Reduced Respiratory Syncytial Virus Load, Symptoms, and Infections: A Human Challenge Trial of MVA-BN-RSV Vaccine.

BACKGROUND: Respiratory syncytial virus (RSV) causes significant disease burden in older adults. MVA-BN-RSV is a novel poxvirus-vectored vaccine encoding internal and external RSV proteins. METHODS: In a phase 2a randomized double-blind, placebo-controlled trial, healthy participants aged 18 to 50 years received MVA-BN-RSV or placebo, then were challenged 4 weeks later with RSV-A Memphis 37b. Viral load was assessed from nasal washes. RSV symptoms were collected. Antibody titers and cellular markers were assessed before and after vaccination and challenge. RESULTS: After receiving MVA-BN-RSV or placebo, 31 and 32 participants, respectively, were challenged. Viral load areas under the curve from nasal washes were lower (P = .017) for MVA-BN-RSV (median = 0.00) than placebo (median = 49.05). Total symptom scores also were lower (median = 2.50 and 27.00, respectively; P = .004). Vaccine efficacy against symptomatic, laboratory-confirmed or culture-confirmed infection was 79.3% to 88.5% (P = .022 and .013). Serum immunoglobulin A and G titers increased approximately 4-fold after MVA-BN-RSV vaccination. Interferon-γ-producing cells increased 4- to 6-fold after MVA-BN-RSV in response to stimulation with the encoded RSV internal antigens. Injection site pain occurred more frequently with MVA-BN-RSV. No serious adverse events were attributed to vaccination. CONCLUSIONS: MVA-BN-RSV vaccination resulted in lower viral load and symptom scores, fewer confirmed infections, and induced humoral and cellular responses. CLINICAL TRIALS REGISTRATION: NCT04752644.

Deciphering the metabolic response of Mycobacterium tuberculosis to nitrogen stress.

A key component to the success of Mycobacterium tuberculosis as a pathogen is the ability to sense and adapt metabolically to the diverse range of conditions encountered in vivo, such as oxygen tension, environmental pH and nutrient availability. Although nitrogen is an essential nutrient for every organism, little is known about the genes and pathways responsible for nitrogen assimilation in M. tuberculosis. In this study we have used transcriptomics and chromatin immunoprecipitation and high-throughput sequencing to address this. In response to nitrogen starvation, a total of 185 genes were significantly differentially expressed (96 up-regulated and 89 down regulated; 5% genome) highlighting several significant areas of metabolic change during nitrogen limitation such as nitrate/nitrite metabolism, aspartate metabolism and changes in cell wall biosynthesis. We identify GlnR as a regulator involved in the nitrogen response, controlling the expression of at least 33 genes in response to nitrogen limitation. We identify a consensus GlnR binding site and relate its location to known transcriptional start sites. We also show that the GlnR response regulator plays a very different role in M. tuberculosis to that in non-pathogenic mycobacteria, controlling genes involved in nitric oxide detoxification and intracellular survival instead of genes involved in nitrogen scavenging.

Genome wide analysis of the complete GlnR nitrogen-response regulon in Mycobacterium smegmatis.

BACKGROUND: Nitrogen is an essential element for bacterial growth and an important component of biological macromolecules. Consequently, responding to nitrogen limitation is critical for bacterial survival and involves the interplay of signalling pathways and transcriptional regulation of nitrogen assimilation and scavenging genes. In the soil dwelling saprophyte Mycobacterium smegmatis the OmpR-type response regulator GlnR is thought to mediate the transcriptomic response to nitrogen limitation. However, to date only ten genes have been shown to be in the GlnR regulon, a vastly reduced number compared to other organisms. RESULTS: We investigated the role of GlnR in the nitrogen limitation response and determined the entire GlnR regulon, by combining expression profiling of M. smegmatis wild type and glnR deletion mutant, with GlnR-specific chromatin immunoprecipitation and high throughput sequencing. We identify 53 GlnR binding sites during nitrogen limitation that control the expression of over 100 genes, demonstrating that GlnR is the regulator controlling the assimilation and utilisation of nitrogen. We also determine a consensus GlnR binding motif and identify key residues within the motif that are required for specific GlnR binding. CONCLUSIONS: We have demonstrated that GlnR is the global nitrogen response regulator in M. smegmatis, directly regulating the expression of more than 100 genes. GlnR controls key nitrogen stress survival processes including primary nitrogen metabolism pathways, the ability to utilise nitrate and urea as alternative nitrogen sources, and the potential to use cellular components to provide a source of ammonium. These studies further our understanding of how mycobacteria survive nutrient limiting conditions.

