Efficacy of Alum-Adjuvanted Peptide and Carbohydrate Conjugate Vaccine Candidates against Group A Streptococcus Pharyngeal Infection in a Non-Human Primate Model.

Vaccine development against group A Streptococcus (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking N-acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group.

Increasing stability of a native freshwater fish assemblage following flow rehabilitation.

Stream restorations are increasingly critical for managing and recovering freshwater biodiversity in human-dominated landscapes. However, few studies have quantified how rehabilitative actions promulgate through aquatic communities over decades. Here, a long-term dataset is analyzed for fish assemblage change, incorporating data pre- and post-restoration periods, and testing the extent to which native assemblage stability has increased over time. In the late 1950s, a large capacity dam was installed on Putah Creek (Solano County, CA, USA), which altered the natural flow regime, channel structure, geomorphic processes, and overall ecological function. Notably, downstream flows were reduced (especially during summer months) resulting in an aquatic assemblage dominated by warm-water nonnative species, while endemic native species subsisted at low levels as subordinates. A court-mediated Accord was ratified in 2000, providing a more natural flow regime, specifically for native and anadromous fishes in the stream. The richness of nonnative species decreased at every site following the Accord, while the richness of native species increased or stayed constant. At the three most upstream sites, native species richness increased over time and ultimately exceeded nonnative richness. Native assemblage recovery was strongest upriver, closer to flow releases and habitat restoration activities, and decreased longitudinally downstream. Rank-abundance curves through time revealed that, while species evenness was low throughout the study, dominance shifted from nonnative to native species in the upstream sites coincident with rehabilitation efforts. Mean rank shifts decreased following flow rehabilitation; thus the assemblage became increasingly stable over time following flow rehabilitation. Putah Creek's rehabilitation may represent a model for others interested in improving endemic freshwater communities in degraded ecosystems.

Development of a novel angiotensin converting enzyme 2 stimulator with broad implications in SARS-CoV2 and type 1 diabetes.

Angiotensin-converting enzyme 2 (ACE2) is protective in cardiovascular disease, lung injury and diabetes yet paradoxically underlies our susceptibility to SARs-CoV2 infection and the fatal heart and lung disease it can induce. Furthermore, diabetic patients have chronic, systemic inflammation and altered ACE2 expression resulting in increased risk of severe COVID-19 and the associated mortality. A drug that could increase ACE2 activity and inhibit cellular uptake of severe acute respiratory syndrome coronavirus 2 (SARs-CoV2), thus decrease infection, would be of high relevance to cardiovascular disease, diabetes and SARs-CoV2 infection. While the need for such a drug lead was highlighted over a decade ago receiving over 600 citations,1 to date, no such drugs are available.2 Here, we report the development of a novel ACE2 stimulator, designated '2A'(international PCT filed), which is a 10 amino acid peptide derived from a snake venom, and demonstrate its in vitro and in vivo efficacy against SARs-CoV2 infection and associated lung inflammation. Peptide 2A also provides remarkable protection against glycaemic dysregulation, weight loss and disease severity in a mouse model of type 1 diabetes. No untoward effects of 2A were observed in these pre-clinical models suggesting its strong clinical translation potential.

Taxonomy of the Speckled Dace Species Complex (Cypriniformes: Leuciscidae, Rhinichthys) in California, USA.

The Speckled Dace, Rhinichthys osculus (Girard), is a small species of fish (Cypriniformes, Leuciscidae) that has the widest geographic range of any freshwater dispersing fish in western North America. The dynamic geologic history of the region has produced many isolated watersheds with endemic fish species. However, Speckled Dace from these watersheds cannot be differentiated readily by morphometrics and meristics. This has led to the widely accepted hypothesis that the dace's adaptability and ability to cross geologic barriers has resulted in interbreeding among neighboring populations, maintaining the dace as a single species. We investigate this hypothesis by looking at Speckled Dace populations in California which are the result of at least three separate colonization events of isolated watersheds. We synthesize results from taxonomic, genetic, and zoogeographic studies in combination with the findings of a recent genomics study, to show that there are distinctive evolutionary lineages within the Speckled Dace complex. These lineages are used to designate multiple species and subspecies. We back up these designations by examining how well these lineages fit with the geologic history of the isolated basins they inhabit and with the presence of other endemic fishes. We conclude the following nine taxa can be recognized within the Speckled Dace complex in California.

