MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T Cells Promotes Mucosal Tolerance and Enforces Commensalism.

Commensal microbiota promote mucosal tolerance in part by engaging regulatory T (Treg) cells via Toll-like receptors (TLRs). We report that Treg-cell-specific deletion of the TLR adaptor MyD88 resulted in deficiency of intestinal Treg cells, a reciprocal increase in T helper 17 (Th17) cells and heightened interleukin-17 (IL-17)-dependent inflammation in experimental colitis. It also precipitated dysbiosis with overgrowth of segmented filamentous bacteria (SFB) and increased microbial loads in deep tissues. The Th17 cell dysregulation and bacterial dysbiosis were linked to impaired anti-microbial intestinal IgA responses, related to defective MyD88 adaptor- and Stat3 transcription factor-dependent T follicular regulatory and helper cell differentiation in the Peyer's patches. These findings establish an essential role for MyD88-dependent microbial sensing by Treg cells in enforcing mucosal tolerance and maintaining commensalism by promoting intestinal Treg cell formation and anti-commensal IgA responses.

Two human antibodies to a meningococcal serogroup B vaccine antigen enhance binding of complement Factor H by stabilizing the Factor H binding site.

Microbial pathogens bind host complement regulatory proteins to evade the immune system. The bacterial pathogen Neisseria meningitidis, or meningococcus, binds several complement regulators, including human Factor H (FH). FH binding protein (FHbp) is a component of two licensed meningococcal vaccines and in mice FHbp elicits antibodies that inhibit binding of FH to FHbp, which defeat the bacterial evasion mechanism. However, humans vaccinated with FHbp develop antibodies that enhance binding of FH to the bacteria, which could limit the effectiveness of the vaccines. In the present study, we show that two vaccine-elicited antibody fragments (Fabs) isolated from different human subjects increase binding of complement FH to meningococcal FHbp by ELISA. The two Fabs have different effects on the kinetics of FH binding to immobilized FHbp as measured by surface plasmon resonance. The 1.7- and 2.0-Å resolution X-ray crystal structures of the Fabs in complexes with FHbp illustrate that the two Fabs bind to similar epitopes on the amino-terminal domain of FHbp, adjacent to the FH binding site. Superposition models of ternary complexes of each Fab with FHbp and FH show that there is likely minimal contact between the Fabs and FH. Collectively, the structures reveal that the Fabs enhance binding of FH to FHbp by altering the conformations and mobilities of two loops adjacent to the FH binding site of FHbp. In addition, the 1.5 Å-resolution structure of one of the isolated Fabs defines the structural rearrangements associated with binding to FHbp. The FH-enhancing human Fabs, which are mirrored in the human polyclonal antibody responses, have important implications for tuning the effectiveness of FHbp-based vaccines.

Critical role of IL-25-ILC2-IL-5 axis in the production of anti-Francisella LPS IgM by B1 B cells.

B1 cells, a subset of B lymphocytes whose developmental origin, phenotype, and function differ from that of conventional B2 cells, are the main source of "natural" IgM but can also respond to infection by rapidly producing pathogen-specific IgM directed against T-independent antigens. Francisella tularensis (Ft) is a Gram-negative bacterium that causes tularemia. Infection with Ft Live Vaccine Strain activates B1 cells for production of IgM directed against the bacterial LPS in a process incompletely understood. Here we show that immunization with purified Ft LPS elicits production of LPS-specific IgM and IgG3 by B1 cells independently of TLR2 or MyD88. Immunization, but not infection, generated peritoneum-resident memory B1 cells that differentiated into LPS-specific antibody secreting cells (ASC) upon secondary challenge. IL-5 was rapidly induced by immunization with Ft LPS and was required for production of LPS-specific IgM. Antibody-mediated depletion of ILC2 indicated that these cells were the source of IL-5 and were required for IgM production. IL-25, an alarmin that strongly activates ILC2, was rapidly secreted in response to immunization or infection and its administration to mice significantly increased IgM production and B1 cell differentiation to ASC. Conversely, mice lacking IL-17RB, the IL-25 receptor, showed impaired IL-5 induction, IgM production, and B1 ASC differentiation in response to immunization. Administration of IL-5 to Il17rb-/- mice rescued these B1 cells-mediated responses. Il17rb-/- mice were more susceptible to infection with Ft LVS and failed to develop immunity upon secondary challenge suggesting that LPS-specific IgM is one of the protective adaptive immune mechanisms against tularemia. Our results indicated that immunization with Ft LPS triggers production of IL-25 that, through stimulation of IL-5 release by ILC2, promotes B1 cells activation and differentiation into IgM secreting cells. By revealing the existence of an IL-25-ILC2-IL-5 axis our results suggest novel strategies to improve vaccination against T-independent bacterial antigens.