Deciphering the response of Mycobacterium smegmatis to nitrogen stress using bipartite active modules.

BACKGROUND: The ability to adapt to environments with fluctuating nutrient availability is vital for bacterial survival. Although essential for growth, few nitrogen metabolism genes have been identified or fully characterised in mycobacteria and nitrogen stress survival mechanisms are unknown. RESULTS: A global transcriptional analysis of the mycobacterial response to nitrogen stress, showed a significant change in the differential expression of 16% of the Mycobacterium smegmatis genome. Gene expression changes were mapped onto the metabolic network using Active Modules for Bipartite Networks (AMBIENT) to identify metabolic pathways showing coordinated transcriptional responses to the stress. AMBIENT revealed several key features of the metabolic response not identified by KEGG enrichment alone. Down regulated reactions were associated with the general reduction in cellular metabolism as a consequence of reduced growth rate. Up-regulated modules highlighted metabolic changes in nitrogen assimilation and scavenging, as well as reactions involved in hydrogen peroxide metabolism, carbon scavenging and energy generation. CONCLUSIONS: Application of an Active Modules algorithm to transcriptomic data identified key metabolic reactions and pathways altered in response to nitrogen stress, which are central to survival under nitrogen limiting environments.

The Quest for a Respiratory Syncytial Virus Vaccine for Older Adults: Thinking beyond the F Protein.

Respiratory syncytial virus (RSV) is a common cause of paediatric respiratory tract infection and causes a significant health burden in older adults. Natural immunity to RSV is incomplete, permitting recurrent symptomatic infection over an individual's lifespan. When combined with immunosenescence, this increases older adults' susceptibility to more severe disease symptoms. As RSV prophylaxis is currently limited to infants, older adults represent an important target population for RSV vaccine development. The relationship between RSV and our immune systems is complex, and these interactions require deeper understanding to tailor an effective vaccine candidate towards older adults. To date, vaccine candidates targeting RSV antigens, including pre-F, F, G (A), G (B), M2-1, and N, have shown efficacy against RSV infection in older adults in clinical trial settings. Although vaccine candidates have demonstrated robust neutralising IgG and cellular responses, it is important that research continues to investigate the RSV immune response in order to further understand how the choice of antigenic target site may impact vaccine effectiveness. In this article, we discuss the Phase 3 vaccine candidates being tested in older adults and review the hurdles that must be overcome to achieve effective protection against RSV.

Free glucosylglycerate is a novel marker of nitrogen stress in Mycobacterium smegmatis.

Nitrogen is an essential element for bacterial growth, and as such, bacteria have evolved several pathways to assimilate nitrogen and adapt to situations of nitrogen limitation. However, the adaptation of mycobacteria to nitrogen stress and the regulation of the stress response pathways is unknown. Identification of key metabolites produced by mycobacteria during nitrogen stress could therefore provide important insights into mycobacterial survival strategies. Here we used NMR-based metabolomics to monitor and quantify intracellular and extracellular metabolite levels (metabolic footprinting) in Mycobacterium smegmatis grown under nitrogen-limiting and nitrogen-rich conditions. There were several metabolic differences between the two conditions: following nitrogen run-out, there was an increase in intracellular α-ketoglutarate and a decrease in intracellular glutamine and glutamate levels. In addition, a sugar-derived compound accumulated in nitrogen-starved cells that was subsequently assigned as glucosylglycerate (GGA). Free GGA production was responsive to nitrogen stress in M. smegmatis but not to oxidative or osmotic stress; lack of a functional GGA synthesis pathway slightly reduced growth and decreased ammonium uptake rates under nitrogen-limiting conditions. Hence, GGA could contribute to the fitness of mycobacteria under nitrogen limitation.

