{"paper_id":"6813599e-ec36-4d4b-85fa-4eeae2cf6496","body_text":"A λ Phage Platform for Successful Therapeutic Display of Protein Antigens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article A λ Phage Platform for Successful Therapeutic Display of Protein Antigens Meredith Bush, Xintian Li, Manoj Rajaure, Donald Court, Sankar Adhya This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8435997/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract We have developed a vector platform for delivery of foreign peptides by genetic modification of the temperate lambda (λ) bacteriophage. This delivery platform is capable of displaying peptides or proteins on either terminus of the structural λ head protein D, present in ~ 420 copies per phage particle, and λ side tail fiber (Stf), present at 12 copies per phage particle. Proteins and peptides can be easily fused for display through the low-cost and high-efficiency methods of recombineering and λ prophage induction for recombinant phage preparation described here. To improve this vector technology for use in antigen selection and immunotherapy, we introduced several mutations in the bacterial host and resident prophage λ that improve engineering, induction, phage stability, yield, fusion protein accommodation capacity, and longevity in animal systems. We tested the ability of this λ display system to identify useful antigens and generate antibodies in a mouse model. We report its success as a new technology for both applications: the selection and delivery of therapeutic peptides and proteins. Biological sciences/Biotechnology/Peptide delivery Biological sciences/Biotechnology/Molecular engineering/Protein design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Bacteriophages have been used since the 1990s to display foreign peptides or proteins on the surface of their virion structures 1 , 2 . The bacteriophage used here as a platform for display is lambda (λ), an Escherichia coli (E. coli) bacteriophage composed of two major structural elements (Fig. 1A): the phage capsid or head, which contains the genome, and the phage tail, which includes proteins for binding the bacterial receptor (LamB) and triggering genome injection 3 . When λ infects the bacterium E. coli , it may take one of two developmental paths. One is the lytic path whereupon λ usurps the host machinery to express functions required to replicate from progeny phage, lyse the cell, and release particles into the environment 4 . The alternative path is the lysogenic development following infection where the phage DNA integrates into the bacterial chromosome and produces the phage CI repressor protein to prevent expression of lytic genes. This dual-lifecycle feature allows λ to be genetically manipulated with ease while integrated in the bacterial chromosome and then induced to lytic phase to produce mature engineered phage particles. The λ capsid is composed of two major structural proteins, gpE and gpD (gene product), each present in ~ 420 copies per particle 3 , 5 . In the λ genome, the D and E genes are adjacent and expressed coordinately, with the gpD (gene product D) protein trimers stabilizing the primary gpE hexameric capsid structure 6 (Fig. 1B). Functional λ particles have been made in which non-phage proteins have been fused to either the N or C terminus of gpD 1 , 7 . Both termini protrude away from the head, allowing the display of fusions on the surface of the capsid 8 , as seen in the AlphaFold3 generated λD trimer structure 9 , 10 (Fig. 1C). In most previous examples, the gpD-fusion protein was generated by expressing the D-fusion from a plasmid during infection by a λ mutant containing an inactive D gene. The D-fusion protein made in trans then assembles with the other phage components 11 , 12 . Here we created fusions to either terminus of the D gene in vivo on a λ prophage where the D fusion gene is in its normal position adjacent to gene E in the phage genome and expressed coordinately with gene E (Fig. 2). Stf of λ that attaches to the end of the phage tail can also be used to display foreign proteins, especially in longer segments than are tolerated by λD. Stf (copy number = 12) is integral for λ binding to the E. coli LamB receptor during infection 13 , however, the Stf protein is non-essential for λ assembly from the prophage 14 , which is the extent of our use here. Thus, we can display foreign proteins on the distal exposed carboxyl end of Stf without interrupting essential processes. Our cloning results indicate that adding protein segments at the Stf C terminus generates high-titer phage lysates for display applications. The ability of λ to display foreign proteins has been utilized in a variety of ways: to create large peptide libraries, to identify antigenic proteins, to investigate ligand-receptor associations, to characterize proteins, to isolate peptide molecules, and to study peptide interactions, along with many other uses 11 , 15 – 19 . The λ platform developed and tested here is an efficient display system for any of these applications. This study provides evidence for this novel display technology to be used specifically as an antigen selection system and as a vector for immune stimulation against specific passenger antigens. While other groups have reported the use of λD display to stimulate antibodies 20 – 24 , our modifications improve the engineering and function of the λ display vector by fusing a foreign peptide to a λ protein in its prophage state. Several mutations have been made to facilitate the ease and quality of fusion phage engineering, prophage induction, and testing in animal studies, which are discussed below: The classical λ cI857 temperature sensitive mutation in the cI repressor gene causes the CI repressor to become inactive at an elevated temperature of 42° C 25 . Employing this mutation, a λ cI857 lysogenic culture growing at 32° C can be induced to produce phage particles and lyse the cell by simply shifting the culture temperature to 42° C. This avoids using methods of induction by agents such as mitomycin or UV, which damage DNA. The Sam7 mutation was introduced to the prophage to increase phage yield. Normally the time from λ prophage temperature induction to subsequent lysis and phage release is ~ 50 minutes, at which time ~ 100 particles of progeny phage per cell are released. Lysis is dependent upon the phage S gene encoded holin, which generates micron-scale holes in the cytoplasmic membrane after ~ 50 minutes of prophage induction 26 . These holes allow the endolysin to escape from the cytoplasm and degrade the cell’s peptidoglycan layer, causing complete cell lysis. If the holin protein is inactivated, then cytoplasmic endolysin cannot escape to cause lysis. However, λ continues to replicate within the cell and ultimately produces ~ 1000 phage particles per cell. Here we have used the Sam7 mutation in the prophage to prevent holin synthesis 27 . Phage lysis is caused later by the addition of chloroform or freezing and thawing the culture, thereby releasing the increased phage burst size (~ 10-fold increase). We deleted the 4,285-base-pair b2 region to increase the λ phage packaging capacity. A λ virion consists of a tail and a 48.5 kb dsDNA genome tightly packaged within an icosahedral protein shell. λ phage DNA lengths that fall outside the optimal packaging range of 38–52 kilobase pairs (kb) are generally not incorporated into functional, infectious phage particles 28 , 29 . The b2 region is a large, non-essential segment of the genome (approximately 5.7 kb). Deleting it does not prevent the phage from undergoing the lytic cycle. Further, its deletion increases the capacity of the vector, allowing the cloning of larger DNA fragments into the phage genome while keeping the total size within the optimal packaging range of 38–52 kb 30 . The LamB λ receptor protein encoding gene was deleted in the host chromosome to prevent λ binding and reduced yield after lysis. Initial lysates after chloroform treatment contain bacterial cell debris including isolated external cell surface λ receptors. Receptors present in the cellular debris of the lysate can bind to λ, causing ejection of the genome into the lysate and thereby reducing the number of active phage 31 . We eliminated the latter event of yield reduction by deleting the lamB gene from the host E. coli. 5. For half-life extension of the λ phage in the mammalian circulatory system, the major capsid E gene has been modified from Glu158 (GAG) to Lys158 (AAG), a single amino acid change. This change enables λ particles to have a longer presence in the mammalian circulatory system, which has the potential to increase immunogenicity 32 . A λ phage-based vaccine is an attractive therapeutic option because it confers several advantages over traditional immune-stimulating platforms. It is inexpensive to develop, and displays can be quickly engineered for countering new or evolving diseases. The phage particles themselves are stable at ambient temperatures, providing convenient shipping and storage of a λ phage display therapeutic 33 . The λ phage itself acts as an adjuvant, stimulating the body’s immune response against the high copy-number displayed antigen without requiring an added immune stimulant 22 , 23 , 34 , 35 . With the establishment of a reliable and effective λ platform, a variety of antigen-displaying therapies could be quickly utilized to treat or prevent disease. In this article, we describe the development of our specialized λ phage display vector and the methodology of prophage engineering and induction, as well as show data supporting the use of λ display as an antigen selection system and therapeutic vector platform. Results After generating E. coli host strains with prophage optimized for phage display in three different constructions, we compared the stability of phage displaying a series of melanoma neo-epitopes in all three constructs. Additionally, we demonstrated the use of the C-terminal λD phage display construction for epitope library generation, antigen selection, and immune stimulation in a mouse model against the SARS-CoV-2 spike protein. Construct stability by titer For the purpose of testing which of three melanoma neo-epitope display constructions would best slow the growth of melanoma tumors, we generated thirty phages displaying foreign peptides. Each of ten melanoma antigens were fused to λ in three display constructs: C-terminal Stf, N-terminal λD, and C-terminal λD. The differing titers of these three groups of phage lysates, which carry the same set of foreign peptides, illuminate phage stability by fusion construction (Table 1 A-C). The melanoma antigens used here originated from the detection of ten distinct mutations identified in mouse melanoma cell line B2905 by whole exome sequencing 36 . To create fusion antigens, each mutation was flanked on either side by 12 amino acids from its local genetic region in cell line B2905. These ten 25-aa sequences containing melanoma mutations were recombineered into the λ prophage in the three different constructions by counter-selection recombineering described in the methods (Table 2 ). The fusion phage were then induced, filtered, and the lysates titered on strain XTL1212. All 30 lysates were produced with identical technique. Titering phage samples involved the serial dilution of a phage lysate, plating on a suitable bacterial host (XTL1212), and plaque counting after overnight incubation at 39° C. Stf melanoma fusions produced highest titer phages with all but two samples reaching more than 1x10 12 pfu/ml. C-terminal λD constructions had the second highest stability with most samples titering between 10 10 and 10 11 pfu/ml. N-terminal lysates had the lowest average titers among the three constructions with most samples achieving 10 9 and 10 10 pfu/ml. The N-terminal group also yielded the lowest titer among the 30 fusion phage produced with the Top2A epitope phage lysate titering at only 2x10 8 pfu/ml. While titer among constructions may vary with the identity and length of the fusion antigen, these trends can be considered when choosing a construct for other diseases and display applications. λD antigen detection Here we describe the utility of the λ system using disease target COVID-19. To demonstrate the use of phage λ as an antigen selection tool, we chose to define an antigenic region of the SARS-CoV-2 spike protein through generating spike protein epitope λ displays. SARS-CoV-2, the coronavirus that causes the disease COVID-19 (Coronavirus disease 2019), has a dual subunit spike protein of 1,273 amino acids 37 . This surface glycoprotein functions to make contact with the host cell surface receptor ACE2 (Angiotensin-converting enzyme 2) and complete viral fusion to invade the cell 38 . Subunit 1 is primarily responsible for binding the human ACE2 cell-surface receptor through the spike protein’s receptor binding domain (RBD), while subunit 2 encodes proteins to accomplish the viral fusion process by which the virus gains access to the human cell 39 , 40 . The spike protein is the primary protein to interact with human cells, and therefore, is an ideal target for identifying a useful antigen against COVID-19. Our goal was to define an antigenic region of the spike protein small enough to create a stable, high-titer λ ~ spike protein display to use in a mouse immunogenicity study. The process by which we defined a suitable antigen was to create a series of overlapping spike protein epitopes up to 133 amino acids in length, fuse the peptides to λ through C-terminal λD display, and screen the display phages with human serum post SARS-CoV-2 infection. By western blotting, we were able to define a region of the spike protein with affinity for antibodies raised by natural SARS-CoV-2 infection. Within this larger antigenic region, we also defined a small epitope of strongest antigenicity and tested a phage displaying it in a mouse antibody-generation study. To use λ as a detection system for a SARS-CoV-2 spike protein epitope, we first segmented the spike protein gene into 12 overlapping epitopes of ~ 133 amino acids per segment and a few smaller epitopes (Table 3 and Fig. 3A). Using the methodology of λ prophage recombineering, we fused these genetic epitopes to λD in the C-terminal construction and raised a lysate of each distinct fusion phage. This created a λ display library of the spike protein, which was confirmed through blotting these phage samples and the vector phage against an anti-λD antibody (Fig. 3B). Wild-type λD has a molecular weight of 11.4 8 kDa, seen in lane 1 (1026) in both Fig. 3B blots. Fusions to λD increase the overall size of the protein, raising the location of the band on the western blot to reflect the higher kDa weight of the fusion protein. Therefore, λD carrying fusions of various sizes appears as a higher band than wild-type λD in all lanes besides the vector, which contains unmodified λD protein only. Also included in Fig. 3B are phages displaying an epitope from the SARS-CoV-2 E protein (XTL1324) and two epitopes from the SARS-CoV-2 N protein (XTL1293 and XTL1294). These epitope candidates were not useful antigens and were not investigated further. The rest of the antigen investigation narrowed to only the spike protein epitopes. This library of spike protein phage-displayed epitopes was then blotted against human serum collected after a SARS-CoV-2 infection (Fig. 3C). Only phage strain XTL1322, displaying a 133 aa peptide from the second subunit of the spike protein, reacted with COVID+ serum. This epitope consists of spike protein