Human Monoclonal Antibodies to Escherichia coli Outer Membrane Protein A Porin Domain Cause Aggregation but Do Not Alter In Vivo Bacterial Burdens in a Murine Sepsis Model.

Escherichia coli is one of the most frequent human pathogens, increasingly exhibits antimicrobial resistance, and has complex interactions with the host immune system. E. coli exposure or infection can result in the generation of antibodies specific for outer membrane protein A (OmpA), a multifunctional porin. We identified four OmpA-specific naturally occurring antibodies from healthy human donor B cells and assessed their interactions with E. coli and OmpA. These antibodies are highly specific for OmpA, exhibiting no cross-reactivity to a strain lacking ompA and retaining binding to both laboratory and clinical isolates of E. coli in enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays. One monoclonal antibody (Mab), designated ECOL-11, is specific for the extracellular N-terminal porin domain of OmpA and induces growth phase-specific bacterial aggregation. This aggregation is not induced by the fragment antigen binding (Fab) form of the MAb, suggesting the importance of bivalency for this aggregating activity. ECOL-11 decreases adhesion and phagocytosis of E. coli by RAW 264.7 macrophage-like cells, possibly by inhibiting the adhesion functions of OmpA. Despite this in vitro phenotype, organ E. coli burdens were not altered by antibody prophylaxis in a murine model of lethal E. coli septic shock. Our findings support the importance of OmpA at the host-pathogen interface and begin to explore the implications and utility of E. coli-specific antibodies in human hosts.

Generation of a recombinant antibody for sensitive detection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen that causes nosocomial infections and often exhibits antibiotic resistance. Therefore, the development of an accurate method for detecting P. aeruginosa is required to control P. aeruginosa-related outbreaks. In this study, we established an enzyme-linked immunosorbent assay method for the sensitive detection of three P. aeruginosa strains, UCBPP PA14, ATCC 27853, and multidrug-resistant ATCC BAA-2108. We produced a recombinant antibody (rAb) against P. aeruginosa V-antigen (PcrV), which is a needle tip protein of the type III secretion system of P. aeruginosa using mammalian cells with high yield and purity, and confirmed its P. aeruginosa binding efficiency. The rAb was paired with commercial anti-P. aeruginosa Ab for a sandwich ELISA, resulting in an antigen-concentration-dependent response with a limit of detection value of 230 CFU/mL. These results suggest that the rAb produced herein can be used for the sensitive detection of P. aeruginosa with a wide range of applications in clinical diagnosis and point-of-care testing.

Accessibility of O Antigens Shared between Salmonella Serovars Determines Antibody-Mediated Cross-Protection.

Pathogenic Salmonella serovars produce clinical manifestations ranging from systemic infection typhoid to invasive nontyphoidal Salmonella disease in humans. These serovars share a high degree of homology at the genome and the proteome level. However, whether infection or immunization with one serovar provides protection against other serovars has not been well studied. We show in this study that immunization of mice with live typhoidal serovar, Salmonella Typhi, generates cross-reactive immune responses, which provide far greater resistance against challenge with nontyphoidal serovar Salmonella Enteritidis than with another nontyphoidal serovar, Salmonella Typhimurium. Splenic T cells from these immunized mice produced similar levels of IL-2 and IFN-γ upon ex vivo stimulation with Ags prepared from S Enteritidis and S Typhimurium. In contrast, Abs against S Typhi interacted with live intact S Enteritidis but did not bind intact S Typhimurium. These pathogen-reactive Abs were largely directed against oligosaccharide (O)-antigenic determinant of LPS that S Typhi shares with S Enteritidis. Abs against the O determinant, which S Typhi shares with S Typhimurium, were present in the sera of immunized mice but did not bind live intact Salmonella because of surface inaccessibility of this determinant. Similar accessibility-regulated interaction was seen with Abs generated against S Typhimurium and S Enteritidis. Our results suggest that the ability of protective Abs elicited with one Salmonella serovar to engage with and consequently provide protection against another Salmonella serovar is determined by the accessibility of shared O Ags. These findings have significant and broader implications for immunity and vaccine development against pathogenic Salmonellae.