Sortase A Inhibitor Protein Nanoparticle Formulations Demonstrate Antibacterial Synergy When Combined with Antimicrobial Peptides.

Sortase A (SrtA) is an enzyme which attaches proteins, including virulence factors, to bacterial cell walls. It is a potential target for developing anti-virulence agents against pathogenic and antimicrobial resistant bacteria. This study aimed to engineer 𝛽-lactoglobulin protein nanoparticles (PNPs) for encapsulating safe and inexpensive natural SrtA inhibitors (SrtAIs; trans-chalcone (TC), curcumin (CUR), quercetin (QC), and berberine (BR)) to improve their poor aqueous dispersibility, to screen for synergy with antimicrobial peptides (AMPs), and to reduce the cost, dose, and toxicity of AMPs. Minimum inhibitory concentration (MIC), checkerboard synergy, and cell viability assays were performed for SrtAI PNPs against Gram-positive (methicillin-sensitive and -resistant S. aureus) and Gram-negative (E. coli, P. aeruginosa) bacteria alone and combined with leading AMPs (pexiganan, indolicidin, and a mastoparan derivative). Each SrtAI PNP inhibited Gram-positive (MIC: 62.5-125 µg/mL) and Gram-negative (MIC: 31.3-500 µg/mL) bacterial growth. TC PNPs with pexiganan demonstrated synergy against each bacteria, while BR PNPs with pexiganan or indolicidin provided synergy towards S. aureus. Each SrtAI PNP inhibited SrtA (IC50: 25.0-81.8 µg/mL), and did not affect HEK-293 cell viability at their MIC or optimal synergistic concentrations with AMPs. Overall, this study provides a safe nanoplatform for enhancing antimicrobial synergy to develop treatments for superbug infections.

Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed.

Cryptic Species of Freshwater Sculpin (Cottidae: Cottus) in California, USA.

The Riffle Sculpin (Cottus gulosus) is a small, bottom-dwelling fish regarded as widespread in the cool-water streams that flow into Californias Central Valley and into streams of the central California coast. Using population genomics, supported by other genetic, distributional, and meristic studies, we demonstrate that C. gulosus consists of three cryptic species with four subspecies (five lineages), all but one entirely endemic to California: Cottus pitensis, Pit Sculpin Bailey and Bond 1963 Cottus gulosus, Inland Riffle Sculpin (Girard 1854) g. gulosus: San Joaquin Riffle Sculpin (Girard 1854), nominate subspecies g. wintu: Sacramento Riffle Sculpin, Moyle and Campbell 2022, new subspecies Cottus ohlone, Coastal Riffle Sculpin Moyle and Campbell 2022, new species o. ohlone, Ohlone Riffle Sculpin Moyle and Campbell 2022, nominate subspecies o. pomo, Pomo Riffle Sculpin Moyle and Campbell 2022, new subspecies. The three species are endemic to California watersheds although the range of C. pitensis extends into southeastern Oregon. All are confined to cool headwater streams or to rivers with cold water releases below dams. Their populations are increasingly isolated from one another because of anthropogenic changes to Californias river systems and some are threatened with extinction. Providing taxonomic recognition of the distinct forms will improve conservation efforts on their behalf. This study also demonstrates how genomics can be used to resolve situations where signals from mitochondrial and nuclear DNA are in conflict.

Formulation and Biological Evaluation of Mesoporous Silica Nanoparticles Loaded with Combinations of Sortase A Inhibitors and Antimicrobial Peptides.

