Bactericidal Response Research Articles

Sialic Acid-Siglec-E Interactions During Pseudomonas aeruginosa Infection of Macrophages Interferes With Phagosome Maturation by Altering Intracellular Calcium Concentrations.

Pseudomonas aeruginosa (PA) is commonly associated with nosocomial and chronic infections of lungs. We have earlier demonstrated that an acidic sugar, sialic acid, is present in PA which is recognized and bound by sialic acid binding immunoglobulin type lectins (siglecs) expressed on neutrophils. Here, we have tried to gain a detailed insight into the immunosuppressive role of sialic acid-siglec interactions in macrophage-mediated clearance of sialylated PA (PA+Sia). We have demonstrated that PA+Sia shows enhanced binding (~1.5-fold) to macrophages due to additional interactions between sialic acids and siglec-E and exhibited more phagocytosis. However, internalization of PA+Sia is associated with a reduction in respiratory burst and increase in anti-inflammatory cytokines secretion which is reversed upon desialylation of the bacteria. Phagocytosis of PA+Sia is also associated with reduced intracellular calcium ion concentrations and altered calcium-dependent signaling which negatively affects phagosome maturation. Consequently, although more PA+Sia was localized in early phagosomes (Rab5 compartment), only fewer bacteria reach into the late phagosomal compartment (Rab7). Possibly, this leads to reduced phagosome lysosome fusion where reduced numbers of PA+Sia are trafficked into lysosomes, compared to PA−Sia. Thus, internalized PA+Sia remain viable and replicates intracellularly in macrophages. We have also demonstrated that such siglec-E-sialic acid interaction recruited SHP-1/SHP-2 phosphatases which modulate MAPK and NF-κB signaling pathways. Disrupting sialic acid-siglec-E interaction by silencing siglec-E in macrophages results in improved bactericidal response against PA+Sia characterized by robust respiratory burst, enhanced intracellular calcium levels and nuclear translocation of p65 component of NF-κB complex leading to increased pro-inflammatory cytokine secretion. Taken together, we have identified that sialic acid-siglec-E interactions is another pathway utilized by PA in order to suppress macrophage antimicrobial responses and inhibit phagosome maturation, thereby persisting as an intracellular pathogen in macrophages.

Estimated susceptibility of Canadian meningococcal B isolates to a meningococcal serogroup B vaccine (MenB-FHbp)

BackgroundInvasive meningococcal disease caused by Neisseria meningitidis serogroup B (MenB) remains a health risk in Canada and globally. Two MenB vaccines are now approved for use. An understanding of the genotype of Canadian strains and the potential strain coverage conferred by the MenB-FHbp vaccine is needed to inform immunization policies. MethodsSerogroup B Neisseria meningitidis strains responsible for meningococcal disease in Canada from 2006 to 2012 were collected as part of the Canadian Immunization Monitoring Program Active surveillance network. Genotypic analysis was done on MenB isolates from 2006 to 2012 with determination of fHbp surface expression for a subset of isolates: those occurring from 2010 to 2012. ResultsTwo clonal complexes (cc269 and cc41/44) were observed in 68.8% of the 276 isolates. A total of 50 different fHbp peptides were identified among isolates from 2006 to 2012. Surface expression of fHbp was detected on 95% of MenB isolates from 2010 to 2012 and 91% of isolates expressed fHbp at levels that are predicted to be susceptible to the bactericidal immune response elicited by the MenB-FHbp vaccine. Some regional differences were observed, particularly in isolates from British Columbia and Quebec. ConclusionThe majority of MenB isolates responsible for meningococcal disease in Canada expressed fHbp at levels predicted to be sufficient for complement mediated bactericidal activity in the presence of MenB-FHbp induced serum antibodies.

'Template-free' hierarchical MoS2 foam as a sustainable 'green' scavenger of heavy metals and bacteria in point of use water purification.