Opsonization-Enhanced Antigen Presentation by MR1 Activates Rapid Polyfunctional MAIT Cell Responses Acting as an Effector Arm of Humoral Antibacterial Immunity.

Mucosa-associated invariant T (MAIT) cells are innate-like antimicrobial T cells recognizing a breadth of important pathogens via presentation of microbial riboflavin metabolite Ags by MHC class Ib-related (MR1) molecules. However, the interaction of human MAIT cells with adaptive immune responses and the role they may play in settings of vaccinology remain relatively little explored. In this study we investigated the interplay between MAIT cell-mediated antibacterial effector functions and the humoral immune response. IgG opsonization of the model microbe Escherichia coli with pooled human sera markedly enhanced the capacity of monocytic APC to stimulate MAIT cells. This effect included greater sensitivity of recognition and faster response kinetics, as well as a markedly higher polyfunctionality and magnitude of MAIT cell responses involving a range of effector functions. The boost of MAIT cell responses was dependent on strongly enhanced MR1-mediated Ag presentation via increased FcγR-mediated uptake and signaling primarily mediated by FcγRI. To investigate possible translation of this effect to a vaccine setting, sera from human subjects before and after vaccination with the 13-valent-conjugated Streptococcus pneumoniae vaccine were assessed in a MAIT cell activation assay. Interestingly, vaccine-induced Abs enhanced Ag presentation to MAIT cells, resulting in more potent effector responses. These findings indicate that enhancement of Ag presentation by IgG opsonization allows innate-like MAIT cells to mount a faster, stronger, and qualitatively more complex response and to function as an effector arm of vaccine-induced humoral adaptive antibacterial immunity.

The importance of polyreactive antibodies in protection against pneumococcal infection.

Antibodies are a key element of the immune response. They can bind their molecular targets with exquisite sensitivity and specificity, providing protection against a multitude of pathogens. They have long been understood to be markers of a successful response to vaccination, and are now widely manufactured as highly specific and robust immunotherapeutic agents. Less well understood are the polyreactive antibodies, found in serum, which are able to bind more than one target molecule. Here, we highlight new research into these naturally occurring polyreactive antibodies, which demonstrates their importance for protection against Streptococcus pneumoniae, a common cause of airway infection.

Do Long-Lived Plasma Cells Maintain a Healthy Microbiota in the Gut?

Disruptions to the gut microbiota have been associated with a variety of diseases. Understanding the underlying mechanisms that regulate the maintenance of a healthy microbiota may therefore have therapeutic implications. Secretory IgA play a unique role in immune-microbiota crosstalk by directly binding to bacteria in the gut lumen. Microbe-specific IgA responses co-develop with the assembly of the gut microbiota during infancy, and resemble those of adults by 2 years postnatally in the healthy host. We propose here that microbiota-specific IgA-producing gut plasma cells generated during infancy live for many decades and contribute to a stable microbiota community. We furthermore suggest that members of the microbiota that induce long-lasting IgA responses in the gut are putative targets for therapeutic interventions.

Oral vaccination of Nile tilapia (Oreochromis niloticus) against francisellosis elevates specific antibody titres in serum and mucus.

Although Nile tilapia (Oreochromis niloticus) is a well-established aquaculture species globally, there are a limited number of commercial vaccines available or are used for this species. The majority of diseases affecting farmed tilapia are bacterial, with antibiotics frequently used to treat fish. The current study was performed to optimise the use of mucosal vaccines for tilapia by adapting an existing bacterin vaccine against Francisella noatunensis subsp. orientalis (Fno) as a proof of concept. This vaccine has previously provided excellent protection by injection, however, the preference for tilapia farmers would be to vaccinate fish by immersion or orally, due to the lower cost and ease of application. These vaccination routes, however, are often less efficacious probably due to the lack of adjuvants in immersion and oral vaccines. The aims of this study, therefore, were to optimise the formulation and dose of the Fno vaccine with mucosal adjuvants for oral and immersion delivery. Tilapia fry (av. 6 g) were given three concentrations (high, medium, low; i.e. 1×109, 1×108 and 1×107 CFU mL-1) of antigen combined with the oral adjuvant by oral gavage, to optimise the dose needed to induce an immune response to Fno, and the immune response obtained compared with fish vaccinated by immersion (with and without an immersion adjuvant). Fry were boosted by the same route at 420 degree days (DD), and samples (serum, mucus ) taken at 840 DD for specific antibody responses measured by ELISA and western blotting. Specific IgM titres were significantly elevated in serum and mucus of fish given the high dose adjuvanted vaccine by gavage. In addition, by western blotting with serum, a significant immunogenic reaction was evident between 20 and 37 kDa in the fish given the high dose oral vaccine by gavage. As protection against Fno provided by the injection vaccine was correlated with specific antibody responses these findings suggest the oral vaccine also has potential to provide protection. Further studies are needed to optimise delivery of the vaccine via feed.

