Recombinant Phage Research Articles

Novel SARS-COV2 poly epitope phage-based candidate vaccine and its immunogenicity

Background and purpose: The global emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted widespread concern. Bacteriophages have recently gained attention as a cost-effective and stable alternative for vaccine development due to their adjuvant properties. This study aimed to design and validate a poly epitope composed of viral proteins. Experimental approach: SARS-CoV-2 proteins (spike, nucleocapsid, membrane, envelope, papain-like protease, and RNA-dependent RNA polymerase) were selected for analysis. Immunoinformatic methods were employed to predict B and T cell epitopes, assessing their antigenicity, allergenicity, and toxicity. Epitopes meeting criteria for high antigenicity, non-allergenicity, and non-toxicity were linked to form poly epitopes. These sequences were synthesized and cloned into pHEN4 plasmids to generate Poly1 and Poly2 phagemid vectors. Recombinant Poly1 and Poly2 phages were produced by transforming M13ΔIII plasmids and phagemid vectors into E. coli TG1. Female Balb/c mice were immunized with a cocktail of Poly1 and Poly2 phages, and their serum was collected for ELISA testing. Interferon-gamma (IFN-γ) testing was performed on spleen-derived lymphocytes to evaluate immune system activation. Findings/Results: Recombinant Poly1 and Poly2 phages were produced, and their titer was determined as 1013 PFU/mL. Efficient humoral immune responses and cellular immunity activation in mice were achieved following phage administration. Conclusion and implication: Poly epitopes displayed on phages exhibit adjuvant properties, enhancing humoral and cellular immunity in mice. This suggests that phages could serve as adjuvants to bolster immunity against SARS-Cov-2. Recombinant phages could be applied as effective candidates for injectable and oral vaccine development strategies.

Comparing Methods to Genetically Engineer Bacteriophage and Increase Host Range.

Antibacterial resistance is an emerging problem in military medicine. Disruptions to the health care systems in war-torn countries that result from ongoing conflict can potentially exacerbate this problem and increase the risk to U.S. forces in the deployed environment. Therefore, novel therapies are needed to mitigate the impact of these potentially devastating infections on military operations. Bacteriophages are viruses that infect and kill bacteria. They can be delivered as therapeutic agents and offer a promising alternative to traditional antibiotic chemotherapy. There are several potential benefits to their use, including high specificity and comparative ease of use in the field setting. However, the process of engineering phages for military medical applications can be a laborious and time-consuming endeavor. This review examines available techniques and compares their efficacy. This review evaluates the scientific literature on the development and application of four methods of bacteriophage genome engineering and their consideration in the context of military applications. Preffered Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed for a systematic review of available literature that met criteria for analysis and inclusion. The research completed for this review article originated from the United States Military Academy's library "Scout" search engine, which compiles results from 254 available databases (including PubMed, Google Scholar, and SciFinder). Particular attention was focused on identifying useful mechanistic insight into the nature of the engineering technique, the ease of use, and the applicability of the technique to countering the problem of antimicrobial resistance in the military setting. A total of 52 studies were identified that met inclusion criteria following PRISMA guidelines. The bioengineering techniques analyzed included homologous recombination (12 articles), in vivo recombineering (9 articles), bacteriophage recombineering of electroporated DNA (7 articles), and the CRISPR-Cas system (10 articles). Rates of success and fidelity varied across each platform, and comparative benefits and drawbacks are considered. Each of the phage engineering techniques addressed herein varies in amount of effort and overall success rate. CRISPR-Cas-facilitated modification of phage genomes presents a highly efficient method that does not require a lengthy purification and screening process. It therefore appears to be the method best suited for military medical applications.

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 μg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 μg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 μg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 μg ml-1) and P. aeruginosa P2307 (65.00 μg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Designing molecules: directing stem cell differentiation.