Molybdenum disulfide (MoS2), with its unique optical and electrical properties, has been explored for a variety of applications in the recent past. Still, its capabilities in point-of-use heavy metal ion removal remain to be explored. Herein, for the first time using a facile approach, we fabricated three-dimensional (3D) MoS2 foam from exfoliated single to few-layered MoS2 sheets for the selective exclusion of heavy metals and stringent bactericidal response. This foam was able to exclude 99.9% of Pb(ii) and 98.7% of As(iii) instantaneously and reduced more than 98% of bacteria E. coli. Moreover, the foam exhibits selective toxicity towards bacterial cells while showing no observable toxicity towards mammalian cells. The foam can be recycled and reused for at least five cycles under accelerated conditions and thus can be used for a promising non-cytotoxic, facile, and environmentally benign process for inline water remediation to remove heavy metal ions from the feed and as a potential antibacterial agent.

Re-growth of Mycobacterium tuberculosis populations exposed to antibiotic combinations is due to the presence of isoniazid and not bacterial growth rate.

Modulation of growth rate in Mycobacterium tuberculosis is key to its survival in the host; particularly with regard to its adaptation during chronic infection when the growth rate is very slow. The resulting physiological changes will influence the way this pathogen interacts with the host and responds to antibiotics. Therefore, it is important that we understand how growth rate impacts antibiotic efficacy, particularly with respect to recovery/relapse. This is the first study that has asked how growth rates influence the mycobacterial responses to combinations of frontline antimycobacterials, isoniazid (INH), rifampicin (RIF), and pyrazinamide (PZA), using continuous cultures. Time-course profiles of log-transformed total viable counts for cultures, controlled at either a fast growth rate (23.1. mean generation time (MGT)) or slow growth rate (69.3h MGT), were analysed with the fitting of a mathematical model by nonlinear regression that accounted for the dilution rate in the chemostat, and profiled kill rates and recovery in culture. Using this approach, we show that populations growing more slowly were generally less susceptible to all treatments. We observed a higher kill rate associated with INH (compared to RIF or PZA) and the appearance of re-growth. In line with this observation, re-growth was not observed with RIF-exposure, which provided a slower bactericidal response. The sequential additions of RIF and PZA did not eliminate re-growth. We consider here that faster, early bactericidal activity is not what is required for successful sterilisation of M. tuberculosis, but instead slower elimination of bacilli followed by reduced recovery of the bacterial population.

Host defense mechanisms against Mycobacterium tuberculosis.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of death worldwide from a single infectious pathogen. Mtb is a paradigmatic intracellular pathogen that primarily invades the lungs after host inhalation of bacteria-containing droplets via the airway. However, the majority of Mtb-exposed individuals can spontaneously control the infection by virtue of a robust immune defense system. The mucosal barriers of the respiratory tract shape the first-line defense against Mtb through various mucosal immune responses. After arriving at the alveoli, the surviving mycobacteria further encounter a set of host innate immune cells that exert multiple cellular bactericidal functions. Adaptive immunity, predominantly mediated by a range of different T cell and B cell subsets, is subsequently activated and participates in host anti-mycobacterial defense. During Mtb infection, host bactericidal immune responses are exquisitely adjusted and balanced by multifaceted mechanisms, including genetic and epigenetic regulation, metabolic regulation and neuroendocrine regulation, which are indispensable for maintaining host immune efficiency and avoiding excessive tissue injury. A better understanding of the integrated and equilibrated host immune defense system against Mtb will contribute to the development of rational TB treatment regimens especially novel host-directed therapeutics.

Impaired inflammasome activation and bacterial clearance in G6PD deficiency due to defective NOX/p38 MAPK/AP-1 redox signaling