Complement Activation Occurs at the Surface of Platelets Activated by Streptococcal M1 Protein and This Results in Phagocytosis of Platelets.

Platelets circulate the bloodstream and principally maintain hemostasis. Disturbed hemostasis, a dysregulated inflammatory state, and a decreased platelet count are all hallmarks of severe invasive Streptococcus pyogenes infection, sepsis. We have previously demonstrated that the released M1 protein from S. pyogenes activates platelets, and this activation is dependent on the binding of M1 protein, fibrinogen, and M1-specific IgG to platelets in susceptible donors. In this study, we characterize the M1-associated protein interactions in human plasma and investigate the acquisition of proteins to the surface of activated platelets and the consequences for platelet immune function. Using quantitative mass spectrometry, M1 protein was determined to form a protein complex in plasma with statistically significant enrichment of fibrinogen, IgG3, and complement components, especially C1q. Using flow cytometry, these plasma proteins were also confirmed to be acquired to the platelet surface, resulting in complement activation on M1-activated human platelets. Furthermore, we demonstrated an increased phagocytosis of M1-activated platelets by monocytes, which was not observed with other physiological platelet agonists. This reveals a novel mechanism of complement activation during streptococcal sepsis, which contributes to the platelet consumption that occurs in sepsis.

Covalently Immobilized Regenerable Immunoaffinity Layer with Orientation-Controlled Antibodies Based on Z-Domain Autodisplay.

A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation control, antibody-binding Z-domain-autodisplaying Escherichia coli (E. coli) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG)5), were evaluated, and DSS at a concentration of 500 µM was confirmed to be optimal. The E. coli-cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of E. coli cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%.

Generation of digital patients for the simulation of tuberculosis with UISS-TB.

BACKGROUND: The STriTuVaD project, funded by Horizon 2020, aims to test through a Phase IIb clinical trial one of the most advanced therapeutic vaccines against tuberculosis. As part of this initiative, we have developed a strategy for generating in silico patients consistent with target population characteristics, which can then be used in combination with in vivo data on an augmented clinical trial. RESULTS: One of the most challenging tasks for using virtual patients is developing a methodology to reproduce biological diversity of the target population, ie, providing an appropriate strategy for generating libraries of digital patients. This has been achieved through the creation of the initial immune system repertoire in a stochastic way, and through the identification of a vector of features that combines both biological and pathophysiological parameters that personalise the digital patient to reproduce the physiology and the pathophysiology of the subject. CONCLUSIONS: We propose a sequential approach to sampling from the joint features population distribution in order to create a cohort of virtual patients with some specific characteristics, resembling the recruitment process for the target clinical trial, which then can be used for augmenting the information from the physical the trial to help reduce its size and duration.

Characterization of an Antibody Recognizing the Conserved Inner Core of Pseudomonas aeruginosa Lipopolysaccharides.

Bacterial infections are a growing public health threat with carbapenem-resistant Pseudomonas aeruginosa being classified as a Priority 1 critical threat by the World Health Organization. Antibody-based therapeutics can serve as an alternative and in some cases supplement antibiotics for the treatment of bacterial infections. The glycans covering the bacterial cell surface have been proposed as intriguing targets for binding by antibodies; however, antibodies that can engage with high affinity and specificity with glycans are much less common compared to antibodies that engage with protein antigens. In this study, we sought to characterize an antibody that targets a conserved glycan epitope on the surface of Pseudomonas. First, we characterized the breadth of binding of VSX, demonstrating that the VSX is specific to Pseudomonas but can bind across multiple serotypes of the organism. Next, we provide insight into how VSX engages with its target epitope, using a combination of biolayer interferometry and nuclear magnetic resonance, and verify our results using site-directed mutagenesis experiments. We demonstrate that the antibody, with limited somatic hypermutation of the complementarity-determining regions (CDRs) and with a characteristic set of arginines within the CDRs, specifically targets the conserved inner core of Pseudomonas lipopolysaccharides. Our results provide important additional context to antibody-glycan contacts and provide insight useful for the construction of vaccines and therapeutics against Pseudomonas aeruginosa, an important human pathogen.