This study aimed to develop synergistic therapies to treat superbug infections through the encapsulation of sortase A inhibitors (SrtAIs; trans-chalcone (TC), curcumin (CUR), quercetin (QC), or berberine chloride (BR)) into MCM-41 mesoporous silica nanoparticles (MSNs) or a phosphonate-modified analogue (MCM-41-PO3-) to overcome their poor aqueous solubility. A resazurin-modified minimum inhibitory concentration (MIC) and checkerboard assays, to measure SrtAI synergy in combination with leading antimicrobial peptides (AMPs; pexiganan (PEX), indolicidin (INDO), and [I5, R8] mastoparan (MASTO)), were determined against methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The results demonstrated that the MCM-41 and MCM-41-PO3- formulations significantly improved the aqueous solubility of each SrtAI. The MICs for SrtAI/MCM-41-PO3- formulations were lower compared to the SrtAI/MCM-41 formulations against tested bacterial strains, except for the cases of BR/MCM-41 and QC/MCM-41 against P. aeruginosa. Furthermore, the following combinations demonstrated synergy: PEX with TC/MCM-41 (against all strains) or TC/MCM-41-PO3- (against all strains except P. aeruginosa); PEX with BR/MCM-41 or BR/MCM-41-PO3- (against MSSA and MRSA); INDO with QC/MCM-41 or QC/MCM-41-PO3- (against MRSA); and MASTO with CUR/MCM-41 (against E. coli). These combinations also reduced each components' toxicity against human embryonic kidney cells. In conclusion, MCM-41 MSNs provide a platform to enhance SrtAI solubility and demonstrated antimicrobial synergy with AMPs and reduced toxicity, providing novel superbug treatment opportunities.

Growth of Lahontan cutthroat trout from multiple sources re-introduced into Sagehen Creek, CA.

Lahontan cutthroat trout Oncorhynchus clarkii henshawi have experienced massive declines in their native range and are now a threatened species under the US Endangered Species Act. A key management goal for this species is re-establishing extirpated populations using translocations and conservation hatcheries. In California USA, two broodstocks (Pilot Peak and Independence Lake) are available for reintroduction, in addition to translocations from wild and naturalized sources. Pilot Peak and Independence Lake fish are hatchery stocks derived from native fish from the Truckee River basin and used for recovery activities in the western Geographic Management Unit Areas only, specifically within the Truckee River basin. Yet suitability of these sources for re-introduction in different ecosystem types remains an open and important topic. We conducted growth experiments using Lahontan cutthroat trout stocked into Sagehen Creek, CA, USA. Experiments evaluated both available broodstocks and a smaller sample of fish translocated representing a naturalized population of unknown origin from a nearby creek. Fish from the Independence Lake source had significantly higher growth in weight and length compared to the other sources. Further, Independence Lake fish were the only stock that gained weight on average over the duration of the experiment. Our experiments suggest fish from the Independence Lake brood stock should be considered in reintroduction efforts.

YY-11, a camel milk-derived peptide, inhibits TGF-ß-mediated atherogenic signaling in human vascular smooth muscle cells.

Atherosclerosis, the major underlying pathology of cardiovascular disease, commences with the binding and trapping of lipids on modified proteoglycans, with hyperelongated glycosaminoglycan chains. Transforming growth factor (TGF)-ß stimulates glycosaminoglycan elongation in vascular smooth muscle cells. We have recently shown that this TGF-ß signaling pathway involves reactive oxygen species (ROS). YY-11 is a dodecapeptide derived from camel milk and it has antioxidant activity. We have investigated the role of YY-11 in blocking ROS signaling and downstream atherogenic responses. YY-11 inhibited TGF-ß stimulated ROS production and inhibited the expression of genes for glycosaminoglycan chain elongation as a component of an in vitro model of atherosclerosis. This study provides a biochemical mechanism for the role of camel milk as a potential nutritional product to contribute to the worldwide amelioration of cardiovascular disease. PRACTICAL APPLICATIONS: The identification of readily accessible foods with antioxidant properties would provide a convenient and cost-effective approach community wide reducing oxidative stress induced pathologies such as atherosclerosis. We demonstrate that camel milk-derived peptide is an antioxidant that can inhibit growth factor-mediated proteoglycan modification in vitro. As proteoglycan modification is being recognized as one of the earliest atherogenic responses, these data support the notion of camel milk as a suitable nutritional product to contribute to the prevention of early stage of atherosclerosis development.