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway that modulates cellular redox homeostasis via the regeneration of NADPH. G6PD-deficient cells have a reduced ability to induce the innate immune response, thus increasing host susceptibility to pathogen infections. An important part of the immune response is the activation of the inflammasome. G6PD-deficient peripheral blood mononuclear cells (PBMCs) from patients and human monocytic (THP-1) cells were used as models to investigate whether G6PD modulates inflammasome activation. A decreased expression of IL-1β was observed in both G6PD-deficient PBMCs and PMA-primed G6PD-knockdown (G6PD-kd) THP-1 cells upon lipopolysaccharide (LPS)/adenosine triphosphate (ATP) or LPS/nigericin stimulation. The pro-IL-1β expression of THP-1 cells was decreased by G6PD knockdown at the transcriptional and translational levels in an investigation of the expression of the inflammasome subunits. The phosphorylation of p38 MAPK and downstream c-Fos expression were decreased upon G6PD knockdown, accompanied by decreased AP-1 translocation into the nucleus. Impaired inflammasome activation in G6PD-kd THP-1 cells was mediated by a decrease in the production of reactive oxygen species (ROS) by NOX signaling, while treatment with hydrogen peroxide (H2O2) enhanced inflammasome activation in G6PD-kd THP-1 cells. G6PD knockdown decreased Staphylococcus aureus and Escherichia coli clearance in G6PD-kd THP-1 cells and G6PD-deficient PBMCs following inflammasome activation. These findings support the notion that enhanced pathogen susceptibility in G6PD deficiency is, in part, due to an altered redox signaling, which adversely affects inflammasome activation and the bactericidal response.

Central composite design-based optimization and fabrication of benzylisothiocynate-loaded PLGA nanoparticles for enhanced antimicrobial attributes

Benzylisothiocynate also referred to as BITC is a compound which is commonly found in Cruciferae plants. This chemical moiety falls under the category of Isothiocynates (ITCs), and is known to possess superior antibacterial properties. Hence, endeavors have been made in the present research to encapsulate benzyl isothiocynate (BITC) within the framework of polymeric nanoparticles to enhance its potency, stability, safety, and biocompatibility. The study also aimed at fabricating a nanoparticulate system with engineered release characteristics so that the susceptibility of BITC in evoking a bactericidal response towards E. coli can be enhanced. To attain an optimal system with desired critical quality attributes [CQAs (particle size, entrapment efficiency and release)] all the requisite critical material attributes [CMAs (amount of polymer, and concentration of surfactant)] were optimized using central composite design (CCD). An overall assessment of the physicochemical characteristics of fabricated PLGA nanoparticles containing BITC viz. BITC-PLGA NPs were accomplished using DLS, FE-SEM, UV–Vis, and ATR-FTIR spectroscopy. The BITC-PLGA NPs were found to be 106.2 ± 1.78 nm in size, and has a surface charge of − 16.3 ± 0.64 mV. The BITC-PLGA NPs were found to be homogenous as the DLS analysis revealed a PDI value of 0.353 ± 0.008. A significantly high entrapment efficiency and percentage loading (~ 74 and 32%) along with a sustained release of BITC (~ 64.2 (pH 5.5)–(pH 5.5), and 83.8 (pH 7.4) %) was obtained from BITC-PLGA NPs. The antibacterial assay also pointed towards a safe and efficient delivery system which can further prove to be a pioneer in the upcoming times.

Impact of the Dual Deletion of the Mitochondrial Sirtuins SIRT3 and SIRT5 on Anti-microbial Host Defenses.

The sirtuins SIRT3 and SIRT5 are the main mitochondrial lysine deacetylase and desuccinylase, respectively. SIRT3 and SIRT5 regulate metabolism and redox homeostasis and have been involved in age-associated metabolic, neurologic and oncologic diseases. We have previously shown that single deficiency in either SIRT3 or SIRT5 had no impact on host defenses in a large panel of preclinical models of sepsis. However, SIRT3 and SIRT5 may compensate each other considering that they share subcellular location and targets. Here, we generated a SIRT3/5 double knockout mouse line. SIRT3/5 deficient mice multiplied and developed without abnormalities. Hematopoiesis and immune cell development were largely unaffected in SIRT3/5 deficient mice. Whole blood, macrophages and neutrophils from SIRT3/5 deficient mice displayed enhanced inflammatory and bactericidal responses. In agreement, SIRT3/5 deficient mice showed somewhat improved resistance to Listeria monocytogenes infection. Overall, the double deficiency in SIRT3 and SIRT5 has rather subtle impacts on immune cell development and anti-microbial host defenses unseen in single deficient mice, indicating a certain degree of overlap between SIRT3 and SIRT5. These data support the assumption that therapies directed against mitochondrial sirtuins, at least SIRT3 and SIRT5, should not impair antibacterial host defenses.