amino acids 1,038 − 1,170, which includes the subunit 2 connector domain (CD), open reading frame 1069–1162, and the first 7 amino acids of heptad repeat 2 (HR2). Both the CD and HR2 regions assist in viral fusion to the mammalian cell membrane after the spike protein receptor binding domain contacts the ACE2 cell surface receptor 37 . Fusion protein XTL1322 created a low titer lysate, likely due to decreased stability because of the large size of the 133 aa fusion protein. To construct a more stable phage, epitope XTL1322 was further dissected into overlapping DNA sequences encoding smaller peptide fragments (Fig. 3A). These DNA fragments were recombined in-frame at the 3’ end of the λD gene as described previously. While many smaller XTL1322 epitopes were tested against human COVID+ serum, only XTL1379 and XTL1380 reacted. These two epitopes were further probed against serum samples from three COVID+ patients and three healthy donors (Fig. 3D). All COVID+ sera reacted with the phage-displayed epitopes, while the healthy sera did not react with any of the phage samples. The peptides displayed by these two phage strains overlap by 11 amino acids (Fig. 3E). Upon sequence alignment with spike proteins of different variants such as alpha, beta, delta, omicron, as well as SARS-CoV-1, we found that this 11 amino acid overlapping segment between XTL1379 and XTL1380 was conserved among all other variants of SARS-CoV. We proceeded to reengineer a new hybrid strain XTL1382 that displayed the overlapping 11 amino acid peptide flanked by the consecutive 10 amino acids at either end of the peptide. When equal plaque forming units of λ displaying XTL1382, 1380, and 1379 were again probed with COVID+ serum (COVI-SP-0009) (Fig. 3E), we found that the newly engineered hybrid strain XTL1382 strongly reacted with the antibody present in the serum as indicated by the band intensity in Fig. 3E, left. Figure 3E, right, shows the same phage samples blotted against an anti-λD antibody, demonstrating correct fusion identity of display phages compared to the vector phage. While any of the three small displayed epitopes (XTL1382, XTL1380, and XTL1379) were strong candidates for use in a mouse immunogenicity study, we chose to test phage displaying XTL1382 since it gave the most robust signal against COVID+ serum antibodies. Mouse immunogenicity study with λD display phage XTL1382 λD epitope fusion phage display lysate was tested in a mouse study for the phage’s ability to stimulate antibodies against the displayed spike epitope. Groups of mice were immunized with fusion phage and vector phage three times over the course of the 49-day study (Fig. 4A). Post-injection serum samples were tested for antigen-specific antibody production through both ELISA and western blotting. Sera from mice injected with XTL1382 fusion phage demonstrated high XTL1382-specific antibody titers at 100x dilution two weeks after initial vaccination (Fig. 4B). Titer increased further after Day 13 injection but dipped between the second and third injection on Day 34. However, upon re-injection on Day 34, titers rose again to a high level on the final bleed day. Pre-bleed antibody titers as well as all vector phage-injected sera samples remained at a background absorbance level, indicating the specificity of antibody response detected in the experimental serum group. Western blots confirmed that phage displaying the XTL1382 antigen created XTL1382-specific antibodies in mice (Fig. 4C). When vector (lane 1) and XTL1382 phage (lane 2) were blotted with serum from a mouse that received the XTL1382 injection (left), the XTL1382 phage lane showed a strong band at the expected λD fusion protein region of ~ 15 kDa. The vector phage lane was blank, indicating no protein binding with mouse serum antibodies. As a control, both phage samples were blotted against serum from a mouse injected with vector phage (right). No bands appeared in either phage lane, indicating a lack of antibody presence specific to either the vector or XTL1382 fusion phage λD protein. This study was a confirmation of a previous mouse study that likewise injected XTL1382 display phage into a group of mice (N = 3). That study found similar antibody levels in response to phage display injection and yielded the same western blot results confirming presence of XTL1382-specific antibodies (Fig. S1 A-C). These two antibody-detection methods, ELISA and western blotting, show XTL1382-specific antibody production in mice from an injection of λ displaying the epitope, demonstrating the use of the λ vector for immune-stimulation. Discussion The production of an optimized λ phage vector for antigen detection and immune stimulation involved the evolution of strain SJ_XTL175 by several steps of genetic recombineering through the tetA-sacB cassette counter-selection method described in materials and methods 41 . Our system uniquely utilizes this technique to make a direct gene fusion in the prophage chromosome. The mutations to the platform maximize phage titer, increase particle stability, and expedite the engineering process. Additionally, our methodology streamlines prophage induction through the heat-inducible genetic switch and purifies the samples through tangential flow filtration. With ready-to-modify strains for any of the three fusion constructions, a stable lysate of a phage displaying a foreign protein can be made in a week’s time. This simple and rapid methodology allows for the use of λ phage display for many applications. The display platform is suitable for foreign protein display in three constructions: the C or N terminus of the λD capsid protein or the C terminus of the Stf protein. We have shown that protein fusions to any of these locations can produce phage display lysates. When creating a library of phage displaying melanoma antigens, the Stf fusion constructions yielded the highest titer phage lysates, followed by the C-terminal λD fusion constructions (Table 1 ). N-terminal λD fusions yielded the lowest virion stability and titer among the three fusion construction options, which may limit its use. A possible structural explanation for this stability difference is that the first 14 N terminus residues of λD were shown through crystal structure to be disordered and positioned near the three-fold λD trimer axis 8 . Conversely, C-terminal residues had a stable confirmation and protruded from the protein away from the trimer axis. The N terminus’ high flexibility, lack of consistent conformation, and position near an essential assembly point may contribute to particle instability when fusing to this location. N-terminal fusion lower titer may be potentially mitigated by inducing a supply of helper wild-type λD from the host chromosome through arabinose addition during phage assembly (Fig. 6). Additionally, this titer stability trend may differ when fusing other peptides from those tested here. While Stf fusion constructs yielded the highest titer phage lysates, λD fusions package 35x more antigens per phage particle. This difference in protein copy number per phage particle (~ 420 for λD vs. 12 for Stf) may make λD fusions, particularly in the C-terminal construction, more attractive for certain applications where a high copy number is desired. Here, we created a displayed spike protein library to detect segments of the SARS-CoV-2 spike protein which had affinity for human COVID+ serum antibodies. Through the process of library generation and antigenicity testing, we were able to detect a 133 aa region of the spike protein, XTL1322, which consistently reacted against human COVID+ serum (Fig. 3). This technique of generating a foreign peptide display library and running antibody-protein affinity tests, also called biopanning, could be easily replicated with the λ system to identify proteins of interest for a variety of diseases. Additionally, as shown here by the selection of small epitope XTL1382 within the larger XTL1322 epitope, the λ system can also define specific regions of high antigenicity within larger proteins. Initially, we selected overlapping epitopes XTL1379 and XTL1380 for their antibody affinity observed on COVID+ serum western blots. Upon investigation of the 11aa overlapping sequence between the two and discovering that this was a highly conserved coronavirus region, we created an epitope centering this sequence with flanking arms. The resulting epitope had the strongest affinity for COVID+ serum antibodies. This method of testing overlapping protein segments could be useful for defining regions of high antigenicity in other disease targets. To demonstrate λ’s ability to stimulate a strong and specific antibody response against a selected antigen, we performed a mouse study, injecting the same display phage used to detect spike protein antigen XTL1382 along with vector phage into groups of mice (Fig. 4). Antibody levels detected by ELISA and western blotting from study sera indicate that the λ-displayed antigen was able to stimulate a rapid, strong, and specific antibody titer against the XTL1382 peptide. Importantly, this immune-stimulating effect from λ display injections was achieved without the use of any additional adjuvant. The COVID-19 antigenicity study described here is being further investigated by testing the mouse antibodies created by these and other spike protein antigens for their neutralizing ability against SARS-CoV-2 infection. Through several ongoing studies fusing other disease therapeutics to the three fusion locations, we are continuing to investigate the best engineering construction and mouse study conditions of the λ phage display platform, including exploring applications of Stf display, comparing N- and C-terminal λD fusion, testing linkers of differing lengths, and determining the most efficacious routes of injection. The three uses reported here: library generation, antigen detection, and immune stimulation, are only a few of the possible applications of this λ display technology. With the ease, timeliness, and low cost of this antigen display method, the potential uses reach as far as display and vector delivery needs expand. Methods Recombineering All genetic changes made to the host strain (Table 4A) and its prophage λ (Table 4B) were made by the same general method of recombineering 41 . All primers used to make these genetic changes and check accuracy are reported in Table 4C. For all recombineered strains, a tetA-sacB cassette was inserted at the site on the bacterial chromosome or prophage genome to be deleted or altered by selecting for tetracycline resistance and ensuring that the drug-resistant recombinants had also become sensitive to sucrose because of the sacB gene (Fig. 5). Using recombineering technology, the tetA-sacB cassette is replaced using PCR fragments, gene blocks, or single strand oligos containing the coding sequence of the antigenic genes to be fused to λ D or stf . The single strand DNA oligonucleotide is flanked by the homologous sequences to target the chromosome or prophage genome for recombination. These sucrose-resistant recombinants are tetracycline sensitive and are engineered to fuse the antigenic gene to the D or stf gene in the same reading frame. When placed at the λ D N terminus, the fusion DNA has an upstream 5’ ATG start codon, and when placed at the λ D C terminus, the fusion DNA has a downstream 3’ TAA stop codon. Additional DNA coding sequences are included to generate a flexible linker sequence between the λD or Stf protein and the foreign protein from one to three repeats of the amino acid sequence GGGGS 42 . Once fusions are generated, the λ D -fusion or stf- fusion segments are amplified by PCR and sequenced to verify the accuracy of the new gene fusions. E. coli strain evolution The details of strain construction and constructs are adapted from Thomason et al., 2023 43 . SJ_ΧΤL175 : Strain SJ_XTL175 was used as the starting strain for the construction of the host bacterium used here. SJ_XTL175 is a wild-type MG1655 derivative 44 that contains the following genotype: Δ lacI, lacY A177C , Δ araE, Δ araFGH :: spec R , Δ araD :: tetA - sacB - amp . SJ_XTL175 was further modified by recombineering to replace the arabinose operon genes araB araA araD with gene D of λ. The araC gene remains intact, allowing AraC regulation of the λD gene (Fig. 6). In this construct, tetA - sacB - amp DNA is inserted after araA to create a counter-selection mechanism (Fig. 6B). Then, from the start codon of araB , including araA and the insert, to the araD TAA stop codon is replaced with gene D of λ (Fig. 6C). This places D of λ under AraC regulation and arabinose-inducible control. The other mutations (Δ lacI, lacY A177C , Δ araE, Δ araFGH ::specR) already present in SJ_XTL175 confer on this new strain (XTL981) the ability to tune the λD-protein expression level in every cell in response to the added extracellular arabinose concentration 44 . A mixture of wild-type λD and fusion λD has been used before to stabilize λ display particles 45 , but it has not been used previously in a well-controlled system like the tunable arabinose method used here. In our system, wild-type λD protein expression can be turned on at a precise time and directly tuned to the concentration of arabinose added. This allows for a stable, high-titer phage yield from each lysis, which is especially necessary to support the stability of the phage particle when fusing larger display proteins 46 . XTL981: Strain XTL981 described above has been further engineered to optimize λ phage production and the generation of λ particles carrying D-fusion proteins. First, XTL981 was infected with λ cI857 Sam7 phage to generate lysogens, and standard techniques were used to identify a lysogen in which a single phage genome was integrated into its native attachment site ( attB ) on the bacterial chromosome 47 . Next, additional changes were made to the bacterial chromosome and to the prophage λ cI857 Sam7 genome using recombineering technologies. As mentioned above, the host lamB gene was deleted. The fhuA gene, encoding the phage T1 and phage φ80 receptor protein, was deleted to avoid contamination by T1 and φ80 bacteriophage 48 . The nonessential λ genes ea59 and ea31 were precisely removed in a DNA segment of 4,285bp from the b region of the λ prophage 49 . This deletion provides more room in the phage genome for cloning and encapsidation of DNA segments to be fused to the D gene. XTL1026: XTL1026 is the strain containing all the modifications to the host and the λ prophage that are described above. This strain also contains plasmid pKM208, which is a low-copy pSC101-based plasmid that expresses Red functions Exo Bet and Gam from the lac promoter for recombineering 50 . The D gene in the λ prophage is the target for creating D fusions to express conjugated proteins joined at either the N or C terminus of λD. Two strains are derived from XTL1026 in which the λD gene is modified for generating λD gene fusions. The counter-selectable tetA-sacB marker cassette is inserted just upstream or downstream of the D gene in the prophage by selecting for tetracycline resistance. These two Tet R prophage strains are respectively, XTL1059 and XTL1048. Both become sucrose sensitive because of the sacB gene function linked to the tetA marker, which can then be replaced with the sequence of the fusion peptide. Strain XTL1048 was used to create the C-terminal SARS-CoV-2 spike protein λD fusion phage described in the results. XTL1226: This strain was created from XTL1026 to allow for the addition of foreign peptides to the C terminus of λ᾽s Stf