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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)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Harhala, Gembara, Rybicka, Kaźmierczak, Miernikiewicz, Majewska, Budziar, Nasulewicz-Goldeman, Nelson, Owczarek and Dąbrowska.)

Predictors for the risk of severe COVID-19 are crucial for patient care and control of the disease. Other infectious diseases as potential comorbidities in SARS-CoV-2 infection are still poorly understood. Here we identify association between the course of COVID-19 and Lyme disease (borreliosis), caused by Borrelia burgdorferi transmitted to humans by ticks. Exposure to Borrelia was identified by multi-antigenic (19 antigens) serological testing of patients: severe COVID-19 (hospitalized), asymptomatic to mild COVID-19 (home treated or not aware of being infected), and not infected with SARS-CoV-2. Increased levels of Borrelia-specific IgGs strongly correlated with COVID-19 severity and risk of hospitalization. This suggests that a history of tick bites and related infections may contribute to the risks in COVID-19. Though mechanisms of this link is not clear yet, screening for antibodies targeting Borrelia may help accurately assess the odds of hospitalization for SARS-CoV-2 infected patients, supporting efforts for efficient control of COVID-19., (© 2022. The Author(s).)

The immune response and specific antibody production in COVID-19 are among the key factors that determine both prognostics for individual patients and the global perspective for controlling the pandemics. So called "dark figure", that is, a part of population that has been infected but not registered by the health care system, make it difficult to estimate herd immunity and to predict pandemic trajectories. Here we present a follow up study of population screening for hidden herd immunity to SARS-CoV-2 in individuals who had never been positively diagnosed against SARS-CoV-2; the first screening was in May 2021, and the follow up in December 2021. We found that specific antibodies targeting SARS-CoV-2 detected in May as the "dark figure" cannot be considered important 7 months later due to their significant drop. On the other hand, among participants who at the first screening were negative for anti-SARS-CoV-2 IgG, and who have never been diagnosed for SARS-CoV-2 infection nor vaccinated, 26% were found positive for anti-SARS-CoV-2 IgG. This can be attributed to of the "dark figure" of the recent, fourth wave of the pandemic that occurred in Poland shortly before the study in December. Participants who were vaccinated between May and December demonstrated however higher levels of antibodies, than those who undergone mild or asymptomatic (thus unregistered) infection. Only 7% of these vaccinated participants demonstrated antibodies that resulted from infection (anti-NCP). The highest levels of protection were observed in the group that had been infected with SARS-CoV-2 before May 2021 and also fully vaccinated between May and December. These observations demonstrate that the hidden fraction of herd immunity is considerable, however its potential to suppress the pandemics is limited, highlighting the key role of vaccinations., Competing Interests: The authors have declared that no competing interests exist.

Endolysins are bacteriolytic enzymes derived from bacteriophages. They represent an alternative to antibiotics, since they are not susceptible to conventional antimicrobial resistance mechanisms. Since non-human proteins are efficient inducers of specific immune responses, including the IgG response or the development of an allergic response mediated by IgE, we evaluated the general immunogenicity of the highly active antibacterial enzyme, PlyC, in a human population and in a mouse model. The study includes the identification of molecular epitopes of PlyC. The overall assessment of potential hypersensitivity to this protein and PlyC-specific IgE testing was also conducted in mice. PlyC induced efficient IgG production in mice, and the molecular analysis revealed that PlyC-specific IgG interacted with four immunogenic regions identified within the PlyCA subunit. In humans, approximately 10% of the population demonstrated IgG reactivity to the PlyCB subunit only, which is attributed to cross-reactions since this was a naïve serum. Of note, in spite of being immunogenic, PlyC induced a normal immune response, without hypersensitivity, since both the animals challenged with PlyC and in the human population PlyC-specific IgE was not detected.

