A Reliable Multifaceted Solution against Foodborne Viral Infections: The Case of RiLK1 Decapeptide.

Food-borne transmission is a recognized route for many viruses associated with gastrointestinal, hepatic, or neurological diseases. Therefore, it is essential to identify new bioactive compounds with broad-spectrum antiviral activity to exploit innovative solutions against these hazards. Recently, antimicrobial peptides (AMPs) have been recognized as promising antiviral agents. Indeed, while the antibacterial and antifungal effects of these molecules have been widely reported, their use as potential antiviral agents has not yet been fully investigated. Herein, the antiviral activity of previously identified or newly designed AMPs was evaluated against the non-enveloped RNA viruses, hepatitis A virus (HAV) and murine norovirus (MNV), a surrogate for human norovirus. Moreover, specific assays were performed to recognize at which stage of the viral infection cycle the peptides could function. The results showed that almost all peptides displayed virucidal effects, with about 90% of infectivity reduction in HAV or MNV. However, the decapeptide RiLK1 demonstrated, together with its antibacterial and antifungal properties, a notable reduction in viral infection for both HAV and MNV, possibly through direct interaction with viral particles causing their damage or hindering the recognition of cellular receptors. Hence, RiLK1 could represent a versatile antimicrobial agent effective against various foodborne pathogens including viruses, bacteria, and fungi.

The Antimicrobial Peptide 1018-K6 Interacts Distinctly with Eukaryotic and Bacterial Membranes, the Basis of Its Specificity and Bactericidal Activity.

Since penicillin was discovered, antibiotics have been critical in the fight against infections. However, antibiotic misuse has led to drug resistance, which now constitutes a serious health problem. In this context, antimicrobial peptides (AMPs) constitute a natural group of short proteins, varying in structure and length, that act against certain types of bacterial pathogens. The antimicrobial peptide 1018-K6 (VRLIVKVRIWRR- NH2) has significant bactericidal and antibiofilm activity against Listeria monocytogenes isolates, and against different strains and serotypes of Salmonella. Here, the mechanism of action of 1018-K6 was explored further to understand the peptide-membrane interactions relevant to its activity, and to define their determinants. We combined studies with model synthetic membranes (liposomes) and model biological membranes, assessing the absorption maximum and the quenching of 1018-K6 fluorescence in aqueous and lipid environments, the self-quenching of carboxyfluorescein, as well as performing lipid sedimentation assays. The data obtained reflect the differential interactions of the 1018-K6 peptide with eukaryotic and prokaryotic membranes, and the specific interactions and mechanisms of action in the three prokaryotic species studied: Salmonella Typhimurium2GN, Escherichia coli3GN, and Staphylococcus aureus3GP. The AMP 1018-K6 is a candidate to prevent (food preservation) or treat (antibiotic use) infections caused by certain pathogenic bacteria, especially some that are resistant to current antibiotics.

Selective inhibition of acylpeptide hydrolase in SAOS-2 osteosarcoma cells: is this enzyme a viable anticancer target?

Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this, most of the human serine hydrolases remain poorly characterized with respect to their biological functions and substrates and only a limited number of in vivo active inhibitors have been so far identified. Acylpeptide hydrolase (APEH) is a member of the prolyl-oligopeptidase class, with a unique substrate specificity, that has been suggested to have a potential oncogenic role. In this study, a set of peptides was rationally designed from the lead compound SsCEI 4 and in vitro screened for APEH inhibition. Out of these molecules, a dodecapeptide named Ala 3 showed the best inhibitory effects and it was chosen as a candidate for investigating the anti-cancer effects induced by inhibition of APEH in SAOS-2 cell lines. The results clearly demonstrated that Ala 3 markedly reduced cell viability via deregulation of the APEH-proteasome system. Furthermore, flow cytometric analysis revealed that Ala 3 anti-proliferative effects were closely related to the activation of a caspase-dependent apoptotic pathway. Our findings provide further evidence that APEH can play a crucial role in the pathogenesis of cancer, shedding new light on the great potential of this enzyme as an attractive target for the diagnosis and the quest for selective cancer therapies.

Oxidized Substrates of APEH as a Tool to Study the Endoprotease Activity of the Enzyme.