Antibacterial response of Cd-TiO2/PEG/folic acid nanocomposite under ultraviolet, visible light, or ultrasonic irradiation

Synthesis and characterization of Cd-TiO2/PEG/FA nanocomposite as a biomaterial is the main aim of this research. Cd-doped TiO2 nanoparticles (NPs) were synthesized by solvothermal assisted sol-gel method. Then, polyethylene glycol (PEG) was added to the as-synthesized NPs in order to modify their surface and to prevent agglomeration. In the next step, folic acid (FA) was conjugated to Cd-TiO2/PEG nanocomposite. Optimization of FA concentration and influence of ultraviolet (UV) radiation, visible light, or ultrasonic irradiation on antibacterial activity of the synthesized nanocomposite were assessed. Results showed that the influence of FA on the antibacterial performance of the as-synthesized compounds is positive and dose-dependent. FA was used as bait for bacteria and led them to accumulate around the Cd-TiO2 NPs. The bactericidal response under UV or visible light irradiation was maximized at 0.6 weight ratio of FA to Cd-TiO2, whereas, this parameter under ultrasonic irradiation was improved and then became approximately constant.

Phase 1 trial of a 20-valent pneumococcal conjugate vaccine in healthy adults

IntroductionStreptococcus pneumoniae is a leading cause of bacteremia, bacterial pneumonia, and meningitis, and is associated with substantial morbidity and mortality, particularly in those under 2 years of age and those over 65 years of age. While significant progress against S. pneumoniae-related disease has been made as a result of the introduction of pneumococcal conjugate vaccines (PCV7, PCV10 and PCV13), there remains value in further expanding pneumococcal vaccine serotype coverage. Here we present the first report of a 20-valent pneumococcal conjugate vaccine (PCV20) containing capsular polysaccharide conjugates present in PCV13 as well as 7 new serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) which are important contributors to pneumococcal disease. MethodsThis Phase I first-in-human study was a randomized, controlled, observer-blinded study with a two-arm parallel design to assess the safety, tolerability, and immunogenicity of PCV20 in adults. A total of 66 healthy adults 18–49 years of age with no history of pneumococcal vaccination were enrolled and randomized to receive a single dose of PCV20 or a licensed tetanus, diphtheria, acellular pertussis combination vaccine (Tdap) control. Local injection site reactions, select systemic symptoms, laboratory studies, and adverse events were assessed. Opsonophagocytic activity (OPA) titers and IgG concentrations were measured in sera collected prior to, and approximately one month (28–35 days) after vaccination. ResultsVaccination with PCV20 elicited substantial IgG and functional bactericidal immune responses as demonstrated by increases in IgG geometric mean concentrations (GMCs) and OPA geometric mean titers (GMTs) to the 20 vaccine serotypes. The overall safety profile of PCV20 was similar to Tdap, and generally consistent with that observed after PCV13 administration. ConclusionsVaccination with PCV20 was well tolerated and induced substantial functional (OPA) and IgG responses to all vaccine serotypes. There were no safety issues identified in this Phase 1 study, and the data supported further evaluation of PCV20.

Influence of Ag-Cu nanoparticles on the microstructural and bactericidal properties of TiAlN(Ag,Cu) coatings for medical applications deposited by Direct Current (DC) magnetron sputtering

