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Carbamazepine is considered as an emerging pollutant since in aquatic environments is toxic for fish and difficult to degrade. The goal of this study was to isolate yeasts capable of degrading carbamazepine. A hospital wastewater sample was used as an inoculant to carry out three successive transfers in minimal mineral medium added with 50 mg L−1 of carbamazepine. The isolates were characterized by colonial and microscopic morphology. A yeast was identified by sequencing its ITS1-5.8S-ITS2 intergenic region and by constructing of a phylogenetic tree. A kinetics of carbamazepine degradation was done with an initial concentration of 150 mg L−1 and the residual drug was determined by HPLC-UV. A total of 12 isolates were obtained, one of them had the characteristics of yeast (Carba14), the others were bacteria. Yeast isolate Carba 14 grew at all concentrations of carbamazepine tested (50, 100, 150 and 200 mg L−1) and grew better than bacterial isolates. This isolate degraded carbamazepine with an efficiency of 30% within 25 days. Yeast isolate Carba 14 was identified as Candida palmioleophila Carba 14. Our study shows for the first time the isolation of C. palmioleophila Carba 14 with potential to degrade carbamazepine. [ABSTRACT FROM AUTHOR]

We recently reported the isolation and characterization of anti-SARS-CoV-2 antibodies from a phage display library built with the VH repertoire of a convalescent COVID-19 patient, paired with four naïve synthetic VL libraries. One of the antibodies, called IgG-A7, neutralized the Wuhan, Delta (B.1.617.2) and Omicron (B.1.1.529) strains in authentic neutralization tests (PRNT). It also protected 100% transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE-2) from SARS-CoV-2 infection. In this study, the four synthetic VL libraries were combined with the semi-synthetic VH repertoire of ALTHEA Gold Libraries™ to generate a set of fully naïve, general-purpose, libraries called ALTHEA Gold Plus Libraries™. Three out of 24 specific clones for the RBD isolated from the libraries, with affinity in the low nanomolar range and sub-optimal in vitro neutralization in PRNT, were affinity optimized via a method called "Rapid Affinity Maturation" (RAM). The final molecules reached sub-nanomolar neutralization potency, slightly superior to IgG-A7, while the developability profile over the parental molecules was improved. These results demonstrate that general-purpose libraries are a valuable source of potent neutralizing antibodies. Importantly, since general-purpose libraries are "ready-to-use", it could expedite isolation of antibodies for rapidly evolving viruses such as SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Neutralizing antibodies targeting the receptor-binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and/or treat COVID-19. However, as SARS-CoV-2 has evolved into new variants, most of the neutralizing antibodies authorized by the US FDA and/or EMA to treat COVID-19 have shown reduced efficacy or have failed to neutralize the variants of concern (VOCs), particularly B.1.1.529 (Omicron). Previously, we reported the discovery and characterization of antibodies with high affinity for SARS-CoV-2 RBD Wuhan (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) strains. One of the antibodies, called IgG-A7, also blocked the interaction of human angiotensin-converting enzyme 2 (hACE2) with the RBDs of the three strains, suggesting it may be a broadly SARS-CoV-2 neutralizing antibody. Herein, we show that IgG-A7 efficiently neutralizes all the three SARS-CoV-2 strains in plaque reduction neutralization tests (PRNTs). In addition, we demonstrate that IgG-A7 fully protects K18-hACE2 transgenic mice infected with SARS-CoV-2 WT. Taken together, our findings indicate that IgG-A7 could be a suitable candidate for development of antibody-based drugs to treat and/or prevent SARS-CoV-2 VOCs infection. [ABSTRACT FROM AUTHOR]

Canine parvovirus type II (CPV-2) infection induces canine parvoviral enteritis (CPE), which in turn promotes sepsis and systemic inflammatory response syndrome (SIRS). Mortality in this disease is usually registered within 48–72 h post-hospitalization, the critical period of the illness. It has been recently described that the use of an immunomodulator, whose major component is monomeric ubiquitin (mUb) without the last two glycine residues (Ub∆GG), in pediatric human patients with sepsis augments survival. It is known that CXCR4 is the cell receptor of extracellular ubiquitin in humans. This work aimed to explore the effect of one immunomodulator (human Dialyzable Leukocyte Extract-hDLE) as a therapeutic auxiliary in puppies with sepsis and SIRS induced by CPE. We studied two groups of puppies with CPV-2 infection confirmed by polymerase chain reaction. The first group received conventional treatment (CT) and vehicle (V), while the second group received CT plus the immunomodulator (I). We assessed both groups' survival, clinical condition, number of erythrocytes, neutrophils, and lymphocytes during the hospitalization period. In addition, hematocrit, hemoglobin, plasma proteins and cortisol values, as well as norepinephrine/epinephrine and serotonin concentration were determined. Puppies treated with CT + I showed 81% survival, mild clinical signs, and a significant decrease in circulating neutrophils and lymphocytes in the critical period of the treatment. In contrast, the CT + V group presented a survival of 42%, severe clinical status, and no improvement of the parameters evaluated in the critical period of the disease. We determined in silico that human Ub∆GG can bind to dog CXCR4. In conclusion, the administration of a human immunomodulator (0.5 mg/day × 5 days) to puppies with CPE under six months of age reduces the severity of clinical signs, increases survival, and modulates inflammatory cell parameters. Further studies are necessary to take full advantage of these clinical findings, which might be mediated by the human Ub∆GG to canine CXCR4 interaction. [ABSTRACT FROM AUTHOR]