Stem cells have been widely applied in regenerative and therapeutic medicine for their unique regenerative properties. Although much research has shown their potential, it remains tricky in directing stem cell differentiation. The advancement of genetic and therapeutic technologies, however, has facilitated this issue through development of design molecules. These molecules are designed to overcome the drawbacks previously faced, such as unexpected differentiation outcomes and insufficient migration of endogenous or exogenous MSCs. Here, we introduced aptamer, bacteriophage, and biological vectors as design molecules and described their characteristics. The methods of designing/developing discussed include various Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedures, in silico approaches, and non-SELEX methods for aptamers, and genetic engineering methods such as homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), Bacteriophage Recombineering with Infectious Particles (BRIP), and genome rebooting for bacteriophage. For biological vectors, methods such as alternate splicing, multiple promoters, internal ribosomal entry site, CRISPR-Cas9 system and Cre recombinase mediated recombination were used to design viral vectors, while non-viral vectors like exosomes are generated through parental cell-based direct engineering. Besides that, we also discussed the pros and cons, and applications of each design molecule in directing stem cell differentiation to illustrate their great potential in stem cells research. Finally, we highlighted some safety and efficacy concerns to be considered for future studies.

Evaluation of humoral and cellular immune responses against Vibrio cholerae using oral immunization by multi-epitope-phage-based vaccine

IntroductionCholera is a severe gastrointestinal disease that manifests with rapid onset of diarrhea, vomiting, and high mortality rates. Due to its widespread occurrence in impoverished communities with poor water sanitation, there is an urgent demand for a cost-effective and highly efficient vaccine. Multi-epitope vaccines containing dominant immunological epitopes and adjuvant compounds have demonstrated potential in boosting the immune response. Material and methodsB and T epitopes of OMPU, OMPW, TCPA, CTXA, and CTXB proteins were predicted using bioinformatics methods. Subsequently, highly antigenic multi-epitopes that are non-allergenic and non-toxic were synthesized. These multi-epitopes were then cloned into the pCOMB phagemid. A plasmid M13KO7ΔpIII containing all helper phage proteins except pIII was created to produce the recombinant phage. Female Balb/c mice were divided into three groups and immunized accordingly. The mice received the helper phage, recombinant phage or PBS via gavage feeding thrice within two weeks. Serum samples were collected before and after immunization for the ELISA test as well as evaluating immune system induction through ELISpot testing of spleen lymphocytes. ResultsThe titer of the recombinant phage was determined to be 1011 PFU/ml. The presence of the recombinant phage was confirmed through differences in optical density between sample and control groups in the ELISA phage technique, as well as by observing transduction activity, which demonstrated successful production of a recombinant phage displaying the Vibrio multi-epitope on M13 phage pIII. ELISA results revealed significant differences in phage antibodies before and after inoculation, particularly notable in the negative control mice. Mice treated with multi-epitope phages exhibited antibodies against Vibrio cholerae lysate. Additionally, ELISpot results indicated activation of cellular immunity in mice receiving both Vibrio and helper phage. ConclusionThis study emphasizes the potential of multi-epitope on phage to enhance both cellular and humoral immunity in mice, demonstrating how phages can be used as adjuvants to stimulate mucosal immunity and act as promising candidates for oral vaccination.

Controlling Recombination to Evolve Bacteriophages.

Recombination among different phages sometimes facilitates their ability to grow on new hosts. Protocols to direct the evolution of phage host range, as might be used in the application of phage therapy, would then benefit from including steps to enable recombination. Applying mathematical and computational models, in addition to experiments using phages T3 and T7, we consider ways that a protocol may influence recombination levels. We first address coinfection, which is the first step to enabling recombination. The multiplicity of infection (MOI, the ratio of phage to cell concentration) is insufficient for predicting (co)infection levels. The force of infection (the rate at which cells are infected) is also critical but is more challenging to measure. Using both a high force of infection and high MOI (>1) for the different phages ensures high levels of coinfection. We also apply a four-genetic-locus model to study protocol effects on recombinant levels. Recombinants accumulate over multiple generations of phage growth, less so if one phage outgrows the other. Supplementing the phage pool with the low-fitness phage recovers some of this 'lost' recombination. Overall, fine tuning of phage recombination rates will not be practical with wild phages, but qualitative enhancement can be attained with some basic procedures.

