (aHR 0·49, 95%CI 0·32-0·76), disease progression to AIDS (0·83, 0·68-1·00), and virological failure (aIRR 0·78, 95%CI 0·65-0·94), were more likely to receive regular CD4+ cell count (1·08, 1·07-1·10) and HIV viral load monitoring (1·13, 1·12-1·15), were less likely to report missing ART doses (0·54, 0·49-0·61), interrupt ART (0·34, 0·26-0·44), or be lost to follow-up (0·56, 0·49-0·65) compared to WSM. Men who inject drugs were almost twice as likely as women who inject drugs to die (aHR 1·72, 95%CI 1·03-2·85), experience disease progression to AIDS (2·05, 1·18-3·57), virological failure (aIRR 1·81, 95%CI 1·19-2·76), report ART side effects (1·78, 1·43-2·22), and interruptions in ART (2·29, 1·50-3·50). Interpretation: Our findings highlight the importance of identifying potentially at-risk MSW and promoting HIV education and testing among them. Particular attention is warranted among men who inject drugs to improve timely HIV diagnosis, drug interaction management, and retention in treatment. Additional research from rural settings is needed to assess the long-term treatment outcomes and adherence in MSM with HIV

Integrated immunodominant epitope discovery for dual-purpose rapid and economical diagnostic and immunoprotective applications against MRSA

Current diagnostic and preventive strategies against Staphylococcus aureus methicillin-resistant strains (MRSA) remain inadequate. Hence, we aimed to identify candidate epitopes as potential therapeutic targets and diagnostic biomarkers. We focused on clinically validated targets and investigated four antigens (Hla, SEB, MntC, and IsdB) currently incorporated into phase III clinical trials of a recombinant five-antigen vaccine (termed rFSAV) and the recently identified leukocidin LukG. Using convalescent serum samples from patients with clinically confirmed MRSA, we identified 10 immunodominant epitopes through ELISA screening of overlapping 18-mer peptides, seven of which named MntC55-72, MntC121-138, MntC271-285, SEB37-54, LukG30-47, LukG235-252, and LukG246–263 have not been previously reported. Immunoprotection trials showed that five epitopes Hla168–185, IsdB384–401, MntC55–72, SEB37–54, and LukG235–252 elicited effective protection in a BALB/c murine sepsis model infected with MRSA252. The combination of these protective epitopes exhibited broad-spectrum efficacy against both the MRSA252 strain and phylogenetically distinct clinical isolates. Diagnostically, the performance of the epitope panel was superior to that of conventional culture methods with a sensitivity of 0.839 and specificity of 0.826 in a 3-h detection window, thus offering rapid and cost-effective advantages. Notably, bioinformatic analysis showed that all identified B-cell epitopes contained predicted CD4+ T-cell epitope sequences, which suggests the potential to elicit combined T–B cell immune responses through MHC-II presentation. Thus, these immunodominant epitopes with dual functions that integrate both diagnostic and immunoprotective capabilities could function as a novel immunodiagnostic toolkit that enables rapid MRSA detection and aid in establishing a multi-epitope vaccine platform. These findings present an integrated strategy that bridges diagnostic development and vaccine design for MRSA management

Expression, Purification and Characterization of Arginase from Helicobacter pylori in Its Apo Form

Arginase, a manganese-dependent enzyme that widely distributed in almost all creatures, is a urea cycle enzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. Compared with the well-studied arginases from animals and yeast, only a few eubacterial arginases have been characterized, such as those from H. pylori and B. anthracis. However, these enzymes used for arginase activity assay were all expressed with LB medium, as low concentration of Mn2+ was detectable in the medium, protein obtained were partially Mn2+ bonded, which may affect the results of arginase activity assay. In the present study, H. pylori arginase (RocF) was expressed in a Mn2+ and Co2+ free minimal medium, the resulting protein was purified through affinity and gel filtration chromatography and the apo-form of RocF was confirmed by flame photometry analysis. Gel filtration indicates that the enzyme exists as monomer in solution, which was unique as compared with homologous enzymes. Arginase activity assay revealed that apo-RocF had an acidic pH optimum of 6.4 and exhibited metal preference of Co2+>Ni2+>Mn2+. We also confirmed that heat-activation and reducing regents have significant impact on arginase activity of RocF, and inhibits S-(2-boronoethyl)-L-Cysteine (BEC) and Nω-hydroxy-nor-Arginine (nor-NOHA) inhibit the activity of RocF in a dose-dependent manner