Most medical instrumentation is manufactured using AISI 420 martensitic stainless steel, due to its acceptable biocompatibility, good hardenability, adequate hardness and resistance to corrosion. However, this steel can be susceptible to contamination with bacteria, representing a potential risk to the patient's health. This research work focused on the development of titanium‑aluminum-nitride coatings doped with silver and copper nanoparticles, TiAlN (Ag,Cu), with an appropriate bactericidal effect. TiAlN coatings doped with four different contents of Ag and Cu (11 at.% to 20 at.%) were deposited onto AISI 420 steel by means of DC unbalanced magnetron sputtering using two composite targets of Ti/Al and Ag/Cu. The diffusion of the Ag,Cu nanoparticles at the sample surface, as well as their quantity, size, shape and distribution, was controlled by appropriate adjustment of the power applied to the Ag/Cu- target, and the temperature and time of the deposition process. The mechanical, tribological and bactericidal properties of the compound depend on, among other factors, the above-mentioned characteristics of the nanoparticles. The microstructure, surface topography, and chemical and phase composition were analyzed by scanning and transmission electron microscopy, energy dispersive X-ray spectroscopy and X-ray diffraction. To evaluate the bactericidal effect of the coatings, in vitro inhibition and adhesion tests were carried out using selected Staphylococcus aureus and Escherichia coli, two of the major pathogens associated with infections frequently found in hospital environments. The coating without doping presented a structure of columnar growth, which became densified with the increase in the Ag-Cu content and took on a glassy appearance, accompanied by an increased size of the Ag-Cu particles and consequently also in the surface roughness. TiAlN(Ag,Cu) with 17 and 20 at.% Ag-Cu exhibited a higher bactericidal effect than TiAlN, showing high inhibition and less adhesion to both bacteria strains. Another series of techniques that comprehensively describe the relationship between external factors and the bactericidal response of the material were made. Based on the results obtained, the developed nanostructured coating system can be considered very promising for potential applications in surgical and dental instrumentation.

Effect on meningococcal serogroup W immunogenicity when Tdap was administered prior, concurrent or subsequent to the quadrivalent (ACWY) meningococcal CRM197-conjugate vaccine in adult Hajj pilgrims: A randomised controlled trial

Immune responses to the capsular polysaccharide administered in the polysaccharide-protein conjugate vaccines can be either improved or suppressed by the pre-existence of immunity to the carrier protein. Receiving multiple vaccinations is essential for travellers such as Hajj pilgrims, and the use of conjugated vaccines is recommended.We studied the immune response to meningococcal serogroup W upon prior, concurrent and sequential administration of a quadrivalent meningococcal conjugate vaccine (MCV4) conjugated to CRM197 (coadministered with 13 valent pneumococcal vaccine conjugate CRM197 [PCV13]), and tetanus-diphtheria-acellular pertussis (Tdap) vaccine in Australian adults before attending the Hajj pilgrimage in 2014.Participants were randomly assigned, by computer-generated numbers, to three study arms by 1:1:1 ratio. Group A received Tdap followed by MCV4-CRM197 (+PCV13) 3–4 weeks later. Group B received all three vaccines in a single visit. Group C received MCV4-CRM197 (+PCV13) followed by Tdap 3–4 weeks later. Blood samples obtained prior to and 3–4 weeks after immunisation with MCV4-CRM197 were tested for meningococcal serogroup W-specific serum bactericidal antibody responses using baby rabbit complement (rSBA).One hundred and seven participants aged between 18 and 64 (median 40) years completed the study. No significant difference in meningococcal serogroup W rSBA geometric mean titre (GMT) was observed between the study arms post vaccination with MCV-CRM197 but Group A tended to have a slightly lower GMT (A = 404, B = 984 and C = 1235, p = 0.15). No statistical difference was noticed between the groups in proportions of subjects achieving a ≥4-fold rise in rSBA titres or achieving rSBA titre ≥8 post vaccination.In conclusion, receipt of MCV4-CRM197 vaccine prior, concurrent or subsequent to Tdap has similar immunologic response, and hence concurrent administration is both immunogenic and practical. However, further investigation into whether carrier induced suppression is a public health issue is suggested.Clinical trial registration: ANZCTR no. ACTRN12613000536763.

A mycobacteriophage genomics approach to identify novel mycobacteriophage proteins with mycobactericidal properties.

Mycobacteriophages that are specific to mycobacteria are sources of various effector proteins that are capable of eliciting bactericidal responses. We describe a genomics approach in combination with bioinformatics to identify mycobacteriophage proteins that are toxic to mycobacteria upon expression. A genomic library comprising phage genome collections was screened for clones capable of killing Mycobacterium smegmatis strain mc2155. We identified four unique clones: clones 45 and 12N (from the mycobacteriophage D29) and clones 66 and 85 (from the mycobacteriophage Che12). The gene products from clones 66 and 45 were identified as Gp49 of the Che12 phage and Gp34 of the D29 phage, respectively. The gene products of the other two clones, 85 and 12N, utilized novel open reading frames (ORFs) coding for synthetic proteins. These four clones (clones 45, 66, 85 and 12N) caused growth defects in M. smegmatis and Mycobacterium bovis upon expression. Clones with Gp49 and Gp34 also induced growth defects in Escherichia coli, indicating that they target conserved host machineries. Their expression induced various morphological changes, indicating that they affected DNA replication and cell division steps. We predicted that Gp34 is a Xis protein that is required in phage DNA excision from the bacterial chromosome. Gp49 is predicted to have an HTH motif with DNA-bending/twisting properties. We suggest that this methodology is useful to identify new phage proteins with the desired properties without laboriously characterizing the individual phages. It is universal and could be applied to other bacteria-phage systems. We speculate that the existence of a virtually unlimited number of phages with unique gene products could offer a cheaper and less hazardous alternative to explore new antimicrobial molecules.

