Modification of avian pathogenic Escherichia coli χ7122 lipopolysaccharide increases accessibility to glycoconjugate antigens.

BACKGROUND: Worldwide, an estimated 70.7 billion broilers were produced in 2020. With the reduction in use of prophylactic antibiotics as a result of consumer pressure and regulatory oversight alternative approaches, such as vaccination, are required to control bacterial infections. A potential way to produce a multivalent vaccine is via the generation of a glycoconjugate vaccine which consists of an antigenic protein covalently linked to an immunogenic carbohydrate. Protein-glycan coupling technology (PGCT) is an approach to generate glycoconjugates using enzymes that can couple proteins and glycan when produced in bacterial cells. Previous studies have used PGCT to generate a live-attenuated avian pathogenic Escherichia coli (APEC) strain capable of N-glycosylation of target proteins using a chromosomally integrated Campylobacter jejuni pgl locus. However, this proved ineffective against C. jejuni challenge. RESULTS: In this study we demonstrate the lack of surface exposure of glycosylated protein in APEC strain χ7122 carrying the pgl locus. Furthermore, we hypothesise that this may be due to the complex cell-surface architecture of E. coli. To this end, we removed the lipopolysaccharide O-antigen of APEC χ7122 pgl+ via deletion of the wecA gene and demonstrate increased surface exposure of glycosylated antigens (NetB and FlpA) in this strain. We hypothesise that increasing the surface expression of the glycosylated protein would increase the chance of host immune cells being exposed to the glycoconjugate, and therefore the generation of an efficacious immune response would be more likely. CONCLUSIONS: Our results demonstrate an increase in cell surface exposure and therefore accessibility of glycosylated antigens upon removal of lipopolysaccharide antigen from the APEC cell surface.

PglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome.

BACKGROUND: Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni. RESULTS: We generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain. CONCLUSIONS: We determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level.

Multivalent poultry vaccine development using Protein Glycan Coupling Technology.

BACKGROUND: Poultry is the world's most popular animal-based food and global production has tripled in the past 20 years alone. Low-cost vaccines that can be combined to protect poultry against multiple infections are a current global imperative. Glycoconjugate vaccines, which consist of an immunogenic protein covalently coupled to glycan antigens of the targeted pathogen, have a proven track record in human vaccinology, but have yet to be used for livestock due to prohibitively high manufacturing costs. To overcome this, we use Protein Glycan Coupling Technology (PGCT), which enables the production of glycoconjugates in bacterial cells at considerably reduced costs, to generate a candidate glycan-based live vaccine intended to simultaneously protect against Campylobacter jejuni, avian pathogenic Escherichia coli (APEC) and Clostridium perfringens. Campylobacter is the most common cause of food poisoning, whereas colibacillosis and necrotic enteritis are widespread and devastating infectious diseases in poultry. RESULTS: We demonstrate the functional transfer of C. jejuni protein glycosylation (pgl) locus into the genome of APEC χ7122 serotype O78:H9. The integration caused mild attenuation of the χ7122 strain following oral inoculation of chickens without impairing its ability to colonise the respiratory tract. We exploit the χ7122 pgl integrant as bacterial vectors delivering a glycoprotein decorated with the C. jejuni heptasaccharide glycan antigen. To this end we engineered χ7122 pgl to express glycosylated NetB toxoid from C. perfringens and tested its ability to reduce caecal colonisation of chickens by C. jejuni and protect against intra-air sac challenge with the homologous APEC strain. CONCLUSIONS: We generated a candidate glycan-based multivalent live vaccine with the potential to induce protection against key avian and zoonotic pathogens (C. jejuni, APEC, C. perfringens). The live vaccine failed to significantly reduce Campylobacter colonisation under the conditions tested but was protective against homologous APEC challenge. Nevertheless, we present a strategy towards the production of low-cost "live-attenuated multivalent vaccine factories" with the ability to express glycoconjugates in poultry.

Mitigating and monitoring flight crew fatigue on a westward ultra-long-range flight.

