Relative biomembrane fusogenicities of the tumor-selective liposomes of RGDK- and CGKRK-lipopeptides.

Cancer is the second leading cause of death globally after heart diseases. Currently used highly cytotoxic anti-cancer drugs not only kill cancer cells but also often kill non-cancerous healthy body cells, causing adverse side effects. Efforts are now being directed towards developing tumor-selective chemotherapy. Tumor/tumor endothelial cell selective peptide ligands are being covalently grafted onto the exo-surfaces of drug carriers such as liposomes, polymers, etc. A number of prior studies used conjugation of tumor/tumor endothelial cell-selective RGDK- or CGKRK-peptide ligands on the outer surfaces of liposomes, metal-based nanoparticles, single walled carbon nanotubes (SWNTs), etc. However, studies aimed at examining the relative cell membrane fusogenicities and the relative degrees of cellular uptake for the RGDK- and CGKRK-ligand-grafted nanometric drug carriers have not yet been undertaken. Herein, using the widely used liposomes of DOPC, DOPE, DOPS and cholesterol (45 : 25 : 20 : 15, w/w ratio) as the model biomembranes and the fluorescence resonance energy transfer (FRET) assay for measuring membrane fusogenicities, we show that the liposomes of the RGDK-lipopeptide are more biomembrane fusogenic than the liposomes of the CGKRK-lipopeptide. Notably, such FRET assay-derived relative biomembrane fusogenicities of the liposomes of RGDK- and CGKRK-lipopeptides were found to be consistent with their relative degrees of cellular uptake in cultured cancer cells. The present findings open the door for undertaking in-depth in vivo studies aimed at evaluating the relative therapeutic potential of different nanocarriers of drugs/genes/siRNA having tumor-targeting RGDK- and CGKRK-peptides on their exo-surfaces.

Discovery and Derivatization of Tridecaptin Antibiotics with Altered Host Specificity and Enhanced Bioactivity.

The prevalence of multidrug-resistant (MDR) pathogens combined with a decline in antibiotic discovery presents a major challenge for health care. To refill the discovery pipeline, we need to find new ways to uncover new chemical entities. Here, we report the global genome mining-guided discovery of new lipopeptide antibiotics tridecaptin A5 and tridecaptin D, which exhibit unusual bioactivities within their class. The change in the antibacterial spectrum of Oct-TriA5 was explained solely by a Phe to Trp substitution as compared to Oct-TriA1, while Oct-TriD contained 6 substitutions. Metabolomic analysis of producer Paenibacillus sp. JJ-21 validated the predicted amino acid sequence of tridecaptin A5. Screening of tridecaptin analogues substituted at position 9 identified Oct-His9 as a potent congener with exceptional efficacy against Pseudomonas aeruginosa and reduced hemolytic and cytotoxic properties. Our work highlights the promise of tridecaptin analogues to combat MDR pathogens.

Noducyclamides A1-A4, B1, and B2 from the Cyanobacterium Nodularia sp. NIES-3585.

A chemical investigation of the hydrophilic fraction of a cultured Nodularia sp. (NIES-3585) afforded six new cyclic lipopeptides, noducyclamides A1-A4 (1-4) containing 10 amino acid residues and dodecapeptides noducyclamides B1 and B2 (5 and 6). The planar structures of these lipopeptides were elucidated based on the combination of HRMS and 1D and 2D NMR spectroscopic data analyses. These peptides are structurally analogous to laxaphycins and contain the nonproteinogenic amino acids 3-hydroxyvaline and 3-hydroxyleucine and a ß-amino decanoic acid residue. The absolute configurations of the noducyclamides (1-6) were determined by acid hydrolysis, followed by advanced Marfey's analysis. Noducyclamide B1 (5) showed cytotoxic activities against MCF7 breast cancer cell lines with an IC50 value of 3.0 µg/mL (2.2 µM).

Multifunctional Lipidated Protein Carrier with a Built-In Adjuvant as a Universal Vaccine Platform Potently Elevates Immunogenicity of Weak Antigens.