Klebsiella pneumoniae ST258 Negatively Regulates the Oxidative Burst in Human Neutrophils.

The epidemic clone of Klebsiella pneumoniae (Kpn), sequence type 258 (ST258), carbapenamase producer (KPC), commonly infects hospitalized patients that are left with scarce therapeutic option since carbapenems are last resort antibiotics for life-threatening bacterial infections. To improve prevention and treatment, we should better understand the biology of Kpn KPC ST258 infections. Our hypothesis was that Kpn KPC ST258 evade the first line of defense of innate immunity, the polymorphonuclear neutrophil (PMN), by decreasing its functional response. Therefore, our aim was to evaluate how the ST258 Kpn clone affects PMN responses, focusing on the respiratory burst, compared to another opportunistic pathogen, Escherichia coli (Eco). We found that Kpn KPC ST258 was unable to trigger bactericidal responses as reactive oxygen species (ROS) generation and NETosis, compared to the high induction observed with Eco, but both bacterial strains were similarly phagocytized and cause increases in cell size and CD11b expression. The absence of ROS induction was also observed with other Kpn ST258 strains negative for KPC. These results reflect certain selectivity in terms of the functions that are triggered in PMN by Kpn, which seems to evade specifically those responses critical for bacterial survival. In this sense, bactericidal mechanisms evasion was associated with a higher survival of Kpn KPC ST258 compared to Eco. To investigate the mechanisms and molecules involved in ROS inhibition, we used bacterial extracts (BE) and found that BE were able to inhibit ROS generation triggered by the well-known ROS inducer, fMLP. A sequence of experiments led us to elucidate that the polysaccharide part of LPS was responsible for this inhibition, whereas lipid A mediated the other responses that were not affected by bacteria, such as cell size increase and CD11b up-regulation. In conclusion, we unraveled a mechanism of immune evasion of Kpn KPC ST258, which may contribute to design more effective strategies for the treatment of these multi-resistant bacterial infections.

Analysis of the antigenic determinants of the OspC protein of the Lyme disease spirochetes: Evidence that the C10 motif is not immunodominant or required to elicit bactericidal antibody responses

As Ixodes ticks spread to new regions, the incidence of Lyme disease (LD) in companion animals and humans will increase. Preventive strategies for LD in canines center on vaccination and tick control (acaricides). Both subunit and bacterin based LD veterinary vaccines are available. Outer surface protein C (OspC), a potent immunogen and dominant early antigen, has been demonstrated to elicit protective antibody (Ab) responses. However, a single OspC protein elicits a relatively narrow range of protection. There are conflicting reports as to whether the immunodominant epitopes of OspC reside within variable or conserved domains. A detailed understanding of the antigenic determinants of OspC is essential for understanding immune responses to this essential virulence factor and vaccinogen. Here, we investigate the contribution of the conserved C-terminal C10 motif in OspC triggered Ab responses. Using a panel of diverse recombinant full length OspC proteins and their corresponding C10 deletion variants (OspCΔC10), we demonstrate that the C10 motif does not significantly contribute to immunization or infection induced Ab responses in rabbits, rats, canines, horses and non-human primates. Furthermore, the C10 motif is not required to trigger potent bactericidal Ab responses. This study provides insight into the antigenic structure of OspC. The results enhance our understanding of immune responses that develop during infection or upon vaccination and have implications for interpretation of LD diagnostic assays that employ OspC.