Background: The deterioration of the skin accentuates over time, affecting its aesthetic appearance. This is characterized by the weakening of the mechanisms involved in the regeneration and repair of the dermal matrix. Consequently, the skin losses elasticity and smoothness resulting in the formation of wrinkles. The alternatives for facial rejuvenation include surgery, injection of botulinum toxin, and the application of masks. Topic products are less invasive, can be self‐applied, and have an increased benefit/risk relationship. Aim: We developed a liquid formulation containing collagen hydrolyzed and evaluated the product by cutting‐edge technology in order to define proper its quality attributes. Methods: We employed nuclear magnetic resonance (NMR), size‐exclusion chromatography (SEC), and mass spectrometry (MS). Additionally, we analyzed its cosmetical effect in five volunteers and we demonstrate the product safety. Results: Our results demonstrate the following: (a) a stable secondary structure identity associated to the known triple helix arrangement in liquid and solid states; (b) a typical conformational flexibility depending on its hydration state; (c) thermal stability confirmed by liquid‐ and solid‐state nuclear magnetic resonance schemes; and (d) a molecular mass distribution of peptides between 0.5 and 19.5 kDa. The product faded wrinkles in the forehead, an effect that remained after removing the mask. The formula was non‐irritating and hypoallergenic. Conclusion: We characterized, using state‐of‐the‐art methodologies, the quality attributes that are critical for the safety and beneficial effect of a new collagen‐containing formula. [ABSTRACT FROM AUTHOR]

"Transferon Oral" is a peptide-derived product with immunomodulatory properties obtained from the lysis and dialysis of human buffy coat. Its active pharmaceutical ingredient, generically known as Dialyzable Leucocyte Extract, is a mixture of peptide populations with reproducible proportions among batches. "Transferon Oral" modulates IFN-γ, TNF-α, and IL-6 and increases the survival rate in a herpes infection murine model when oropharyngeally (ORO) administered, which correlate with clinical observations where "Transferon Oral" is used as a therapeutic auxiliary in inflammatory diseases. Notwithstanding, how a peptide-derived product elicits systemic modulation of cytokines when ORO administered remains unclear. To shed light on the pharmacology of "Transferon Oral" its peptide components must be known. Ten "Transferon Oral" batches were sequenced by mass spectrometry and the intact peptides were identified. The most abundant peptides were the monomeric human Ubiquitin (Ub), a globular low-molecular mass protein, and an Ub variant which lacks the two-terminal Gly (Ub-GG). Recombinant Ub prevented murine death when ORO administered in a herpes infection murine model. Besides, the percentage of survival increased in groups treated with Transferon Oral+Ub and decreased in groups treated with Ub-depleted "Transferon Oral" respect to the group treated with "Transferon Oral" only. Our findings indicate that the biological properties of "Transferon Oral" are partially associated to the Ub content. They suggest that Ub may activate its extracellular receptor (CXCR-4) in the stomach eliciting systemic immunomodulatory effects via vagus nerve. This is the first report that identifies an active component of "Transferon Oral" with the potential for the development of oral peptide immunomodulators. [ABSTRACT FROM AUTHOR]