protein. Initially, PCR was performed on the T-SACK strain 41 using primers XT1116 and XT1117 (Table 4C) to generate the stf386-tetA-sacB cassette. The recombination of this cassette into the prophage strain inserts the tetA-sacB cassette into the stf gene at position 386 and can be verified by PCR using primers XT1118 and XT40 (expected product: 505 bp). Strain XTL1226 is sucrose sensitive due to the addition of the stf386-tetA-sacB cassette. This strain is then able to recombine with foreign peptides. When replacing the stf386-tetA-sacB cassette with a foreign peptide, we use a gene block that truncates the rest of the stf gene from its 774 amino acid wild-type length to 386 amino acids, placing the fusion peptide at the new stf C terminus. This truncation slightly improves phage titer and was performed to ensure fusion peptide stability to a smaller Stf fusion protein. Full-length Stf is non-essential to phage assembly, lysis, or protein display, so it can be truncated for optimal fusion without disrupting functions necessary to protein display. XTL1226 was used to produce Stf melanoma epitope prophage by the insertion of melanoma epitopes with flaked λ homology at the stf386-tetA-sacB cassette location (Table 1 and Table 2). The prophages were screened using primers XT1118 and XT1119 (expected product: 436 bp). Identity of melanoma epitopes were further confirmed by Sanger sequencing. XTL1439 : This strain completes the truncation of Stf without placing a fusion peptide at the Stf location. Strain XTL1226 is recombineered with a gene block to replace the stf386-tetA-sacB cassette with a stop codon, truncating Stf. A new tetA - sacB cassette is then inserted at the C or N terminus of this strain’s λD gene to create strains XTL1441 and XTL1442. These two strains were used to then make melanoma epitope λD display phages in a strain with a truncated Stf to create an identical genetic background for comparison with the Stf melanoma epitope display phages. Prophage induction procedure Once foreign proteins have been genetically fused to the D or stf gene of the λ lysogen, the prophage can be induced to create a bacterial lysate which may be further purified and used in animal studies. To induce prophage, the E. coli strain containing the fusion prophage is grown at 32° C in LB broth to 2x10 8 cell/ml before quickly shifting to 42° C for 30 min and back to 39° C for 3 hrs (Fig. 7). Additionally, 0.2% total arabinose can be added to induce host chromosomewild-type λD to mitigate potential phage particle instability caused by the fusion (Fig. 6). Due to the Sam7 mutation that inactivates the phage holin protein, a lysate of the induced phage can be prepared by at least two methods. The first is the addition of chloroform, and the second is spinning the cells down into a pellet, resuspending in a small volume, and freezing overnight at -80° C. After freezing, the resuspended pellets are thawed to lyse the cells. The resulting crude lysate is then spun down to form a pellet and the supernatant filtered through 0.45 and 0.2 micron filters to collect the clear phage lysate. Freeze/thaw was the method used for lysis in the studies described here. Whether made through freeze/thaw or chloroform addition, the lysate is then titered on a supF strain (XTL1212) which suppresses the Sam7 mutation to make gpS and allow lysis and plaque formation. Strain XTL1212 also contains the plasmid pLXT42, which produces λD protein to complement D-fusion phage particles that may be somewhat defective in D function and cannot form plaques efficiently on their own. The λD fusion identity can then be confirmed by western blot and DNA sequencing before the phage lysates are purified by tangential flow filtration. Tangential flow filtration Engineered phage lysates were filtered and their titer concentrated by passage through a Pellicon® Mini Cassette 0.1 mm Composite Regenerated Cellulose filter by Millipore. The filter retains the phage particles and allows bacterial waste to pass through and be discarded. Filtration occurs by running 5-7 liters of 10 mM Tris and 50mM MgCl₂ buffer 33 through the filter system to purify phage. Phage are resuspended after filtration in a small volume (5-15 ml) of the same buffer. The MgCl₂ stabilizes phage particles and allows for long-term storage. This buffer can be diluted in PBS before injection into mice to reduce MgCl₂. Endotoxin readings were taken after filtration using the Endosafe® Nextgen-PTS™ (Portable Test System) by Charles River to ensure that the lysate was below an acceptable level of toxin for animal injection. Mouse study protocol The mouse study conducted to test the immunogenicity of XTL1382 display phage utilized six- to eight-week-old Balb/c female mice. Groups of five mice were injected with antigen fusion phage, vector phage, and 10 mM Tris and 10mM MgCl₂ buffer. Mice were injected intraperitoneally with 500 µl doses of 2x10⁹ pfu three times over the course of the 49-day study, on days 0, 13, and 34. Bleeds were taken at five time points on days 0, 13, 27, 34, and terminally on day 49. This study was performed under Integrated Biotherapeutics’ (Rockville, MD) approved IACUC protocol #160805. Analysis: ELISA and western blot Western blot Western Blot identification of SARS-CoV-2 spike protein epitope using SARS-CoV-2 infected and healthy donor human serum: To identify antibody-protein affinity between spike antigen display phage and human serum samples, we ran phage samples on 4-20% SDS gradient gels. The samples were then transferred to a nitrocellulose membrane for standard western blotting. First the membrane was blocked with 2.5% non-fat milk buffer, then it was triple washed with TNE buffer (10mM Tris-HCl, 50mM NaCl, 2.5mM EDTA) prior to incubating with 1000-fold dilution of human serum samples extracted either from a healthy donor or a patient positive for SARS-CoV-2. The membrane was incubated with serum samples for three hours and then washed with a wash buffer (10mM Tris-HCl, 50mM NaCl, 2.5mM EDTA). It was then incubated with 10,000-fold diluted goat anti-human IgG secondary-HRP conjugated antibody for an hour and washed with a wash buffer containing 0.1% tween. The membrane was developed using a clarity enhanced chemiluminescence substrate from Bio-Rad. Western blot protocol for mouse serum antibody affinity: Western blots were conducted to quantify mouse sera antibody affinity to phage sample fusion protein. They were performed by loading fusion phage and vector phage on an SDS 4-20% tris-glycine gel, transferring separated proteins to a nitrocellulose membrane, blocking with 5% milk buffer in 1X TNE, and probing with 1:1000 dilution of mouse serum in milk buffer, followed by 1:10,000-fold goat anti-mouse IgG secondary-HRP in 1X TNE. Membranes were developed with Clarity Max Western ECL substrate and imaged with chemiluminescence. ELISA ELISA Assay protocol adapted from Hong Zhou (NIH/NCI) and Thermo Fisher’s general ELISA protocol: (https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLCD%2Fmanuals%2FD20692~.pdf) Mouse serum was analyzed for the presence of antibodies specific to the injected λD XTL1382 peptide by ELISA (enzyme-linked immunosorbent assay). ELISA demonstrated antibody-protein binding by incubating a synthetic peptide version of the fusion phage peptide on a nunc-immobilon plate at a 20 µg/ml concentration. Once the synthetic peptide was immobilized on the plate, mouse serum was added in several 10-fold dilutions. The synthetic protein/antibody binding interaction was quantified by adding TMB substrate, stopping the reaction after 12 min with 17% H₂SO₄ stop solution, and reading color intensity at 450 nm. Absorbance was read at a 100x dilution. After subtracting the background, collected at 562 nm, data was plotted in GraphPad Prism 10. Error bars were generated in Prism software. Materials Synthetic peptides created by GenScript Inc. Spike Protein Epitope XTL1382: YDPLQPELDSFKEELDKYFKNHTSPDVDLGD Synthetic peptide was custom synthesized by GenScript Inc. Peptide sequence is from the second subunit of the SARS-CoV-2 spike protein. The purity of this peptide was above 95%. It was resuspended in sterile H₂O for use in ELISA at 20 µg/ml in each well for the detection of antibodies. Antibodies Anti-λD antibody: Custom-made polyclonal anti-λD capsid protein antibody raised in mouse against purified λD protein from GenScript Corp. Human serum used in antigen selection: blood serum samples from three healthy donors and three patients positive for SARS-CoV-2 infection were generously gifted by the laboratory of Robert J. Kreitman at NCI/NIH. Healthy Donors: α-COVI-SP-0005 α-COVI-SP-0007 α-COVI-SP-0008 COVID+ Sera α-COVI-SP-0009 α-COVI-SP-0179 α-COVI-SP-0187 Declarations Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Resulting Patents US patent WO 2023/114727 A1 was published on 06/22/2023 as an application in the World Intellectual Property Organization detailing the invention of this lambda phage display technology for the display or foreign peptides and proteins. Table 4A E. coli strains used and/or generated here. Bacterial strains generated in the evolution of a λ display platform. Table includes the initial parental strain, each new strain generated to modify the bacterial host, each strain containing modifications to λ prophage within the host, and the final strains used to create antigen-displaying phages in this study. Methods Recombineering All genetic changes made to the host strain (Table 4A) and its prophage λ (Table 4B) were made by the same general method of recombineering 41 . All primers used to make these genetic changes and check accuracy are reported in Table 4C. For all recombineered strains, a tetA-sacB cassette was inserted at the site on the bacterial chromosome or prophage genome to be deleted or altered by selecting for tetracycline resistance and ensuring that the drug-resistant recombinants had also become sensitive to sucrose because of the sacB gene (Fig. 5). Using recombineering technology, the tetA-sacB cassette is replaced using PCR fragments, gene blocks, or single strand oligos containing the coding sequence of the antigenic genes to be fused to λ D or stf . The single strand DNA oligonucleotide is flanked by the homologous sequences to target the chromosome or prophage genome for recombination. These sucrose-resistant recombinants are tetracycline sensitive and are engineered to fuse the antigenic gene to the D or stf gene in the same reading frame. When placed at the λD N terminus, the fusion DNA has an upstream 5’ ATG start codon, and when placed at the λD C terminus, the fusion DNA has a downstream 3’ TAA stop codon. Additional DNA coding sequences are included to generate a flexible linker sequence between the λD or Stf protein and the foreign protein from one to three repeats of the amino acid sequence GGGGS 42 . Once fusions are generated, the λ D -fusion or stf- fusion segments are amplified by PCR and sequenced to verify the accuracy of the new gene fusions. E. coli strain evolution The details of strain construction and constructs are adapted from Thomason et al., 2023 43 . SJ_ΧΤL175 : Strain SJ_XTL175 was used as the starting strain for the construction of the host bacterium used here. SJ_XTL175 is a wild-type MG1655 derivative 44 that contains the following genotype: Δ lacI, lacY A177C , Δ araE , Δ araFGH :: spec R , Δ araD :: tetA - sacB - amp . SJ_XTL175 was further modified by recombineering to replace the arabinose operon genes araB araA araD with gene D of λ. The araC gene remains intact, allowing AraC regulation of the λD gene (Fig. 6). In this construct, tetA - sacB - amp DNA is inserted after araA to create a counter-selection mechanism (Fig. 6B). Then, from the start codon of araB , including araA and the insert, to the araD TAA stop codon is replaced with gene D of λ (Fig. 6C). This places D of λ under AraC regulation and arabinose-inducible control. The other mutations (Δ lacI, lacY A177C , Δ araE , Δ araFGH ::specR) already present in SJ_XTL175 confer on this new strain (XTL981) the ability to tune the λD-protein expression level in every cell in response to the added extracellular arabinose concentration 44 . A mixture of wild-type λD and fusion λD has been used before to stabilize λ display particles 45 , but it has not been used previously in a well-controlled system like the tunable arabinose method used here. In our system, wild-type λD protein expression can be turned on at a precise time and directly tuned to the concentration of arabinose added. This allows for a stable, high-titer phage yield from each lysis, which is especially necessary to support the stability of the phage particle when fusing larger display proteins 46 . XTL981 Strain XTL981 described above has been further engineered to optimize λ phage production and the generation of λ particles carrying D-fusion proteins. First, XTL981 was infected with λ cI857 Sam7 phage to generate lysogens, and standard techniques were used to identify a lysogen in which a single phage genome was integrated into its native attachment site ( attB ) on the bacterial chromosome 47 . Next, additional changes were made to the bacterial chromosome and to the prophage λ cI857 Sam7 genome using recombineering technologies. As mentioned above, the host lamB gene was deleted. The fhuA gene, encoding the phage T1 and phage φ80 receptor protein, was deleted to avoid contamination by T1 and φ80 bacteriophage 48 . The nonessential λ genes ea59 and ea31 were precisely removed in a DNA segment of 4,285bp from the b region of the λ prophage 49 . This deletion provides more room in the phage genome for cloning and encapsidation of DNA segments to be fused to the D gene. XTL1026 XTL1026 is the strain containing all the modifications to the host and the λ prophage that are described above. This strain also contains plasmid pKM208, which is a low-copy pSC101-based plasmid that expresses Red functions Exo Bet and Gam from the lac promoter for recombineering 50 . The D gene in the λ prophage is the target for creating D fusions to express conjugated proteins joined at either the N or C terminus of λD. Two strains are derived from XTL1026 in which the λD gene is modified for generating λD gene fusions. The counter-selectable tetA-sacB marker cassette is inserted just upstream or downstream of the D gene in the prophage by selecting for tetracycline resistance. These two Tet R prophage strains are respectively, XTL1059 and XTL1048. Both become sucrose sensitive because of the sacB gene function linked to the tetA marker, which can then be replaced with the sequence of the fusion peptide. Strain XTL1048 was used to create the C-terminal SARS-CoV-2 spike protein λD fusion phage described in the results. XTL1439 This strain completes the truncation of Stf without placing a fusion peptide at the Stf location. Strain XTL1226 is recombineered with a gene block to replace the stf386-tetA-sacB cassette with a stop codon, truncating Stf. A new tetA - sacB cassette is then inserted at the C or N terminus of this strain’s λD gene to create strains XTL1441 and XTL1442. These two strains were used to then make melanoma epitope λD display phages in a strain with a truncated Stf to create an identical genetic background for comparison with the Stf melanoma epitope display phages. Prophage induction procedure Once foreign proteins have been genetically fused to the D or stf gene of the λ lysogen, the prophage can be induced to create a bacterial lysate which may be further purified and used in animal studies. To induce prophage, the E. coli strain containing the