Phage Therapy: The Pharmacology of Antibacterial Viruses.

Pharmacology can be differentiated into two key aspects, pharmacodynamics and pharmacokinetics. Pharmacodynamics describes a drug's impact on the body while pharmacokinetics describes the body's impact on a drug. Another way of understanding these terms is that pharmacodynamics is a description of both the positive and negative consequences of drugs attaining certain concentrations in the body while pharmacokinetics is concerned with our ability to reach and then sustain those concentrations. Unlike the drugs for which these concepts were developed, including antibiotics, the bacteriophages (or 'phages') that we consider here are not chemotherapeutics but instead are the viruses of bacteria. Here we review the pharmacology of these viruses, particularly as they can be employed to combat bacterial infections (phage therapy). Overall, an improved pharmacological understanding of phage therapy should allow for more informed development of phages as antibacterial 'drugs', allow for more rational post hoc debugging of phage therapy experiments, and encourage improved design of phage therapy protocols. Contrasting with antibiotics, however, phages as viruses impact individual bacterial cells as single virions rather than as swarms of molecules, and while they are killing bacteria, bacteriophages also can amplify phage numbers, in situ. Explorations of phage therapy pharmacology consequently can often be informed as well by basic principles of the ecological interactions between phages and bacteria as by study of the pharmacology of drugs. Bacteriophages in phage therapy thus can display somewhat unique as well as more traditional pharmacological aspects.

Antibodies specific to SARS-CoV-2 proteins N, S and E in COVID-19 patients in the normal population and in historical samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally; recognition of immune responses to this virus will be crucial for coronavirus disease 2019 (COVID-19) control, prevention and treatment. We comprehensively analysed IgG and IgA antibody responses to the SARS-CoV-2 nucleocapsid protein (N), spike protein domain 1 (S1) and envelope protein (E) in: SARS-CoV-2-infected patient, healthy, historical and pre-epidemic samples, including patients' medical, epidemiological and diagnostic data, virus-neutralizing capability and kinetics. N-specific IgG and IgA are the most reliable diagnostic targets for infection. Serum IgG levels correlate to IgA levels. Half a year after infection, anti-N and anti-S1 IgG decreased, but sera preserved virus-inhibitory potency; thus, testing for IgG may underestimate the protective potential of antibodies. Historical and pre-epidemic sera did not inhibit SARS-CoV-2, thus its circulation before the pandemic and a protective role from antibodies pre-induced by other coronaviruses cannot be confirmed by this study.

Peptide Charge Derivatization as a Tool for Early Detection of Preeclampsia by Mass Spectrometry-A Comparison with the ELISA Test.

Early detection of any preeclampsia biomarkers may lower the risk of mortality, both for a mother and a child. Our study focuses on techniques for preeclampsia biomarker identification by comparing the results of a method using liquid chromatography mass spectrometry in multiple reaction monitoring mode (LC-MS/MS) with those by the enzyme-linked immunosorbent assay (ELISA) test, as well as by comparing the obtained results with clinical data. In the proposed LC-MS/MS method a tryptic digest peptide charge derivatization strategy was used as a tool for sensitive detection of podocin, i.e., a previously discovered preeclampsia biomarker present in urine samples from pregnant women. Urine samples from pregnant women with diagnosed preeclampsia were collected at different stages of pregnancy and from healthy subjects, and then were analyzed by ELISA test and the proposed method with LC-MS/MS. Charge derivatization of the ε amino group of C-terminal lysine residues in tryptic digests by 2,4,6-triphenylpyrylium salt was performed to increase the ionization efficiency in the LC-MS/MS mode. Podocin was identified at the early stage of pregnancy, while its detection using an ELISA test was not possible. The protocol for urine sample preparation was optimized. Our results show that the proposed method by LC-MS/MS in combination with peptide charge derivatization, provides an ultrasensitive tool for diagnosis of preeclampsia, and provides earlier detection than a clinical diagnosis or ELISA test. The proposed solution may revolutionize medical diagnostics.

Phage therapy efficacy: a review of the last 10 years of preclinical studies.