Transferon® is an immunomodulator made of a complex mixture of peptides from human dialyzable leucocyte extracts (hDLEs). Development of surrogate antibodies directed to hDLE is an indispensable tool for studies during process control and preclinical trials. These antibodies are fundamental for different analytical approaches, such as identity test and drug quantitation, as well as to characterize its pharmacokinetic and mechanisms of action. A previous murine study showed the inability of the peptides of Transferon® to induce antibody production by themselves; therefore, in this work, two approaches were tested to increase its immunogenicity: chemical conjugation of the peptides of Transferon® to carrier proteins and the use of a rabbit model. Bioconjugates were generated with Keyhole Limpet Hemocyanin (KLH) or Bovine Serum Albumin (BSA) through maleimide-activated carrier proteins. BALB/c mice and New Zealand rabbits were immunized with Transferon® conjugated to KLH or nonconjugated Transferon®. Animals that were immunized with conjugated Transferon® showed significant production of antibodies as evinced by the recognition of Transferon®-BSA conjugate in ELISA assays. Moreover, rabbits showed higher antibody titers when compared with mice. Neither mouse nor rabbits developed antibodies when immunized with nonconjugated Transferon®. Interestingly, rabbit antibodies were able to partially block IL-2 production in Jurkat cells after costimulation with Transferon®. In conclusion, it is feasible to elicit specific and functional antibodies anti-hDLE with different potential uses during the life cycle of the product. [ABSTRACT FROM AUTHOR]

Collagen hydrolysates are dietary supplements used for nutritional and medical purposes. They are complex mixtures of low‐molecular‐weight peptides obtained from the enzymatic hydrolysis of collagen, which provide intrinsic batch‐to‐batch heterogeneity. In consequence, the quality of these products, which is related to the reproducibility of their mass distribution pattern, should be addressed. Here, we propose an analytical approach to determine the peptide pattern as a quality attribute of Colagenart®, a product containing collagen hydrolysate. In addition, we evaluated the safety by measuring the viability of two cell lines exposed to the product. The consistency of peptide distribution was determined using Size Exclusion Chromatography (SEC), Mass Spectrometry coupled to a reversed phase UPLC system (MS‐RP‐UPLC), and Shaped‐pulse off‐resonance water‐presaturation proton nuclear magnetic resonance spectrometry [1Hwater_presat NMR]. The mass distribution pattern determined by SEC was in a range from 1.35 to 17 kDa, and from 2 to 14 kDa by MS‐RP‐UPLC. [1Hwater_presat NMR] showed the detailed spin‐systems of the collagen hydrolysates components by global assignment of backbone Hα and NH, as well as side‐chain proton resonances. Additionally, short‐range intraresidue connectivity pathways of identified spin‐regions were obtained by a 2D homonuclear shift correlation Shaped‐pulse solvent suppression COSY scheme. Safety analysis of Colagenart® was evaluated in CaCo‐2 and HepG2 cells at 2.5 and 25 μg/mL and no negative effects were observed. The results demonstrated batch‐to‐batch reproducibility, which evinces the utility of this approach to establish the consistency of the quality attributes of collagen hydrolysates. Practical Application: We propose state‐of‐the art analytical methodologies (SEC, MS, and NMR) to evaluate peptide profile and composition of collagen hydrolysates as quality attributes. These methodologies are suitable to be implemented for quality control purposes. [ABSTRACT FROM AUTHOR]

Neutralizing antibodies targeting the receptor-binding domain (RBD) of SARSCoV-2 are among the most promising strategies to prevent and/or treat COVID-19. However, as SARS-CoV-2 has evolved into new variants, most of the neutralizing antibodies authorized by the US FDA and/or EMA to treat COVID-19 have shown reduced efficacy or have failed to neutralize the variants of concern (VOCs), particularly B.1.1.529 (Omicron). Previously, we reported the discovery and characterization of antibodies with high affinity for SARS-CoV-2 RBD Wuhan (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) strains. One of the antibodies, called IgG-A7, also blocked the interaction of human angiotensin-converting enzyme 2 (hACE2) with the RBDs of the three strains, suggesting it may be a broadly SARS-CoV-2 neutralizing antibody. Herein, we show that IgG-A7 efficiently neutralizes all three SARS-CoV-2 strains in plaque reduction neutralization tests (PRNTs). In addition, we demonstrate that IgG-A7 fully protects K18-hACE2 transgenic mice infected with SARS-CoV-2 WT. Our findings indicate that IgG-A7 could be a suitable candidate for developing antibody-based drugs to treat and/ or prevent SARS-CoV-2 VOCs infection. [ABSTRACT FROM AUTHOR]

We describe here the construction of a single domain antibody (sdAb) library consisting of grafting natural human HCDR3 fragments obtained from 200 healthy human donors into a synthetic framework region designed with the human germline IGHV3-23. After three rounds of panning against the hACE-2 receptor binding domain (RBD) of SARS-CoV-2, 90 clones were randomly picked and screened for binding to RBD and BSA. A total of 35 clones specifically bound RBD, with 9 clones showing unique sequences. Out of these unique clones, four blocked RBD:hACE2 interaction.. A Clone, termed sdC3, was expressed in E. coli and purified by Protein A, exhibiting the expected molecular mass of ≈18 kDa. Purified sdC3 had an affinity for RBD comparable to a potent neutralizing scFv and blocked the RBD:ACE2 interaction. Further, heat-treated sdC3 at 80 °C for 10 minutes retained its binding affinity, indicating high thermal stability, an advantageous property typical of sdAbs. Thus, the platform we developed for rapid isolation of fully human sdAbs set the stage for future discovery of diverse molecules with application not only in rapidly evolving viral diseases such as SARS-CoV-2 but also in other applications that require highly stable molecules. [ABSTRACT FROM AUTHOR]