Recombinant bacteriophage T4 displaying key epitopes of the foot-and-mouth disease virus as a novel nanoparticle vaccine

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that has caused significant economic losses in the livestock industry. Peptide vaccines engineered with the protective epitopes of FMDV have provided a safer alternative for disease prevention than the traditional inactivated vaccines. However, the immunogenicity of the peptide is usually poor and therefore an adjuvant is required. Here, we showed that recombinant T4 phages displaying the B-cell epitope of the FMDV VP1 protein (VP1130–158), without additional adjuvants, induced similar levels of antigen-specific IgG1 but higher levels of IgG2a compared to the peptide vaccine. Incorporation of a CD4+ T cell epitope, either 3A21–35 of FMDV 3A protein or P2830–844 of tetanus toxoid, further enhanced the immunogenicity of VP1-T4 phage nanoparticles. Interestingly, the extrinsic adjuvant cannot enhance the immunogenicity of the nanoparticles, indicating the intrinsic adjuvant activities of T4 phage. Furthermore, the recombinant T4 phage can be produced on a large scale within a short period of time at a relatively low-cost using Escherichia coli, heralding its potential in the development of a safe and effective FMDV vaccine.

Genetic Engineering of Therapeutic Phages Using Type III CRISPR-Cas Systems.

The functional characterization of "hypothetical" phage genes is a major bottleneck in basic and applied phage research. To compound this issue, the most suitable phages for therapeutic applications-the strictly lytic variety-are largely recalcitrant to classical genetic techniques due to low recombination rates and lack of selectable markers. Here we describe methods for fast and effective phage engineering that rely upon a Type III-A CRISPR-Cas system. In these methods, the CRISPR-Cas system is used as a powerful counterselection tool to isolate rare phage recombinants.

Synthetic Biology to Engineer Bacteriophage Genomes.

Recent advances in the synthetic biology field have enabled the development of new molecular biology techniques used to build specialized bacteriophages with new functionalities. Bacteriophages have been engineered toward a wide range of applications, including pathogen control and detection, targeted drug delivery, or even assembly of new materials.In this chapter, two strategies that have been successfully used to genetically engineer bacteriophage genomes will be addressed: the bacteriophage recombineering of electroporated DNA (BRED) and the yeast-based phage-engineering platform.

Bacteriophage Engineering for Improved Copper Ion Binding.

In this study, fd viruses are genetically modified to display seven cropped versions (H, HG, HGF, HGFA, HGFAN, HGFANV and HGFANVA) of the previously identified Cu(II) specific peptide (HGFANVA). Atomic force microscopy (AFM) imaging reveals the typical filamentous structures of recombinant phages with thicknesses of ≈2-5nm in dry state. Scanning electron microscopy (SEM) imaging shows that HGFANVA viruses form larger elongated assemblies than H viruses that are deposited with a mineral layer after Cu(II) treatment. C and N peaks are detected for virus samples through Energy dispersive X-ray spectroscopy (EDX) analyses confirming the presence of phage organic material. Cu peak is only detected for engineered viruses after Cu(II) exposure. Enzyme-linked immunosorbent assay (ELISA) analyses show the selective Cu(II) binding of engineered phages. Agarose gel electrophoresis (AGE) and zeta potential analyses reveal negative surface charges of engineered viral constructs. Positively charged Cytopore beads are coated with bacteriophages and used for Cu(II) ion sorption studies. ICP-MS analyses clearly show the improved Cu(II) binding of engineered viruses with respect to wild-type fd phages. Such bottom-up constructed, genetically engineered virus-based biomaterials may be applied in bioremediation studies targeting metal species from environmental samples.

Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C-terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525, was found to contain the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to rhACE2, confirming that a contiguous stretch of residues could adopt the appropriate structural orientation of F6 as an insertion within the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes, improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV.

