Validation of a rapid, saliva-based, and ultra-sensitive SARS-CoV-2 screening system for pandemic-scale infection surveillance.

Without any realistic prospect of comprehensive global vaccine coverage and lasting immunity, control of pandemics such as COVID-19 will require implementation of large-scale, rapid identification and isolation of infectious individuals to limit further transmission. Here, we describe an automated, high-throughput integrated screening platform, incorporating saliva-based loop-mediated isothermal amplification (LAMP) technology, that is designed for population-scale sensitive detection of infectious carriers of SARS-CoV-2 RNA. Central to this surveillance system is the "Sentinel" testing instrument, which is capable of reporting results within 25 min of saliva sample collection with a throughput of up to 3840 results per hour. It incorporates continuous flow loading of samples at random intervals to cost-effectively adjust for fluctuations in testing demand. Independent validation of our saliva-based RT-LAMP technology on an automated LAMP instrument coined the "Sentinel", found 98.7% sensitivity, 97.6% specificity, and 98% accuracy against a RT-PCR comparator assay, confirming its suitability for surveillance screening. This Sentinel surveillance system offers a feasible and scalable approach to complement vaccination, to curb the spread of COVID-19 variants, and control future pandemics to save lives.

A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery.

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell's endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple "Phylomer" CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne's muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.

ß-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides.

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 ß-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length ß-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of ß-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split ß-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the ß-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

GFP-complementation assay to detect functional CPP and protein delivery into living cells.

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell's membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease.

Antibacterial antibody responses associated with the development of asthma in house dust mite-sensitised and non-sensitised children.

BACKGROUND: Infants who develop house dust mite (HDM) allergy and HDM-sensitised children with severe persistent asthma have low antibody responses to the P6 antigen of Haemophilus influenzae. OBJECTIVE: To measure the development of antibody to two ubiquitous bacteria of the respiratory mucosa in a prospective birth cohort at high risk of allergic disease and to assess which responses are associated with asthma and atopy. METHODS: IgG1 and IgG4 antibody to H influenzae (P4 and P6) and Streptoccocus pneumoniae (PspA and PspC) surface antigens was measured in yearly blood samples of children aged 1-5 years. IgE to the P6 antigen was examined for the 5-year group. The children were stratified based on HDM sensitisation and asthma at 5 years of age. RESULTS: HDM-sensitised children had lower IgG1 antibody titres to the bacterial antigens, and early responses (<3 years and before the development of HDM sensitisation and asthma) corrected for multiple antigens were significantly reduced for P4, P6 and PspC (p=0.008, p=0.004 and p=0.028, respectively). Similar associations with asthma were also found (p=0.008, p=0.004 and p=0.032 for P4, P6 and PspC, respectively). The IgG4 antibody titre and prevalence were similar in both HDM-sensitised and non-sensitised groups, but sensitised children had a slower downregulation of the IgG4 response. Children with asthma (27/145 at 5 years) had lower anti-P6 IgE responses (p<0.05). CONCLUSIONS: HDM-sensitised children have early defective antibody responses to bacteria that are associated with asthma. Surprisingly, antibacterial IgE was associated with a reduced risk for asthma.

Maternal antibodies to pneumolysin but not to pneumococcal surface protein A delay early pneumococcal carriage in high-risk Papua New Guinean infants.

Immunization of pregnant women can be an efficient strategy to induce early protection in infants in developing countries. Pneumococcal protein-based vaccines may have the capacity to induce pneumococcal serotype-independent protection. To understand the potential of maternal pneumococcal protein-specific antibodies in infants in high-risk areas, we studied the placental transfer of naturally acquired antibodies to pneumolysin (Ply) and pneumococcal surface protein A family 1 and 2 (PspA1 and PspA2) in relation to onset of pneumococcal nasopharyngeal carriage in infants in Papua New Guinea (PNG). In this study, 76% of the infants carried Streptococcus pneumoniae in the upper respiratory tract within the first month of life, at a median age of 19 days. Maternal and cord blood antibody titers to Ply (rho = 0.824, P < 0.001), PspA1 (rho = 0.746, P < 0.001), and PspA2 (rho = 0.631, P < 0.001) were strongly correlated. Maternal pneumococcal carriage (hazard ratio [HR], 2.60; 95% confidence interval [CI], 1.25 to 5.39) and younger maternal age (HR, 0.74; 95% CI, 0.54 to 1.00) were independent risk factors for early carriage, while higher cord Ply-specific antibody titers predicted a significantly delayed onset (HR, 0.71; 95% CI, 0.52 to 1.00) and cord PspA1-specific antibodies a significantly younger onset of carriage in PNG infants (HR, 1.57; 95% CI, 1.03 to 2.40). Maternal vaccination with a pneumococcal protein-based vaccine should be considered as a strategy to protect high-risk infants against pneumococcal disease by reducing carriage risks in both mothers and infants.

Pyroglyphid house dust mite allergens.

Mites of the family Pyroglyphidae are the most important source of house dust mite allergens. A small number of allergens, namely those of groups 1, 2, 4, 5 and 7 constitute the known major and mid-potency specificities, with possible important contributions of the groups 11, 14 and 15 requiring further definition. Most of the allergens can be identified by sequence homologies and the structures of the major allergens have been solved. There are however challenges in determining the nature of the group 5 and 7 allergens and in obtaining detailed structures of the significant allergens to be used for genetic engineering.

Protein silencing with Phylomers: a new tool for target validation and generating lead biologicals targeting protein interactions.

As the field of proteomics expands, the need for functional annotation of the proteome increases to allow the rational choice of particular targets from amongst a bewildering and increasing set of candidates. Similarly, the description of the interactome will continue to generate a plethora of candidate interactions that will need to be validated in functional assays. Although knockout mice and RNAi knockdowns have proven to be invaluable as primary tools in functional genomics, it should be remembered that these techniques apply at the gene and transcript level respectively and are, therefore, not always suitable for true functional proteomics investigations. Protein levels do not always correlate with RNA levels, particularly where active protein levels are regulated by protein stability or post-translational modifications. Moreover, off-target regulation of genes by RNAi/siRNA can complicate the validation of targets using this approach. In recent years, a myriad of potential protein targets including complexes has been emerging from high-throughput proteomics. Thus, the dilemma facing many investigators is having too many potential targets and too few means of validating them. Therefore, there is an unmet need for the development of technologies which allow the targeted disruption of particular proteins. Intracellular antibodies (intrabodies) and peptide aptamers are beginning to be applied in this area. A new class of peptide, known as Phylomers(®), also has significant potential in the area of disruption of protein-protein interactions. Phylomers, which are derived from protein subdomains, are small enough to synthesise and are of a suitable size for delivery to tissues and even into cells, making them an ideal candidates for a next-generation tool for functional validation of the proteome.

Characterization of a family 11 xylanase from Bacillus subtillis B230 used for paper bleaching.

Enzymes such as family 11 xylanases are increasingly being used for industrial applications. Here, the cloning, structure determination and temperature-stability data of a family 11 xylanase, Xyn11X, from the alkali-tolerant Bacillus subtilis subspecies B230 are reported. This enzyme, which degrades xylan polymers, is being produced on an industrial scale for use in the paper-bleaching industry. Xyn11X adopts the canonical family 11 xylanase fold. It has a greater abundance of side chain to side chain hydrogen bonds compared with all other family 11 xylanase crystal structures. Means by which the thermostability of Xyn11X might be improved are suggested.