Optimized protocols for assessing libraries of poorly soluble sortase A inhibitors for antibacterial activity against medically-relevant bacteria, toxicity and enzyme inhibition.

Increasing antimicrobial resistance is a major global health concern. Conventional antibiotics apply selection pressures, which promote the accumulation of resistant microbes. Anti-virulence strategies, in contrast, are less potent antimicrobials, but are less likely to select for resistance, can be combined with existing antibiotics to improve their activity, and in some cases can overcome antimicrobial resistance towards other antimicrobials. Sortase A inhibitors (SrtAIs) represent an exciting example of this class; however, many reported examples demonstrate poor water solubility, which complicates their biological assessment and activity. This includes reports that use antimicrobial concentrations of organic solvents or conditions that fail to solubilise these compounds for minimal inhibitory concentration (MIC) assessments. Herein, we report the first study to optimise screening processes for a library of prospective SrtAIs (trans-chalcone (TC), berberine (BR), curcumin (CUR), and quercetin (QC)), including comparative assessment of the effects of various co-solvent concentrations, along with comparative assessment of their antimicrobial activities against multiple disease relevant bacterial strains (methicillin-sensitive and resistant S. aureus, E. coli, and P. aeruginosa), inhibition of the sortase A enzyme, and toxicity towards mammalian cells (HEK-293), using these optimised conditions. Optimal solubility with minimal effect on bacterial viability was observed in the presence of 5% (v/v) dimethyl sulfoxide (DMSO)-Mueller-Hinton Broth. Three antimicrobial susceptibility tests (broth microdilution, agar dilution, and disk diffusion) were assessed for their ability to accurately determine minimal inhibitory concentration (MIC) data for each SrtAI. Broth microdilution and agar dilution were both effective; however, the broth microdilution assay required the addition of a colorimetric metabolic indicator (resazurin) to enable simple and reliable MIC determination due to the development of precipitants over time. In contrast, disk diffusion did not provide reliable zone of inhibition data. Identical MIC data was observed with methicillin-sensitive and -resistant S. aureus (MRSA; ATCC43300), with lower potency activity against E. coli and P. aeruginosa. Under these conditions, TC and CUR demonstrated significant toxicity towards human embryonic kidney (HEK-293) cells, with QC showing less toxicity and BR limited-to-no toxicity at its MIC. Overall, the findings of this work provide optimised processes, which will prove useful for the study of other poorly soluble antimicrobial agents and SrtAIs. The obtained data suggests that BR should be considered in preference to the other SrtAIs for the development of new antimicrobial formulations, based on its superior antimicrobial and SrtA inhibition potency, and greatly reduced toxicity.

Biogeochemical processes create distinct isotopic fingerprints to track floodplain rearing of juvenile salmon.