The epidemiology of infectious diseases in Europe in 2020 versus 2017-2019 and the rise of tick-borne encephalitis (1995-2020).

Health control measures instituted in 2020 to mitigate the COVID-19 pandemic decreased the case numbers of many infectious diseases across Europe. One notable exception was tick-borne encephalitis (TBE). In Austria, Germany, Switzerland, Lithuania, and the Czech Republic, the upturn was significantly higher compared to the average of the three years previously (P<0.05), with increases of 88%, 48%, 51%, 28%, and 18%, respectively. Six countries reported TBE incidences of ≥5 cases/100,000, defined as highly endemic by the World Health Organization (WHO). Possible factors contributing to this surge may include increased participation in outdoor activities in endemic regions and increased tick counts/tick activity. In highly endemic regions, the WHO recommends that vaccination be offered to all age groups, including children.

Perceptions of vaccine preventable diseases in Australian healthcare: focus on pertussis.

Adult vaccination in Australia is suboptimal. For instance, as few as one in nine people have received a pertussis vaccine in adolescence or adulthood, despite increasing disease burden and evidence of a positive correlation between older age and hospitalization rates. The objectives of this study were to describe general practitioners' (GPs) and adult consumers' knowledge and attitudes toward adult vaccination, with an emphasis on pertussis. Australian GPs and consumers were recruited in two nationally representative online surveys repeated annually between 2014 and 2018. Vaccination discussions occurred in a minority of adult/GP encounters. Pertussis was among the five most frequently identified vaccine preventable diseases but was unlikely to be proactively discussed with adults not in contact with young children. Among consumers, only one in three recalled ever receiving a pertussis vaccination. GPs are a strong predictor of adults receiving a pertussis vaccine. Possible factors contributing to low uptake are misconceptions around pertussis disease, vaccination requirements and lack of GP recommendation for adult vaccination. GPs have a key role to play in increasing adult vaccination coverage with their recommendation.

Pertussis in high-risk groups: an overview of the past quarter-century.

Infectious diseases can impact chronic medical conditions. However, it is currently not clear how pertussis correlates with preexisting or underlying disorders. We reviewed literature from the last 25 years to describe the burden and impact of pertussis infection in specific risk groups in individuals aged ≥11 years. Our literature search returned 543 hits, of which 18 were eligible for this review. Adolescents and adults with underlying conditions, such as asthma, chronic obstructive pulmonary disease (COPD), or obesity are potentially at increased risk of pertussis infection. Immunodeficiency and smoking have also been associated with worsened pertussis symptoms and an increased pertussis-related hospitalization rate. In patients with pertussis and preexisting asthma or COPD, symptoms were worsened, and health-care costs were consequently increased. Further efforts are needed to close the knowledge gap and to understand the burden of pertussis in at-risk adolescent and adult populations to help inform vaccination strategies and recommendations.

A systematic review of the burden of pertussis disease in infants and the effectiveness of maternal immunization against pertussis.