Weak antigens represented by MUC1 are poorly immunogenic, which greatly constrains the development of relevant vaccines. Herein, we developed a multifunctional lipidated protein as a carrier, in which the TLR1/2 agonist Pam3CSK4 was conjugated to the N-terminus of MUC1-loaded carrier protein BSA through pyridoxal 5'-phosphate-mediated transamination reaction. The resulting Pam3CSK4-BSA-MUC1 conjugate was subsequently incorporated into liposomes, which biomimics the membrane structure of tumor cells. The results indicated that this lipidated protein carrier significantly enhanced antigen uptake by APCs and obviously augmented the retention of the vaccine at the injection site. Compared with the BSA-MUC1 and BSA-MUC1 + Pam3CSK4 groups, Pam3CSK4-BSA-MUC1 evoked 22- and 11-fold increases in MUC1-specific IgG titers. Importantly, Pam3CSK4-BSA-MUC1 elicited robust cellular immunity and significantly inhibited tumor growth. This is the first time that lipidated protein was constructed to enhance antigen immunogenicity, and this universal carrier platform exhibits promise for utilization in various vaccines, holding the potential for further clinical application.

N-Desmethylmajusculamide B, a lipopeptide isolated from the Okinawan cyanobacterium Okeania hirsuta.

A new lipopeptide, N-desmethylmajusculamide B (1), was isolated from the Okinawan cyanobacterium Okeania hirsuta along with 2 known compounds majusculamide A (2) and majusculamide B (3). The planar structure of (1) was elucidated by a detailed analysis of mass spectrometry and nuclear magnetic resonance spectra. The absolute configurations of the amino acid residues were determined using Marfey's analysis. The configuration of C-16 in the α-methyl-ß-keto-decanoyl moiety was determined unambiguously to be S by conducting a semisynthesis of N-desmethylmajusculamide B from 3. The cytotoxicity against mouse L1210 leukemia cells was evaluated for majusculamides (1-3).

Total synthesis of antifungal lipopeptide iturin A analogues and evaluation of their bioactivity against F. graminearum.

The pursuit of novel antifungal agents is imperative to tackle the threat of antifungal resistance, which poses major risks to both human health and to food security. Iturin A is a cyclic lipopeptide, produced by Bacillus sp., with pronounced antifungal properties against several pathogens. Its challenging synthesis, mainly due to the laborious synthesis of the ß-amino fatty acid present in its structure, has hindered the study of its mode of action and the development of more potent analogues. In this work, a facile synthesis of bioactive iturin A analogues containing an alkylated cysteine residue is presented. Two analogues with opposite configurations of the alkylated cysteine residue were synthesized, to evaluate the role of the stereochemistry of the newly introduced amino acid on the bioactivity. Antifungal assays, conducted against F. graminearum, showed that the novel analogues are bioactive and can be used as a synthetic model for the design of new analogues and in structure-activity relationship studies. The assays also highlight the importance of the ß-amino acid in the natural structure and the role of the stereochemistry of the amino fatty acid, as the analogue with the D configuration showed stronger antifungal properties than the one with the L configuration.

Studies of antibacterial activity (in vitro and in vivo) and mode of action for des-acyl tridecaptins (DATs).

Tridecaptins comprise a class of linear cationic lipopeptides with an N-terminal fatty acyl moiety. These 13-mer antimicrobial peptides consist of a combination of d- and l-amino acids, conferring increased proteolytic stability. Intriguingly, they are biosynthesized by non-ribosomal peptide synthetases in the same bacterial species that also produce the cyclic polymyxins displaying similar fatty acid tails. Previously, the des-acyl analog of TriA1 (termed H-TriA1) was found to possess very weak antibacterial activity, albeit it potentiated the effect of several antibiotics. In the present study, two series of des-acyl tridecaptins were explored with the aim of improving the direct antibacterial effect. At the same time, overall physico-chemical properties were modulated by amino acid substitution(s) to diminish the risk of undesired levels of hemolysis and to avoid an impairment of mammalian cell viability, since these properties are typically associated with highly hydrophobic cationic peptides. Microbiology and biophysics tools were used to determine bacterial uptake, while circular dichroism and isothermal calorimetry were used to probe the mode of action. Several analogs had improved antibacterial activity (as compared to that of H-TriA1) against Enterobacteriaceae. Optimization enabled identification of the lead compound 29 that showed a good ADMET profile as well as in vivo efficacy in a variety of mouse models of infection.

Identification of the lipodepsipeptide selethramide encoded in a giant nonribosomal peptide synthetase from a Burkholderia bacterium.