Due to the rise of multidrug-resistant infections in humans, phage therapy is gaining renewed attention in Western medicine. Despite the increasing number of publications focussed on the isolation, characterization and in vitro performance of different phages, there is still a lack of concise pre-clinical information to guide the application of phage therapy in clinical practice. Nevertheless, over the last decade, efforts have been made to conduct more detailed studies of the in vivo efficacy of phages. Here, we review the most relevant in vivo studies performed in the last decade covering phage efficacy in both preclinical and clinical trials. We compare different routes of administration, dosage effect and different animal models of distinct types of infections. Moreover, insights into case studies and results from clinical trials are presented. Challenges and limitations of phage use as evidenced by the current state of research are also discussed in order to improve both the trustworthiness and success of the implementation of phage therapy.

Endocytosis in cellular uptake of drug delivery vectors: Molecular aspects in drug development.

Drug delivery vectors are widely applied to increase drug efficacy while reducing the side effects and potential toxicity of a drug. They allow for patient-tailored therapy, dose titration, and therapeutic drug monitoring. A major part of drug delivery systems makes use of large nanocarriers: liposomes or virus-like particles (VLPs). These systems allow for a relatively large amount of cargo with good stability of vectors, and they offer multiple options for targeting vectors in vivo. Here we discuss endocytic pathways that are available for drug delivery by large nanocarriers. We focus on molecular aspects of the process, including an overview of potential molecular targets for studies of drug delivery vectors and for future solutions allowing targeted drug delivery.

Enrichment of Cysteine-Containing Peptide by On-Resin Capturing and Fixed Charge Tag Derivatization for Sensitive ESI-MS Detection.

High complexity of cell and tissue proteomes limits the investigation of proteomic biomarkers. Therefore, the methods of enrichment of some chemical groups of peptides including thiopeptides are important tools that may facilitate the proteomic analysis by reducing sample complexity and increasing proteome coverage. Here, we present a new method of cysteine-containing tryptic peptide enrichment using commercially available TentaGel R RAM resin modified by the linker containing the maleimide group, allowing thiol conjugation. The captured tryptic peptides containing lysine residue were then tagged by 2,4,6-triphenylpyrylium salt to form 2,4,6-triphenylpyridinium derivatives, which increases the ionization efficiency during mass spectrometry analysis. This makes it possible to conduct an ultrasensitive analysis of the trace amount of compounds. The proposed strategy was successfully applied in the enrichment of model tryptic podocin peptide and podocin tryptic digest.

Phage therapy: What factors shape phage pharmacokinetics and bioavailability? Systematic and critical review.

Bacteriophages are not forgotten viruses anymore: scientists and practitioners seek to understand phage pharmacokinetics in animals and humans, investigating bacteriophages as therapeutics, nanocarriers or microbiome components. This review provides a comprehensive overview of factors that determine phage circulation, penetration, and clearance, and that in consequence determine phage applicability for medicine. It makes use of experimental data collected by the phage community so far (PubMed 1924-2016, including non-English reports), combining elements of critical and systematic review. This study covers phage ability to enter a system by various routes of administration, how (and if) the phage may access various tissues and organs, and finally what mechanisms determine the courses of phage clearance. The systematic review method was applied to analyze (i) phage survival in the gut (gut transit) and (ii) phage ability to enter the mammalian system by many administration routes. Aspects that have not yet been covered by a sufficient number of reports for mathematical analysis, as well as mechanisms underlying trends, are discussed in the form of a critical review. In spite of the extraordinary diversity of bacteriophages and possible phage applications, the analysis revealed that phage morphology, phage specificity, phage dose, presence of sensitive bacteria or the characteristics of treated individuals (age, taxonomy) may affect phage bioavailability in animals and humans. However, once phages successfully enter the body, they reach most organs, including the central nervous system. Bacteriophages are cleared mainly by the immune system: innate immunity removes phages even when no specific response to bacteriophages has yet developed.

ClyJ Is a Novel Pneumococcal Chimeric Lysin with a Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase Catalytic Domain.