Utility of Hybrid Transferrin Binding Protein Antigens for Protection Against Pathogenic Neisseria Species.

The surface transferrin receptor proteins from Neisseria gonorrhoeae have been recognized as ideal vaccine targets due to their critical role in survival in the human male genitourinary tract. Recombinant forms of the surface lipoprotein component of the receptor, transferrin binding protein B (TbpB), can be readily produced at high levels in the Escherichia coli cytoplasm and is suitable for commercial vaccine production. In contrast, the integral outer membrane protein, transferrin binding protein A (TbpA), is produced at relatively low levels in the outer membrane and requires detergents for solubilization and stabilization, processes not favorable for commercial applications. Capitalizing on the core β-barrel structural feature common to the lipoprotein and integral outer membrane protein we engineered the lipoprotein as a scaffold for displaying conserved surface epitopes from TbpA. A stable version of the C-terminal domain of TbpB was prepared by replacing four larger exposed variable loops with short linking peptide regions. Four surface regions from the plug and barrel domains of Neisseria TbpA were transplanted onto this TbpB C-lobe scaffold, generating stable hybrid antigens. Antisera generated in mice and rabbits against the hybrid antigens recognized TbpA at the surface of Neisseria meningitidis and inhibited transferrin-dependent growth at levels comparable or better than antisera directed against the native TbpA protein. Two of the engineered hybrid antigens each elicited a TbpA-specific bactericidal antibody response comparable to that induced by TbpA. A hybrid antigen generated using a foreign scaffold (TbpB from the pig pathogen Haemophilus parasuis) displaying neisserial TbpA loop 10 was evaluated in a model of lower genital tract colonization by N. gonorrhoeae and a model of invasive infection by N. meningitidis. The loop 10 hybrid antigen was as effective as full length TbpA in eliminating N. gonorrhoeae from the lower genital tract of female mice and was protective against the low dose invasive infection by N. meningitidis. These results demonstrate that TbpB or its derivatives can serve as an effective scaffold for displaying surface epitopes of integral outer membrane antigens and these antigens can elicit protection against bacterial challenge.

Persistence and 4-year boosting of the bactericidal response elicited by two- and three-dose schedules of MenB-FHbp: A phase 3 extension study in adolescents

Persistence and 4-year boosting of the bactericidal response elicited by two- and three-dose schedules of MenB-FHbp: A phase 3 extension study in adolescents

Safety and Immunogenicity of Two Doses of a Quadrivalent Meningococcal Polysaccharide Diphtheria Toxoid Conjugate Vaccine in Indian and Russian Children Aged 9 to 17 Months

Evaluation of tolerability, safety and immunogenicity of a two-dose series of a quadrivalent meningococcal polysaccharide diptheria toxoid conjugate (ACYW-D) vaccine in Indian and Russian infants/toddlers. Open-label, single-arm, phase III multi-national trial. 300 children aged 9-17 months, previously unvaccinated against meningococcal disease from four sites each in India (n=200) and the Russian Federation (n=100). Two 0.5 mL doses of ACYW-D by intramuscular injection, 3-6 months apart. Meningococcal antibody titers to serogroups A, C, W-135 and Y, determined using a serum bactericidal assay in the presence of human complement before vaccination and 28 days after the second vaccination. Titers ≥1:8 against either/all of the A, C, W-135 or Y were considered sero-protective. After dose 2, 95.7-99.5% and 92.9-99.0% of infants/toddlers achieved seroprotection across the four serogroups in India and the Russian Federation, respectively. No immediate adverse events were reported after any dose of ACYW-D. Solicited reactions were reported in 49.2% of participants, and were mainly of Grade 1 severity, and resolved within three days. Unsolicited adverse events were reported in 19.1% of infants: one event (Grade 3 diarrhea, resolving within one day) was considered related to study vaccine. No non-serious adverse events led to premature withdrawal from the study. Four serious adverse events were reported; none were considered related to study vaccine. No deaths occurred during the study. A two-dose series of ACYW-D vaccine in Indian and Russian children (9-17 month) was well-tolerated with no safety concerns, and induced robust bactericidal antibody responses against the meningococcal serogroups contained in the vaccine.