APEH is a ubiquitous and cytosolic serine protease belonging to the prolyl oligopeptidase (POP) family, playing a critical role in the processes of degradation of proteins through both exo- and endopeptidase events. Endopeptidase activity has been associated with protein oxidation; however, the actual mechanisms have yet to be elucidated. We show that a synthetic fragment of GDF11 spanning the region 48-64 acquires sensitivity to the endopeptidase activity of APEH only when the methionines are transformed into the corresponding sulphoxide derivatives. The data suggest that the presence of sulphoxide-modified methionines is an important prerequisite for the substrates to be processed by APEH and that the residue is crucial for switching the enzyme activity from exo- to endoprotease. The cleavage occurs on residues placed on the C-terminal side of Met(O), with an efficiency depending on the methionine adjacent residues, which thereby may play a crucial role in driving and modulating APEH endoprotease activity.

Key Physicochemical Determinants in the Antimicrobial Peptide RiLK1 Promote Amphipathic Structures.

Antimicrobial peptides (AMPs) represent a skilled class of new antibiotics, due to their broad range of activity, rapid killing, and low bacterial resistance. Many efforts have been made to discover AMPs with improved performances, i.e., high antimicrobial activity, low cytotoxicity against human cells, stability against proteolytic degradation, and low costs of production. In the design of new AMPs, several physicochemical features, such as hydrophobicity, net positive charge, propensity to assume amphipathic conformation, and self-assembling properties, must be considered. Starting from the sequence of the dodecapeptide 1018-K6, we designed a new 10-aminoacid peptide, namely RiLK1, which is highly effective against both fungi and Gram-positive and -negative bacteria at low micromolar concentrations without causing human cell cytotoxicity. In order to find the structural reasons explaining the improved performance of RiLK1 versus 1018-K6, a comparative analysis of the two peptides was carried out with a combination of CD, NMR, and fluorescence spectroscopies, while their self-assembling properties were analyzed by optical and atomic force microscopies. Interestingly, the different spectroscopic and microscopic profiles exhibited by the two peptides, including the propensity of RiLK1 to adopt helix arrangements in contrast to 1018-K6, could explain the improved bactericidal, antifungal, and anti-biofilm activities shown by the new peptide against a panel of food pathogens.

A Safe and Multitasking Antimicrobial Decapeptide: The Road from De Novo Design to Structural and Functional Characterization.

Antimicrobial peptides (AMPs) are excellent candidates to fight multi-resistant pathogens worldwide and are considered promising bio-preservatives to control microbial spoilage through food processing. To date, designing de novo AMPs with high therapeutic indexes, low-cost synthesis, high resistance, and bioavailability, remains a challenge. In this study, a novel decapeptide, named RiLK1, was rationally designed starting from the sequence of the previously characterized AMP 1018-K6, with the aim of developing short peptides, and promoting higher selectivity over mammalian cells, antibacterial activity, and structural resistance under different salt, pH, and temperature conditions. Interestingly, RiLK1 displayed a broad-spectrum of bactericidal activity against Gram-positive and Gram-negative bacteria, including multidrug resistant clinical isolates of Salmonella species, with Minimal Bactericidal Concentration (MBC) values in low micromolar range, and it was effective even against two fungal pathogens with no evidence of cytotoxicity on human keratinocytes and fibroblasts. Moreover, RiLK1-activated polypropylene films were revealed to efficiently prevent the growth of microbial spoilage, possibly improving the shelf life of fresh food products. These results suggested that de novo designed peptide RiLK1 could be the first candidate for the development of a promising class of decameric and multitask antimicrobial agents to overcome drug-resistance phenomena.

Functionalized Polymeric Materials with Bio-Derived Antimicrobial Peptides for "Active" Packaging.