Floodplains represent critical nursery habitats for a variety of fish species due to their highly productive food webs, yet few tools exist to quantify the extent to which these habitats contribute to ecosystem-level production. Here we conducted a large-scale field experiment to characterize differences in food web composition and stable isotopes (δ¹³C, δ¹5N, δ³4S) for salmon rearing on a large floodplain and adjacent river in the Central Valley, California, USA. The study covered variable hydrologic conditions including flooding (1999, 2017), average (2016), and drought (2012-2015). In addition, we determined incorporation rates and tissue fractionation between prey and muscle from fish held in enclosed locations (experimental fields, cages) at weekly intervals. Finally, we measured δ³4S in otoliths to test if these archival biominerals could be used to reconstruct floodplain use. Floodplain-reared salmon had a different diet composition and lower δ13C and δ³4S (δ¹³C = -33.02±2.66‰, δ³4S = -3.47±2.28‰; mean±1SD) compared to fish in the adjacent river (δ¹³C = -28.37±1.84‰, δ³4S = +2.23±2.25‰). These isotopic differences between habitats persisted across years of extreme droughts and floods. Despite the different diet composition, δ¹5N values from prey items on the floodplain (δ¹5N = 7.19±1.22‰) and river (δ¹5N = 7.25±1.46‰) were similar, suggesting similar trophic levels. The food web differences in δ13C and δ³4S between habitats were also reflected in salmon muscle tissue, reaching equilibrium between 24-30 days (2014, δ¹³C = -30.74±0.73‰, δ³4S = -4.6±0.68‰; 2016, δ¹³C = -34.74 ±0.49‰, δ³4S = -5.18±0.46‰). δ³4S measured in sequential growth bands in otoliths recorded a weekly time-series of shifting diet inputs, with the outermost layers recording time spent on the floodplain (δ³4S = -5.60±0.16‰) and river (δ³4S = 3.73±0.98‰). Our results suggest that δ¹³C and δ³4S can be used to differentiate floodplain and river rearing habitats used by native fishes, such as Chinook Salmon, across different hydrologic conditions and tissues. Together these stable isotope analyses provide a toolset to quantify the role of floodplains as fish habitats.

Developing GLP-1 Conjugated Self-Assembling Nanofibers Using Copper-Catalyzed Alkyne-Azide Cycloaddition and Evaluation of Their Biological Activity.

Glucagon-like peptide-1 GLP-1 is a gut-derived peptide secreted from pancreatic ß-cells that reduces blood glucose levels and body weight; however, native GLP-1 (GLP-1(7-36)-NH2 and GLP-1(7-37)) have short in vivo circulation half-lives (∼2 min) due to proteolytic degradation and rapid renal clearance due to its low molecular weight (MW; 3297.7 Da). This study aimed to improve the proteolytic stability and delivery properties of glucagon-like peptide-1 (GLP-1) through modifications that form nanostructures. For this purpose, N- (NtG) and C-terminal (CtG), and Lys26 side chain (K26G) alkyne-modified GLP-1 analogues were conjugated to an azide-modified lipidic peptide (L) to give N-L, C-L, and K-26-L, respectively; or CtG was conjugated with a fibrilizing self-assembling peptide (SAP) (AEAEAKAK)3 to yield C-S, using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). N-L demonstrated the best serum stability (t1/2 > 48 h) compared to K-26-L (44 h), C-L (20 h), C-S (27 h), and the parental GLP-1(7-36;A8G)-NH2 (A8G) (19 h) peptides. Each conjugate demonstrated subnanomolar hGLP-1RA potency, and none demonstrated toxicity toward PC-3 cells at concentrations up to 1 µM. Each analogue was observed by transmission electron microscopy to form fibrils in solution. K-26-L demonstrated among the best human serum stability (t1/2 = 44 h) and similar hGLP-1RA potency (EC50 48 pM) to C-S. In conclusion, this study provided an alternative to lipid modification, i.e., fibrillizing peptides, that could improve pharmacokinetic parameters of GLP-1.

Sortase A (SrtA) inhibitors as an alternative treatment for superbug infections.

Virulence factor, sortase A (SrtA), has crucial roles in the pathogenesis of Gram-positive superbugs. SrtA is a bacterial cell membrane enzyme that anchors crucial virulence factors to the cell wall surface of Gram-positive bacteria. SrtA is not necessary for bacterial growth and viability and is conveniently accessible in the cell membrane; therefore, it is an ideal target for antivirulence drug development. In this review, we focus on antimicrobial resistance (AMR)-expressing bacteria and SrtA as a potential target for overcoming AMR. The mechanism of action of SrtA and its inhibition by various types of inhibitors, such as synthetic small molecules, peptides, and natural products, are provided. Future SrtA research perspectives for alternative drug development to antibiotics are also proposed.

Neutralisation of adeno-associated virus transduction by human vitreous humour.