Bacterial natural products have found many important industrial applications. Yet traditional discovery pipelines often prioritize individual natural product families despite the presence of multiple natural product biosynthetic gene clusters in each bacterial genome. Systematic characterization of talented strains is a means to expand the known natural product space. Here, we report genomics, epigenomics, and metabolomics studies of Burkholderia sp. FERM BP-3421, a soil isolate and known producer of antitumor spliceostatins. Its genome is composed of two chromosomes and two plasmids encoding at least 29 natural product families. Metabolomics studies showed that FERM BP-3421 also produces antifungal aminopyrrolnitrin and approved anticancer romidepsin. From the orphan metabolome features, we connected a lipopeptide of 1,928 Da to an 18-module nonribosomal peptide synthetase encoded as a single gene in chromosome 1. Isolation and structure elucidation led to the identification of selethramide which contains a repeating pattern of serine and leucine and is cyclized at the side chain oxygen of the one threonine residue at position 13. A (R)-3-hydroxybutyric acid moiety decorates the N-terminal serine. Initial attempts to obtain deletion mutants to probe the role of selethramide failed. After acquiring epigenome (methylome) data for FERM BP-3421, we employed a mimicry by methylation strategy that improved DNA transfer efficiency. Mutants defective in selethramide biosynthesis showed reduced surfactant activity and impaired swarming motility that could be chemically complemented with selethramide. This work unveils a lipopeptide that promotes surface motility, establishes improved DNA transfer efficiency, and sets the stage for continued natural product identification from a prolific strain.

Quaternary Ammonium Salts of Cationic Lipopeptides with Lysine Residues - Synthesis, Antimicrobial, Hemolytic and Cytotoxic Activities.

Ultrashort cationic lipopeptides (USCLs) and quaternary ammonium salts constitute two groups of cationic surfactants with high antimicrobial activity. This study aimed to investigate the influence of quaternization of the amino group of the lysine side chain in USCLs on their antimicrobial, hemolytic and cytotoxic activities. To do this, two series of lipopeptides were synthesized, USLCs and their quaternized analogues containing trimethylated lysine residues - qUSCLs (quaternized ultrashort cationic lipopeptides). Quaternization was performed on a resin during a standard solid-phase peptide synthesis with CH3I as the methylating agent. According to our knowledge, this is the first study presenting on-resin peptide quaternization. The lipopeptides were tested for their antibacterial and antifungal activities against the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella aerogenes) bacteria and Candida glabrata yeast-like fungus. Most of the compounds proved to be active antimicrobial agents with enhanced activity against Gram-positive strains and fungi and a lower against Gram-negative species. In addition, the antimicrobial activity of lipopeptides was increasing with an increase in hydrophobicity but qUSCLs exhibited usually a poorer antimicrobial activity than their parent molecules. Furthermore, the toxicity against red blood cells and human keratinocytes was assessed. It's worth emphasizing that qUSCLs were less toxic than the parent molecules of comparative hydrophobicity. The results of the study proved that qUSCLs can offer a higher selectivity to pathogens over human cells than that of USCLs. Last but not least, quaternization of the peptides could increase their solubility and therefore their bioavailability and utility.

Enhanced Uptake of Anticancer C6-Pep Dimer in a Model Membrane Caused by Differential pKa in Acidic Microenvironment.

Acidic tumor microenvironment (TME) presents a challenge for the action of antitumor drugs by acting as an additional barrier for the passive crossing of the cell membrane by chemotherapic agents playing a critical role in the proliferation of tumor cells. Anticancer lipopeptide C6-Pep dimer containing the leucine zipper motif shows an increased uptake into the model tumor membrane in TME, and application of external heat might lead to the uncoiling of the zipper, which could result in cell lysis. This work investigated the cause of this increased uptake of C6-Pep dimer into the bilayer model in TME. Accurate protonation states of all the titratable residues of the C6-Pep dimer in TME were determined using constant pH molecular dynamics. In TME, except for two terminal Glu5 residues, all other Glu residues in the C6-Pep dimer were permanently protonated. The remaining Glu5 residues had differential pKa values, leading to the construction of four possible dimers with different fixed protonation states, and molecular dynamics was used to study their interaction with the anionic bilayer. Except for the dimer at a physiological pH, the other dimers were positively charged and could readily adsorb on the membrane surface. The free energy of insertion of these dimers in the bilayer was lower for single and double protonated Glu5-containing dimers than for the others. After the insertion of the lipopeptides into the membrane, thinning of the bilayer in the vicinity of dimers and an increase in area per lipid of the bilayer were observed for all systems, indicating destabilization of the bilayer due to this intercalation. This study shows that the anticancer lipopeptide C6-Pep utilizes the TME around a tumor cell for insertion into the membrane.