Single domain antibodies specific for HER2 dimerization domain effectively disrupts HER2 dimerization

Dimer-dependent phosphorylation of HER2 receptor is a key event for the signal transduction of HER family of receptors which correlates with tumor invasion and metastasis. New generation of therapies based on dimerization domain inhibition using monoclonal or fragment antibodies was introduced. A potent method for manufacturing antibodies and antibody fragments is the phage display antibody library method. A recombinant phage was generated using the phage display method from synthetic dAb library. Subtractive biopanning was performed on sepharose 4b resin. Evaluation of success of subtractive biopanning was confirmed by the PCR fingerprinting after the fourth round of biopanning. The fourth round of biopanning results in the isolation of several dimerization domain reactive clones based on the polyclonal phage ELISA results. Monoclonal phage cell ELISA was used to select the positive clones with the highest affinity, and they were subsequently employed for functional tests. Cell-ELISA, MTT assay and dimerization inhibition test revealed that the reactivity and specificity of the selected monoclonal phage to dimerization domain of HER2. Further, Annexin V/PI staining and gene expression analysis showed that increased apoptosis rates. Also, in silico binding of the selected clones to conformational structure of HER2 was applied, using protein–protein docking tool of the ICM-Pro software, and showed sdAbs were specifically interacted with dimerization domain of the receptor. In conclusion, we have identified a single domain targeting HER2 dimerization, which represents a promising therapeutic and diagnostic candidate for HER2-positive cancers. Purified sdAb needs to more research to evaluate it both in vivo and in vitro via functional tests to determine if it can be applied for treatment and diagnostics.

Characterization of novel recombinant mycobacteriophages derived from homologous recombination between two temperate phages.

Comparative analyses of mycobacteriophage genomes reveals extensive genetic diversity in genome organization and gene content, contributing to widespread mosaicism. We previously reported that the prophage of mycobacteriophage Butters (cluster N) provides defense against infection by Island3 (subcluster I1). To explore the anti-Island3 defense mechanism, we attempted to isolate Island3 defense escape mutants on a Butters lysogen, but only uncovered phages with recombinant genomes comprised of regions of Butters and Island3 arranged from left arm to right arm as Butters-Island3-Butters (BIBs). Recombination occurs within two distinct homologous regions that encompass lysin A, lysin B, and holin genes in one segment, and RecE and RecT genes in the other. Structural genes of mosaic BIB genomes are contributed by Butters while the immunity cassette is derived from Island3. Consequently, BIBs are morphologically identical to Butters (as shown by transmission electron microscopy) but are homoimmune with Island3. Recombinant phages overcome antiphage defense and silencing of the lytic cycle. We leverage this observation to propose a stratagem to generate novel phages for potential therapeutic use.

T4 bacteriophage nanoparticles engineered through CRISPR provide a versatile platform for rapid development of flu mucosal vaccines

Vaccines that trigger mucosal immune responses at the entry portals of pathogens are highly desired. Here, we showed that antigen-decorated nanoparticle generated through CRISPR engineering of T4 bacteriophage can serve as a universal platform for the rapid development of mucosal vaccines. Insertion of Flu viral M2e into phage T4 genome through fusion to Soc (Small Outer Capsid protein) generated a recombinant phage, and the Soc-M2e proteins self-assembled onto phage capsids to form 3M2e-T4 nanoparticles during propagation of T4 in E. coli. Intranasal administration of 3M2e-T4 nanoparticles maintains antigen persistence in the lungs, resulting in increased uptake and presentation by antigen-presenting cells. M2e-specific secretory IgA, effector (TEM), central (TCM), and tissue-resident memory CD4+ T cells (TRM) were efficiently induced in the local mucosal sites, which mediated protections against divergent influenza viruses. Our studies demonstrated the mechanisms of immune protection following 3M2e-T4 nanoparticles vaccination and provide a versatile T4 platform that can be customized to rapidly develop mucosal vaccines against future emerging epidemics.