Population immunity (herd immunity) to SARS-CoV-2 derives from two sources: vaccinations or cases of infection with the virus. Infections can be diagnosed as COVID-19 and registered, or they can be asymptomatic, oligosymptomatic, or even full-blown but undiagnosed and unregistered when patients recovered at home. Estimation of population immunity to SARS-CoV-2 is difficult and remains a subject of speculations. Here we present a population screening for SARS-CoV-2 specific IgG and IgA antibodies in Polish citizens (N = 501) who had never been positively diagnosed with or vaccinated against SARS-CoV-2. Serum samples were collected in Wrocław (Lower Silesia) on 15th and 22nd May 2021. Sera from hospitalized COVID-19 patients (N = 22) or from vaccinated citizens (N = 14) served as positive controls. Sera were tested with Microblot-Array COVID-19 IgG and IgA (quantitative) that contain specific SARS-CoV-2 antigens: NCP, RBD, Spike S2, E, ACE2, PLPro protein, and antigens for exclusion cross-reactivity with other coronaviruses: MERS-CoV, SARS-CoV, HCoV 229E Np, HCoV NL63 Np. Within the investigated population of healthy individuals who had never been positively diagnosed with or vaccinated against SARS-CoV-2, we found that 35.5% (178 out of 501) were positive for SARS-CoV-2-specific IgG and 52.3% (262 out of 501) were positive for SARS-CoV-2-specific IgA; 21.2% of the investigated population developed virus-specific IgG or IgA while being asymptomatic. Anti-RBD IgG, which represents virus-neutralizing potential, was found in 25.6% of individuals (128 out of 501). These patients, though positive for anti-SARS-CoV-2 antibodies, cannot be identified in the public health system as convalescents due to undiagnosed infections, and they are considered unaffected by SARS-CoV-2. Their contribution to population immunity against COVID-19 should however be considered in predictions and modeling of the COVID-19 pandemic. Of note, the majority of the investigated population still lacked anti-RBD IgG protection (74.4%); thus vaccination against COVID-19 is still of the most importance for controlling the pandemic., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Increasing number of deaths from multi-drug resistant bacterial infections has caused both the World Health Organization and the Centers for Disease Control and Prevention to repeatedly call for development of new, non-traditional antibacterial treatments. Antimicrobial enzymes, including those derived from bacteriophages, known as endolysins or enzybiotics, are considered promising solutions among the emerging therapies. These naturally occurring proteins specifically destroy bacterial cell walls (peptidoglycan) and as such, are capable of killing several logs of bacteria within minutes. Some endolysins cause lysis of a wide range of susceptible bacteria, including both Gram-positive and Gram-negative organisms, whereas other endolysins are species- or even strain-specific. To make wide use of endolysins as antibacterial agents, some basic research issues remain to be clarified or addressed. Currently available methods for testing endolysin kinetics are indirect, require large numbers of bacteria, long incubation times and are affected by technical problems or limited reproducibility. Also, available methods are focused more on enzymatic activity rather than killing efficiency which is more relevant from a medical perspective. Results: We show a novel application of a DNA dye, SYTOX Green. It can be applied in comprehensive, real-time and rapid measurement of killing efficiency, lytic activity, and susceptibility of a bacterial population to lytic enzymes. Use of DNA dyes shows improved reaction times, higher sensitivity in low concentrations of bacteria, and independence of bacterial growth. Our data show high precision in lytic activity and enzyme efficiency measurements. This solution opens the way to the development of new, high throughput, precise measurements and tests in variety of conditions, thus unlocking new possibilities in development of novel antimicrobials and analysis of bacterial samples., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Harhala, Gembara, Miernikiewicz, Owczarek, Kaźmierczak, Majewska, Nelson and Dąbrowska.)

Antibodies specific to phage virions have been observed many times, both in animals and in humans. Phages induce the T-dependent type of immune response, which is fundamental for immunological memory and long retention of abilities to recognize and respond to foreign epitopes. Experimental models have shown that phage-specific antibodies can be devastating for a phage in vivo. Observations from phage treatment in humans demonstrate however that antibodies do not necessarily hinder phage therapy. Controlling effective doses may help in controlling the immune response to phage and to create 'therapeutic windows' for the phage. Phage therapy design may also benefit from understanding phage antigenicity and predicting possible cross-reactions between antibodies specific to different phages., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Bacteriophages may induce specific antibodies after natural exposure to phages or after phage therapy. As such, phage-specific antibodies might impact phage bioavailability in vivo , although limited non-neutralizing or insignificant effects have also been reported. Materials and Methods: Here, we report antibody induction against PB1-related phages ( Pseudomonas viruses LMA2, F8, DP1) in mice over an 80-day period, for a healthy population of humans, and in patients undergoing phage therapy (oral and/or topical treatment). Results: All phages effectively induced specific immunoglobulin M and immunoglobulin G in mice. Phage-specific antibodies were observed in humans, whereas recombinant virion proteins (PB1 gp22, gp29) did not induce phage-neutralizing antibodies, either in mice or in humans. The healthy human population was differentiated for frequency of phage-neutralizing antibodies. Conclusions: These data can hold key considerations for phage therapy cocktail design, as highly similar phages can still be highly complementary in cases where specific immune response hinders therapeutic use of phages., Competing Interests: No other competing financial interests exist., (Copyright 2020, Mary Ann Liebert, Inc., publishers.)