fusion prophage is grown at 32° C in LB broth to 2x10 8 cell/ml before quickly shifting to 42° C for 30 min and back to 39° C for 3 hrs (Fig. 7). Additionally, 0.2% total arabinose can be added to induce host chromosome wild-type λD to mitigate potential phage particle instability caused by the fusion (Fig. 6). Due to the Sam7 mutation that inactivates the phage holin protein, a lysate of the induced phage can be prepared by at least two methods. The first is the addition of chloroform, and the second is spinning the cells down into a pellet, resuspending in a small volume, and freezing overnight at -80° C. After freezing, the resuspended pellets are thawed to lyse the cells. The resulting crude lysate is then spun down to form a pellet and the supernatant filtered through 0.45 and 0.2 micron filters to collect the clear phage lysate. Freeze/thaw was the method used for lysis in the studies described here. Whether made through freeze/thaw or chloroform addition, the lysate is then titered on a supF strain (XTL1212) which suppresses the Sam7 mutation to make gpS and allow lysis and plaque formation. Strain XTL1212 also contains the plasmid pLXT42, which produces λD protein to complement D-fusion phage particles that may be somewhat defective in D function and cannot form plaques efficiently on their own. The λD fusion identity can then be confirmed by western blot and DNA sequencing before the phage lysates are purified by tangential flow filtration. Tangential flow filtration Engineered phage lysates were filtered and their titer concentrated by passage through a Pellicon® Mini Cassette 0.1 mm Composite Regenerated Cellulose filter by Millipore. The filter retains the phage particles and allows bacterial waste to pass through and be discarded. Filtration occurs by running 5–7 liters of 10 mM Tris and 50mM MgCl₂ buffer 33 through the filter system to purify phage. Phage are resuspended after filtration in a small volume (5–15 ml) of the same buffer. The MgCl₂ stabilizes phage particles and allows for long-term storage. This buffer can be diluted in PBS before injection into mice to reduce MgCl₂. Endotoxin readings were taken after filtration using the Endosafe® Nextgen-PTS™ (Portable Test System) by Charles River to ensure that the lysate was below an acceptable level of toxin for animal injection. Mouse study protocol The mouse study conducted to test the immunogenicity of XTL1382 display phage utilized six- to eight-week-old Balb/c female mice. Groups of five mice were injected with antigen fusion phage, vector phage, and 10 mM Tris and 10mM MgCl₂ buffer. Mice were injected intraperitoneally with 500 µl doses of 2x10⁹ pfu three times over the course of the 49-day study, on days 0, 13, and 34. Bleeds were taken at five time points on days 0, 13, 27, 34, and terminally on day 49. This study was performed under Integrated Biotherapeutics’ (Rockville, MD) approved IACUC protocol #160805. Acknowledgment This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute (NCI), and the Center for Cancer Research. (CCR). The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. We would like to thank Hong Zhou for performing ELISA assays and collecting data found in supplemental Fig. 1. We also thank all other members of our lab for their helpful assistance. Data Availability Statement: Relevant data are included in the manuscript. 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Biol. 4 , 11 (2003) Tables Table 1A Table 1B Table 1C Stf strain name Antigen Titer λD C-terminal strain name Antigen Titer λD N-terminal strain name Antigen Titer YXTL1429 Atr 4x10 12 YXTL1443 Atr 1.2x10 12 YXTL1453 Atr 6x10 9 YXTL1430 Dennd6b 2x10 11 YXTL1444 Dennd6b 2x10 11 YXTL1454 Dennd6b 8x10 10 YXTL1431 Gnaq 8x10 11 YXTL1445 Gnaq 6x10 10 YXTL1455 Gnaq 1x10 10 YXTL1432 Kras 2x10 12 YXTL1446 Kras 2x10 9 YXTL1456 Kras 1.4x10 9 YXTL1433 Mctp1 1.6x10 12 YXTL1447 Mctp1 9x10 11 YXTL1457 Mctp1 1.4x10 9 YXTL1434 Mfn1 1.2x10 12 YXTL1448 Mfn1 4x10 10 YXTL1458 Mfn1 2.4x10 11 YXTL1435 nt5dc1 1.2x10 12 YXTL1449 nt5dc1 4x10 11 YXTL1459 nt5dc1 2.8x10 11 YXTL1436 Prpf38a 1.8x10 12 YXTL1450 Prpf38a 2x10 11 YXTL1460 Prpf38a 6x10 9 YXTL1437 Top2a 2x10 12 YXTL1451 Top2a 4x10 11 YXTL1461 Top2a 2x10 8 YXTL1438 Mtmr10 2.2x10 12 YXTL1452 Mtmr10 2.4x10 11 YXTL1462 Mtmr10 3x10 10 Table 1: Titers of λ phage lysates displaying melanoma antigens in three engineering constructions: Stf, λD C-terminal, and λD N-terminal. Phage titers from each of thirty phage lysates prepared by induction and freeze/thaw lysis. Each phage displays one of ten antigens found in mouse melanoma cell line B2905 in three differing fusion constructions: A: Stf, B: C-terminal λD, and C: N-terminal λD. Prophage genotypes are listed in Table 2. Titers were determined by plating serial phage lysate dilutions on supF strain XTL1212 and counting resulting plaques after overnight incubation. Table 2 Prophage Strain Name Strain Genotype Melanoma Neo-Epitope YXTL1429 XTL1226 (stf386~Melanoma Atr-neo-epitope) Atr - RMKEAYTHAQIAKNNELKDTLILTT YXTL1430 XTL1226 (stf386~Melanoma Dennd6b-neo-epitope) Dennd6b - PPNVVLGVTNPFIIKTLQHWPHVLR YXTL1431 XTL1226 (stf386~Melanoma Gnaq-neo-epitope) Gnaq - QSVIFRMVDVGGLRSERRKWIHCFE YXTL1432 XTL1226 (stf386~Melanoma Kras-neo-epitope) Kras - MTEYKLVVVGADGVGKSALTIQLI YXTL1433 XTL1226 (stf386~Melanoma Mctp1-neo-epitope) Mctp1 - MYQLDITLRRGQGLAARDRGGTSDP YXTL1434 XTL1226 (stf386~Melanoma Mfn1-neo-epitope) Mfn1 - CSDFQEDIVFRFFLGWSSLVHRFLG YXTL1435 XTL1226 (stf386~Melanoma Nt5dc1-neo-epitope) Nt5dc1 - DKPGWYSQGNAALLYELLKKMTSKP YXTL1436 XTL1226 (stf386~Melanoma Prpf38a-neo-epitope) Prpf38a - GLTAELVVDKAMKLKFVGGVYGGNI YXTL1437 XTL1226 (stf386~Melanoma Top2a-neo-epitope) Top2a - IAGSTKDVKVFLSGNSLPVKGFRSY YXTL1438 XTL1226 (stf386~Melanoma Mtmr10-neo-epitope) Mtmr10 - DPYFRTITGFQSLIQKEWVWRAISS (containing single amino acid deletion) YXTL1443 XTL1441 (λD~Melanoma Atr-neo-epitope) Atr - RMKEAYTHAQIAKNNELKDTLILTT YXTL1444 XTL1441 (λD~Melanoma Dennd6b-neo-epitope) Dennd6b - PPNVVLGVTNPFIIKTLQHWPHVLR YXTL1445 XTL1441 (λD~Melanoma Gnaq-neo-epitope) Gnaq - QSVIFRMVDVGGLRSERRKWIHCFE YXTL1446 XTL1441 (λD~Melanoma Kras-neo-epitope) Kras - MTEYKLVVVGADGVGKSALTIQLI YXTL1447 XTL1441 (λD~Melanoma Mctp1-neo-epitope) Mctp1 - MYQLDITLRRGQGLAARDRGGTSDP YXTL1448 XTL1441 (λD~Melanoma Mfn1-neo-epitope) Mfn1 - CSDFQEDIVFRFFLGWSSLVHRFLG YXTL1449 XTL1441 (λD~Melanoma Nt5dc1-neo-epitope) Nt5dc1 - DKPGWYSQGNAALLYELLKKMTSKP YXTL1450 XTL1441 (λD~Melanoma Prpf38a-neo-epitope) Prpf38a - GLTAELVVDKAMKLKFVGGVYGGNI YXTL1451 XTL1441 (λD~Melanoma Top2a-neo-epitope) Top2a - IAGSTKDVKVFLSGNSLPVKGFRSY YXTL1452 XTL1441 (λD~Melanoma Mtmr10-neo-epitope) Mtmr10 - DPYFRTITGFQSLIQKEWVWRAISS (containing single amino acid deletion) YXTL1453 XTL1442 (Melanoma Atr-neo-epitope~λD) Atr - RMKEAYTHAQIAKNNELKDTLILTT YXTL1454 XTL1442 (Melanoma Dennd6b-neo-epitope~λD) Dennd6b - PPNVVLGVTNPFIIKTLQHWPHVLR YXTL1455 XTL1442 (Melanoma Gnaq-neo-epitope~λD) Gnaq - QSVIFRMVDVGGLRSERRKWIHCFE YXTL1456 XTL1442 (Melanoma Kras-neo-epitope~λD) Kras - MTEYKLVVVGADGVGKSALTIQLI YXTL1457 XTL1442 (Melanoma Mctp1-neo-epitope~λD) Mctp1 - MYQLDITLRRGQGLAARDRGGTSDP YXTL1458 XTL1442 (Melanoma Mfn1-neo-epitope~λD) Mfn1 - CSDFQEDIVFRFFLGWSSLVHRFLG YXTL1459 XTL1442 (Melanoma Nt5dc1-neo-epitope~λD) Nt5dc1 - DKPGWYSQGNAALLYELLKKMTSKP YXTL1460 XTL1442 (Melanoma Prpf38a-neo-epitope~λD) Prpf38a - GLTAELVVDKAMKLKFVGGVYGGNI YXTL1461 XTL1442 (Melanoma Top2a-neo-epitope~λD) Top2a - IAGSTKDVKVFLSGNSLPVKGFRSY YXTL1462 XTL1442 (Melanoma Mtmr10-neo-epitope~λD) Mtmr10 - DPYFRTITGFQSLIQKEWVWRAISS (containing single amino acid deletion) Red letter in neo-epitope sequence denotes single amino acid mutation found in mouse melanoma cell line B2905 36 . * ~ symbol refers to a genetic fusion Y= temperature sensitive cI857 mutation Table 2: Melanoma prophage strains . Prophage strains developed to generate phage displaying each of ten melanoma neo-epitopes at three fusion locations: C terminus of Stf, C terminus of λD, and the N terminus of λD. Parental strains and melanoma epitopes listed for each prophage developed. Table 3 Prophage Strain Strain Genotype Spike Protein Antigen Sequence Length (aa) Spike Protein Domain Boundary YXTL1312 XTL1048 D~linker~spike epitope Linker and antigen are inserted at λD gene TAA stop. TNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAG 47 602-648 YXTL1313 Same as above SSSGWTAGAAAYYVGYLQPRTFLL 24 254-277 YXTL1314 Same as above MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATN 125 1-125 YXTL1315 Same as above NATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDS 133 122-254 YXTL1316 Same as above DSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPT 133 253-385 YXTL1317 Same as above PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFE 133 384-516 YXTL1318 Same as above FELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRA 133 515-647 YXTL1319 Same as above RAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNT 133 646-778 YXTL1320 Same as above NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGI 133 777-909 YXTL1321 Same as above NGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKR 133 907-1039 YXTL1322 Same as above KRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDIS 133 1038-1170 YXTL1323 Same as above LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 108 1166-1273 YXTL1375 Same as above KRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPA 33 1038-1070 YXTL1376 Same as above VVFLHVTYVPAQEKNFTTAPAICHDGKAHFPRE 33 1060-1092 YXTL1377 Same as above CHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI 33 1082-1114 YXTL1378 Same as above VTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNT 33 1104-1136 YXTL1379 Same as above CDVVIGIVNNTVYDPLQPELDSFKEELDKYFKN 33 1126-1158 YXTL1380 Same as above FKEELDKYFKNHTSPDVDLGDIS 23 1148-1170 YXTL1381 Same as above MSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIV 84 1049-1133 YXTL1382 Same as above YDPLQPELDSFKEELDKYFKNHTSPDVDLGD 31 1138-1168 * ~ symbol refers to a genetic fusion Y= temperature sensitive cI857 mutation Table 3: SARS-CoV-2 spike protein prophage strains and their displayed epitopes. Prophage strains developed for use in detecting a suitable SARS-CoV-2 spike protein antigen. Each strain is a modification of YXTL1048, containing a partial sequence of the spike protein fused to the C-terminal of the prophage’s λ D gene with a single unit linker in the sequence: GGGGS. Highlighted sequences are those displayed antigens which produced a strong response against COVID+ donor serum. Table 4A Bacterial Strains Relevant Genetic Markers Source Prophage LE392 tyrT(supF58) glnV(supE44) hsdR514(r-m+) lacY1 galK2 galT22 metB1 trpR55 Lynn Enquist _ XTL1212 LE392 pΔ BAD :: λD * /pLXT42** This study _ SJ_XTL175 MG1655, lacY A177C , ΔaraFGH::spec R , Δ lacI, Δ araE , ΔaraD:: tetA-sacB-amp Li, Xt., Jun, Y., Erickstad, M. et al. tCRISPRi: tunable and reversible, one-step control of gene expression. Sci Rep 6, 39076 (2016). _ XTL981 SJ_XTL175 araC + ΔaraBAD :: λD This study _ XTL1026 XTL981 Δ lamB , Δ fhuA /pKM208*** This study YXTL1026 XTL1048 XTL1026 D ~linker:: tetA - sacB*, The tetA-sacB cassette is inserted with a linker coding sequence at λD gene TAA stop. This study YXTL1048 XTL1059 XTL1026 tetA - sacB ::linker~ D, The tetA-sacB cassette is inserted with a linker coding sequence at λD gene ATG start. This study YXTL1059 XTL1226 XTL1026 stf386-tetA-sacB insertion This study YXTL1226 XTL1439 XTL1226 stf386 truncation. tetA-sacB is replaced by sequence to truncate stf. This study YXTL1439 XTL1441 XTL1439 D ~linker:: tetA - sacB, the tetA-sacB cassette is inserted with a linker coding sequence at λD gene ATG start. Used to create melanoma C-terminal λD fusion phage in a strain that included Stf truncation. This study YXTL1441 XTL1442 XTL1439 tetA - sacB ::linker~ D, the tetA-sacB cassette is inserted with a linker coding sequence at λD gene ATG start. Used to create melanoma N-terminal λD fusion phage in a strain that included Stf truncation. This study YXTL1442 * :: indicates that the gene preceding the double colon is replaced by the gene following it. **pLXT42 is a plasmid that expresses wild-type λD under the tunable pBAD promoter, inducible by arabinose. It also contains the following elements: AmpR geneand pMBori. ***pKM208 is a pSC101 based plasmid that expresses Red functions exo bet and gam from the lac promoter for recombineering. Obtained from Ken Murphy. * ~ symbol refers to a genetic fusion. Y= temperature sensitive cI857 mutation Table 4A: E. coli strains used and/or generated here. Bacterial strains generated in the evolution of a λ display platform. Table includes the initial parental strain, each new strain generated to modify the bacterial host, each strain containing modifications to λ prophage within the host, and the final strains used to create antigen-displaying phages in this study. Table 4B Prophage Name Prophage Genotype Source YXTL1026 λ cI857, Sam7, Δ fhuA, Δb2, E E158K This study YXTL1048 YXTL1026 D ~linker:: tetA - sacB*, The tetA-sacB cassette is inserted with a linker coding sequence at λD gene TAA stop This study YXTL1059 YXTL1026 tetA - sacB ::linker~ D, The tetA-sacB cassette is inserted with a linker coding sequence at λD gene ATG start This study YXTL1226 XTL1026 stf386-tetA-sacB This study YXTL1439 XTL1226 stf386 truncation. TetA-sacB is replaced by sequence to truncate stf. This study YXTL1441 XTL1439 D ~linker:: tetA - sacB, the tetA-sacB cassette is inserted with a linker coding sequence at λD gene ATG start. Used to create melanoma C-terminal λD fusion phage in a strain that included Stf truncation. This study YXTL1442 XTL1439 tetA - sacB ::linker~ D, the tetA-sacB cassette is inserted with a linker coding sequence at λD gene ATG start. Used to create melanoma N-terminal λD fusion phage in a strain that included Stf truncation. This study * ~ symbol refers to a genetic fusion. Y= temperature sensitive cI857 mutation Table 4B: λ prophage used and/or generated here . Complementary prophage strains to Table 4A, listing the genetic modifications of each prophage strain that was used to generate display phages in this study. Table 4C Oligo name Oligo sequence Purpose XT928 CGGCGTAAACGCCTTATCCGGCCCAGGTTTTGCTATCTATCTCCTGAGTCATTGCTTTTCTTTTTTCACA lamB deletion XT924 AAATGGGCACGGAAATCCGTGCCCCAAAAGAGAAATGGTATATCTCTGATGTAAAGTGAATGATAACGTA fhuA deletion XT992 CTGGCTCAACCGTTATTGCTGAATTTGAATCGCTGGGTCAGTGCATTCACCACCACAAGCAGATAATAAC att80 deletion XT957 ATTGCGGTTATTTTAATAAAATTAAGGGTTACTATTGAGTCATAGAACTTCCATTATTCTCCTGAAGATA λ ea59 ea31 b deletion XT934 ACTCCGTGCCGCCGGACTGCGTGATGTTATTCT CCT TACTGCGGCCCATATCCACCTCAACCGGATCGAAGGC λ gene E mutation XT914 CGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATAGGAGGATGAAACGatgacgagcaaagaaacctttacc ara P BAD λ gene D XT915 TGAGTATAGCCTGGTTTCGTTTGATTGGCTGTGGTTTTATACAGTCATTAaacgatgctgattgccgttccg ara P BAD λ gen e D XT1021 CAGCCAATCAAACGAAACCAGGCTATACTCAAGCCTGGTTTTTTGATGGAgttccggatctgcatcgcag ara P BAD λD retrieval XT1022 TGCAGAATCCCTGCTTCGTCCATTTGACAGGCACATTATGCAAGCATTGCtgtgataaactaccgcattaaagc ara P BAD λD retrieval XT1115 GCCGCACAGGCGGCCTTTAGTGATGAAGGGTAAAGCCCTTACACTGGTGTGTTCAGCAAATCGTTAACGG Homology arms for λD deletion XT918 attcttgcggttgctgctgac Primer to check λD C-terminal fusion gene. XT919 ataacctcaccggaaacaatcg Primer to check λD C-terminal fusion gene. XT1039 cgccgcattctggccgcagc Primer to check λD N-terminal fusion gene. XT1040 tggtgccatcccacgcaacc Primer to check λD N-terminal fusion gene. XT40 AAGTAATCCAGCGATGCGCC Primer on tet gene XT1116 CCAACAGCACGTCTGAAACGCTTGCTGCAACGCCAAAGGCGGTTAAGGTCggtggcggagggagtTCCTAATTTTTGTTGACACTCTATC Primer used to make XTL1026 stf386_tetA-sacB XT1117 ATTATAAATTTTTATGGTCCGTGGTTGTTCACTCATTCTGAATGCCATTAATCAAAGGGAAAACTGTCCATATGC Primer used to make XTL1026 stf386_tetA-sacB XT1118 GCAATACGTGCAAAAAATTCGGC Primer used to verify XTL1026 stf386_tetA-sacB XT1119 GCCGGAATATCTGGCGGTGC Primer used to verify XTL1026 stf386_tetA-sacB XT1387 GGGGAGTGCGTCAACGGCAT Primer used to check stf fusion genes in strain XTL1226 XT1388 GGTAACGGACCGAGTTCAGAA Primer used to check stf fusion genes in strain XTL1226 Table 4C: Oligonucleotides used to generate bacterial and prophage strains in this study . Designed for the deletion, modification, and addition of genes or to check by PCR and Sanger Sequencing the identity of modified sequences. Additional Declarations There is NO Competing Interest. Supplementary Files XXDisplaySystemSupplementalFigure1.pdf Supplementary Figure 1 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8435997\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Biological Sciences - Article\",\"associatedPublications\":[],\"authors\":[{\"id\":582376684,\"identity\":\"3bed3fe8-daca-48c3-9829-efa54ff748ca\",\"order_by\":0,\"name\":\"Meredith 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legend\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"XXDisplaySystemFigures7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8435997/v1/f3069c2e3acf0476e64e1b86.png\"},{\"id\":104783661,\"identity\":\"aee0f66e-6c41-46e9-a346-1baafd869035\",\"added_by\":\"auto\",\"created_at\":\"2026-03-17 08:03:10\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2559674,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8435997/v1/56896427-ba54-4c29-82a9-a673a66ffd78.pdf\"},{\"id\":103582915,\"identity\":\"47b15ecc-4d86-4cc9-a57a-8ebc077a4d85\",\"added_by\":\"auto\",\"created_at\":\"2026-02-27 10:36:51\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":889271,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplementary Figure 1\",\"description\":\"\",\"filename\":\"XXDisplaySystemSupplementalFigure1.