Crystal Structure of EHEC Intimin: Insights into the Complementarity between EPEC and EHEC

Enterohaemorrhagic E. coli (EHEC) O157:H7 is a primary food-borne bacterial pathogen capable of causing life-threatening human infections which poses a serious challenge to public health worldwide. Intimin, the bacterial outer-membrane protein, plays a key role in the initiating process of EHEC infection. This activity is dependent upon translocation of the intimin receptor (Tir), the intimin binding partner of the bacteria-encoded host cell surface protein. Intimin has attracted considerable attention due to its potential function as an antibacterial drug target. Here, we report the crystal structure of the Tir-binding domain of intimin (Int188) from E. coli O157:H7 at 2.8 Å resolution, together with a mutant (IntN916Y) at 2.6 Å. We also built the structural model of EHEC intimin-Tir complex and analyzed the key binding residues. It suggested that the binding pattern of intimin and Tir between EHEC and Enteropathogenic E. coli (EPEC) adopt a similar mode and they can complement with each other. Detailed structural comparison indicates that there are four major points of structural variations between EHEC and EPEC intimins: one in Domain I (Ig-like domain), the other three located in Domain II (C-type lectin-like domain). These variations result in different binding affinities. These findings provide structural insight into the binding pattern of intimin to Tir and the molecular mechanism of EHEC O157: H7

Crystal Structure of EHEC Intimin: Insights into the Complementarity between EPEC and EHEC

Enterohaemorrhagic E. coli (EHEC) O157:H7 is a primary food-borne bacterial pathogen capable of causing life-threatening human infections which poses a serious challenge to public health worldwide. Intimin, the bacterial outer-membrane protein, plays a key role in the initiating process of EHEC infection. This activity is dependent upon translocation of the intimin receptor (Tir), the intimin binding partner of the bacteria-encoded host cell surface protein. Intimin has attracted considerable attention due to its potential function as an antibacterial drug target. Here, we report the crystal structure of the Tir-binding domain of intimin (Int188) from E. coli O157:H7 at 2.8 Å resolution, together with a mutant (IntN916Y) at 2.6 Å. We also built the structural model of EHEC intimin-Tir complex and analyzed the key binding residues. It suggested that the binding pattern of intimin and Tir between EHEC and Enteropathogenic E. coli (EPEC) adopt a similar mode and they can complement with each other. Detailed structural comparison indicates that there are four major points of structural variations between EHEC and EPEC intimins: one in Domain I (Ig-like domain), the other three located in Domain II (C-type lectin-like domain). These variations result in different binding affinities. These findings provide structural insight into the binding pattern of intimin to Tir and the molecular mechanism of EHEC O157: H7

A dominant CD4+ T cell response to <i>Helicobacter pylori</i> is associated with decreased risk of gastric diseases in human (P4328)