BACKGROUND: This study examined the uptake and effectiveness of fatigue mitigation guidance material including sleep recommendations for a trip with a westward ultra-long-range flight and return long-range flight. METHODS: There were 52 flight crew (4-pilot crews, mean age 55 yr) who completed a sleep/duty diary and wore an actigraph prior to, during, and after the trip. Primary crew flew the takeoff and landing, while relief crew flew the aircraft during the Primary crew's breaks. At key times in flight, crewmembers rated their fatigue (Samn-Perelli fatigue scale) and sleepiness (Karolinska Sleepiness Scale) and completed a 5-min Psychomotor Vigilance Task. RESULTS: Napping was common prior to the outbound flight (54%) and did not affect the quantity or quality of in-flight sleep (mean 4.3 h). Primary crew obtained a similar amount on the inbound flight (mean 4.0 h), but Secondary crew had less sleep (mean 2.9 h). Subjective fatigue and sleepiness increased and performance slowed across flights. Performance was faster on the outbound than inbound flight. On both flights, Primary crew were less fatigued and sleepy than Secondary crew, particularly at top of descent and after landing. Crewmembers slept more frequently and had more sleep in the first 24 h of the layover than the last, and had shifted their main sleep to the local night by the second night. DISCUSSION: The suggested sleep mitigations were employed by the majority of crewmembers. Fatigue levels were no worse on the outbound ultra-long-range flight than on the return long-range flight.

A "suicide" BCG strain provides enhanced immunogenicity and robust protection against Mycobacterium tuberculosis in macaques.

Intravenous (IV) BCG delivery provides robust protection against Mycobacterium tuberculosis (Mtb) in macaques but poses safety challenges. Here, we constructed two BCG strains (BCG-TetON-DL and BCG-TetOFF-DL) in which tetracyclines regulate two phage lysin operons. Once the lysins are expressed, these strains are cleared in immunocompetent and immunocompromised mice, yet induced similar immune responses and provided similar protection against Mtb challenge as wild type BCG. Lysin induction resulted in release of intracellular BCG antigens and enhanced cytokine production by macrophages. In macaques, cessation of doxycycline administration resulted in rapid elimination of BCG-TetOFF-DL. However, IV BCG-TetOFF-DL induced increased pulmonary CD4 T cell responses compared to WT BCG and provided robust protection against Mtb challenge, with sterilizing immunity in 6 of 8 macaques, compared to 2 of 8 macaques immunized with WT BCG. Thus, a "suicide" BCG strain provides an additional measure of safety when delivered intravenously and robust protection against Mtb infection.

Monitoring and Managing Cabin Crew Sleep and Fatigue During an Ultra-Long Range Trip.

BACKGROUND: The aims of this study were to monitor cabin crew fatigue, sleep, and performance on an ultra-long range (ULR) trip and to evaluate the appropriateness of applying data collection methods developed for flight crew to cabin crew operations under a fatigue risk management system (FRMS). METHODS: Prior to, throughout, and following the ULR trip (outbound flight ULR; mean layover duration=52.6 h; inbound flight long range), 55 cabin crew (29 women; mean age 36.5 yr; 25 men; mean age 36.6 yr; one missing data) completed a sleep/duty diary and wore an actigraph. Across each flight, crewmembers rated their fatigue (Samn-Perelli Crew Status Check) and sleepiness (Karolinska Sleepiness Scale) and completed a 5-min Psychomotor Vigilance Task (PVT) at key times. RESULTS: Of crewmembers approached, 73% (N=134) agreed to participate and 41% (N=55) provided data of suitable quality for analysis. In the 24 h before departure, sleep averaged 7.0 h and 40% took a preflight nap. All crewmembers slept in flight (mean total sleep time=3.6 h outbound, 2.9 h inbound). Sleepiness and fatigue were lower, and performance better, on the longer outbound flight than on the inbound flight. Post-trip, crewmembers slept more on day 1 (mean=7.9 h) compared to baseline days, but there was no difference from day 2 onwards. DISCUSSION: The present study demonstrates that cabin crew fatigue can be managed effectively on a ULR flight and that FRMS data collection is feasible for cabin crew, but operational differences between cabin crew and flight crew need to be considered.

Comparative genomics of aeschynomene symbionts: insights into the ecological lifestyle of nod-independent photosynthetic bradyrhizobia.

Tropical aquatic species of the legume genus Aeschynomene are stem- and root-nodulated by bradyrhizobia strains that exhibit atypical features such as photosynthetic capacities or the use of a nod gene-dependent (ND) or a nod gene-independent (NI) pathway to enter into symbiosis with legumes. In this study we used a comparative genomics approach on nine Aeschynomene symbionts representative of their phylogenetic diversity. We produced draft genomes of bradyrhizobial strains representing different phenotypes: five NI photosynthetic strains (STM3809, ORS375, STM3847, STM4509 and STM4523) in addition to the previously sequenced ORS278 and BTAi1 genomes, one photosynthetic strain ORS285 hosting both ND and NI symbiotic systems, and one NI non-photosynthetic strain (STM3843). Comparative genomics allowed us to infer the core, pan and dispensable genomes of Aeschynomene bradyrhizobia, and to detect specific genes and their location in Genomic Islands (GI). Specific gene sets linked to photosynthetic and NI/ND abilities were identified, and are currently being studied in functional analyses.