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8435997/v1/86ed1caf52a53b7d20049492.pdf\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"A λ Phage Platform for Successful Therapeutic Display of Protein Antigens\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eBacteriophages have been used since the 1990s to display foreign peptides or proteins on the surface of their virion structures\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. The bacteriophage used here as a platform for display is lambda (λ), an \\u003cem\\u003eEscherichia coli (E. coli)\\u003c/em\\u003e bacteriophage composed of two major structural elements (Fig.\\u0026nbsp;1A): the phage capsid or head, which contains the genome, and the phage tail, which includes proteins for binding the bacterial receptor (LamB) and triggering genome injection\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eWhen λ infects the bacterium \\u003cem\\u003eE. coli\\u003c/em\\u003e, it may take one of two developmental paths. One is the lytic path whereupon λ usurps the host machinery to express functions required to replicate from progeny phage, lyse the cell, and release particles into the environment\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. The alternative path is the lysogenic development following infection where the phage DNA integrates into the bacterial chromosome and produces the phage CI repressor protein to prevent expression of lytic genes. This dual-lifecycle feature allows λ to be genetically manipulated with ease while integrated in the bacterial chromosome and then induced to lytic phase to produce mature engineered phage particles.\\u003c/p\\u003e \\u003cp\\u003eThe λ capsid is composed of two major structural proteins, gpE and gpD (gene product), each present in ~\\u0026thinsp;420 copies per particle\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. In the λ genome, the \\u003cem\\u003eD\\u003c/em\\u003e and \\u003cem\\u003eE\\u003c/em\\u003e genes are adjacent and expressed coordinately, with the gpD (gene product D) protein trimers stabilizing the primary gpE hexameric capsid structure\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e (Fig.\\u0026nbsp;1B). Functional λ particles have been made in which non-phage proteins have been fused to either the N or C terminus of gpD\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e. Both termini protrude away from the head, allowing the display of fusions on the surface of the capsid\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e, as seen in the AlphaFold3 generated λD trimer structure\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e (Fig.\\u0026nbsp;1C). In most previous examples, the gpD-fusion protein was generated by expressing the D-fusion from a plasmid during infection by a λ mutant containing an inactive \\u003cem\\u003eD\\u003c/em\\u003e gene. The D-fusion protein made \\u003cem\\u003ein trans\\u003c/em\\u003e then assembles with the other phage components\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e. Here we created fusions to either terminus of the \\u003cem\\u003eD\\u003c/em\\u003e gene \\u003cem\\u003ein vivo\\u003c/em\\u003e on a λ prophage where the \\u003cem\\u003eD\\u003c/em\\u003e fusion gene is in its normal position adjacent to gene \\u003cem\\u003eE\\u003c/em\\u003e in the phage genome and expressed coordinately with gene \\u003cem\\u003eE\\u003c/em\\u003e (Fig.\\u0026nbsp;2).\\u003c/p\\u003e \\u003cp\\u003eStf of λ that attaches to the end of the phage tail can also be used to display foreign proteins, especially in longer segments than are tolerated by λD. Stf (copy number\\u0026thinsp;=\\u0026thinsp;12) is integral for λ binding to the \\u003cem\\u003eE. coli\\u003c/em\\u003e LamB receptor during infection\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e, however, the Stf protein is non-essential for λ assembly from the prophage\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e, which is the extent of our use here. Thus, we can display foreign proteins on the distal exposed carboxyl end of Stf without interrupting essential processes. Our cloning results indicate that adding protein segments at the Stf C terminus generates high-titer phage lysates for display applications.\\u003c/p\\u003e \\u003cp\\u003eThe ability of λ to display foreign proteins has been utilized in a variety of ways: to create large peptide libraries, to identify antigenic proteins, to investigate ligand-receptor associations, to characterize proteins, to isolate peptide molecules, and to study peptide interactions, along with many other uses \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e,\\u003cspan additionalcitationids=\\\"CR16 CR17 CR18\\\" citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e. The λ platform developed and tested here is an efficient display system for any of these applications. This study provides evidence for this novel display technology to be used specifically as an antigen selection system and as a vector for immune stimulation against specific passenger antigens. While other groups have reported the use of λD display to stimulate antibodies\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR21 CR22 CR23\\\" citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e, our modifications improve the engineering and function of the λ display vector by fusing a foreign peptide to a λ protein in its prophage state. Several mutations have been made to facilitate the ease and quality of fusion phage engineering, prophage induction, and testing in animal studies, which are discussed below:\\u003c/p\\u003e \\u003cp\\u003e \\u003col\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003eThe classical λ \\u003cem\\u003ecI857\\u003c/em\\u003e temperature sensitive mutation in the \\u003cem\\u003ecI\\u003c/em\\u003e repressor gene causes the CI repressor to become inactive at an elevated temperature of 42\\u0026deg; C\\u003csup\\u003e25\\u003c/sup\\u003e. Employing this mutation, a λ \\u003cem\\u003ecI857\\u003c/em\\u003e lysogenic culture growing at 32\\u0026deg; C can be induced to produce phage particles and lyse the cell by simply shifting the culture temperature to 42\\u0026deg; C. This avoids using methods of induction by agents such as mitomycin or UV, which damage DNA.\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003eThe \\u003cem\\u003eSam7\\u003c/em\\u003e mutation was introduced to the prophage to increase phage yield. Normally the time from λ prophage temperature induction to subsequent lysis and phage release is ~\\u0026thinsp;50 minutes, at which time\\u0026thinsp;~\\u0026thinsp;100 particles of progeny phage per cell are released. Lysis is dependent upon the phage \\u003cem\\u003eS\\u003c/em\\u003e gene encoded holin, which generates micron-scale holes in the cytoplasmic membrane after ~\\u0026thinsp;50 minutes of prophage induction\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. These holes allow the endolysin to escape from the cytoplasm and degrade the cell\\u0026rsquo;s peptidoglycan layer, causing complete cell lysis. If the holin protein is inactivated, then cytoplasmic endolysin cannot escape to cause lysis. However, λ continues to replicate within the cell and ultimately produces\\u0026thinsp;~\\u0026thinsp;1000 phage particles per cell. Here we have used the \\u003cem\\u003eSam7\\u003c/em\\u003e mutation in the prophage to prevent holin synthesis\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e. Phage lysis is caused later by the addition of chloroform or freezing and thawing the culture, thereby releasing the increased phage burst size (~\\u0026thinsp;10-fold increase).\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003eWe deleted the 4,285-base-pair \\u003cem\\u003eb2\\u003c/em\\u003e region to increase the λ phage packaging capacity. A λ virion consists of a tail and a 48.5 kb dsDNA genome tightly packaged within an icosahedral protein shell. λ phage DNA lengths that fall outside the optimal packaging range of 38\\u0026ndash;52 kilobase pairs (kb) are generally not incorporated into functional, infectious phage particles\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. The \\u003cem\\u003eb2\\u003c/em\\u003e region is a large, non-essential segment of the genome (approximately 5.7 kb). Deleting it does not prevent the phage from undergoing the lytic cycle. Further, its deletion increases the capacity of the vector, allowing the cloning of larger DNA fragments into the phage genome while keeping the total size within the optimal packaging range of 38\\u0026ndash;52 kb\\u003csup\\u003e30\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003e The \\u003cem\\u003eLamB\\u003c/em\\u003e λ receptor protein encoding gene was deleted in the host chromosome to prevent λ binding and reduced yield after lysis. Initial lysates after chloroform treatment contain bacterial cell debris including isolated external cell surface λ receptors. Receptors present in the cellular debris of the lysate can bind to λ, causing ejection of the genome into the lysate and thereby reducing the number of active phage\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e. We eliminated the latter event of yield reduction by deleting the \\u003cem\\u003elamB\\u003c/em\\u003e gene from the host \\u003cem\\u003eE. coli.\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003cspan\\u003e \\u003cli\\u003e \\u003cp\\u003e5. For half-life extension of the λ phage in the mammalian circulatory system, the major capsid \\u003cem\\u003eE\\u003c/em\\u003e gene has been modified from Glu158 (GAG) to Lys158 (AAG), a single amino acid change. This change enables λ particles to have a longer presence in the mammalian circulatory system, which has the potential to increase immunogenicity\\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/li\\u003e \\u003c/span\\u003e \\u003c/ol\\u003e \\u003c/p\\u003e \\u003cp\\u003eA λ phage-based vaccine is an attractive therapeutic option because it confers several advantages over traditional immune-stimulating platforms. It is inexpensive to develop, and displays can be quickly engineered for countering new or evolving diseases. The phage particles themselves are stable at ambient temperatures, providing convenient shipping and storage of a λ phage display therapeutic\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. The λ phage itself acts as an adjuvant, stimulating the body\\u0026rsquo;s immune response against the high copy-number displayed antigen without requiring an added immune stimulant\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e. With the establishment of a reliable and effective λ platform, a variety of antigen-displaying therapies could be quickly utilized to treat or prevent disease. In this article, we describe the development of our specialized λ phage display vector and the methodology of prophage engineering and induction, as well as show data supporting the use of λ display as an antigen selection system and therapeutic vector platform.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eAfter generating \\u003cem\\u003eE. coli\\u003c/em\\u003e host strains with prophage optimized for phage display in three different constructions, we compared the stability of phage displaying a series of melanoma neo-epitopes in all three constructs. Additionally, we demonstrated the use of the C-terminal \\u0026lambda;D phage display construction for epitope library generation, antigen selection, and immune stimulation in a mouse model against the SARS-CoV-2 spike protein.\\u003c/p\\u003e\\n\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003eConstruct stability by titer\\u003c/h2\\u003e\\n\\u003cp\\u003eFor the purpose of testing which of three melanoma neo-epitope display constructions would best slow the growth of melanoma tumors, we generated thirty phages displaying foreign peptides. Each of ten melanoma antigens were fused to \\u0026lambda; in three display constructs: C-terminal Stf, N-terminal \\u0026lambda;D, and C-terminal \\u0026lambda;D. The differing titers of these three groups of phage lysates, which carry the same set of foreign peptides, illuminate phage stability by fusion construction (Table\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA-C).\\u003c/p\\u003e\\n\\u003cp\\u003eThe melanoma antigens used here originated from the detection of ten distinct mutations identified in mouse melanoma cell line B2905 by whole exome sequencing\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e. To create fusion antigens, each mutation was flanked on either side by 12 amino acids from its local genetic region in cell line B2905. These ten 25-aa sequences containing melanoma mutations were recombineered into the \\u0026lambda; prophage in the three different constructions by counter-selection recombineering described in the methods (Table\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). The fusion phage were then induced, filtered, and the lysates titered on strain XTL1212. All 30 lysates were produced with identical technique. Titering phage samples involved the serial dilution of a phage lysate, plating on a suitable bacterial host (XTL1212), and plaque counting after overnight incubation at 39\\u0026deg; C.