Lipopeptides as tools in catalysis, supramolecular, materials and medicinal chemistry.

Lipopeptides are amphiphilic peptides in which an aliphatic chain is attached to either the C or N terminus of peptides. Their self-assembly - into micelles, vesicles, nanotubes, fibres or nanobelts - leads to applications in nanotechnology, catalysis or medicinal chemistry. Self-organization of lipopeptides is dependent on both the length of the lipid tail and the amino acid sequence, in which the chirality of the peptide sequence can be transmitted into the supramolecular species. This Review describes the use of lipopeptides to design synthetic advanced dynamic supramolecular systems, nanostructured materials or self-responsive delivery systems in the area of medical biotechnology. We examine the influence of external stimuli, the ability of lipopeptide-derived structures to adapt over time and their application as medicinal agents with antibacterial, antifungal, antiviral or anticancer activities. Finally, we discuss the catalytic efficiency of lipopeptides, with the aim of building minimal synthetic enzymes, and recent efforts to incorporate metals into lipopeptide assemblies.

Synthesis, evaluation and molecular dynamics study of human toll-like receptor 2/6 specific monoacyl lipopeptides as candidate immunostimulants.

TLR2 agonists typified by the S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (Pam2CS) motif have exhibited powerful immunostimulatory activities. Based on simplified monoacyl lipopeptide (Carbamate-linked N-Ac PamCS), we describe interesting SAR investigations where modifications are done to alter the size of substituents on the cysteine amine, introduce ionizable groups to the terminal and insert aromatic substitutions to the aliphatic chain. Our structural modifications have led to a highly specific human TLR2/6 agonist 14a (EC50 = 0.424 nM), which behaves like Pam2CSK4 by inducing NF-κB activation to trigger downstream signaling pathways, such as subsequent phosphorylation of related proteins (p65, p38) and production of key inflammatory cytokines (IL-6, IL-1ß, TNF-α). Importantly, the ability to stimulate enhanced T cell response compared to Carbamate-linked N-Ac PamCS makes compound 14a a further potential candidate immunostimulant.

Lipopeptides development in cosmetics and pharmaceutical applications: A comprehensive review.

Lipopeptides are surface active, natural products of bacteria, fungi and green-blue algae origin, having diverse structures and functionalities. In analogy, a number of chemical synthesis techniques generated new designer lipopeptides with desirable features and functions. Lipopetides are self-assembly guided, supramolecular compounds which have the capacity of high-density presentation of the functional epitopes at the surface of the nanostructures. This feature contributes to their successful application in several industry sectors, including food, feed, personal care, and pharmaceutics. In this comprehensive review, the novel class of ribosomally synthesized lipopeptides is introduced alongside the more commonly occuring non-ribosomal lipopeptides. We highlight key representatives of the most researched as well as recently described lipopeptide families, with emphasis on structural features, self-assembly and associated functions. The common biological, chemical and hybrid production routes of lipopeptides, including prominent analogues and derivatives are also discussed. Furthermore, genetic engineering strategies aimed at increasing lipopeptide yields, diversity and biological activity are summarized and exemplified. With respect to application, this work mainly details the potential of lipopeptides in personal care and cosmetics industry as cleansing agents, moisturizer, anti-aging/anti-wrinkling, skin whitening and preservative agents as well as the pharmaceutical industry as anitimicrobial agents, vaccines, immunotherapy, and cancer drugs. Given that this review addresses human applications, we conclude on the topic of safety of lipopeptide formulations and their sustainable production.

Production of biosurfactant by salt-resistant Bacillus in lead-supplemented media: application and toxicity.

A group of biosurfactants are lipopeptides that are produced by some microorganisms, especially Bacillus strains. They are new bioactive agents with anticancer, antibacterial, antifungal, and antiviral activities. Also, they are used in sanitation industries. In this study, a lead-resistant strain of Bacillus halotolerans was isolated for lipopeptide production. This isolate exhibited metal resistance (lead, calcium, chromium, nickel, copper, manganese, and mercury), salt tolerance (12%), and antimicrobial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Saccharomyces cerevisiae. The production of lipopeptide was optimized, concentrated, and then extracted from the polyacrylamide gel in a simple way for the first time. The nature of the purified lipopeptide was determined by FTIR, GC/MS, and HPLC analyses. The purified lipopeptide indicated significant antioxidant properties (90.38% at a concentration of 0.8 mg ml-1). Also, it had anticancer activity by apoptosis (flow cytometry analysis) in MCF-7 cells, while it had no cytotoxicity on HEK-293 normal cells. Therefore, Bacillus halotolerans lipopeptide has the potential to be used as an antioxidant, antimicrobial, or anticancer agent in the medical and food industries.

