Intracellular functions and interactions of age-related macular degeneration-associated variant B cystatin C.

Age related macular degeneration (AMD) is the leading cause of blindness among the elderly population. It occurs in two forms, a nonexudative "dry" form which can lead to geographic atrophy of the retinal pigment epithelial (RPE) cells in the region of the macula and an exudative "wet" form which involves neovascularisation from the choroid, through Bruch's membrane and the RPE layer and into the photoreceptor layer causing significant damage. Cystatin C is a cysteine protease inhibitor known to inhibit cathepsin activity. It is a highly abundant transcript in the RPE and is known to be processed through the secretory pathway . A variant form of this protein with an amino acid substitution in its signal sequence results in the protein being retained intracellularly and is found localised with the mitochondria. This variant form has been associated with increased risk of developing exudative AMD. In this study a number of key cellular processes were examined to elucidate the effect the variant B protein has on the RPE. These processes were respiration, apoptosis, autophagy and oxidative stress; all of which have been implicated in AMD pathology or ageing. In addition the protein-protein interactions of variant B cystatin C were assessed through mass spectrometry analysis of pulled-down cystatin C protein from transfected cell lysates. No effect on respiration, apoptosis or autophagy was identified. However a statistically significant difference in oxidative stress was identified as a result of overexpression of either wild type cystatin C or variant B cystatin C. Mass spectrometry analysis resulted in two highly promising proteins that were found interacting with variant B cystatin C at a statistically significant level, prohibitin and voltage-dependent anion-selective channel protein 1 (VDAC1). Although oxidative stress was found for both proteins given the nature of the expression (driven by a CMV promoter) it seems likely that the oxidative stress response is due to a high level of intracellular cystatin C. This suggests that the retention of variant B protein within the cell would lead to increased oxidative stress levels. It can be speculated that this response could be a contributory factor to development of AMD. VDAC1 and prohibitin may offer an explanation of what happens to the variant B protein within RPE cells. VDAC1 is the major pore-forming protein in the mitochondrial membrane, disruption of its functioning by the binding of variant B protein might be expected to have detrimental effects. Prohibitin is an even more promising target as it has been associated with oxidative stress. In addition it is known to translocate between the nucleus and the mitochondria, offering the tantalising possibility of a complete explanation for the mitochondrial mislocalisation

Gross solids from combined sewers in dry weather and storms, elucidating production, storage and social factors

Variation in rates of sanitary hygiene products, toilet tissue and faeces occurring in sewers are presented for dry and wet weather from three steep upstream urban catchments with different economic, age and ethnic profiles. Results show, for example, that total daily solids per capita from the low income and ageing populations are almost twice that from high income or ethnic populations. Relative differences are verified through independent questionnaires. The relationship between solids stored in sewers prior to storms, antecedent dry weather period and the proportion of roof to total catchment area is quantified. A full solids' flush occurs when storm flows exceed three times the peak dry weather flow. The data presented will assist urban drainage designers in managing pollution caused by the discharge of sewage solids

A feasibility study of controlled human infection with intradermal Bacillus Calmette–Guérin (BCG) injection: Pilot BCG controlled human infection model

Tuberculosis (TB) caused 1.5 million deaths in 2020, making it the leading infectious killer after COVID-19. Bacille Calmette-Guerin (BCG) is the only licensed vaccine against TB but has sub-optimal efficacy against pulmonary TB and reduced effectiveness in regions close to the equator with high burden. Efforts to find novel vaccines are hampered due to the need for large-scale, prolonged, and costly clinical trials. Controlled human infection models (CHIMs) for TB may be used to accelerate vaccine development by ensuring only the most promising vaccine candidates are selected for phase 3 trials, but it is not currently possible to give participants Mycobacterium tuberculosis as a challenge agent. This study aims to replicate and refine an established BCG CHIM at the Liverpool School of Tropical Medicine. Participants will receive an intradermal injection with licensed BCG vaccine (Statens Serum Institut strain). In phase A, participants will undergo punch biopsy two weeks after administration, paired with minimally invasive methods of skin sampling (skin swab, microbiopsy, skin scrape). BCG detection by classical culture and molecular methods will be compared between these techniques and gold standard punch biopsy. Techniques meeting our pre-defined sensitivity and specificity criteria will be applied in Phase B to longitudinally assess intradermal BCG growth two, seven and fourteen days after administration. We will also measure compartmental immune responses in skin, blood and respiratory mucosa in Phase B. This feasibility study will transfer and refine an existing and safe model of BCG controlled human infection. Longitudinal BCG quantification has the potential to increase model sensitivity to detect vaccine and therapeutic responses. If successful, we aim to transfer the model to Malawi in future studies, a setting with endemic TB disease, to accelerate development of vaccines and therapeutics relevant for underserved populations who stand to benefit the most. Registration: ISRCTN: ISRCTN94098600 and ClinicalTrials.gov: NCT058205

Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications

Clinical trials of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapeutics often include virological secondary endpoints to compare viral clearance and viral load reduction between treatment and placebo arms. This is typically achieved using quantitative reverse-transcriptase PCR (RT-qPCR), which cannot differentiate replicant competent virus from non-viable virus or free RNA, limiting its utility as an endpoint. Culture-based methods for SARS-CoV-2 exist; however, these are often insensitive and poorly standardized for use as clinical trial endpoints. We report optimization of a culture-based approach evaluating three cell lines, three detection methods, and key culture parameters. We show that Vero-angiotensin-converting enzyme 2-transmembrane serine protease 2 cells in combination with RT-qPCR of culture supernatants from the first passage provides the greatest overall detection of Delta viral replication (22 of 32, 68.8%), being able to identify viable virus in 83.3% (20 of 24) of clinical samples with initial Ct values of <30. Likewise, we demonstrate that RT-qPCR using culture supernatants from the first passage of Vero human signaling lymphocytic activation molecule cells provides the highest overall detection of Omicron viral replication (9 of 31, 29%), detecting live virus in 39.1% (9 of 23) of clinical samples with initial Ct values of <25. This assessment demonstrates that combining RT-qPCR with virological endpoint analysis has utility in clinical trials of therapeutics for SARS-CoV-2; however, techniques may require optimization based on dominant circulating strain. IMPORTANCE RT-qPCR is commonly used for virological endpoints during clinical trials for antiviral therapy to determine the quantity and presence of virus in a sample. However, RT-qPCR identifies viral RNA and cannot determine if viable virus is present. Existing culture-based techniques for SARS-CoV-2 are insensitive and not sufficiently standardized to be employed as clinical study endpoints. The use of a culture system to monitor replicating viruses could mitigate the possibility of molecular techniques identifying viral RNA from inactive or lysed viral particles. The methodology optimized in this study for detecting infectious viruses may have application as a secondary virological endpoint in clinical trials of therapeutics for SARS-CoV-2 in addition to numerous research processes

Protocol for a phase IV double-blind randomised controlled trial to investigate the effect of the 13-valent pneumococcal conjugate vaccine and the 23-valent pneumococcal polysaccharide vaccine on pneumococcal colonisation using the experimental human pneumococcal challenge model in healthy adults (PREVENTING PNEUMO 2)

Introduction: Despite widely available vaccinations, Streptococcus pneumoniae (SPN) remains a major cause of morbidity and mortality worldwide, causing community-acquired pneumonia, meningitis, otitis media, sinusitis and bacteraemia. Here, we summarise an ethically approved protocol for a double-blind, randomised controlled trial investigating the effect of the 13-valent pneumococcal conjugate vaccine (PCV13) and the 23-valent pneumococcal polysaccharide vaccine (PPV23) on pneumococcal nasopharyngeal colonisation acquisition, density and duration using experimental human pneumococcal challenge (EHPC). Methods and analysis: Healthy adult participants aged 18–50 years will be randomised to receive PCV13, PPV23 or placebo and then undergo one or two EHPCs involving intranasal administration of SPN at 1-month post-vaccination with serotype 3 (SPN3) and 6 months with serotype 6B (SPN6B). Participants randomised to PCV13 and placebo will also be randomised to one of two clinically relevant SPN3 strains from distinct lineages within clonal complex 180, clades Ia and II, creating five study groups. Following inoculation, participants will be seen on days 2, 7, 14 and 23. During the follow-up period, we will monitor safety, colonisation status, density and duration, immune responses and antigenuria. The primary outcome of the study is comparing the rate of SPN3 acquisition between the vaccinated (PCV13 or PPV23) and unvaccinated (placebo) groups as defined by classical culture. Density and duration of colonisation, comparison of acquisition rates using molecular methods and evaluation of the above measurements for individual SPN3 clades and SPN6B form the secondary objectives. Furthermore, we will explore the immune responses associated with these vaccines, their effect on colonisation and the relationship between colonisation and urinary pneumococcal antigen detection. Ethics and dissemination: The study is approved by the NHS Research and Ethics Committee (Reference: 20/NW/0097) and by the Medicines and Healthcare products Regulatory Agency (Reference: CTA 25753/0001/001–0001). Findings will be published in peer-reviewed journals

Detection of SARS-CoV-2 infection by saliva and nasopharyngeal sampling in frontline healthcare workers: An observational cohort study