\\u003c/p\\u003e\\n\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u0026nbsp;\\u003c/div\\u003e\\n\\u003cp\\u003eStf melanoma fusions produced highest titer phages with all but two samples reaching more than 1x10\\u003csup\\u003e12\\u003c/sup\\u003e pfu/ml. C-terminal \\u0026lambda;D constructions had the second highest stability with most samples titering between 10\\u003csup\\u003e10\\u003c/sup\\u003e and 10\\u003csup\\u003e11\\u003c/sup\\u003e pfu/ml. N-terminal lysates had the lowest average titers among the three constructions with most samples achieving 10\\u003csup\\u003e9\\u003c/sup\\u003e and 10\\u003csup\\u003e10\\u003c/sup\\u003epfu/ml. The N-terminal group also yielded the lowest titer among the 30 fusion phage produced with the Top2A epitope phage lysate titering at only 2x10\\u003csup\\u003e8\\u003c/sup\\u003e pfu/ml. While titer among constructions may vary with the identity and length of the fusion antigen, these trends can be considered when choosing a construct for other diseases and display applications.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003e\\u0026lambda;D antigen detection\\u003c/h3\\u003e\\n\\u003cp\\u003eHere we describe the utility of the \\u0026lambda; system using disease target COVID-19. To demonstrate the use of phage \\u0026lambda; as an antigen selection tool, we chose to define an antigenic region of the SARS-CoV-2 spike protein through generating spike protein epitope \\u0026lambda; displays. SARS-CoV-2, the coronavirus that causes the disease COVID-19 (Coronavirus disease 2019), has a dual subunit spike protein of 1,273 amino acids\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e. This surface glycoprotein functions to make contact with the host cell surface receptor ACE2 (Angiotensin-converting enzyme 2) and complete viral fusion to invade the cell\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e. Subunit 1 is primarily responsible for binding the human ACE2 cell-surface receptor through the spike protein\\u0026rsquo;s receptor binding domain (RBD), while subunit 2 encodes proteins to accomplish the viral fusion process by which the virus gains access to the human cell\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e,\\u003cspan class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e. The spike protein is the primary protein to interact with human cells, and therefore, is an ideal target for identifying a useful antigen against COVID-19.\\u003c/p\\u003e\\n\\u003cp\\u003eOur goal was to define an antigenic region of the spike protein small enough to create a stable, high-titer \\u0026lambda;\\u0026thinsp;~\\u0026thinsp;spike protein display to use in a mouse immunogenicity study. The process by which we defined a suitable antigen was to create a series of overlapping spike protein epitopes up to 133 amino acids in length, fuse the peptides to \\u0026lambda; through C-terminal \\u0026lambda;D display, and screen the display phages with human serum post SARS-CoV-2 infection. By western blotting, we were able to define a region of the spike protein with affinity for antibodies raised by natural SARS-CoV-2 infection. Within this larger antigenic region, we also defined a small epitope of strongest antigenicity and tested a phage displaying it in a mouse antibody-generation study.\\u003c/p\\u003e\\n\\u003cp\\u003eTo use \\u0026lambda; as a detection system for a SARS-CoV-2 spike protein epitope, we first segmented the spike protein gene into 12 overlapping epitopes of ~\\u0026thinsp;133 amino acids per segment and a few smaller epitopes (Table\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e and Fig.\\u0026nbsp;3A). Using the methodology of \\u0026lambda; prophage recombineering, we fused these genetic epitopes to \\u0026lambda;D in the C-terminal construction and raised a lysate of each distinct fusion phage. This created a \\u0026lambda; display library of the spike protein, which was confirmed through blotting these phage samples and the vector phage against an anti-\\u0026lambda;D antibody (Fig.\\u0026nbsp;3B). Wild-type \\u0026lambda;D has a molecular weight of 11.4\\u003csup\\u003e8\\u003c/sup\\u003e kDa, seen in lane 1 (1026) in both Fig.\\u0026nbsp;3B blots. Fusions to \\u0026lambda;D increase the overall size of the protein, raising the location of the band on the western blot to reflect the higher kDa weight of the fusion protein. Therefore, \\u0026lambda;D carrying fusions of various sizes appears as a higher band than wild-type \\u0026lambda;D in all lanes besides the vector, which contains unmodified \\u0026lambda;D protein only.\\u003c/p\\u003e\\n\\u003cdiv class=\\\"gridtable\\\"\\u003e\\n\\u003cdiv class=\\\"colspec\\\" align=\\\"left\\\"\\u003eAlso included in Fig.\\u0026nbsp;3B are phages displaying an epitope from the SARS-CoV-2 E protein (XTL1324) and two epitopes from the SARS-CoV-2 N protein (XTL1293 and XTL1294). These epitope candidates were not useful antigens and were not investigated further. The rest of the antigen investigation narrowed to only the spike protein epitopes.\\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003eThis library of spike protein phage-displayed epitopes was then blotted against human serum collected after a SARS-CoV-2 infection (Fig.\\u0026nbsp;3C). Only phage strain XTL1322, displaying a 133 aa peptide from the second subunit of the spike protein, reacted with COVID+ serum. This epitope consists of spike protein amino acids 1,038\\u0026thinsp;\\u0026minus;\\u0026thinsp;1,170, which includes the subunit 2 connector domain (CD), open reading frame 1069\\u0026ndash;1162, and the first 7 amino acids of heptad repeat 2 (HR2). Both the CD and HR2 regions assist in viral fusion to the mammalian cell membrane after the spike protein receptor binding domain contacts the ACE2 cell surface receptor\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003eFusion protein XTL1322 created a low titer lysate, likely due to decreased stability because of the large size of the 133 aa fusion protein. To construct a more stable phage, epitope XTL1322 was further dissected into overlapping DNA sequences encoding smaller peptide fragments (Fig.\\u0026nbsp;3A). These DNA fragments were recombined in-frame at the 3\\u0026rsquo; end of the \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene as described previously. While many smaller XTL1322 epitopes were tested against human COVID+ serum, only XTL1379 and XTL1380 reacted. These two epitopes were further probed against serum samples from three COVID+ patients and three healthy donors (Fig.\\u0026nbsp;3D). All COVID+ sera reacted with the phage-displayed epitopes, while the healthy sera did not react with any of the phage samples.\\u003c/p\\u003e\\n\\u003cp\\u003eThe peptides displayed by these two phage strains overlap by 11 amino acids (Fig.\\u0026nbsp;3E). Upon sequence alignment with spike proteins of different variants such as alpha, beta, delta, omicron, as well as SARS-CoV-1, we found that this 11 amino acid overlapping segment between XTL1379 and XTL1380 was conserved among all other variants of SARS-CoV. We proceeded to reengineer a new hybrid strain XTL1382 that displayed the overlapping 11 amino acid peptide flanked by the consecutive 10 amino acids at either end of the peptide. When equal plaque forming units of \\u0026lambda; displaying XTL1382, 1380, and 1379 were again probed with COVID+ serum (COVI-SP-0009) (Fig.\\u0026nbsp;3E), we found that the newly engineered hybrid strain XTL1382 strongly reacted with the antibody present in the serum as indicated by the band intensity in Fig.\\u0026nbsp;3E, left. Figure\\u0026nbsp;3E, right, shows the same phage samples blotted against an anti-\\u0026lambda;D antibody, demonstrating correct fusion identity of display phages compared to the vector phage.\\u003c/p\\u003e\\n\\u003cp\\u003eWhile any of the three small displayed epitopes (XTL1382, XTL1380, and XTL1379) were strong candidates for use in a mouse immunogenicity study, we chose to test phage displaying XTL1382 since it gave the most robust signal against COVID+ serum antibodies.\\u003c/p\\u003e\\n\\u003ch3\\u003eMouse immunogenicity study with \\u0026lambda;D display phage\\u003c/h3\\u003e\\n\\u003cp\\u003eXTL1382 \\u0026lambda;D epitope fusion phage display lysate was tested in a mouse study for the phage\\u0026rsquo;s ability to stimulate antibodies against the displayed spike epitope. Groups of mice were immunized with fusion phage and vector phage three times over the course of the 49-day study (Fig.\\u0026nbsp;4A). Post-injection serum samples were tested for antigen-specific antibody production through both ELISA and western blotting.\\u003c/p\\u003e\\n\\u003cp\\u003eSera from mice injected with XTL1382 fusion phage demonstrated high XTL1382-specific antibody titers at 100x dilution two weeks after initial vaccination (Fig.\\u0026nbsp;4B). Titer increased further after Day 13 injection but dipped between the second and third injection on Day 34. However, upon re-injection on Day 34, titers rose again to a high level on the final bleed day. Pre-bleed antibody titers as well as all vector phage-injected sera samples remained at a background absorbance level, indicating the specificity of antibody response detected in the experimental serum group.\\u003c/p\\u003e\\n\\u003cp\\u003eWestern blots confirmed that phage displaying the XTL1382 antigen created XTL1382-specific antibodies in mice (Fig.\\u0026nbsp;4C). When vector (lane 1) and XTL1382 phage (lane 2) were blotted with serum from a mouse that received the XTL1382 injection (left), the XTL1382 phage lane showed a strong band at the expected \\u0026lambda;D fusion protein region of ~\\u0026thinsp;15 kDa. The vector phage lane was blank, indicating no protein binding with mouse serum antibodies. As a control, both phage samples were blotted against serum from a mouse injected with vector phage (right). No bands appeared in either phage lane, indicating a lack of antibody presence specific to either the vector or XTL1382 fusion phage \\u0026lambda;D protein.\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was a confirmation of a previous mouse study that likewise injected XTL1382 display phage into a group of mice (N\\u0026thinsp;=\\u0026thinsp;3). That study found similar antibody levels in response to phage display injection and yielded the same western blot results confirming presence of XTL1382-specific antibodies (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eA-C). These two antibody-detection methods, ELISA and western blotting, show XTL1382-specific antibody production in mice from an injection of \\u0026lambda; displaying the epitope, demonstrating the use of the \\u0026lambda; vector for immune-stimulation.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe production of an optimized \\u0026lambda; phage vector for antigen detection and immune stimulation involved the evolution of strain SJ_XTL175 by several steps of genetic recombineering through the \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette counter-selection method described in materials and methods\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e. Our system uniquely utilizes this technique to make a direct gene fusion in the prophage chromosome. The mutations to the platform maximize phage titer, increase particle stability, and expedite the engineering process. Additionally, our methodology streamlines prophage induction through the heat-inducible genetic switch and purifies the samples through tangential flow filtration. With ready-to-modify strains for any of the three fusion constructions, a stable lysate of a phage displaying a foreign protein can be made in a week\\u0026rsquo;s time. This simple and rapid methodology allows for the use of \\u0026lambda; phage display for many applications.\\u003c/p\\u003e\\n\\u003cp\\u003eThe display platform is suitable for foreign protein display in three constructions: the C or N terminus of the \\u0026lambda;D capsid protein or the C terminus of the Stf protein. We have shown that protein fusions to any of these locations can produce phage display lysates. When creating a library of phage displaying melanoma antigens, the Stf fusion constructions yielded the highest titer phage lysates, followed by the C-terminal \\u0026lambda;D fusion constructions (Table\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). N-terminal \\u0026lambda;D fusions yielded the lowest virion stability and titer among the three fusion construction options, which may limit its use. A possible structural explanation for this stability difference is that the first 14 N terminus residues of \\u0026lambda;D were shown through crystal structure to be disordered and positioned near the three-fold \\u0026lambda;D trimer axis\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. Conversely, C-terminal residues had a stable confirmation and protruded from the protein away from the trimer axis. The N terminus\\u0026rsquo; high flexibility, lack of consistent conformation, and position near an essential assembly point may contribute to particle instability when fusing to this location. N-terminal fusion lower titer may be potentially mitigated by inducing a supply of helper wild-type \\u0026lambda;D from the host chromosome through arabinose addition during phage assembly (Fig.\\u0026nbsp;6). Additionally, this titer stability trend may differ when fusing other peptides from those tested here. While Stf fusion constructs yielded the highest titer phage lysates, \\u0026lambda;D fusions package 35x more antigens per phage particle. This difference in protein copy number per phage particle (~\\u0026thinsp;420 for \\u0026lambda;D vs. 12 for Stf) may make \\u0026lambda;D fusions, particularly in the C-terminal construction, more attractive for certain applications where a high copy number is desired.\\u003c/p\\u003e\\n\\u003cdiv class=\\\"gridtable\\\"\\u003e\\n\\u003cdiv class=\\\"colspec\\\" align=\\\"left\\\"\\u003eHere, we created a displayed spike protein library to detect segments of the SARS-CoV-2 spike protein which had affinity for human COVID+ serum antibodies. Through the process of library generation and antigenicity testing, we were able to detect a 133 aa region of the spike protein, XTL1322, which consistently reacted against human COVID+ serum (Fig.\\u0026nbsp;3). This technique of generating a foreign peptide display library and running antibody-protein affinity tests, also called biopanning, could be easily replicated with the \\u0026lambda; system to identify proteins of interest for a variety of diseases. Additionally, as shown here by the selection of small epitope XTL1382 within the larger XTL1322 epitope, the \\u0026lambda; system can also define specific regions of high antigenicity within larger proteins. Initially, we selected overlapping epitopes XTL1379 and XTL1380 for their antibody affinity observed on COVID+ serum western blots. Upon investigation of the 11aa overlapping sequence between the two and discovering that this was a highly conserved coronavirus region, we created an epitope centering this sequence with flanking arms. The resulting epitope had the strongest affinity for COVID+ serum antibodies. This method of testing overlapping protein segments could be useful for defining regions of high antigenicity in other disease targets.\\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003eTo demonstrate \\u0026lambda;\\u0026rsquo;s ability to stimulate a strong and specific antibody response against a selected antigen, we performed a mouse study, injecting the same display phage used to detect spike protein antigen XTL1382 along with vector phage into groups of mice (Fig.\\u0026nbsp;4). Antibody levels detected by ELISA and western blotting from study sera indicate that the \\u0026lambda;-displayed antigen was able to stimulate a rapid, strong, and specific antibody titer against the XTL1382 peptide. Importantly, this immune-stimulating effect from \\u0026lambda; display injections was achieved without the use of any additional adjuvant.