Transferon, a human dialyzable leukocyte extract (hDLE), is a biotherapeutic that comprises a complex mixture of low-molecular-weight peptides (< 10 kDa) and is used to treat diseases with an inflammatory component. Some biotherapeutics, including those composed of peptides, can induce anti-drug antibodies (ADA) that block or diminish their therapeutic effect. Nevertheless, few studies have evaluated peptide-derived drug immunogenicity. In this study, the immunogenicity of Transferon was examined in a murine model during an immunization scheme using the following adjuvants: Al(OH)3, incomplete Freund's adjuvant (IFA), or Titermax Gold. The inoculation scheme entailed three routes of administration (intraperitoneal, Day 1; subcutaneous, Day 7; and intramuscular, Day 14) using 200 μg Transferon/inoculation. Serum samples were collected on Day 21. Total IgG levels were quantitated by affinity chromatography, and specific antibodies against components of Transferon were analyzed by dot-blot and ELISA. Ovalbumin (OVA, 44 kDa) and peptides from hydrolyzed collagen (PFHC, < 17 kDa) were used as positive and negative controls, respectively, in the same inoculation scheme and analyses for Transferon. OVA, PFHC, and Transferon increased total IgG concentrations in mice. However, only IgG antibodies against OVA were detected. Based on the results, it is concluded that Transferon does not induce generation of specific antibodies against its components in this model, regardless of adjuvant and route of administration. These results support the safety of Transferon by confirming its inability to induce ADA in this animal model. [ABSTRACT FROM PUBLISHER]

Transferon, a biotherapeutic agent that has been used for the past 2 decades for diseases with an inflammatory component, has been approved by regulatory authorities in Mexico (COFEPRIS) for the treatment of patients with herpes infection. The active pharmaceutical ingredient (API) of Transferon is based on polydispersion of peptides that have been extracted from lysed human leukocytes by a dialysis process and a subsequent ultrafiltration step to select molecules below 10 kDa. To physicochemically characterize the drug product, we developed chromatographic methods and an SDS-PAGE approach to analyze the composition and the overall variability of Transferon. Reversed-phase chromatographic profiles of peptide populations demonstrated batch-to-batch consistency from 10 representative batches that harbored 4 primary peaks with a relative standard deviation (RSD) of less than 7%. Aminogram profiles exhibited 17 proteinogenic amino acids and showed that glycine was the most abundant amino acid, with a relative content of approximately 18%. Further, based on their electrophoretic migration, the peptide populations exhibited a molecular mass of about 10 kDa. Finally, we determined the Transferon fingerprint using a mass spectrometry tool. Because each batch was produced from independent pooled buffy coat samples from healthy donors, supplied by a local blood bank, our results support the consistency of the production of Transferon and reveal its peptide identity with regard to its physicochemical attributes. [ABSTRACT FROM AUTHOR]

Human dialyzable leukocyte extracts (DLEs) are heterogeneous mixtures of low-molecular-weight peptides that are released on disruption of peripheral blood leukocytes from healthy donors. DLEs improve clinical responses in infections, allergies, cancer, and immunodeficiencies. Transferon is a human DLE that has been registered as a hemoderivate by Mexican health authorities and commercialized nationally. To develop an animal model that could be used routinely as a quality control assay for Transferon, we standardized and validated a murine model of cutaneous HSV-1 infection. Using this model, we evaluated the activity of 27 Transferon batches. All batches improved the survival of HSV-1-infected mice, wherein average survival rose from 20.9% in control mice to 59.6% in Transferon-treated mice. The activity of Transferon correlated with increased serum levels of IFN-γ and reduced IL-6 and TNF-α concentrations. Our results demonstrate that (i) this mouse model of cutaneous herpes can be used to examine the activity of DLEs, such as Transferon; (ii) the assay can be used as a routine test for batch release; (iii) Transferon is produced with high homogeneity between batches; (iv) Transferon does not have direct virucidal, cytoprotective, or antireplicative effects; and (v) the protective effect of Transferon in vivo correlates with changes in serum cytokines. [ABSTRACT FROM AUTHOR]