Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

Current Applications of Plant-Based Drug Delivery Nano Systems for Leishmaniasis Treatment.

Leishmania is a trypanosomatid that causes leishmaniasis. It is transmitted to vertebrate hosts during the blood meal of phlebotomine sandflies. The clinical manifestations of the disease are associated with several factors, such as the Leishmania species, virulence and pathogenicity, the host-parasite relationship, and the host's immune system. Although its causative agents have been known and studied for decades, there have been few advances in the chemotherapy of leishmaniasis. The urgency of more selective and less toxic alternatives for the treatment of leishmaniasis leads to research focused on the study of new pharmaceuticals, improvement of existing drugs, and new routes of drug administration. Natural resources of plant origin are promising sources of bioactive substances, and the use of ethnopharmacology and folk medicine leads to interest in studying new medications from phytocomplexes. However, the intrinsic low water solubility of plant derivatives is an obstacle to developing a therapeutic product. Nanotechnology could help overcome these obstacles by improving the availability of common substances in water. To contribute to this scenario, this article provides a review of nanocarriers developed for delivering plant-extracted compounds to treat clinical forms of leishmaniasis and critically analyzing them and pointing out the future perspectives for their application.

In Vitro and In Vivo Effect of pH-Sensitive PLGA-TPGS-Based Hybrid Nanoparticles Loaded with Doxorubicin for Breast Cancer Therapy.

Doxorubicin (DOX) is an antineoplastic agent clinically employed for treating breast cancer patients. Despite its effectiveness, its inherent adverse toxic side effects often limit its clinical application. To overcome these drawbacks, lipid-polymer hybrid nanoparticles (LPNP) arise as promising nanoplatforms that combine the advantages of both liposomes and polymeric nanoparticles into a single delivery system. Alpha-tocopherol succinate (TS) is a derivative of vitamin E that shows potent anticancer mechanisms, and it is an interesting approach as adjuvant. In this study, we designed a pH-sensitive PLGA-polymer-core/TPGS-lipid-shell hybrid nanoparticle, loaded with DOX and TS (LPNP_TS-DOX). Nanoparticles were physicochemically and morphologically characterized. Cytotoxicity studies, migration assay, and cellular uptake were performed in 4T1, MCF-7, and MDA-MB-231 cell lines. Antitumor activity in vivo was evaluated in 4T1 breast tumor-bearing mice. In vitro studies showed a significant reduction in cell viability, cell migration, and an increase in cellular uptake for the 4T1 cell line compared to free DOX. In vivo antitumor activity showed that LPNP-TS-DOX was more effective in controlling tumor growth than other treatments. The high cellular internalization and the pH-triggered payload release of DOX lead to the increased accumulation of the drugs in the tumor area, along with the synergic combination with TS, culminating in greater antitumor efficacy. These data support LPNP-TS-DOX as a promising drug delivery system for breast cancer treatment.

Synbiotic Supplementation Modulates Gut Microbiota, Regulates ß-Catenin Expression and Prevents Weight Gain in ob/ob Mice: Preliminary Findings.

BACKGROUND: Obesity is one of the main health problems in the world today, and dysbiosis seems to be one of the factors involved. The aim of this study was to examine the impact of synbiotic supplementation on obesity and the microbiota in ob/ob mice. Twenty animals were divided into four groups: obese treated (OT), obese control (OC), lean treated (LT) and lean control (LC). All animals received a standard diet for 8 weeks. The treated groups received a synbiotic (Simbioflora-Invictus Farmanutrição Ltd., Sao Paulo, Brazil) in water, while the nontreated groups received only water. After 8 weeks, all animals were sacrificed, and gut tissue and stool samples were collected for mRNA isolation and microbiota analysis, respectively. ß-Catenin, occludin, cadherin and zonulin in the gut tissue were analyzed via RT-qPCR. Microbiome DNA was extracted from stool samples and sequenced using an Ion PGM Torrent platform. RESULTS: Synbiotic supplementation reduced body weight gain in the OT group compared with the OC group (p = 0.0398) and was associated with an increase in Enterobacteriaceae (p = 0.005) and a decrease in Cyanobacteria (p = 0.047), Clostridiaceae (p = 0.026), Turicibacterales (p = 0.005) and Coprococcus (p = 0.047). On the other hand, a significant reduction in Sutterella (p = 0.009) and Turicibacter (p = 0.005) bacteria was observed in the LT group compared to the LC group. Alpha and beta diversities were different among all treated groups. ß-Catenin gene expression was significantly decreased in the gut tissue of the OT group (p ≤ 0.0001) compared to the other groups. No changes were observed in occludin, cadherin or zonulin gene expression in the gut tissue. CONCLUSIONS: Synbiotic supplementation prevents excessive weight gain, modulates the gut microbiota, and reduces ß-catenin expression in ob/ob mice.