\\u003c/p\\u003e\\n\\u003cp\\u003eThe COVID-19 antigenicity study described here is being further investigated by testing the mouse antibodies created by these and other spike protein antigens for their neutralizing ability against SARS-CoV-2 infection. Through several ongoing studies fusing other disease therapeutics to the three fusion locations, we are continuing to investigate the best engineering construction and mouse study conditions of the \\u0026lambda; phage display platform, including exploring applications of Stf display, comparing N- and C-terminal \\u0026lambda;D fusion, testing linkers of differing lengths, and determining the most efficacious routes of injection.\\u003c/p\\u003e\\n\\u003cp\\u003eThe three uses reported here: library generation, antigen detection, and immune stimulation, are only a few of the possible applications of this \\u0026lambda; display technology. With the ease, timeliness, and low cost of this antigen display method, the potential uses reach as far as display and vector delivery needs expand.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003ch3\\u003e\\u003cstrong\\u003eRecombineering\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eAll genetic changes made to the host strain (Table 4A) and its prophage \\u0026lambda; (Table 4B) were made by the same general method of recombineering\\u003csup\\u003e41\\u003c/sup\\u003e. All primers used to make these genetic changes and check accuracy are reported in Table 4C. For all recombineered strains, a \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette was inserted at the site on the bacterial chromosome or prophage genome to be deleted or altered by selecting for tetracycline resistance and ensuring that the drug-resistant recombinants had also become sensitive to sucrose because of the \\u003cem\\u003esacB\\u003c/em\\u003e gene (Fig. 5). Using recombineering technology, the \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette is replaced using PCR fragments, gene blocks, or single strand oligos containing the coding sequence of the antigenic genes to be fused to \\u0026lambda;\\u003cem\\u003eD\\u003c/em\\u003eor \\u003cem\\u003estf\\u003c/em\\u003e. The single strand DNA oligonucleotide is flanked by the homologous sequences to target the chromosome or prophage genome for recombination. These sucrose-resistant recombinants are tetracycline sensitive and are engineered to fuse the antigenic gene to the \\u003cem\\u003eD\\u003c/em\\u003e or \\u003cem\\u003estf\\u0026nbsp;\\u003c/em\\u003egene in the same reading frame. When placed at the\\u0026nbsp;\\u003cem\\u003e\\u0026lambda;\\u003c/em\\u003e\\u003cem\\u003eD\\u003c/em\\u003e N terminus, the fusion DNA has an upstream 5\\u0026rsquo; ATG start codon, and when placed at the \\u003cem\\u003e\\u0026lambda;\\u003c/em\\u003e\\u003cem\\u003eD\\u003c/em\\u003e C terminus, the fusion DNA has a downstream 3\\u0026rsquo; TAA stop codon. Additional DNA coding sequences are included to generate a flexible linker sequence between the \\u0026lambda;D or Stf protein and the foreign protein from one to three repeats of the amino acid sequence GGGGS\\u003csup\\u003e42\\u003c/sup\\u003e. Once fusions are generated, the \\u0026lambda;\\u003cem\\u003eD\\u003c/em\\u003e-fusion or \\u003cem\\u003estf-\\u003c/em\\u003efusion segments are amplified by PCR and sequenced to verify the accuracy of the new gene fusions.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;strain evolution\\u0026nbsp;\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u0026nbsp;The details of strain construction and constructs are adapted from Thomason et al., 2023\\u003c/em\\u003e\\u003cem\\u003e\\u003csup\\u003e43\\u003c/sup\\u003e\\u003c/em\\u003e\\u003cem\\u003e.\\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eSJ_\\u0026Chi;\\u0026Tau;L175\\u003c/u\\u003e\\u003c/em\\u003e: Strain SJ_XTL175 was used as the starting strain for the construction of the host bacterium used here. SJ_XTL175 is a wild-type MG1655 derivative\\u003csup\\u003e44\\u003c/sup\\u003e that contains the following genotype: \\u0026Delta;\\u003cem\\u003elacI,\\u003c/em\\u003e \\u003cem\\u003elacY\\u003c/em\\u003e\\u003csub\\u003eA177C\\u003c/sub\\u003e , \\u0026Delta;\\u003cem\\u003earaE,\\u003c/em\\u003e \\u0026Delta;\\u003cem\\u003earaFGH\\u003c/em\\u003e::\\u003cem\\u003espec\\u003csup\\u003eR\\u003c/sup\\u003e\\u003c/em\\u003e, \\u0026Delta;\\u003cem\\u003earaD\\u003c/em\\u003e::\\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB\\u003c/em\\u003e-\\u003cem\\u003eamp\\u003c/em\\u003e. SJ_XTL175 was further modified by recombineering to replace the arabinose operon genes \\u003cem\\u003earaB\\u003c/em\\u003e \\u003cem\\u003earaA\\u003c/em\\u003e \\u003cem\\u003earaD\\u003c/em\\u003e with gene \\u003cem\\u003eD\\u003c/em\\u003e of \\u0026lambda;. The \\u003cem\\u003earaC\\u003c/em\\u003e gene remains intact, allowing \\u003cem\\u003eAraC\\u003c/em\\u003e regulation of the \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene (Fig. 6). In this construct, \\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB\\u003c/em\\u003e-\\u003cem\\u003eamp\\u003c/em\\u003e DNA is inserted after \\u003cem\\u003earaA\\u0026nbsp;\\u003c/em\\u003eto create a counter-selection mechanism (Fig. 6B). Then, from the start codon of \\u003cem\\u003earaB\\u003c/em\\u003e, including \\u003cem\\u003earaA\\u003c/em\\u003e and the insert, to the \\u003cem\\u003earaD\\u003c/em\\u003e TAA stop codon is replaced with gene \\u003cem\\u003eD\\u003c/em\\u003e of \\u0026lambda; (Fig. 6C). This places \\u003cem\\u003eD\\u003c/em\\u003e of \\u0026lambda; under \\u003cem\\u003eAraC\\u003c/em\\u003e regulation and arabinose-inducible control. The other mutations (\\u0026Delta;\\u003cem\\u003elacI,\\u003c/em\\u003e \\u003cem\\u003elacY\\u003c/em\\u003e\\u003csub\\u003eA177C\\u003c/sub\\u003e , \\u0026Delta;\\u003cem\\u003earaE,\\u003c/em\\u003e \\u0026Delta;\\u003cem\\u003earaFGH\\u003c/em\\u003e::specR) already present in SJ_XTL175 confer on this new strain (XTL981) the ability to tune the \\u0026lambda;D-protein expression level in every cell in response to the added extracellular arabinose concentration\\u003csup\\u003e44\\u003c/sup\\u003e. A mixture of wild-type \\u0026lambda;D and fusion \\u0026lambda;D has been used before to stabilize \\u0026lambda; display particles\\u003csup\\u003e45\\u003c/sup\\u003e, but it has not been used previously in a well-controlled system like the tunable arabinose method used here. In our system, wild-type \\u0026lambda;D protein expression can be turned on at a precise time and directly tuned to the concentration of arabinose added. This allows for a stable, high-titer phage yield from each lysis, which is especially necessary to support the stability of the phage particle when fusing larger display proteins\\u003csup\\u003e46\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eXTL981:\\u003c/u\\u003e\\u003c/em\\u003eStrain XTL981 described above has been further engineered to optimize \\u0026lambda; phage production and the generation of \\u0026lambda; particles carrying D-fusion proteins. First, XTL981 was infected with \\u0026lambda;\\u003cem\\u003ecI857\\u003c/em\\u003e \\u003cem\\u003eSam7\\u0026nbsp;\\u003c/em\\u003ephage to generate lysogens, and standard techniques were used to identify a lysogen in which a single phage genome was integrated into its native attachment site (\\u003cem\\u003eattB\\u003c/em\\u003e) on the bacterial chromosome\\u003csup\\u003e47\\u003c/sup\\u003e. Next, additional changes were made to the bacterial chromosome and to the prophage \\u0026lambda;\\u003cem\\u003ecI857\\u003c/em\\u003e \\u003cem\\u003eSam7\\u003c/em\\u003e genome using recombineering technologies. As mentioned above, the host \\u003cem\\u003elamB\\u003c/em\\u003e gene was deleted. The \\u003cem\\u003efhuA\\u003c/em\\u003e gene, encoding the phage T1 and phage \\u0026phi;80 receptor protein, was deleted to avoid contamination by T1 and \\u0026phi;80 bacteriophage\\u003csup\\u003e48\\u003c/sup\\u003e. The nonessential \\u0026lambda; genes \\u003cem\\u003eea59\\u003c/em\\u003e and \\u003cem\\u003eea31\\u003c/em\\u003e were precisely removed in a DNA segment of 4,285bp from the \\u003cem\\u003eb\\u003c/em\\u003e region of the \\u0026lambda; prophage\\u003csup\\u003e49\\u003c/sup\\u003e. This deletion provides more room in the phage genome for cloning and encapsidation of DNA segments to be fused to the \\u003cem\\u003eD\\u003c/em\\u003e gene.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eXTL1026:\\u003c/u\\u003e\\u003c/em\\u003e XTL1026 is the strain containing all the modifications to the host and the \\u0026lambda; prophage that are described above. This strain also contains plasmid pKM208, which is a low-copy pSC101-based plasmid that expresses Red functions Exo Bet and Gam from the \\u003cem\\u003elac\\u003c/em\\u003e promoter for recombineering\\u003csup\\u003e50\\u003c/sup\\u003e. The \\u003cem\\u003eD\\u0026nbsp;\\u003c/em\\u003egene in the \\u0026lambda; prophage is the target for creating \\u003cem\\u003eD\\u003c/em\\u003e fusions to express conjugated proteins joined at either the N or C terminus of \\u0026lambda;D. Two strains are derived from XTL1026 in which the \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene is modified for generating \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene fusions. The counter-selectable \\u003cem\\u003etetA-sacB\\u003c/em\\u003e marker cassette is inserted just upstream or downstream of the \\u003cem\\u003eD\\u003c/em\\u003e gene in the prophage by selecting for tetracycline resistance. These two Tet\\u003csup\\u003eR\\u003c/sup\\u003e prophage strains are respectively, XTL1059 and XTL1048. Both become sucrose sensitive because of the \\u003cem\\u003esacB\\u003c/em\\u003e gene function linked to the \\u003cem\\u003etetA\\u003c/em\\u003e marker, which can then be replaced with the sequence of the fusion peptide. Strain XTL1048 was used to create the C-terminal SARS-CoV-2 spike protein \\u0026lambda;D fusion phage\\u0026nbsp;described in the results.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eXTL1226:\\u003c/u\\u003e\\u003c/em\\u003e This strain was created from XTL1026 to allow for the addition of foreign peptides to the C terminus of \\u0026lambda;᾽s Stf protein. Initially, PCR was performed on the T-SACK strain\\u003csup\\u003e41\\u003c/sup\\u003e using primers XT1116 and XT1117 (Table 4C) to generate the \\u003cem\\u003estf386-tetA-sacB\\u003c/em\\u003e cassette. The recombination of this cassette into the prophage strain inserts the \\u003cem\\u003etetA-sacB\\u0026nbsp;\\u003c/em\\u003ecassette into the \\u003cem\\u003estf\\u003c/em\\u003e gene at position 386 and can be verified by PCR using primers XT1118 and XT40 (expected product: 505 bp). Strain XTL1226 is sucrose sensitive due to the addition of the \\u003cem\\u003estf386-tetA-sacB\\u003c/em\\u003e cassette.\\u003c/p\\u003e\\n\\u003cp\\u003eThis strain is then able to recombine with foreign peptides. When replacing the \\u003cem\\u003estf386-tetA-sacB\\u003c/em\\u003e cassette with a foreign peptide, we use a gene block that truncates the rest of the \\u003cem\\u003estf\\u003c/em\\u003e gene from its 774 amino acid wild-type length to 386 amino acids, placing the fusion peptide at the new \\u003cem\\u003estf\\u0026nbsp;\\u003c/em\\u003eC terminus. This truncation slightly improves phage titer and was performed to ensure fusion peptide stability to a smaller Stf fusion protein. Full-length Stf is non-essential to phage assembly, lysis, or protein display, so it can be truncated for optimal fusion without disrupting functions necessary to protein display. XTL1226 was used to produce Stf melanoma epitope prophage by the insertion of melanoma epitopes with flaked \\u0026lambda; homology at the \\u003cem\\u003estf386-tetA-sacB\\u003c/em\\u003e cassette location\\u0026nbsp;(Table 1 and Table 2). The prophages were screened using primers XT1118 and XT1119 (expected product: 436 bp). Identity of melanoma epitopes were further confirmed by Sanger sequencing.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eXTL1439\\u003c/u\\u003e\\u003c/em\\u003e\\u003cem\\u003e:\\u003c/em\\u003e This strain completes the truncation of Stf without placing a fusion peptide at the Stf location. Strain XTL1226 is recombineered with a gene block to replace the \\u003cem\\u003estf386-tetA-sacB\\u003c/em\\u003e cassette with a stop codon, truncating Stf. A new \\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB\\u0026nbsp;\\u003c/em\\u003ecassette is then inserted at the C or N terminus of this strain\\u0026rsquo;s \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene to create strains XTL1441 and XTL1442. These two strains were used to then make melanoma epitope \\u0026lambda;D display phages in a strain with a truncated Stf to create an identical genetic background for comparison with the Stf melanoma epitope display phages.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003eProphage induction procedure\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eOnce foreign proteins have been genetically fused to the \\u003cem\\u003eD\\u003c/em\\u003e or \\u003cem\\u003estf\\u0026nbsp;\\u003c/em\\u003egene of the \\u0026lambda; lysogen, the prophage can be induced to create a bacterial lysate which may be further purified and used in animal studies. To induce prophage, the \\u003cem\\u003eE. coli\\u003c/em\\u003e strain containing the fusion prophage is grown at 32\\u0026deg; C in LB broth to 2x10\\u003csup\\u003e8\\u003c/sup\\u003e cell/ml before quickly shifting to 42\\u0026deg; C for 30 min and back to 39\\u0026deg; C for 3 hrs (Fig. 7). Additionally, 0.2% total arabinose can be added to induce host chromosomewild-type \\u0026lambda;D to mitigate potential phage particle instability caused by the fusion (Fig. 6). Due to the \\u003cem\\u003eSam7\\u003c/em\\u003e mutation that inactivates the phage holin protein, a lysate of the induced phage can be prepared by at least two methods. The first is the addition of chloroform, and the second is spinning the cells down into a pellet, resuspending in a small volume, and freezing overnight at -80\\u0026deg; C. After freezing, the resuspended pellets are thawed to lyse the cells. The resulting crude lysate is then spun down to form a pellet and the supernatant filtered through 0.45 and 0.2 micron filters to collect the clear phage lysate. Freeze/thaw was the method used for lysis in the studies described here. Whether made through freeze/thaw or chloroform addition, the lysate is then titered on a \\u003cem\\u003esupF\\u003c/em\\u003e strain (XTL1212) which suppresses the \\u003cem\\u003eSam7\\u003c/em\\u003e mutation to make gpS and allow lysis and plaque formation. Strain XTL1212 also contains the plasmid pLXT42, which produces \\u0026lambda;D protein to complement D-fusion phage particles that may be somewhat defective in D function and cannot form plaques efficiently on their own. The \\u0026lambda;D fusion identity can then be confirmed by western blot and DNA sequencing before the phage lysates are purified by tangential flow filtration.