Evolved distal tail protein of skunaviruses facilitates adsorption to exopolysaccharide-encoding lactococci.

Aim: Lactococcal skunaviruses are diverse and problematic in the industrial dairy environment. Host recognition involves the specific interaction of phage-encoded proteins with saccharidic host cell surface structures. Lactococcal plasmid pEPS6073 encodes genes required for the biosynthesis of a cell surface-associated exopolysaccharide (EPS), designated 6073-like. Here, the impact of this EPS on Skunavirus sensitivity was assessed. Methods: Conjugal transfer of pEPS6073 into two model strains followed by phage plaque assays and adsorption assays were performed to assess its effect on phage sensitivity. Phage distal tail proteins were analyzed bioinformatically using HHpred and modeling with AlphaFold. Construction of recombinant phages carrying evolved Dits was performed by supplying a plasmid-encoded template for homologous recombination. Results: pEPS6073 confers resistance against a subset of skunaviruses via adsorption inhibition. IFF collection skunaviruses that infect strains encoding the 6073-like eps gene cluster carry insertions in their distal tail protein-encoding (dit) genes that result in longer Dit proteins (so-called evolved Dits), which encode carbohydrate-binding domains. Three skunaviruses with classical Dits (no insertion) were unable to fully infect their hosts following the conjugal introduction of pEPS6073, showing reductions in both adsorption and efficiency of plaquing. Cloning the evolved Dit into these phages enabled full infectivity on their host strains, both wild type and transconjugant carrying pEPS6073, with recombinant phages adsorbing slightly better to the EPS+ host than wild type. Conclusion: The 6073-like EPS potentially occludes the phage receptor for skunaviruses that encode a classical Dit protein. Skunaviruses that infect strains encoding the 6073-like EPS harbor evolved Dits, which likely help promote phage adsorption rather than just allow the phage to circumvent the putative EPS barrier. This work furthers our knowledge of phage-host interactions in Lactococcus and proposes a role for insertions in the Dit proteins of a subset of skunaviruses.

Evolutionary Qβ Phage Displayed Nanotag Library and Peptides for Biosensing.

We selected a novel biotin-binding peptide for sensing biotin, biotinylated proteins, and nucleotides. From a 15-mer library displayed on the RNA coliphage Qβ, a 15-amino acid long peptide (HGHGWQIPVWPWGQG) hereby referred to as a nanotag was identified to selectively bind biotin. The target selection was achieved through panning with elution by infection. The selected peptide was tested as a transducer for an immunogenic epitope of the foot-and-mouth disease virus (FMDV) on Qβ phage platform separated by a linker. The biotin-tag showed no significant influence on the affinity of the epitope to its cognate antibody (SD6). The nanotag-bound biotin selectively fused either to the C- or N-terminus of the epitope. The epitope would not bind or recognize SD6 while positioned at the N-terminus of the nanotag. Additionally, the biotin competed linearly with the SD6 antibody in a competitive ELISA. Competition assays using the selected recombinant phage itself as a probe or transducer enable the operationalization of this technology as a biosensor toolkit to sense and quantify SD6 analyte. Herein, the published Strep II nanotag (DVEWLDERVPLVET) was used as a control and has similar functionalities to our proposed novel biotin-tag thereby providing a new platform for developing devices for diagnostic purposes.

Localization and identification of denatured antigenic sites of glycinin A3 subunit after using two processing technologies

Glycinin is an important allergen in soybeans. In this study, molecular cloning and recombinant phage construction were performed to explore the antigenic sites of the glycinin A3 subunit that were denatured during processing. Next, the A-1-a fragment was located as the denatured antigenic sites by indirect ELISA. The combined UHP heat treatment showed better denaturation of this subunit than the single heat treatment assay. In addition, identification of the synthetic peptide showed that the A-1-a fragment was an amino acid sequence containing a conformational and linear IgE site, in which the first synthetic peptide (P1) being both an antigenic and allergenic site. The results of alanine-scanning showed that the key amino acids affecting antigenicity and allergenicity of A3 subunit were S28, K29, E32, L35 and N13. Our results could provide the basis for further development of more efficient methods to reduce the allergenicity of soybeans.