Simultaneous Production of Antibacterial Protein and Lipopeptides in Bacillus tequilensis, Detected by MALDI-TOF and GC Mass Analyses.

As antibiotic resistance is nowadays one of the important challenges, efforts are crucial for the discovery of novel antibacterial drugs. This study aimed to evaluate antimicrobial/anticancerous activities of halophilic bacilli from the human microbiota. A spore-forming halotolerant bacterium with antibacterial effect against Staphylococcus aureus was isolated from healthy human feces. The antibacterial protein components of the extracted supernatant were identified by SDS-PAGE and zymography. The MALDI-TOF, GC mass, and FTIR analyses were used for peptide and lipopeptide identification, respectively. The stability, toxicity, and anticancerous effects were investigated using MTT and Flow cytometry methods. According to the molecular analysis, the strain was identified as Bacillus tequilensis and showed potential probiotic properties, such as bile and acid resistance, as well as eukaryotic cell uptake. SDS-PAGE and zymography showed that 15 and 10-kDa fragments had antibacterial effects. The MALDI-TOF mass analysis indicated that the 15-kDa fragment was L1 ribosomal protein, which was the first report of the RpL1 in bacilli. GC-mass and FTIR analyses confirmed the lipopeptide nature of the 10-kDa fragment. Both the extracted fractions (precipitation or "P" and chloroform or "C" fractions) were stable at < 100 °C for 10 min, and their antibacterial effects were preserved for more than 6 months. Despite its non-toxicity, the P fraction had anticancer activities against MCF7 cells. The anticancer and antibacterial properties of B. tequilensis, along with its non-toxicity and stability, have made it a potential candidate for studying the beneficial probiotic properties for humans and drug production.

Synthesis and Antimicrobial Activity of Short Analogues of the Marine Antimicrobial Peptide Turgencin A: Effects of SAR Optimizations, Cys-Cys Cyclization and Lipopeptide Modifications.

We have synthesised short analogues of the marine antimicrobial peptide Turgencin A from the colonial Arctic ascidian Synoicum turgens. In this study, we focused on a central, cationic 12-residue Cys-Cys loop region within the sequence. Modified (tryptophan- and arginine-enriched) linear peptides were compared with Cys-Cys cyclic derivatives, and both linear and Cys-cyclic peptides were N-terminally acylated with octanoic acid (C8), decanoic acid (C10) or dodecanoic acid (C12). The highest antimicrobial potency was achieved by introducing dodecanoic acid to a cyclic Turgencin A analogue with low intrinsic hydrophobicity, and by introducing octanoic acid to a cyclic analogue displaying a higher intrinsic hydrophobicity. Among all tested synthetic Turgencin A lipopeptide analogues, the most promising candidates regarding both antimicrobial and haemolytic activity were C12-cTurg-1 and C8-cTurg-2. These optimized cyclic lipopeptides displayed minimum inhibitory concentrations of 4 µg/mL against Staphylococcus aureus, Escherichia coli and the fungus Rhodothorula sp. Mode of action studies on bacteria showed a rapid membrane disruption and bactericidal effect of the cyclic lipopeptides. Haemolytic activity against human erythrocytes was low, indicating favorable selective targeting of bacterial cells.

Sustainable Production of Biosurfactant from Agro-Industrial Oil Wastes by Bacillus subtilis and Its Potential Application as Antioxidant and ACE Inhibitor.