Streptococcus pneumoniae is one of the leading pathogens that cause a variety of mucosal and invasive infections. With the increased emergence of multidrug-resistant S. pneumoniae, new antimicrobials with mechanisms of action different from conventional antibiotics are urgently needed. In this study, we identified a putative lysin (gp20) encoded by the Streptococcus phage SPSL1 using the LytA autolysin as a template. Molecular dissection of gp20 revealed a binding domain (GPB) containing choline-binding repeats (CBRs) that are high specificity for S. pneumoniae By fusing GPB to the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain of the PlyC lysin, we constructed a novel chimeric lysin, ClyJ, with improved activity to the pneumococcal Cpl-1 lysin. No resistance was observed in S. pneumoniae strains after exposure to incrementally doubling concentrations of ClyJ for 8 continuous days in vitro In a mouse bacteremia model using penicillin G as a control, a single intraperitoneal injection of ClyJ improved the survival rate of lethal S. pneumoniae-infected mice in a dose-dependent manner. Given its high lytic activity and safety profile, ClyJ may represent a promising alternative to combat pneumococcal infections.

Podocyturia as an early diagnostic marker of preeclampsia: a literature review.

CONTEXT: Preeclampsia (PE) is a pregnancy-related disease, and it is a leading cause of maternal and neonatal morbidity and mortality. It is characterized by the new onset of hypertension after 20 weeks of gestation together with signs of organ damage, most commonly the kidneys. The treatment of PE is symptomatic and final intervention requires delivery, regardless of the gestational age of the foetus. Furthermore, PE is a risk factor for developing cardiovascular disease and chronic kidney disease - even many years after the delivery. OBJECTIVE: Current research of PE has revealed that detection of podocytes in urine (podocyturia) could be a useful method for both confirmation of PE diagnosis and for the prediction of the severity of the disease. CONCLUSION: The main aim of this review is to summarize the current state of available methods for podocyte detection and to discuss their relevance in clinical practice.

Aggregation/dispersion transitions of T4 phage triggered by environmental ion availability.

BACKGROUND: Bacteriophage survives in at least two extremes of ionic environments: bacterial host (high ionic-cytosol) and that of soil (low ionic-environmental water). The impact of ionic composition in the micro- and macro-environments has not so far been addressed in phage biology. RESULTS: Here, we discovered a novel mechanism of aggregation/disaggregation transitions by phage virions. When normal sodium levels in phage media (150 mM) were lowered to 10 mM, advanced imaging by scanning electron microscopy, atomic force microscopy and dynamic light scattering all revealed formation of viral packages, each containing 20-100 virions. When ionic strength was returned from low to high, the aggregated state of phage reversed to a dispersed state, and the change in ionic strength did not substantially affect infectivity of the phage. By providing the direct evidence, that lowering of the sodium ion below the threshold of 20 mM causes rapid aggregation of phage while returning Na+ concentration to the values above this threshold causes dispersion of phage, we identified a biophysical mechanism of phage aggregation. CONCLUSIONS: Our results implicate operation of group behavior in phage and suggest a new kind of quorum sensing among its virions that is mediated by ions. Loss of ionic strength may act as a trigger in an evolutionary mechanism to improve the survival of bacteriophage by stimulating aggregation of phage when outside a bacterial host. Reversal of phage aggregation is also a promising breakthrough in biotechnological applications, since we demonstrated here the ability to retain viable virion aggregates on standard micro-filters.

Immunogenicity studies of proteins forming the T4 phage head surface.