Salting-out extraction of recombinant κ-carrageenase and phage T7 released from Escherichia coli cells.

Traditional technology of cell disruption has become one of the bottlenecks restricting the industrialization of genetic engineering products due to its high cost and low efficiency. In this study, a novel bioprocess of phage lysis coupled with salting-out extraction (SOE) was evaluated. The lysis effect of T7 phage on genetically engineered Escherichia coli expressing κ-carrageenase was investigated at different multiplicity of infection (MOI), meanwhile the phage and enzyme released into the lysate were separated by SOE. It was found that T7 phage could lyse 99.9% of host cells at MOI=1 and release more than 90.0% of enzyme within 90min. After phage lysis, 87.1% of T7 phage and 71.2% of κ-carrageenase could be distributed at the middle phase and the bottom phase, respectively, in the SOE system composed of 16% ammonium sulfate and 20% ethyl acetate (w/w). Furthermore, κ-carrageenase in the bottom phase could be salted out by ammonium sulfate with a yield of 40.1%. Phage lysis exhibits some advantages, such as mild operation conditions and low cost. While SOE can efficiently separate phage and intracellular products. Therefore, phage lysis coupled with SOE is expected to become a viable alternative to the classical cell disruption and intracellular product recovery.

A Recombinant RBD-Based Phage Vaccine Report: A Solution to the Prevention of New Diseases?

The safety, inherent immunogenicity, stability, and low-cost production of bacteriophages make them an ideal platform for vaccine development. Most vaccination strategies against COVID-19 have targeted the spike protein of SARS-CoV-2 to generate neutralizing antibodies. P1, a truncated RBD-derived spike protein, has been shown to induce virus-neutralizing antibodies in preclinical studies. In this study, we first investigated whether recombinant phages displaying P1 on the M13 major protein could immunize mice against COVID-19, and second, whether inoculation with 50 µg of purified P1 in addition to the recombinant phages would stimulate the immune systems of the animals. The results showed that the mice that received recombinant phages were immunized against the phage particles, but did not have anti-P1 IgG. In contrast, compared with the negative control, the group that received a combination of P1 protein and recombinant phage was immunized against the P1 protein. In both groups, CD4+ and CD8+ T cells appeared in the lung tissue. These results suggest that the number of antigens on the phage body plays a crucial role in stimulating the immune system against the bacteriophage, although it is immunogenic enough to function as a phage vaccine.

Characterization of a lytic Pseudomonas aeruginosa phage vB_PaeP_ASP23 and functional analysis of its lysin LysASP and holin HolASP.

In this study, we isolated a lytic Pseudomonas aeruginosa phage (vB_PaeP_ASP23) from the sewage of a mink farm, characterized its complete genome and analyzed the function of its putative lysin and holin. Morphological characterization and genome annotation showed that phage ASP23 belonged to the Krylovirinae family genus Phikmvvirus, and it had a latent period of 10 min and a burst size of 140 pfu/infected cell. In minks challenged with P. aeruginosa, phage ASP23 significantly reduced bacterial counts in the liver, lung, and blood. The whole-genome sequencing showed that its genome was a 42,735-bp linear and double-stranded DNA (dsDNA), with a G + C content of 62.15%. Its genome contained 54 predicted open reading frames (ORFs), 25 of which had known functions. The lysin of phage ASP23 (LysASP), in combination with EDTA, showed high lytic activity against P. aeruginosa L64. The holin of phage ASP23 was synthesized by M13 phage display technology, to produce recombinant phages (HolASP). Though HolASP exhibited a narrow lytic spectrum, it was effective against Staphylococcus aureus and Bacillus subtilis. However, these two bacteria were insensitive to LysASP. The findings highlight the potential of phage ASP23 to be used in the development of new antibacterial agents.