Introduction Infants too young to be fully immunized are the most vulnerable to severe pertussis disease. To close this susceptibility gap, passive infant immunization through vaccination of pregnant women against pertussis was first introduced in 2011 in the United States and has been extended since then to more than 40 countries. Areas covered We conducted two systematic literature searches to describe the worldwide burden of pertussis disease in infants <6 months of age since 2005, and the effectiveness and impact of maternal pertussis vaccination in preventing infant pertussis since 2011. Expert opinion Pertussis disease incidence rates in infants aged <2-3 months were substantial in all countries with available data, exceeding 1000 cases per 100,000 population during outbreaks. Virtually all pertussis deaths occurred in this age group. Data from Africa, Eastern Mediterranean, and Asia were limited, but suggest a similar or higher disease burden than in Europe or the Americas. Estimates of effectiveness of second/third trimester pertussis vaccination in preventing pertussis disease in <2-3 months old infants were consistently high (69%-93%) across the observational studies reviewed, conducted in various settings with different designs. Maternal vaccination programs appear to be achieving their goal of reducing the burden of disease in very young infants. Plain language summary What is the context? Pertussis, also known as whooping cough, is a highly contagious disease of the respiratory tract. Infants too young to be fully vaccinated are at the highest risk of severe pertussis disease, hospitalization, and death. Vaccinating pregnant women against pertussis with a Tdap vaccine is recommended in more than 40 countries as a safe and effective strategy to protect infants for the first months of life. What is new? This review summarizes recent literature describing the burden of pertussis disease in infants worldwide prior to the introduction of maternal vaccination programs; pertussis disease incidence rates in infants aged <2-3 months were substantial in all countries with available data, exceeding 1000 cases per 100,000 population during outbreaks. Immunization of pregnant women with a Tdap vaccine can prevent about 70-90% of pertussis disease and up to 90.5% of pertussis hospitalizations in infants under 3 months of age. What is the impact? Limited available data suggest that incidence rates of pertussis disease after the introduction of Tdap maternal immunization have declined in infants. Current knowledge supports the implementation of Tdap maternal immunization programs.

Pharmacokinetic-Pharmacodynamic modelling of intracellular Mycobacterium tuberculosis growth and kill rates is predictive of clinical treatment duration.

Tuberculosis (TB) treatment is long and complex, typically involving a combination of drugs taken for 6 months. Improved drug regimens to shorten and simplify treatment are urgently required, however a major challenge to TB drug development is the lack of predictive pre-clinical tools. To address this deficiency, we have adopted a new high-content imaging-based approach capable of defining the killing kinetics of first line anti-TB drugs against intracellular Mycobacterium tuberculosis (Mtb) residing inside macrophages. Through use of this pharmacokinetic-pharmacodynamic (PK-PD) approach we demonstrate that the killing dynamics of the intracellular Mtb sub-population is critical to predicting clinical TB treatment duration. Integrated modelling of intracellular Mtb killing alongside conventional extracellular Mtb killing data, generates the biphasic responses typical of those described clinically. Our model supports the hypothesis that the use of higher doses of rifampicin (35 mg/kg) will significantly reduce treatment duration. Our described PK-PD approach offers a much needed decision making tool for the identification and prioritisation of new therapies which have the potential to reduce TB treatment duration.

Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation.

PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation.

Aspartate D48 is essential for the GlnR-mediated transcriptional response to nitrogen limitation in Mycobacterium smegmatis.

Nitrogen is an essential element required for bacterial growth and consequently bacteria must adapt to situations of nitrogen limitation for survival. The transcriptional response to nitrogen limitation in Mycobacterium smegmatis is thought to be regulated by GlnR, although, to date, only five nitrogen metabolism genes have been shown to be under its direct control. GlnR belongs to the OmpR family of two-component response regulators that are typically activated by phosphorylation of a conserved aspartate residue. The M. smegmatis GlnR protein contains the highly conserved aspartate residue (D48) corresponding to the phosphorylation sites identified in other OmpR family regulators. In this study, we replaced GlnR D48 with alanine and constructed a GlnR deletion mutant. Under nitrogen-limiting conditions, both the GlnR_D48A and GlnR deletion mutants exhibited reduced growth rates compared with wild type. Transcriptional analysis showed both mutants failed to up-regulate the expression of GlnR-controlled genes under nitrogen-limiting conditions. We therefore demonstrate that the GlnR aspartate (D48) residue is essential for its function as a nitrogen-stress transcriptional response regulator in M. smegmatis.