UNLABELLED: Advances in phage therapy and novel applications of phages in biotechnology encourage interest in phage impact on human and animal immunity. Here we present comparative studies of immunogenic properties of T4 phage head surface proteins gp23*, gp24*, Hoc, and Soc, both as elements of the phage capsid and as isolated agents. Studies comprise evaluation of specific antibodies in the human population, analysis of the proteins' impact on the primary and secondary responses in mice, and the effect of specific antibodies on phage antibacterial activity in vitro and in vivo in mice. In humans, natural antibodies specific to T4-like phages were abundant (81% of investigated sera). Among those, significantly elevated levels of IgG antibodies only against major head protein (gp23*) were found, which probably reflected cross-reactions of T4 with antibodies induced by other T4-like phages. Both IgM and IgG antibodies were induced mostly by gp23* and Hoc, while weak (gp24*) and very weak (Soc) reactivities of other head proteins were noticed. Thus, T4 head proteins that markedly contribute to immunological memory to the phage are highly antigenic outer capsid protein (Hoc) and major capsid protein (gp23*). Specific anti-gp23* and anti-Hoc antibodies substantially decreased T4 phage activity in vitro and to some extent in vivo. Cooperating with antibodies, the immune complement system also contributed to annihilating phages. IMPORTANCE: Current descriptions of phage immunogenicity and its biological consequences are still vague and incomplete; thus, the central problem of this work is timely and may have strong practical implications. Here is presented the very first description of the contribution of bacteriophage proteins to immunological memory of the phage. Understanding of interactions between phages and mammalian immunology may help in biotechnological adaptations of phages for therapeutic requirements as well as for better appreciation of phage ecology and their role in the biosphere.

T4 bacteriophage as a phage display platform.

Analysis of molecular events in T4-infected Escherichia coli has revealed some of the most important principles of biology, including relationships between structures of genes and their products, virus-induced acquisition of metabolic function, and morphogenesis of complex structures through sequential gene product interaction rather than sequential gene activation. T4 bacteriophages and related strains were applied in the first formulations of many fundamental biological concepts. These include the unambiguous recognition of nucleic acids as the genetic material, the definition of the gene by fine-structure mutation, recombinational and functional analyses, the demonstration that the genetic code is triplet, the discovery of mRNA, the importance of recombination and DNA replications, light-dependent and light-independent DNA repair mechanisms, restriction and modification of DNA, self-splicing of intron/exon arrangement in prokaryotes, translation bypassing and others. Bacteriophage T4 possesses unique features that make it a good tool for a multicomponent vaccine platform. Hoc/Soc-fused antigens can be assembled on the T4 capsid in vitro and in vivo. T4-based phage display combined with affinity chromatography can be applied as a new method for bacteriophage purification. The T4 phage display system can also be used as an attractive approach for cancer therapy. The data show the efficient display of both single and multiple HIV antigens on the phage T4 capsid and offer insights for designing novel particulate HIV or other vaccines that have not been demonstrated by other vector systems.

Interaction of Bacteriophages with the Immune System: Induction of Bacteriophage-Specific Antibodies.

In all cases when a bacteriophage makes a direct contact with a mammalian organism, it may challenge the mammalian immunological system. Its major consequence is the production of antibodies specific to the bacteriophage, particularly IgM, IgG, and IgA as the typical response. Here we present protocols applicable in studies of the ability of bacteriophage to induce specific antibodies; immunization to whole virions or to isolated phage proteins has been included. The protocols have been divided into three parts: purification, immunization, and detection (enzyme-linked immunosorbent assay, ELISA).

Biological Properties of 12 Newly Isolated Acinetobacter baumannii-Specific Bacteriophages.

Infections with the opportunistic Gram-negative bacterium Acinetobacter baumannii pose a serious threat today, which is aggravated by the growing problem of multi-drug resistance among bacteria, caused by the overuse of antibiotics. Treatment of infections caused by antibiotic-resistant A. baumannii strains with the use of phage therapy is not only a promising alternative, but sometimes the only option. Therefore, phages specific for clinical multi-drug resistant A. baumannii were searched for in environmental, municipal, and hospital wastewater samples collected from different locations in Poland. The conducted research allowed us to determine the biological properties and morphology of the tested phages. As a result of our research, 12 phages specific for A. baumannii, 11 of which turned out to be temperate and only one lytic, were isolated. Their lytic spectra ranged from 11 to 75%. The plaques formed by most phages were small and transparent, while one of them formed relatively large plaques with a clearly marked 'halo' effect. Based on Transmission Electron Microscopy (TEM), most of our phages have been classified as siphoviruses (only one phage was classified as a podovirus). All phages have icosahedral capsid symmetry, and 11 of them have a long tail. Optimal multiplicity of infections (MOIs) and the adsorption rate were also determined. MOI values varied depending on the phage-from 0.001 to 10. Based on similarities to known bacteriophages, our A. baumannii-specific phages have been proposed to belong to the Beijerinckvirinae and Junivirinae subfamilies. This study provides an additional tool in the fight against this important pathogen and may boost the interest in phage therapy as an alternative and supplement to the current antibiotics.