Anti-EF-Tu IgG titers increase with age and may contribute to protection against the respiratory pathogen Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) is a pathogen that commonly colonizes the nasopharynx of preschool children, causing opportunistic infections including acute otitis media (AOM). Patients suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized with NTHi and occasionally suffer from exacerbations by the bacterium leading to increased morbidity. Elongation-factor thermo unstable (EF-Tu), a protein critical for bacterial protein synthesis, has been found to moonlight on the surface of several bacteria. Here, we show that antibodies against NTHi EF-Tu were present in children already at 18 months of age, and that the IgG antibody titers increased with age. Children harboring NTHi in the nasopharynx also displayed significantly higher IgG concentrations. Interestingly, children suffering from AOM had significantly higher anti-EF-Tu IgG levels when NTHi was the causative agent. Human sera recognized mainly the central and C-terminal part of the EF-Tu molecule and peptide-based epitope mapping confirmed similar binding patterns for sera from humans and immunized mice. Immunization of BALB/c and otitis-prone Junbo (C3H/HeH) mice promoted lower infection rates in the nasopharynx and middle ear, respectively. In conclusion, our results suggest that IgG directed against NTHi EF-Tu may play an important role in the host immune response against NTHi.

Generation of recombinant baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies.

BACKGROUND: Clostridium perfringens is the causative agent of several diseases and enteric infections in animals and humans. The virulence of C. perfringens is largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1-250), and the C-terminal region (aa residues 251-370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced in E. coli were protected against C. perfringens infections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms. RESULTS: In the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250-363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of the L21 consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250-363H6 neutralized the phospholipase C activity of full-length PLC. CONCLUSIONS: The L21 leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.

Protein A-Mediated Binding of Staphylococcus spp. to Antibodies in Flow Cytometric Assays and Reduction of This Binding by Using Fc Receptor Blocking Reagent.

Staphylococcus aureus and other coagulase-positive Staphylococcus spp. bind the Fc region of IgG antibodies through expression of protein A (SpA). These species have consequently been a source of false-positive signals in antibody-based assays designed to detect other target bacteria. Here, flow cytometry was used to study the influence of a number of factors on the SpA-mediated binding of single cells to an anti-human IgG antibody, including strain, heat killing, overnight storage, growth phase, cell physiology, surface adhesion, and growth in model food systems. Through the costaining of antibody-stained cells with the permeability dye propidium iodide and calcein violet AM, the cell physiological status was related to SpA-mediated antibody binding. Generally, permeabilized cells lacking esterase activity did not strongly bind antibody. The binding of a number of commercially available polyclonal IgG antibodies to non-Staphylococcus spp. was also characterized. Not all SpA-expressing species showed strong binding of mouse IgG, and one species not known to express SpA showed strong binding. Most SpA-expressing strains bound rabbit IgG antibodies to some extent, whereas only one strain bound goat IgG. To reduce or eliminate SpA-mediated IgG binding, the following products were evaluated as blocking reagents and applied prior to staining with primary or secondary antibody: normal rabbit serum, mouse IgG isotype control, goat IgG, and a commercial FcR blocking reagent. Only the FcR blocking reagent consistently reduced SpA-mediated binding of Staphylococcus spp. to antibodies against other species and could be recommended as a blocking reagent in immunoassays designed to detect non-Staphylococcus species.IMPORTANCE This study characterizes a widespread but little-studied problem associated with the antibody-based detection of microbes-the Staphylococcus protein A (SpA)-mediated binding of IgG antibodies-and offers a solution: the use of commercial FcR blocking reagent. A common source of false-positive signals in the detection of microbes in clinical, food, or environmental samples can be eliminated by applying this study's findings. Using flow cytometry, the authors demonstrate the extent of heterogeneity in a culture's SpA-mediated binding of antibodies and that the degree of SpA-mediated antibody binding is strain, growth phase, and food matrix dependent and influenced by simulated food processing treatments and cell adherence. In addition, our studies of SpA-mediated binding of Staphylococcus spp. to antibodies against other bacterial species produced a very nuanced picture, leading us to recommend testing against multiple strains of S. aureus and S. hyicus of all antibodies to be incorporated into any immunoassay designed to detect a non-Staphylococcus spp.

Effect of deletion of gene cluster involved in synthesis of Enterobacterial common antigen on virulence and immunogenicity of live attenuated Salmonella vaccine when delivering heterologous Streptococcus pneumoniae antigen PspA.