Food packaging is not only a simple protective barrier, but a real "active" component, which is expected to preserve food quality, safety and shelf-life. Therefore, the materials used for packaging production should show peculiar features and properties. Specifically, antimicrobial packaging has recently gained great attention with respect to both social and economic impacts. In this paper, the results obtained by using a polymer material functionalized by a small synthetic peptide as "active" packaging are reported. The surface of Polyethylene Terephthalate (PET), one of the most commonly used plastic materials in food packaging, was plasma-activated and covalently bio-conjugated to a bactenecin-derivative peptide named 1018K6, previously characterized in terms of antimicrobial and antibiofilm activities. The immobilization of the peptide occurred at a high yield and no release was observed under different environmental conditions. Moreover, preliminary data clearly demonstrated that the "active" packaging was able to significantly reduce the total bacterial count together with yeast and mold spoilage in food-dairy products. Finally, the functionalized-PET polymer showed stronger efficiency in inhibiting biofilm growth, using a Listeria monocytogenes strain isolated from food products. The use of these "active" materials would greatly decrease the risk of pathogen development and increase the shelf-life in the food industry, showing a real potential against a panel of microorganisms upon exposure to fresh and stored products, high chemical stability and re-use possibility.

Small Synthetic Peptides Bioconjugated to Hybrid Gold Nanoparticles Destroy Potentially Deadly Bacteria at Submicromolar Concentrations.

Synthetic antibacterial peptides are advanced weapons that scientists design and produce to confront current threats of harmful and mortal pathogens, which could affect humans in everyday life. Recently, many small amino acid sequences, greatly efficient in their antibacterial action, have been reported in the literature. To date, only a few synthetic peptides, acting at micromolar or even tenths of micromolar concentrations, are on the market as commercial products, mainly because of their high cost of production. In this context, materials science can provide fundamental help by engineering small synthetic peptides, powered by hybrid gold nanoparticles, which have been found to strongly enhance antimicrobial activity against bacterial infections. Submicromolar concentrations of the 1018K6 peptide, bioconjugated to hybrid polymer-gold nanoparticles, kill almost 100% of pathogen bacteria, such as Listeria and Salmonella genera, paving the way for economically sustainable commercial products based on this synthetic nanocomplex.

Isolation and characterization from solar salterns of North Algeria of a haloarchaeon producing a new halocin.

Halophilic archaea, thriving in hypersaline environments, synthesize antimicrobial substances with an unknown role, called halocins. It has been suggested that halocin production gives transient competitive advantages to the producer strains and represents one of the environmental factors influencing the microbial community composition. Herein, we report on the antibacterial activity of a new haloarchaeon selected from solar salterns of the northern coast of Algeria. A total of 81 halophilic strains, isolated from the microbial consortia, were screened for the production of antimicrobial compounds by interspecies competition test and against a collection of commercial haloarchaea. On the basis of the partial 16S rRNA sequencing, the most efficient halocin producer was recognized as belonging to Haloferax (Hfx) sp., while the best indicator microorganism, showing high sensitivity toward halocin, was related to Haloarcula genus. The main morphological, physiological and biochemical properties of Hfx were investigated and a partial purification of the produced halocin was allowed to identify it as a surface membrane protein with a molecular mass between 30 and 40 kDa. Therefore, in this study, we isolated a new strain belonging to Haloferax genus and producing a promising antimicrobial compound useful for applications in health and food industries.

Unusual Antioxidant Properties of 26S Proteasome Isolated from Cold-Adapted Organisms.

The oxidative challenge represents an important factor affecting the adaptive strategies in Antarctic fish, but their impact on the protein degradation machinery still remains unclear. The previous analysis of the first 26S proteasome from the Antarctic red-blooded fish Trematomus bernacchii, evidenced improved antioxidant functions necessary to counteract the environmental pro-oxidant conditions. The purpose of this work was to carry out a study on 26S proteasomes from the temperate red-blooded Dicenthrarcus labrax and the icefish Chionodraco hamatus in comparison with the isoform already described from T. bernacchii, to better elucidate the cold-adapted physiological functions of this complex. Therefore, the 26S isoforms were isolated and the complementary DNAs (cDNAs) codifying the catalytic subunits were cloned. The biochemical characterization of Antarctic 26S proteasomes revealed their significantly higher structural stability and resistance to H2O2 with respect to that of the temperate counterpart, as also suggested by a comparative modeling analysis of the catalytic subunits. Moreover, in contrast to that observed in T. bernacchii, the 26S systems from C. hamatus and D. labrax were incapable to hydrolyze oxidized proteins in a ubiquitin-independent manner. Therefore, the 'uncommon' properties displayed by the Antarctic 26S proteasomes can mirror the impact exercised by evolutionary pressure in response to richly oxygenated environments.