Formulation of Amphotericin B in PEGylated Liposomes for Improved Treatment of Cutaneous Leishmaniasis by Parenteral and Oral Routes.

Liposomal amphotericin B (AmB) or AmBisome® is the most effective and safe therapeutic agent for visceral leishmaniasis (VL), but its clinical efficacy is limited in cutaneous leishmaniasis (CL) and HIV/VL co-infection. The aim of this work was to develop a formulation of AmB in PEGylated liposomes and compare its efficacy to AmBisome® in a murine model of CL. Formulations of AmB in conventional and PEGylated liposomes were characterized for particle size and morphology, drug encapsulation efficiency and aggregation state. Those were compared to AmBisome® in Leishmania amazonensis-infected BALB/c mice for their effects on the lesion size growth and parasite load. The conventional and PEGylated formulations showed vesicles with 100-130 nm diameter and low polydispersity, incorporating more than 95% of AmB under the non-aggregated form. Following parenteral administration in the murine model of CL, the PEGylated formulation of AmB significantly reduced the lesion size growth and parasite load, in comparison to control groups, in contrast to conventional liposomal AmB. The PEGylated formulation of AmB was also effective when given by oral route on a 2-day regimen. This work reports for the first time that PEGylated liposomal AmB can improve the treatment of experimental cutaneous leishmaniasis by both parenteral and oral routes.

Memantine-Derived Schiff Bases as Transdermal Prodrug Candidates.

Condensation reactions of salicylaldehyde, 2-pyridinecarboxaldehyde, and pyridoxaldehyde with memantine (Me) produced novel memantine-derived Schiff bases (1-3). Speciation predictions and calculations of Log P, Log D, and of the percentage (%) of neutral species for (1-3) were carried out. In comparison with Me, the Schiff bases presented increased log P and log D in all cases and pH values, suggesting higher hydrophobicity. The determined solubilities in n-octanol were 34.7 mg/mL for memantine hydrochloride and 67.3 mg/mL for (3). According to the molecular weights and calculated logP, compounds (1-3) are suitable for transdermal administration, especially compound (3). In addition, hydrolysis of 3 with the release of pyridoxal, a daily cofactor in human metabolism, was observed. The results suggested that 3 is the most promising compound and that formation of the pyridoxal Schiff base with Me might be an effective strategy to obtain a prodrug candidate with increased lipophilicity, which would be able to passively cross biological barriers during transdermal delivery and might have applications in the treatment of Alzheimer's disease and other neurological disorders.

Stepwise Protocols for Preparation and Use of Porcine Ear Skin for in Vitro Skin Permeation Studies Using Franz Diffusion Cells.

The skin, the largest organ of the body, is an attractive route of topical and systemic drug administration. During the development of topical formulations, in vitro skin permeation studies using biological membranes mounted in Franz diffusion cells are a useful tool to assess the permeation of substances through the skin, and are recommended by the Organization for Economic Cooperation and Development (OECD). Among the types of biological membranes used in such studies, porcine ear skin has been identified as the most promising, due to its similarities to human skin and its greater accessibility as compared to human skin. To standardize techniques for the preparation and use of porcine ear skin as biological membrane, here we present systematic procedures for the selection of porcine ears, their cleaning, the removal of skin from cartilage, its transformation into membranes, and its use for the in vitro assessment of the permeation of drugs from topical formulations. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Obtaining porcine ear membranes Basic Protocol 2: Preparation of membranes from porcine ear skin and use of membranes for in vitro skin permeation studies.

Microbial associations for bioremediation. What does "microbial consortia" mean?