Neutralising antibodies (NAbs), caused by past adeno-associated virus (AAV) infection, represent a critical challenge for AAV-mediated gene therapy, with even low NAb titres capable of inhibiting gene transfer, however in protein-rich environments such as the vitreous it is expected that other constituents could also interact with the transduction process. Inhibition of AAV2/2, AAV2/5, AAV2/6 and AAV2/8 transduction by human vitreous humour (VH) obtained from 80 post-mortem eye cups was investigated in this report, with clinically relevant vitreous dilutions as low as 1:2. Unexpectedly, the highest prevalence of inhibition of transduction was observed against AAV2/6, with 66% of tested samples displaying neutralisation at a 1:2 VH dilution. Only two samples showed inhibition of AAV2/8, indicating this serotype is an attractive vector for use in non-vitrectomised eyes of unscreened individuals. Levels of anti-AAV NAbs observed in the VH were much lower than previously observed in serum of a similar Australian population. Among ten tested eye cup pairs, we observed only small variation in anti-AAV NAbs levels between the left and right eye cups. Interaction with 1:2 diluted VH had an augmentation effect on AAV2/8 transduction (p = 0.004), a phenomenon which was not due to albumin or transferrin and which, if developed, might benefit the use of AAV2/8 in clinical settings.

Supercritical fluid assembly of albendazole liposomes targeting gastrin-releasing peptide receptor overexpressing tumors.

Aim: To develop albendazole (ABZ)-loaded bombesin(6-14) (BBN(6-14)) functionalized liposomes for targeting GRPR to enhance delivery to cancer cells. Materials & methods: ABZ-loaded liposomes were formulated using supercritical CO2 technology; functionalized with a GRPR-targeted lipid-anchored BBN(6-14) peptide; and evaluated for effects on cell viability, particle size and targeted cell uptake. Results: BBN(6-14)-coated ABZ liposomes decreased cell viability compared with nonfunctionalized ABZ liposomes. The level of GRPR expression positively correlated with intracellular uptake and decreased cell viability. The reduced cell viability, higher cell uptake and GRPR expression were observed in the order PC-3 > Caco-2 > HepG2 cells. Conclusion: BBN(6-14)-functionalized ABZ liposomes showed enhanced reduction in cell viability compared with nonfunctionalized ABZ liposomes.

Optimized Methods for the Production and Bioconjugation of Site-Specific, Alkyne-Modified Glucagon-like Peptide-1 (GLP-1) Analogs to Azide-Modified Delivery Platforms Using Copper-Catalyzed Alkyne-Azide Cycloaddition.

This study aimed to develop and optimize chemistries to produce alkyne-modified glucagon-like peptide-1(7-36)-amide (GLP-1(7-36)-NH2) libraries, which could be rapidly and efficiently conjugated to other components and screened to identify compounds with the best drug delivery properties, as potential treatments for type 2 diabetes or obesity. For this purpose, the Lys26 (K26) side-chain, and the amino (N)- and carboxy (C)-termini of a dipeptidyl peptidase 4 (DPPIV)-resistant GLP-1 sequence (GLP-1(7-36;A8G)-NH2), were modified with an alkyne (4-pentynoic acid or propiolic acid). These analogs were characterized with respect to human GLP-1 receptor (hGLP-1R) agonist activity, effects on cell viability and human serum stability, revealing that these modifications maintained low (N-terminal; EC50 1.5 × 10-9 M) to subnanomolar (C-terminal and K26, ∼4 × 10-10 M) agonist activity toward hGLP-1, had no effect on cell viability, and for the N-terminal and K26 modifications, increased human serum proteolytic stability (t1/2 > 24 h). Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction conditions were investigated using the C-terminal modified GLP-1 analog and an azide-modified model lipid peptide, with respect to the effects of altering the azide/alkyne ratio, cosolvents, temperature, reducing agents, Cu(I)-stabilizing ligand, copper source, and the concentrations of reagents/reactants, in order to identify general conditions that provide fast reactions and high yields. A 1:2 azide-alkyne (lipid:GLP-1 peptide) and 4:1 sodium ascorbate/copper sulfate molar ratio in 65% v/v DMSO-water at room temperature, in the absence of Cu(I)-stabilizing ligands (THPTA or l-histidine) and buffers (phosphate, pH 7), provided the best yields. This work reports a library of characterized GLP-1 analogs and chemistries for their attachment to other species, providing useful tools to improve GLP-1 delivery and pharmacology (e.g., through conjugation to other species that lower blood glucose, increase the duration of action, or enable delivery via a nonparenteral route).