Adaptive response activated by dietary cis9, trans11 conjugated linoleic acid prevents distinct signs of gliadin-induced enteropathy in mice.

PURPOSE: The beneficial effects of conjugated linoleic acid (CLA) mixture (cis9, trans11, c9; trans10, cis12, t10) against gliadin-induced toxicity in HLA-DQ8-transgenic mice (DQ8) have been associated with improved duodenal cytoprotective mechanisms [nuclear factor-E2-related factor-2, Nrf2; acylpeptide hydrolase (APEH)/proteasome]. The present study was aimed at investigating the ability of individual CLA isomers to improve the efficacy of these defensive mechanisms and to protect against duodenal injury caused by the combined administration of gliadin and indomethacin (GI). METHODS: Gluten-mediated enteropathy was induced in DQ8 mice by three intra-gastric administration of gliadin (20 mg kg(-1)/bw) and indomethacin (15 mg L(-1)) in drinking water for 10 days (GI). C9 or t10 CLA (520 mg kg(-1)/bw/day) were orally administered for 2 weeks. Pro-oxidant and toxic effects associated with GI treatment, anti-oxidant/detoxifying ability of c9 or t10-CLA and the protective effect induced by c9 pre-treatment (c9 + GI) were evaluated in DQ8 mice duodenum by combining enzymatic, immunoblotting, histological evaluation and quantitative real-time PCR assays. RESULTS: GI treatment produces the time-dependent decline of the considered detoxifying mechanisms thus leading to pro-apoptotic and pro-oxidant effects. APEH/proteasome pathway was not markedly affected by individual CLA isomers, but duodenal redox status and activity/mRNA levels of Nrf2-activated enzymes were significantly improved by c9 administration. c9 pre-treatment protects against GI-mediated accumulation of oxidative stress markers, and histological examination reveals the increase of goblet cells number in mouse duodenum but induces only a partial recovery of APEH/proteasome activity. CONCLUSIONS: The activation of and adaptive response by low doses of c9 supplementation prevents distinct signs of gliadin-induced enteropathy in DQ8 mice.

APEH Inhibition Affects Osteosarcoma Cell Viability via Downregulation of the Proteasome.

The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH-proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-κB, p21(Waf1), and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies.

Gliadin intake alters the small intestinal mucosa in indomethacin-treated HLA-DQ8 transgenic mice.

Celiac disease (CD) is an enteropathy caused by the ingestion of wheat gluten in genetically susceptible individuals. A complete understanding of the pathogenic mechanisms in CD has been hindered because of the lack of adequate in vivo models. In the present study, we explored the events after the intragastric administration of gliadin and of the albumin/globulin fraction from wheat in human leukocyte antigen-DQ8 transgenic mice (DQ8 mice) treated with indomethacin, an inhibitor of cyclooxygenases (COXs). After 10 days of treatment, mice showed a significant reduction of villus height, increased crypt depth, increased number of lamina propria-activated macrophages, and high basal interferon-γ secretion in mesenteric lymph nodes, all of which were specifically related to gliadin intake, whereas the albumin/globulin fraction of wheat was unable to induce similar changes. Cotreatment with NS-398, a specific inhibitor of COX-2, also induced the intestinal lesion. Enteropathy onset was further characterized by high levels of oxidative stress markers, similar to CD. Biochemical assessment of the small intestine revealed the specific activation of matrix metalloproteinases 2 and 9, high caspase-3 activity, and a significant increase of tissue transglutaminase protein levels associated with the intestinal lesion. Notably, after 30 days of treatment, enteropathic mice developed serum antibodies toward gliadin (IgA) and tissue transglutaminase (IgG). We concluded that gliadin intake in combination with COX inhibition caused a basal inflammatory status and an oxidative stress condition in the small intestine of DQ8 mice, thus triggering the mucosal lesion and, subsequently, an antigen-specific immunity.

A new pepstatin-insensitive thermopsin-like protease overproduced in peptide-rich cultures of Sulfolobus solfataricus.