Microbial associations arise as useful tools in several biotechnological processes. Among them, bioremediation of contaminated environments usually takes advantage of these microbial associations. Despite being frequently used, these associations are indicated using a variety of expressions, showing a lack of consensus by specialists in the field. The main idea of this work is to analyze the variety of microbial associations referred to as "microbial consortia" (MC) in the context of pollutants biodegradation and bioremediation. To do that, we summarize the origin of the term pointing out the features that an MC is expected to meet, according to the opinion of several authors. An analysis of related bibliography was done seeking criteria to rationalize and classify MC in the context of bioremediation. We identify that the microbe's origin and the level of human intervention are usually considered as a category to classify them as natural microbial consortia (NMC), artificial microbial consortia (AMC), and synthetic microbial consortia (SMC). In this sense, NMC are those associations composed by microorganisms obtained from a single source while AMC members come from different sources. SMC are a class of AMC in which microbial composition is defined to accomplish a certain specific task. We propose that the effective or potential existence of the interaction among MC members in the source material should be considered as a category in the classification as well, in combination with the origin of the source and level of intervention. Cross-kingdom MC and new developments were also considered. Finally, the existence of grey zones in the limits between each proposed microbial consortia category is addressed. KEY POINTS: • Microbial consortia for bioremediation can be obtained through different methods. • The use of the term "microbial consortia" is unclear in the specialized literature. • We propose a simplified classification for microbial consortia for bioremediation.

Liposomal Amphotericin B for Treatment of Leishmaniasis: From the Identification of Critical Physicochemical Attributes to the Design of Effective Topical and Oral Formulations.

The liposomal amphotericin B (AmB) formulation, AmBisome®, still represents the best therapeutic option for cutaneous and visceral leishmaniasis. However, its clinical efficacy depends on the patient's immunological status, the clinical manifestation and the endemic region. Moreover, the need for parenteral administration, its side effects and high cost significantly limit its use in developing countries. This review reports the progress achieved thus far toward the understanding of the mechanism responsible for the reduced toxicity of liposomal AmB formulations and the factors that influence their efficacy against leishmaniasis. It also presents the recent advances in the development of more effective liposomal AmB formulations, including topical and oral liposome formulations. The critical role of the AmB aggregation state and release rate in the reduction of drug toxicity and in the drug efficacy by non-invasive routes is emphasized. This paper is expected to guide future research and development of innovative liposomal formulations of AmB.

Metagenomic strategies identify diverse integron-integrase and antibiotic resistance genes in the Antarctic environment.

The objective of this study is to identify and analyze integrons and antibiotic resistance genes (ARGs) in samples collected from diverse sites in terrestrial Antarctica. Integrons were studied using two independent methods. One involved the construction and analysis of intI gene amplicon libraries. In addition, we sequenced 17 metagenomes of microbial mats and soil by high-throughput sequencing and analyzed these data using the IntegronFinder program. As expected, the metagenomic analysis allowed for the identification of novel predicted intI integrases and gene cassettes (GCs), which mostly encode unknown functions. However, some intI genes are similar to sequences previously identified by amplicon library analysis in soil samples collected from non-Antarctic sites. ARGs were analyzed in the metagenomes using ABRIcate with CARD database and verified if these genes could be classified as GCs by IntegronFinder. We identified 53 ARGs in 15 metagenomes, but only four were classified as GCs, one in MTG12 metagenome (Continental Antarctica), encoding an aminoglycoside-modifying enzyme (AAC(6´)acetyltransferase) and the other three in CS1 metagenome (Maritime Antarctica). One of these genes encodes a class D ß-lactamase (blaOXA-205) and the other two are located in the same contig. One is part of a gene encoding the first 76 amino acids of aminoglycoside adenyltransferase (aadA6), and the other is a qacG2 gene.

Nanoencapsulated Doxorubicin Prevents Mucositis Development in Mice.

Doxorubicin (DOX), a chemotherapy drug successfully used in the therapy of various types of cancer, is currently associated with the mucositis development, an inflammation that can cause ulcerative lesions in the mucosa of the gastrointestinal tract, abdominal pain and secondary infections. To increase the safety of the chemotherapy, we loaded DOX into nanostructured lipid carriers (NLCs). The NLC-DOX was characterized by HPLC, DLS, NTA, Zeta potential, FTIR, DSC, TEM and cryogenic-TEM. The ability of NLC-DOX to control the DOX release was evaluated through in vitro release studies. Moreover, the effect of NLC-DOX on intestinal mucosa was compared to a free DOX solution in C57BL/6 mice. The NLC-DOX showed spherical shape, high drug encapsulation efficiency (84.8 ± 4.6%), high drug loading (55.2 ± 3.4 mg/g) and low average diameter (66.0-78.8 nm). The DSC and FTIR analyses showed high interaction between the NLC components, resulting in controlled drug release. Treatment with NLC-DOX attenuated DOX-induced mucositis in mice, improving shortening on villus height and crypt depth, decreased inflammatory parameters, preserved intestinal permeability and increased expression of tight junctions (ZO-1 and Ocludin). These results indicated that encapsulation of DOX in NLCs is viable and reduces the drug toxicity to mucosal structures.