Development of an Enzyme-Mediated, Site-Specific Method to Conjugate Toll-Like Receptor 2 Agonists onto Protein Antigens: Toward a Broadly Protective, Four Component, Group A Streptococcal Self-Adjuvanting Lipoprotein-Fusion Combination Vaccine.

Subunit vaccines composed of protein antigens covalently attached to Toll-like receptor (TLR) agonists elicit superior immune responses compared to mixtures of antigens and TLR agonists. Among different conjugation approaches, enzyme-mediated ligation is one of the few that provides an opportunity for the generation of homogeneous, molecularly defined products in which protein antigens are maintained with native structures, which is most critical to elicit protective immune responses upon vaccination. Four highly conserved protein antigens from Group A Streptococcus (GAS) have the potential to be safe and efficacious vaccine candidates. After a TLR2 agonist fibroblast-stimulating lipopeptide-1 (FSL-1) was successfully attached onto each antigen using sortase A and techniques for their purification were developed, a combination vaccine containing interleukin 8 (IL-8) protease (Streptococcus pyogenes cell envelope proteinase [SpyCEP]), Group A Streptococcal C5a peptidase (SCPA), anchorless virulence factor arginine deiminase (ADI), and trigger factor (TF)-TLR2 conjugates was produced. This combination was assessed for immunity in mice and compared with mixtures of the four antigens with FSL-1 or alum. High titer antigen-specific IgG antibodies were detected from all vaccine groups, with antibodies elicited from FSL-1 conjugates around 10-fold higher compared to the FSL-1 mixture group. Furthermore, the FSL-1 conjugates afforded a more balanced TH1/TH2 immune response than the alum-adjuvanted group, suggesting that this combination vaccine represents a promising candidate for the prevention of GAS diseases. Thus, we established a conjugation platform that allows for the production of defined, site-specific antigen-adjuvant conjugates, which maintain the native three-dimensional structure of antigens and can be potentially applied to a variety of protein antigens.

A Self-Adjuvanting Vaccine Platform: Optimization of Site-Specific Sortase A Mediated Conjugation of Toll-Like Receptor 2 Ligands onto the Carboxyl or Amino terminus of Recombinant Protein Antigens.

Self-adjuvanting vaccines, consisting of recombinant protein antigens and covalently attached Toll-like receptor (TLR) agonists, have the ability to simultaneously and efficiently deliver antigen and TLR adjuvant to antigen presenting cells (APCs). Here, an enzyme-mediated ligation approach was used to overcome difficulties in producing homogeneous, molecularly defined self-adjuvanting vaccine products under native conditions. This process was optimized to allow the incorporation of the lipopeptide TLR2 agonist fibroblast-stimulating lipopeptide (FSL)-1 onto the N- or C-termini of recombinant protein antigens, employing the enzyme Staphylococcus aureus sortase A (SrtAsa) penta mutant. In addition, because SrtAsa-mediated ligations are reversible, a tryptophan zipper derived sequence was introduced into both reactants, which was demonstrated to improve ligation efficiency through the formation of a ß-hairpin structure that hinders the reverse reaction. Finally, it was demonstrated that N- or C-terminal conjugation, and the incorporation of the ß-hairpin structure, did not affect the TLR2-agonist activities of protein antigen TLR agonist conjugates. Overall, this SrtAsa-mediated ligation platform enabled production of antigen TLR2 agonist conjugates with enhanced ligation efficiency, with the conjugates demonstrating potent TLR2 signaling activation (EC50 <1nM).