In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease), while the less abundant (named SsMTP-1) one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50-90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

Surface-exposed glycoproteins of hyperthermophilic Sulfolobus solfataricus P2 show a common N-glycosylation profile.

Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the N-glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of S. solfataricus, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex4(GlcNAc)2 plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of S. acidocaldarius, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of S. solfataricus are N-glycosylated by the same sulfated oligosaccharide and we identified 56 N-glycosylation sites in this subproteome.

A strategy to recover a poor-quality ligase product.

Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation. We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5' end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.

A new versatile peroxidase with extremophilic traits over-produced in MicroTom cell cultures.

Peroxidases are widespread key antioxidant enzymes that catalyse the oxidation of electron donor substrates in parallel with the decomposition of H2O2. In this work, a novel tomato peroxidase, named SAAP2, was isolated from MicroTom cell cultures, purified, and characterised. The enzyme was identified with 64% sequence coverage as the leprx21 gene product (suberization-associated anionic peroxidase 2-like) from Solanum lycopersicum, 334 amino acids long. Compared to other plant peroxidases, SAAP2 was more active at elevated temperatures, with the optimal temperature and pH at 90 °C and 5.0, respectively. Furthermore, the enzyme retained more than 80% of its maximal activity over the range of 70-80 °C and the presence of NaCl (1.0-4.5 M). It also exhibited broad pH versatility (65% relative activity over the pH range 2.0-7.0), acid-tolerance (80% residual activity after 22 h at pH 2.0-7.0), high thermostability (50% residual activity after 2 h at 80 °C) and proteolytic resistance. SAAP2 exhibited exceptional resistance under thermo-acidic conditions compared to the horseradish peroxidase benchmark, suggesting that it may find potential applications as a supplement or anti-pollution agent in the food industry.

Antimicrobial activity, membrane interaction and structural features of short arginine-rich antimicrobial peptides.

Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its ß-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.

In Vitro Assays for the Bifunctional Acylpeptide Hydrolase (APEH) Enzyme from Antarctic Fish.

The bifunctional enzyme acylpeptide hydrolase (APEH) is involved in important metabolic processes both as an exopeptidase and as an endopeptidase. Hence, the growing interest in the study of this protein and the need to set up in vitro assays for its characterization. This chapter describes two in vitro assays able to detect the activities of APEH, one for the exopeptidase activity and one for the endopeptidase activity. In particular, these assays have been set up on the two APEH isoforms from Antarctic fish, characterized by a distinct functionality and marked exo- and endopeptidase activities.

In-silico evaluation of adenoviral COVID-19 vaccination protocols: Assessment of immunological memory up to 6 months after the third dose.

Background: The immune response to adenoviral COVID-19 vaccines is affected by the interval between doses. The optimal interval is unknown. Aim: We aim to explore in-silico the effect of the interval between vaccine administrations on immunogenicity and to analyze the contribution of pre-existing levels of antibodies, plasma cells, and memory B and T lymphocytes. Methods: We used a stochastic agent-based immune simulation platform to simulate two-dose and three-dose vaccination protocols with an adenoviral vaccine. We identified the model's parameters fitting anti-Spike antibody levels from individuals immunized with the COVID-19 vaccine AstraZeneca (ChAdOx1-S, Vaxzevria). We used several statistical methods, such as principal component analysis and binary classification, to analyze the correlation between pre-existing levels of antibodies, plasma cells, and memory B and T cells to the magnitude of the antibody response following a booster dose. Results and conclusions: We find that the magnitude of the antibody response to a booster depends on the number of pre-existing memory B cells, which, in turn, is highly correlated to the number of T helper cells and plasma cells, and the antibody titers. Pre-existing memory T cytotoxic cells and antibodies directly influence antigen availability hence limiting the magnitude of the immune response. The optimal immunogenicity of the third dose is achieved over a large time window, spanning from 6 to 16 months after the second dose. Interestingly, after any vaccine dose, individuals can be classified into two groups, sustainers and decayers, that differ in the kinetics of decline of their antibody titers due to differences in long-lived plasma cells. This suggests that the decayers may benefit from a tailored boosting schedule with a shorter interval to avoid the temporary loss of serological immunity.