Background The SARS-CoV-2 pandemic has caused an unprecedented strain on healthcare systems worldwide, including the United Kingdom National Health Service (NHS). We conducted an observational cohort study of SARS-CoV-2 infection in frontline healthcare workers (HCW) working in an acute NHS Trust during the first wave of the pandemic, to answer emerging questions surrounding SARS-CoV-2 infection, diagnosis, transmission and control. Methods Using self-collected weekly saliva and twice weekly combined oropharyngeal/nasopharyngeal (OP/NP) samples, in addition to self-assessed symptom profiles and isolation behaviours, we retrospectively compared SARS-CoV-2 detection by RT-qPCR of saliva and OP/NP samples. We report the association with contemporaneous symptoms and isolation behaviour. Results Over a 12-week period from 30th March 2020, 40·0% (n = 34/85, 95% confidence interval 31·3–51·8%) HCW had evidence of SARS-CoV-2 infection by surveillance OP/NP swab and/or saliva sample. Symptoms were reported by 47·1% (n = 40) and self-isolation by 25·9% (n = 22) participants. Only 44.1% (n = 15/34) participants with SARS-CoV-2 infection reported any symptoms within 14 days of a positive result and only 29·4% (n = 10/34) reported self-isolation periods. Overall agreement between paired saliva and OP/NP swabs was 93·4% (n = 211/226 pairs) but rates of positive concordance were low. In paired samples with at least one positive result, 35·0% (n = 7/20) were positive exclusively by OP/NP swab, 40·0% (n = 8/20) exclusively by saliva and in only 25·0% (n = 5/20) were the OP/NP and saliva result both positive. Conclusions HCW are a potential source of SARS-CoV-2 transmission in hospitals and symptom screening will identify the minority of infections. Without routine asymptomatic SARS-CoV-2 screening, it is likely that HCW with SARS-CoV-2 infection would continue to attend work. Saliva, in addition to OP/NP swab testing, facilitated ascertainment of symptomatic and asymptomatic SARS-CoV-2 infections. Combined saliva and OP/NP swab sampling would improve detection of SARS-CoV-2 for surveillance and is recommended for a high sensitivity strateg

A Randomised Controlled Trial of Nasal Immunisation with Live Virulence Attenuated Streptococcus pneumoniae Strains Using Human Infection Challenge

Rationale: Pneumococcal pneumonia remains a global health problem. Pneumococcal colonization increases local and systemic protective immunity, suggesting that nasal administration of live attenuated Streptococcus pneumoniae (Spn) strains could help prevent infections. Objectives: We used a controlled human infection model to investigate whether nasopharyngeal colonization with attenuated S. pneumoniae strains protected against recolonization with wild-type (WT) Spn (SpnWT). Methods: Healthy adults aged 18-50 years were randomized (1:1:1:1) for nasal administration twice (at a 2-wk interval) with saline solution, WT Spn6B (BHN418), or one of two genetically modified Spn6B strains, SpnA1 (Δfhs/piaA) or SpnA3 (ΔproABC/piaA) (Stage I). After 6 months, participants were challenged with SpnWT to assess protection against the homologous serotype (Stage II). Measurements and Main Results: 125 participants completed both study stages per intention to treat. No serious adverse events were reported. In Stage I, colonization rates were similar among groups: SpnWT, 58.1% (18 of 31); SpnA1, 60% (18 of 30); and SpnA3, 59.4% (19 of 32). Anti-Spn nasal IgG levels after colonization were similar in all groups, whereas serum IgG responses were higher in the SpnWT and SpnA1 groups than in the SpnA3 group. In colonized individuals, increases in IgG responses were identified against 197 Spn protein antigens and serotype 6 capsular polysaccharide using a pangenome array. Participants given SpnWT or SpnA1 in Stage I were partially protected against homologous challenge with SpnWT (29% and 30% recolonization rates, respectively) at stage II, whereas those exposed to SpnA3 achieved a recolonization rate similar to that in the control group (50% vs. 47%, respectively). Conclusions: Nasal colonization with genetically modified live attenuated Spn was safe and induced protection against recolonization, suggesting that nasal administration of live attenuated Spn could be an effective strategy for preventing pneumococcal infections. Clinical trial registered with the ISRCTN registry (ISRCTN22467293)

Human Infection Challenge with Serotype 3 Pneumococcus

Rationale: Streptococcus pneumoniae serotype 3 (SPN3) is a cause of invasive pneumococcal disease and associated with low carriage rates. Following the introduction of pediatric 13-valent pneumococcal conjugate vaccine (PCV13) programmes, SPN3 declines are less than other vaccine serotypes and incidence has increased in some populations coincident with a shift in predominant circulating SPN3 clade, from I to II. A human challenge model provides an effective means for assessing the impact of PCV13 on SPN3 in the upper airway. Objectives: To establish SPN3’s ability to colonise the nasopharynx using different inoculum clades and doses and the safety of an SPN3 challenge model. Methods: In a human challenge study involving three well characterised and antibiotic sensitive SPN3 isolates (PFESP306 [clade Ia], PFESP231 [no clade] and PFESP505 [clade II]), inoculum doses (10,000, 20,000, 80,000, 160,000 CFU/100μL) were escalated until maximal colonisation rates were achieved, with concurrent acceptable safety. Outcome measures: Presence and density of experimental SPN3 nasopharyngeal colonisation in nasal wash samples, assessed using microbiological culture and molecular methods, on days 2, 7 and 14 post-inoculation. Results: 96 healthy participants (median age 21, interquartile range 19-25) were inoculated (n=6-10 per dose group, 10 groups). Colonisation rates ranged from 30.0-70.0% varying with dose and isolate. 30.0% (29/96) reported mild symptoms (82.8% sore throat, [24/29]), one developed otitis media requiring antibiotics. No serious adverse events occurred. Conclusions: An SPN3 human challenge model is feasible and safe with comparable carriage rates to an established SPN6B human challenge model. SPN3 carriage may cause mild upper respiratory symptoms