Neutralisation of SARS-CoV-2 lineage P.1 by antibodies elicited through natural SARS-CoV-2 infection or vaccination with an inactivated SARS-CoV-2 vaccine: an immunological study.

BACKGROUND: Mutations accrued by SARS-CoV-2 lineage P.1-first detected in Brazil in early January, 2021-include amino acid changes in the receptor-binding domain of the viral spike protein that also are reported in other variants of concern, including B.1.1.7 and B.1.351. We aimed to investigate whether isolates of wild-type P.1 lineage SARS-CoV-2 can escape from neutralising antibodies generated by a polyclonal immune response. METHODS: We did an immunological study to assess the neutralising effects of antibodies on lineage P.1 and lineage B isolates of SARS-CoV-2, using plasma samples from patients previously infected with or vaccinated against SARS-CoV-2. Two specimens (P.1/28 and P.1/30) containing SARS-CoV-2 lineage P.1 (as confirmed by viral genome sequencing) were obtained from nasopharyngeal and bronchoalveolar lavage samples collected from patients in Manaus, Brazil, and compared against an isolate of SARS-CoV-2 lineage B (SARS.CoV2/SP02.2020) recovered from a patient in Brazil in February, 2020. Isolates were incubated with plasma samples from 21 blood donors who had previously had COVID-19 and from a total of 53 recipients of the chemically inactivated SARS-CoV-2 vaccine CoronaVac: 18 individuals after receipt of a single dose and an additional 20 individuals (38 in total) after receipt of two doses (collected 17-38 days after the most recent dose); and 15 individuals who received two doses during the phase 3 trial of the vaccine (collected 134-230 days after the second dose). Antibody neutralisation of P.1/28, P.1/30, and B isolates by plasma samples were compared in terms of median virus neutralisation titre (VNT50, defined as the reciprocal value of the sample dilution that showed 50% protection against cytopathic effects). FINDINGS: In terms of VNT50, plasma from individuals previously infected with SARS-CoV-2 had an 8·6 times lower neutralising capacity against the P.1 isolates (median VNT50 30 [IQR <20-45] for P.1/28 and 30 [<20-40] for P.1/30) than against the lineage B isolate (260 [160-400]), with a binominal model showing significant reductions in lineage P.1 isolates compared with the lineage B isolate (p≤0·0001). Efficient neutralisation of P.1 isolates was not seen with plasma samples collected from individuals vaccinated with a first dose of CoronaVac 20-23 days earlier (VNT50s below the limit of detection [<20] for most plasma samples), a second dose 17-38 days earlier (median VNT50 24 [IQR <20-25] for P.1/28 and 28 [<20-25] for P.1/30), or a second dose 134-260 days earlier (all VNT50s below limit of detection). Median VNT50s against the lineage B isolate were 20 (IQR 20-30) after a first dose of CoronaVac 20-23 days earlier, 75 (<20-263) after a second dose 17-38 days earlier, and 20 (<20-30) after a second dose 134-260 days earlier. In plasma collected 17-38 days after a second dose of CoronaVac, neutralising capacity against both P.1 isolates was significantly decreased (p=0·0051 for P.1/28 and p=0·0336 for P.1/30) compared with that against the lineage B isolate. All data were corroborated by results obtained through plaque reduction neutralisation tests. INTERPRETATION: SARS-CoV-2 lineage P.1 might escape neutralisation by antibodies generated in response to polyclonal stimulation against previously circulating variants of SARS-CoV-2. Continuous genomic surveillance of SARS-CoV-2 combined with antibody neutralisation assays could help to guide national immunisation programmes. FUNDING: São Paulo Research Foundation, Brazilian Ministry of Science, Technology and Innovation and Funding Authority for Studies, Medical Research Council, National Council for Scientific and Technological Development, National Institutes of Health. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil.

Cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Manaus, Brazil, resurged in late 2020 despite previously high levels of infection. Genome sequencing of viruses sampled in Manaus between November 2020 and January 2021 revealed the emergence and circulation of a novel SARS-CoV-2 variant of concern. Lineage P.1 acquired 17 mutations, including a trio in the spike protein (K417T, E484K, and N501Y) associated with increased binding to the human ACE2 (angiotensin-converting enzyme 2) receptor. Molecular clock analysis shows that P.1 emergence occurred around mid-November 2020 and was preceded by a period of faster molecular evolution. Using a two-category dynamical model that integrates genomic and mortality data, we estimate that P.1 may be 1.7- to 2.4-fold more transmissible and that previous (non-P.1) infection provides 54 to 79% of the protection against infection with P.1 that it provides against non-P.1 lineages. Enhanced global genomic surveillance of variants of concern, which may exhibit increased transmissibility and/or immune evasion, is critical to accelerate pandemic responsiveness.

Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier

Since the discovery of SARS-CoV-2, several antigens have been proposed to be part of COVID-19 vaccines. The receptor binding domain (RBD) of Spike protein is one of the promising candidates to develop effective vaccines since it can induce potent neutralizing antibodies. We previously reported the production of RBD in Pichia pastoris and showed it is structurally identical to the protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer construct with the purpose of increasing RBD immunogenicity. We produced multimeric particles by a transpeptidation reaction between the RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric protein of 170 kDa. We vaccinated mice with two doses 30 days apart, and then we measured humoral immune response. When the number of RBD copies coupled to BLS was high (6-7 RBD molecules per BLS decamer, in average), the immune response was significantly better than that elicited by RBD alone or even by RBD-BLS comprising low number of RBD copies (1-2 RBD molecules per BLS decamer). Remarkably, the construct with high number of RBD copies induced high IgG titers with high neutralizing capacity. Furthermore, a superior immune response was observed when Al(OH)3 adjuvant was added to this formulation, exhibiting a higher titer of neutralizing antibodies. Altogether our results suggest that RBD covalent coupled to BLS forming a multimer-particle shows an advantageous architecture to the antigen-presentation to the immune system which enhances immune responses. This new antigen should be considered a potent candidate for a protein-based vaccine.

Genomics and epidemiology of a novel SARS-CoV-2 lineage in Manaus, Brazil.

Cases of SARS-CoV-2 infection in Manaus, Brazil, resurged in late 2020, despite high levels of previous infection there. Through genome sequencing of viruses sampled in Manaus between November 2020 and January 2021, we identified the emergence and circulation of a novel SARS-CoV-2 variant of concern, lineage P.1, that acquired 17 mutations, including a trio in the spike protein (K417T, E484K and N501Y) associated with increased binding to the human ACE2 receptor. Molecular clock analysis shows that P.1 emergence occurred around early November 2020 and was preceded by a period of faster molecular evolution. Using a two-category dynamical model that integrates genomic and mortality data, we estimate that P.1 may be 1.4-2.2 times more transmissible and 25-61% more likely to evade protective immunity elicited by previous infection with non-P.1 lineages. Enhanced global genomic surveillance of variants of concern, which may exhibit increased transmissibility and/or immune evasion, is critical to accelerate pandemic responsiveness.

Pharmacogenomic Profile and Adverse Drug Reactions in a Prospective Therapeutic Cohort of Chagas Disease Patients Treated with Benznidazole.

Chagas disease remains a major social and public health problem in Latin America. Benznidazole (BZN) is the main drug with activity against Trypanosoma cruzi. Due to the high number of adverse drug reactions (ADRs), BZN is underprescribed. The goal of this study was to evaluate the genetic and transcriptional basis of BZN adverse reactions. METHODS: A prospective cohort with 102 Chagas disease patients who underwent BZN treatment was established to identify ADRs and understand their genetic basis. The patients were classified into two groups: those with at least one ADR (n = 73), and those without ADRs (n = 29). Genomic analyses were performed comparing single nucleotide polymorphisms between groups. Transcriptome data were obtained comparing groups before and after treatment, and signaling pathways related to the main ADRs were evaluated. RESULTS: A total of 73 subjects (71.5%) experienced ADRs. Dermatological symptoms were most frequent (45.1%). One region of chromosome 16, at the gene LOC102724084 (rs1518601, rs11861761, and rs34091595), was associated with ADRs (p = 5.652 × 10-8). Transcriptomic data revealed three significantly enriched signaling pathways related to BZN ADRs. CONCLUSIONS: These data suggest that part of adverse BZN reactions might be genetically determined and may facilitate patient risk stratification prior to starting BZN treatment.