BACKGROUND: Enterobacterial common antigen (ECA) is a family-specific surface antigen shared by all members of the Enterobacteriaceae family. Previous studies showed that the loss of ECA results in Salmonella attenuation, indicating its usefulness as a vaccine candidate for Salmonella infection, but no studies have shown whether the mutation resulting from the deletion of the ECA operon in conjunction with other mutations could be used as an antigen vehicle for heterologous protein antigen delivery. RESULTS: In this study, we introduced a nonpolar, defined ECA operon deletion into wild-type S. Typhimurium χ3761 and an attenuated vaccine strain χ9241, obtaining two isogenic ECA operon mutants, namely, χ12357 and χ12358, respectively. A number of in vitro and in vivo properties of the mutants were analyzed. We found that the loss of ECA did not affect the growth, lipopolysaccharide (LPS) production and motility of S. Typhimurium wild type strain χ3761 and its attenuated vaccine strain χ9241 but significantly affected the virulence when administered orally to BALB/c mice. Furthermore, the effects of the ECA mutation on the immunogenicity of a recombinant S. Typhimurium vaccine strain χ9241 when delivering the pneumococcal antigen PspA were determined. The result showed that the total anti-PspA IgG level of χ12358 (pYA4088) was slightly lower than that of χ9241 (pYA4088), but the protection rate was not compromised. CONCLUSIONS: ECA affects virulence and benefits the Th2 immunity of Salmonella Typhimurium, therefore, it is feasible to use a reversible ECA mutant mode to design future Salmonella vaccine strains for heterologous protective antigens.

Th17 cells mediate clade-specific, serotype-independent mucosal immunity.

The interleukin-17 (IL-17) family of cytokines phylogenetically predates the evolution of T cells in jawed vertebrates, suggesting that the ontogeny of the Th17 cell lineage must have arisen to confer an evolutionary advantage to the host over innate sources of IL-17. Utilizing a model of mucosal immunization with the encapsulated bacteria Klebsiella pneumoniae, we found that B cells, which largely recognized polysaccharide capsular antigens, afforded protection to only the vaccine strain. In contrast, memory Th17 cells proliferated in response to conserved outer membrane proteins and conferred protection against several serotypes of K. pneumoniae, including the recently described multidrug resistant New Dehli metallolactamase strain. Notably, this heterologous, clade-specific protection was antibody independent, demonstrating the Th17 cell lineage confers a host advantage by providing heterologous mucosal immunity independent of serotype-specific antibody.

Screening and evaluation of Mycobacterium tuberculosis diagnostic antigens.

In recent years, the prevalence of tuberculosis worldwide has increased, and with it, the number of drug-resistant tuberculosis strains. This has brought new challenges towards prevention and control of the disease. Therefore, it is urgent to find reliable and rapid diagnostic methods for tuberculosis in general, and for the drug-resistant forms of the disease. To this aim, we assessed 17 tuberculosis-specific protein candidates for the detection of tuberculosis-specific antibodies. First, we established an indirect ELISA method to detect anti-Mycobacterium tuberculosis IgM and IgG. We tested 453 sera and analyzed the efficacy of the protein candidates for diagnosis of tuberculosis. Next, we screened antigens rich in T cell epitopes for their ability to induce high levels of IFN-γ, in order to define their suitability does develop detection tests based on IFN-γ release assay (IGRAs). The antigens CFP-10, PPE57, 38kDa, and Rv3807 showed higher diagnostic potential for the detection of anti-tuberculosis IgM, whereas PPE57, Ag85B, CFP-10, Rv0220, and 38kDa antigens performed better for anti-tuberculosis IgG detection. Worth noting is that CFP-10, 38kDa, and PPE57 detected efficiently both IgM and IgG. Rv1987, Rv3807, PPE57, Rv0220, and MPT64 proteins alone and combinations of Rv1987 + Rv3807, 16kDa + Rv0220, and MPT64 + Rv1986 tested in IGRAs displayed a good correlation with the positive control constituted by a cocktail of ESAT-6 + CFP-10 + TB7.7 (ECT), known for their stimulating properties (r > 0.5, p < 0.01). Among these antigen candidates, Rv0220 and Rv1987 + Rv3807 were the most potent. We discovered CFP-10, 38kDa, and PPE57 for the detection of anti-M. tuberculosis IgM and IgG, and Rv0220 alone or the combination Rv1987 + Rv3807 as the strongest stimulators in IGRAs. These antigens provide new references for the screening of tuberculosis-specific antibodies and effective stimulation in IGRAs.