The microbial conversion of agro-industrial oil wastes into biosurfactants shows promise as a biomass refinery approach. In this study, Bacillus subtilis #309 was applied to produce surfactin using rapeseed and sunflower cakes, the most common oil processing side products in Europe. Studies of the chemical composition of the substrates were performed, to determine the feasibility of oil cakes for surfactin production. Initially, screening of proteolytic and lipolytic activity was performed to establish the capability of B. subtilis #309 for substrate utilization and hence effective surfactin production. B. subtilis #309 showed both proteolytic and lipolytic activity. The process of surfactin production was carefully analyzed by measurement of the surfactin concentration, pH, surface tension (ST) and emulsification index (E24). The maximal surfactin concentration in the sunflower and rapeseed cake medium reached 1.19 ± 0.03 and 1.45 ± 0.09 g/L, respectively. At the same time, a progressive decrease in the surface tension and increase in emulsification activity were observed. The results confirmed the occurrence of various surfactin homologues, while the surfactin C15 was the dominant one. Finally, the analysis of surfactin biological function exhibited antioxidant activity and significant angiotensin-converting enzyme (ACE)-inhibitory activity. The half-maximal inhibitory concentration (IC50) value for ACE inhibition was found to be 0.62 mg/mL for surfactin. Molecular docking of the surfactin molecule to the ACE domains confirmed its inhibitory activity against ACE. Several interactions, such as hydrophobic terms, hydrogen bonds and van der Waals interactions, were involved in the complex stabilization. To the best of our knowledge, this is the first report describing the effect of a lipopeptide biosurfactant, surfactin, produced by B. subtilis for multifunctional properties in vitro, namely the ACE-inhibitory activity and the antioxidant properties, using different assays, such as 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). Thus, the ACE-inhibitory lipopeptide biosurfactant shows promise to be used as a natural antihypertensive agent.

Anti-proliferative activity of surfactins on human cancer cells and their potential use in therapeutics.

Surfactins are cyclic lipopeptides that are isolated from various Bacillus strains. They are made up of heptapeptides and ß-hydroxy fatty acids of variable chain lengths of carbon atoms. Therapeutically they are known to inhibit invasion, migration, and colony formation of human breast carcinoma cells. The role of surfactins is also known as anti-proliferative agents against human cancer cells through induction of apoptosis, arrest of the cell cycle, or suppression of survival signaling. The cytotoxic activity of surfactins is also perceived against human chronic myelogenous leukemia cells, human colon cancer cells, and hepatic carcinoma cells. Considering the wide spectrum of targets, the molecular effects of surfactins are diverse in different cancer cells and they can serve as promising chemotherapeutic agents for the treatment of cancer. Surfactins are being delivered to the targeted cancer cells through nano-carriers or nano-formulations. The present review article provides insight on different types and variations of surfactins, their molecular effect on different cancer cells, and their therapeutic use in the treatment of human cancer.

Remediation and detoxification of oil contaminated marine intertidal sites through lipopeptide assisted washing strategy: An experimental and kinetic validation approach.

This paper presents a tightly coupled experimental and kinetic approach for efficient remediation of oil spill from contaminated marine intertidal zone surface through a methodical strategy that deals with biosurfactant mediated washing strategy. The study deals with production, optimization and characterization of lipopeptide biosurfactant from Bacillus subtilis T1 and its application in remediation of oil contaminants from mimic model system of various marine intertidal zone i.e. woodland-Group1, saltmarsh-Group2, mangrove-Group3 and mudflats-Group4. Results demonstrates enhanced washing performance with oil desorption rate of 35 % in Group 4, 17.22 %, 15.6 % and 11 % in Group 3, 2 and 1 along with bio surfactant recovery rate of 41 %, 48.7 %, 51.71 % and 50.3 % respectively. Further, the washing strategy was efficient in soil detoxification with highest rate in Group 4. The kinetic validation depicts good match among experimental data and Lagergren pseudo second order data.

RBD conjugate vaccine with a built-in TLR1/2 agonist is highly immunogenic against SARS-CoV-2 and variants of concern.

The coronavirus 2019 (COVID-19) pandemic is causing serious impacts in the world, and safe and effective vaccines and medicines are the best methods to combat the disease. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in interacting with the angiotensin-converting enzyme 2 (ACE2) receptor, and is regarded as an important target of vaccines. Herein, we constructed the adjuvant-protein conjugate Pam3CSK4-RBD as a vaccine candidate, in which the N-terminal of the RBD was site-selectively oxidized by transamination and conjugated with the TLR1/2 agonist Pam3CSK4. This demonstrated that the conjugation of Pam3CSK4 significantly enhanced the anti-RBD antibody response and cellular response. In addition, sera from the Pam3CSK4-RBD immunized group efficiently inhibited the binding of the RBD to ACE2 and protected cells from SARS-CoV-2 and four variants of concern (alpha, beta, gamma and delta), indicating that this adjuvant strategy could be one of the effective means for protein vaccine development.