Abstract BACKGROUND and AIMS: Immunodominance has been well-demonstrated as a key feature in antiviral, antitumoral and antibacterial cellular immune responses but much less so in the immune responses against Helicobacter pylori (H. pylori). Antigen-specific CD4+ T cells have been reported to play a pivotal role in protecting against H. pylori in murine systems. We investigated the immunodominant CD4+ T cell response to HpaA (also called neuraminyllactose-binding hemagglutinin), a conserved, H. pylori-specific colonization factor which has been considered as a promising vaccine candidate. METHODS: HpaA-specific CD4+ T cells were expanded with autologous peripheral blood mononuclear cells (PBMCs) loaded with recombinant HpaA and further characterized with overlapping synthetic peptides. The percentage of HpaA88-100-specific CD4+ T cells restricted to HLA-DRB1*1501 from 59 H. pylori-infected subjects with different diseases were compared. RESULTS: Several novel, immunodominant CD4+ T cell epitopes were identified and characterized. The immunodominant CD4+ T cell responses specific to HpaA88-100 were observed in most of the H. pylori-infected individuals who expressed HLA-DRB1*1501 and they were significantly more abundant in patients with less severe diseases (P&amp;lt;0.05). CONCLUSIONS: The HLA-DRB1*1501-restricted immunodominant CD4+ T cell response specific to HpaA88-100 is associated with decreased risk of severe gastric diseases. Further study of these and other similar immunodominant CD4+ T cell responses to H. pylori will help the field to gain critical insight into the mechanism of protective immunity against H. pylori in humans and the knowledge gained will improve potential vaccine design.</jats:p

IL-22-producing Th cells regulate myeloid-derived suppressor cells in <i>Helicobacter pylori</i>-associated immunopathogenesis (IRC5P.456)

Abstract Th cell responses are critical for the immunopathogenesis of gastritis induced by persistent infection with Helicobacter pylori (H. pylori). IL-22-producing Th cells represent a new T-cell subset, but their response, regulation, function, and clinical relevance in H. pylori-induced gastritis are presently unknown. We found that IL-22-producing Th cells accumulated in gastric mucosa of cytotoxin-associated Ag (CagA)+ H. pylori-infected patients and mice. CagA+ H. pylori-activated DCs secreted IL-23, which favored IL-22-producing Th cell polarization. IL-22-producing Th cell-derived IL-22 induced CXCL2 chemokine production by gastric epithelial cells that were induced to express IL-22R1 by CagA+ H. pylori, which in turn promoted CXCR2-dependent migration of myeloid-derived suppressor cells (MDSCs). Subsequently, IL-22 induced MDSCs to produce proinflammatory proteins S100A8 and S100A9, and MDSCs itself exerted immunosuppressive roles by suppressing Th1 cell response, in doing so, contribute to the development of H. pylori-associated gastritis. Collectively, this study identifies a novel regulatory network involving IL-22-producing Th cells, which in conjunction with DCs, gastric epithelial cells, and MDSCs exert an inflammatory and immunosuppressive mixed effect within H. pylori-induced gastric microenvironment. Efforts to inhibit IL-22-producing Th cell-MDSC pathway might therefore prove a valuable strategy in the therapy of H. pylori-associated gastritis.</jats:p

Identification of antigen-specific antibody responses associated with upper genital tract pathology in mice infected with Chlamydia muridarum (166.3)

Abstract Urogenital infection with C. trachomatis in some women can lead to upper genital tract pathologies such as hydrosalpinx, potentially affecting fertility. In the current study, 27 of 40 mice intravaginally infected with C. muridarum developed visible hydrosalpinges in the oviduct while the remaining 13 did not although all infected mice displayed a similar infection time course. Antisera from the 40 mice recognized 130 out of 257 C. muridarum proteins as antigens and 17 as immunodominant antigens. Importantly, the 27 mice with hydrosalpinx preferentially recognized two C. muridarum proteins (TC0582 &amp; TC0912; thus, designated as pathology-associated antigens) while the 13 mice with no hydrosalpinx preferentially recognized 10 proteins designated as non-pathology antigens. The preferential recognition was validated by absorption and independently confirmed in Western blot. A fragment of TC0912 was found to improve the differentiation of hydrosalpinx from non-hydrosalpinx mice. TC0582 is a highly conserved ATP synthase and it may contribute to chlamydial pathogenesis via similar mechanisms hypothesized for the highly conserved HSP60. Thus, we have identified chlamydial antigens and epitopes that are associated with either susceptibility or resistance to upper genital tract pathology, which will help us to further understand chlamydial pathogenesis and develop anti-Chlamydia subunit vaccines.</jats:p