Evolution of the T4 phage virion is driven by selection pressure from non-bacterial factors.

Bacteriophages colonize animal and human bodies, propagating on sensitive bacteria that are symbionts, commensals, or pathogens of animals and humans. T4-like phages are dependent on abundant symbionts such as Escherichia coli, commonly present in animal and human gastrointestinal (GI) tracts. Bacteriophage T4 is one of the most complex viruses, and its intricate structure, particularly the capsid head protecting the phage genome, likely contributes substantially to the overall phage fitness in diverse environments. We investigated how individual head proteins-gp24, Hoc, and Soc-affect T4 phage survival under pressure from non-bacterial factors. We constructed a panel of T4 phage variants defective in these structural proteins: T4∆Soc, T4∆24byp24, T4∆Hoc∆Soc, T4∆Hoc∆24byp24, T4∆Soc∆24byp24, and T4∆Hoc∆Soc∆24byp24 (byp = bypass). These variants were investigated for their sensitivity to selected environmental conditions relevant to the microenvironment of the GI tract, including pH, temperature, and digestive enzymes. The simple and "primitive" structure of the phage capsid (∆24byp24) was significantly less stable at low pH and more sensitive to inactivation by digestive enzymes, and the simultaneous lack of gp24 and Soc resulted in a notable decrease in phage activity at 37°C. Gp24 was also found to be highly resistant to thermal and chemical denaturation. Thus, gp24, which was acquired relatively late in evolution, seems to play a key role in T4 withstanding environmental conditions, including those related to the animal/human GI tract, and Soc is a molecular glue that enhances this protective effect. IMPORTANCE Bacteriophages are important components of animal and human microbiota, particularly in the gastrointestinal tract, where they dominate the viral community and contribute to shaping microbial balance. However, interactions with bacterial hosts are not the only element of the equation in phage survival-phages inhabiting the GI tract are constantly exposed to increased temperature, pH fluctuations, or digestive enzymes, which raises the question of whether and how the complex structure of phage capsids contributes to their persistence in the specific microenvironment of human/animal bodies. Here we address this phage-centric perspective, identifying the role of individual head proteins in T4 phage survival in GI tract conditions. The selection pressure driving the evolution of T4-like phages could have come from the external environment that affects phage virions with increased temperature and variable pH; it is possible that in the local microenvironment along the GI tract, the phage benefits from stability-protecting proteins.

New Phage-Derived Antibacterial Enzyme PolaR Targeting Rothia spp.

Rothia is an opportunistic pathogen, particularly life-threatening for the immunocompromised. It is associated with pneumonia, endocarditis, peritonitis and many other serious infections, including septicemia. Of note, Rothia mucilaginousa produces metabolites that support and increase overgrowth of Pseudomonas aeruginosa, one of the ESKAPE bacteria. Endolysins are considered as antibacterial enzymes derived from bacteriophages that selectively and efficiently kill susceptible bacteria without harming human cells or the normal microbiome. Here, we applied a computational analysis of metagenomic sequencing data of the gastric mucosa phageome extracted from human patients' stomach biopsies. A selected candidate anti-Rothia sequence was produced in an expression system, purified and confirmed as a Rothia mucilaginosa- and Rothia dentocariosa-specific endolysin PolaR, able to destroy bacterial cells even when aggregated, as in a biofilm. PolaR had no cytotoxic or antiproliferative effects on mammalian cells. PolaR is the first described endolysin selectively targeting Rothia species, with a high potential to combat infections caused by Rothia mucilaginosa and Rothia dentocariosa, and possibly other bacterial groups. PolaR is the first antibacterial enzyme selected from the gastric mucosa phageome, which underlines the biological complexity and probably underestimated biological role of the phageome in the human gastric mucosa.

Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses.

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs.

Experimental Identification of Cross-Reacting IgG Hotspots to Predict Existing Immunity Evasion of SARS-CoV-2 Variants by a New Biotechnological Application of Phage Display.

Multiple pathogens are competing against the human immune response, leading to outbreaks that are increasingly difficult to control. For example, the SARS-CoV-2 virus continually evolves, giving rise to new variants. The ability to evade the immune system is a crucial factor contributing to the spread of these variants within the human population. With the continuous emergence of new variants, it is challenging to comprehend all the possible combinations of previous infections, various vaccination types, and potential exposure to new variants in an individual patient. Rather than conducting variant-to-variant comparisons, an efficient approach could involve identifying key protein regions associated with the immune evasion of existing immunity against the virus. In this study, we propose a new biotechnological application of bacteriophages, the phage display platform for experimental identification of regions (linear epitopes) that may function as cross-reacting IgG hotspots in SARS-CoV-2 structural proteins. A total of 34,949 epitopes derived from genomes of all SARS-CoV-2 variants deposited prior to our library design were tested in a single assay. Cross-reacting IgG hotspots are protein regions frequently recognized by cross-reacting antibodies in many variants. The assay facilitated the one-step identification of immunogenic regions of proteins that effectively induced specific IgG in SARS-CoV-2-infected patients. We identified four regions demonstrating both significant immunogenicity and the activity of a cross-reacting IgG hotspot in protein S (located at NTD, RBD, HR1, and HR2/TM domains) and two such regions in protein N (at 197-280 and 358-419 aa positions). This novel method for identifying cross-reacting IgG hotspots holds promise for informing vaccine design and serological diagnostics for COVID-19 and other infectious diseases.

Assessment of Clinical Indicators Registered on Admission to the Hospital Related to Mortality Risk in Cancer Patients with COVID-19.

BACKGROUND: Oncology patients are a particularly vulnerable group to the severe course of COVID-19 due to, e.g., the suppression of the immune system. The study aimed to find links between parameters registered on admission to the hospital and the risk of later death in cancer patients with COVID-19. METHODS: The study included patients with a reported history of malignant tumor (n = 151) and a control group with no history of cancer (n = 151) hospitalized due to COVID-19 between March 2020 and August 2021. The variables registered on admission were divided into categories for which we calculated the multivariate Cox proportional hazards models. RESULTS: Multivariate Cox proportional hazards models were successfully obtained for the following categories: Patient data, Comorbidities, Signs recorded on admission, Medications used before hospitalization and Laboratory results recorded on admission. With the models developed for oncology patients, we identified the following variables that registered on patients' admission were linked to significantly increased risk of death. They are: male sex, presence of metastases in neoplastic disease, impaired consciousness (somnolence or confusion), wheezes/rhonchi, the levels of white blood cells and neutrophils. CONCLUSION: Early identification of the indicators of a poorer prognosis may serve clinicians in better tailoring surveillance or treatment among cancer patients with COVID-19.

Endocytosis of Bacteriophages.

Endocytosis is used by eukaryotic cells for ingesting external objects. Importantly, endocytosis is a major process that determines phage pharmacokinetics in vivo. Either dissemination of phages throughout the system or phage clearance engages cellular uptake of phage particles. Here we discuss phage uptake by mammalian cells, focusing on mechanisms and pathways involved. Of note, cellular uptake of phage virions was first observed in professional phagocytes, such as macrophages or granulocytes. For this reason, it was historically referred to as 'phagocytosis'. The modern definition of phagocytosis, however, identifies this process as a type of endocytosis within a larger repertoire of endocytic pathways, such as macropinocytosis, clathrin-mediated endocytosis, and caveolar endocytosis, which have all been included in the scope of this review.