The bivalent factor H binding protein meningococcal serogroup B vaccine elicits bactericidal antibodies against representative non-serogroup B meningococci

MenB-FHbp (Trumenba®; bivalent rLP2086) is a meningococcal serogroup B vaccine containing 2 variants of the recombinant lipidated factor H binding protein (FHbp) antigen. The expression of FHbp, an outer membrane protein, is not restricted to serogroup B strains of Neisseria meningitidis (MenB). This study investigated whether antibodies elicited by MenB-FHbp vaccination also protect against non-MenB strains. Immunological responses were assessed in serum bactericidal assays using human complement (hSBAs) with non-MenB disease-causing test strains from Europe, Africa, and the United States. Importantly, FHbp variant distribution varies among meningococcal serogroups; therefore, strains that code for serogroup-specific prevalent variants (ie, representative of the 2 antigenically distinct FHbp subfamilies, designated subfamily A and subfamily B) and with moderate levels of FHbp surface expression were selected for testing by hSBA. After 2 or 3 doses of MenB-FHbp, 53% to 100% of individuals had bactericidal responses (hSBA titers ≥ 1:8) against meningococcal serogroup C, W, Y, and X strains, and 20% to 28% had bactericidal responses against serogroup A strains; in fact, these bactericidal responses elicited by MenB-FHbp antibodies against non-MenB strains, including strains associated with emerging disease, were greater than the serological correlate of protection for meningococcal disease (ie, hSBA titers ≥ 1:4). This is in comparison to a quadrivalent polysaccharide conjugate vaccine, MCV4 (Menactra®, targeting meningococcal serogroups A, C, W, and Y), which elicited bactericidal responses of 90% to 97% against the serogroup A, C, W, and Y strains and had no activity against serogroup X. Together, these results provide clinical evidence that MenB-FHbp may protect against meningococcal disease regardless of serogroup.

Methionine and choline supply during the peripartal period alter polymorphonuclear leukocyte immune response and immunometabolic gene expression in Holstein cows

Polymorphonuclear leukocytes (PMNL) are the first responders upon pathogen invasion and hence play an important role in inflammatory and immune responses. Rumen-protected methionine (MET) and choline (CHOL) during the peripartal period affect the immune response and inflammatory status in dairy cows to different extents. We aimed to examine the effect of MET and CHOL supply on expression of genes regulating key PMNL functions and associations with whole-blood immune challenge. Thirty multiparous Holstein cows from a larger cohort randomly assigned from -21 to 30 d relative to parturition to a basal control (CON) diet, CON plus MET at a rate of 0.08% of dry matter, or CON plus CHOL at 60 g/d were used. Blood was sampled at -10, 7, and 30 d relative to parturition for inflammatory biomarker analyses and PMNL isolation. Neutrophil and monocyte phagocytosis and oxidative burst in vitro were assessed in whole blood at 1, 7, and 28 d. Although neutrophil and monocyte phagocytosis did not differ, oxidative burst in neutrophils and monocytes was greater in MET-supplemented cows relative to CON cows. Compared with CON, PMNL adhesion and migration-related genes (ITGAM, ITGB2, ITGA4) were downregulated in response to MET and CHOL. Expression of CADM1 and SELL was also lower in MET-supplemented cows compared with CON cows but not in CHOL cows. In contrast, compared with CON cows, the expression of ICAM1 was lower in CHOL but not MET cows. Similar to adhesion and migration-related genes, cows receiving MET- or CHOL-supplemented diets had lower expression of inflammation-related genes (IL1β, IL10RA, NFKB1, STAT3, TLR2). However, expression of IRAK1 and TLR4 was lower in MET- but not CHOL-supplemented cows. Plasma taurine concentration was greater in MET cows compared with CHOL and CON cows, suggesting a better redox status in plasma. In agreement with plasma taurine, oxidative stress-related genes (CBS, CTH, GPX1, GSS, SOD2) in PMNL were lower in response to MET and to CHOL supply. Overall, immunometabolic gene expression profile and blood biomarker analyses suggest an overall better redox status in PMNL during the transition period in response to MET and CHOL supply. These adaptations in PMNL might be beneficial for mounting a better bactericidal response upon challenge.