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003eTangential flow filtration\\u0026nbsp;\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eEngineered phage lysates were filtered and their titer concentrated by passage through a Pellicon\\u0026reg; Mini Cassette 0.1 mm Composite Regenerated Cellulose filter by Millipore. The filter retains the phage particles and allows bacterial waste to pass through and be discarded. Filtration occurs by running 5-7 liters of 10 mM Tris and 50mM MgCl₂ buffer\\u003csup\\u003e33\\u003c/sup\\u003e through the filter system to purify phage. Phage are resuspended after filtration in a small volume (5-15 ml) of the same buffer. The MgCl₂ stabilizes phage particles and allows for long-term storage. This buffer can be diluted in PBS before injection into mice to reduce MgCl₂. Endotoxin readings were taken after filtration using the Endosafe\\u0026reg; Nextgen-PTS\\u0026trade; (Portable Test System) by Charles River to ensure that the lysate was below an acceptable level of toxin for animal injection.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003eMouse study protocol\\u0026nbsp;\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;The mouse study conducted to test the immunogenicity of XTL1382 display phage utilized six- to eight-week-old Balb/c female mice. Groups of five mice were injected with antigen fusion phage, vector phage, and 10 mM Tris and 10mM MgCl₂ buffer. Mice were injected intraperitoneally with 500 \\u0026micro;l doses of 2x10⁹ pfu three times over the course of the 49-day study, on days 0, 13, and 34. Bleeds were taken at five time points on days 0, 13, 27, 34, and terminally on day 49. This study was performed under Integrated Biotherapeutics\\u0026rsquo; (Rockville, MD) approved IACUC protocol #160805.\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003eAnalysis: ELISA and western blot\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eWestern blot\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003col\\u003e\\n \\u003cli\\u003eWestern Blot identification of SARS-CoV-2 spike protein epitope using SARS-CoV-2 infected and healthy donor human serum:\\u003c/li\\u003e\\n\\u003c/ol\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;To identify antibody-protein affinity between spike antigen display phage and human serum samples, we ran phage samples on 4-20% SDS gradient gels. The samples were then transferred to a nitrocellulose membrane for standard western blotting. First the membrane was blocked with 2.5% non-fat milk buffer, then it was triple washed with TNE buffer (10mM Tris-HCl, 50mM NaCl, 2.5mM EDTA) prior to incubating with 1000-fold dilution of human serum samples extracted either from a healthy donor or a patient positive for SARS-CoV-2. The membrane was incubated with serum samples for three hours and then washed with a wash buffer (10mM Tris-HCl, 50mM NaCl, 2.5mM EDTA). It was then incubated with 10,000-fold diluted goat anti-human IgG secondary-HRP conjugated antibody for an hour and washed with a wash buffer containing 0.1% tween. The membrane was developed using a clarity enhanced chemiluminescence substrate from Bio-Rad.\\u003c/p\\u003e\\n\\u003col start=\\\"2\\\"\\u003e\\n \\u003cli\\u003eWestern blot protocol for mouse serum antibody affinity:\\u003c/li\\u003e\\n\\u003c/ol\\u003e\\n\\u003cp\\u003eWestern blots were conducted to quantify mouse sera antibody affinity to phage sample fusion protein. They were performed by loading fusion phage and vector phage on an SDS 4-20% tris-glycine gel, transferring separated proteins to a nitrocellulose membrane, blocking with 5% milk buffer in 1X TNE, and probing with 1:1000 dilution of mouse serum in milk buffer, followed by 1:10,000-fold goat anti-mouse IgG secondary-HRP in 1X TNE. Membranes were developed with Clarity Max Western ECL substrate and imaged with chemiluminescence.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eELISA\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u0026nbsp;ELISA Assay protocol adapted from Hong Zhou (NIH/NCI) and Thermo Fisher\\u0026rsquo;s general ELISA protocol:\\u003c/em\\u003e(https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLCD%2Fmanuals%2FD20692~.pdf)\\u003c/p\\u003e\\n\\u003cp\\u003eMouse serum was analyzed for the presence of antibodies specific to the injected \\u0026lambda;D XTL1382 peptide by ELISA (enzyme-linked immunosorbent assay). ELISA demonstrated antibody-protein binding by incubating a synthetic peptide version of the fusion phage peptide on a nunc-immobilon plate at a 20 \\u0026micro;g/ml concentration. Once the synthetic peptide was immobilized on the plate, mouse serum was added in several 10-fold dilutions. The synthetic protein/antibody binding interaction was quantified by adding TMB substrate, stopping the reaction after 12 min with 17%\\u0026nbsp;H₂SO₄\\u0026nbsp;stop solution, and reading color intensity at 450 nm. Absorbance was read at a 100x dilution. After subtracting the background, collected at 562 nm, data was plotted in GraphPad Prism 10. Error bars were generated in Prism software.\\u003c/p\\u003e\\n\\u003ch1\\u003e\\u003cstrong\\u003eMaterials\\u003c/strong\\u003e\\u003c/h1\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSynthetic peptides created by GenScript Inc.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cul\\u003e\\n \\u003cli\\u003eSpike Protein Epitope XTL1382: YDPLQPELDSFKEELDKYFKNHTSPDVDLGD\\u003c/li\\u003e\\n\\u003c/ul\\u003e\\n\\u003cp\\u003eSynthetic peptide was custom synthesized by GenScript Inc. Peptide sequence is from the second subunit of the SARS-CoV-2 spike protein. The purity of this peptide was above 95%. It was resuspended in sterile H₂O for use in ELISA at 20 \\u0026micro;g/ml in each well for the detection of antibodies.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAntibodies\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cul\\u003e\\n \\u003cli\\u003eAnti-\\u0026lambda;D antibody: Custom-made polyclonal anti-\\u0026lambda;D capsid protein antibody raised in mouse against purified \\u0026lambda;D protein from GenScript Corp.\\u0026nbsp;\\u003c/li\\u003e\\n \\u003cli\\u003eHuman serum used in antigen selection: blood serum samples from three healthy donors and three patients positive for SARS-CoV-2 infection were generously gifted by the laboratory of Robert J. Kreitman at NCI/NIH.\\u0026nbsp;\\u003cul\\u003e\\n \\u003cli\\u003eHealthy Donors:\\u0026nbsp;\\u003cul\\u003e\\n \\u003cli\\u003e\\u0026alpha;-COVI-SP-0005\\u003c/li\\u003e\\n \\u003cli\\u003e\\u0026alpha;-COVI-SP-0007\\u003c/li\\u003e\\n \\u003cli\\u003e\\u0026alpha;-COVI-SP-0008\\u003c/li\\u003e\\n \\u003c/ul\\u003e\\n \\u003c/li\\u003e\\n \\u003cli\\u003eCOVID+ Sera\\u003cul\\u003e\\n \\u003cli\\u003e\\u0026alpha;-COVI-SP-0009\\u003c/li\\u003e\\n \\u003cli\\u003e\\u0026alpha;-COVI-SP-0179\\u003c/li\\u003e\\n \\u003cli\\u003e\\u0026alpha;-COVI-SP-0187\\u003c/li\\u003e\\n \\u003c/ul\\u003e\\n \\u003c/li\\u003e\\n \\u003c/ul\\u003e\\n \\u003c/li\\u003e\\n\\u003c/ul\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003ch2\\u003eConflicts of Interest:\\u003c/h2\\u003e \\u003cp\\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eResulting Patents\\u003c/strong\\u003e \\u003cp\\u003eUS patent WO 2023/114727 A1 was published on 06/22/2023 as an application in the World Intellectual Property Organization detailing the invention of this lambda phage display technology for the display or foreign peptides and proteins.\\u003c/p\\u003e \\u003c/p\\u003e\\u003cp\\u003e \\u003ch2\\u003e \\u003cb\\u003eTable\\u0026nbsp;4A\\u003c/b\\u003e \\u003c/h2\\u003e \\u003cp\\u003e \\u003cb\\u003eE. coli\\u003c/b\\u003e \\u003cb\\u003estrains used and/or generated here.\\u003c/b\\u003e Bacterial strains generated in the evolution of a λ display platform. Table includes the initial parental strain, each new strain generated to modify the bacterial host, each strain containing modifications to λ prophage within the host, and the final strains used to create antigen-displaying phages in this study.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003e \\u003cb\\u003eMethods\\u003c/b\\u003e \\u003c/h2\\u003e \\u003cp\\u003e \\u003cstrong\\u003eRecombineering\\u003c/strong\\u003e \\u003cp\\u003eAll genetic changes made to the host strain (Table\\u0026nbsp;4A) and its prophage λ (Table\\u0026nbsp;4B) were made by the same general method of recombineering\\u003csup\\u003e\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e. All primers used to make these genetic changes and check accuracy are reported in Table\\u0026nbsp;4C. For all recombineered strains, a \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette was inserted at the site on the bacterial chromosome or prophage genome to be deleted or altered by selecting for tetracycline resistance and ensuring that the drug-resistant recombinants had also become sensitive to sucrose because of the \\u003cem\\u003esacB\\u003c/em\\u003e gene (Fig.\\u0026nbsp;5). Using recombineering technology, the \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette is replaced using PCR fragments, gene blocks, or single strand oligos containing the coding sequence of the antigenic genes to be fused to λ\\u003cem\\u003eD\\u003c/em\\u003e or \\u003cem\\u003estf\\u003c/em\\u003e. The single strand DNA oligonucleotide is flanked by the homologous sequences to target the chromosome or prophage genome for recombination. These sucrose-resistant recombinants are tetracycline sensitive and are engineered to fuse the antigenic gene to the \\u003cem\\u003eD\\u003c/em\\u003e or \\u003cem\\u003estf\\u003c/em\\u003e gene in the same reading frame. When placed at the \\u003cem\\u003eλD\\u003c/em\\u003e N terminus, the fusion DNA has an upstream 5\\u0026rsquo; ATG start codon, and when placed at the \\u003cem\\u003eλD\\u003c/em\\u003e C terminus, the fusion DNA has a downstream 3\\u0026rsquo; TAA stop codon. Additional DNA coding sequences are included to generate a flexible linker sequence between the λD or Stf protein and the foreign protein from one to three repeats of the amino acid sequence GGGGS\\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u003c/sup\\u003e. Once fusions are generated, the λ\\u003cem\\u003eD\\u003c/em\\u003e-fusion or \\u003cem\\u003estf-\\u003c/em\\u003efusion segments are amplified by PCR and sequenced to verify the accuracy of the new gene fusions.\\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eE. coli strain evolution\\u003c/strong\\u003e \\u003cp\\u003e \\u003cem\\u003eThe details of strain construction and constructs are adapted from Thomason et al., 2023\\u003c/em\\u003e \\u003csup\\u003e \\u003cem\\u003e43\\u003c/em\\u003e \\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cspan type=\\\"ItalicUnderline\\\" class=\\\"ItalicUnderline\\\" name=\\\"Emphasis\\\"\\u003eSJ_ΧΤL175\\u003c/span\\u003e: Strain SJ_XTL175 was used as the starting strain for the construction of the host bacterium used here. SJ_XTL175 is a wild-type MG1655 derivative\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e that contains the following genotype: Δ\\u003cem\\u003elacI, lacY\\u003c/em\\u003e\\u003csub\\u003eA177C\\u003c/sub\\u003e, Δ\\u003cem\\u003earaE\\u003c/em\\u003e, Δ\\u003cem\\u003earaFGH\\u003c/em\\u003e::\\u003cem\\u003espec\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eR\\u003c/em\\u003e\\u003c/sup\\u003e, Δ\\u003cem\\u003earaD\\u003c/em\\u003e::\\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB\\u003c/em\\u003e-\\u003cem\\u003eamp\\u003c/em\\u003e. SJ_XTL175 was further modified by recombineering to replace the arabinose operon genes \\u003cem\\u003earaB araA araD\\u003c/em\\u003e with gene \\u003cem\\u003eD\\u003c/em\\u003e of λ. The \\u003cem\\u003earaC\\u003c/em\\u003e gene remains intact, allowing \\u003cem\\u003eAraC\\u003c/em\\u003e regulation of the \\u003cem\\u003eλD\\u003c/em\\u003e gene (Fig.\\u0026nbsp;6). In this construct, \\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB\\u003c/em\\u003e-\\u003cem\\u003eamp\\u003c/em\\u003e DNA is inserted after \\u003cem\\u003earaA\\u003c/em\\u003e to create a counter-selection mechanism (Fig.\\u0026nbsp;6B). Then, from the start codon of \\u003cem\\u003earaB\\u003c/em\\u003e, including \\u003cem\\u003earaA\\u003c/em\\u003e and the insert, to the \\u003cem\\u003earaD\\u003c/em\\u003e TAA stop codon is replaced with gene \\u003cem\\u003eD\\u003c/em\\u003e of λ (Fig.\\u0026nbsp;6C). This places \\u003cem\\u003eD\\u003c/em\\u003e of λ under \\u003cem\\u003eAraC\\u003c/em\\u003e regulation and arabinose-inducible control. The other mutations (Δ\\u003cem\\u003elacI, lacY\\u003c/em\\u003e\\u003csub\\u003eA177C\\u003c/sub\\u003e, Δ\\u003cem\\u003earaE\\u003c/em\\u003e, Δ\\u003cem\\u003earaFGH\\u003c/em\\u003e::specR) already present in SJ_XTL175 confer on this new strain (XTL981) the ability to tune the λD-protein expression level in every cell in response to the added extracellular arabinose concentration\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e. A mixture of wild-type λD and fusion λD has been used before to stabilize λ display particles\\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e, but it has not been used previously in a well-controlled system like the tunable arabinose method used here. In our system, wild-type λD protein expression can be turned on at a precise time and directly tuned to the concentration of arabinose added. This allows for a stable, high-titer phage yield from each lysis, which is especially necessary to support the stability of the phage particle when fusing larger display proteins\\u003csup\\u003e\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/p\\u003e\\u003cp\\u003e \\u003ch2\\u003eXTL981\\u003c/h2\\u003e \\u003cp\\u003eStrain XTL981 described above has been further engineered to optimize λ phage production and the generation of λ particles carrying D-fusion proteins. First, XTL981 was infected with λ\\u003cem\\u003ecI857 Sam7\\u003c/em\\u003e phage to generate lysogens, and standard techniques were used to identify a lysogen in which a single phage genome was integrated into its native attachment site (\\u003cem\\u003eattB\\u003c/em\\u003e) on the bacterial chromosome\\u003csup\\u003e\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e\\u003c/sup\\u003e. Next, additional changes were made to the bacterial chromosome and to the prophage λ\\u003cem\\u003ecI857 Sam7\\u003c/em\\u003e genome using recombineering technologies. As mentioned above, the host \\u003cem\\u003elamB\\u003c/em\\u003e gene was deleted. The \\u003cem\\u003efhuA\\u003c/em\\u003e gene, encoding the phage T1 and phage φ80 receptor protein, was deleted to avoid contamination by T1 and φ80 bacteriophage\\u003csup\\u003e\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u003c/sup\\u003e. The nonessential λ genes \\u003cem\\u003eea59\\u003c/em\\u003e and \\u003cem\\u003eea31\\u003c/em\\u003e were precisely removed in a DNA segment of 4,285bp from the \\u003cem\\u003eb\\u003c/em\\u003e region of the λ prophage\\u003csup\\u003e\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u003c/sup\\u003e. This deletion provides more room in the phage genome for cloning and encapsidation of DNA segments to be fused to the \\u003cem\\u003eD\\u003c/em\\u003e gene.\\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eXTL1026\\u003c/strong\\u003e \\u003cp\\u003eXTL1026 is the strain containing all the modifications to the host and the λ prophage that are described above. This strain also contains plasmid pKM208, which is a low-copy pSC101-based plasmid that expresses Red functions Exo Bet and Gam from the \\u003cem\\u003elac\\u003c/em\\u003e promoter for recombineering\\u003csup\\u003e\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e\\u003c/sup\\u003e. The \\u003cem\\u003eD\\u003c/em\\u003e gene in the λ prophage is the target for creating \\u003cem\\u003eD\\u003c/em\\u003e fusions to express conjugated proteins joined at either the N or C terminus of λD. Two strains are derived from XTL1026 in which the \\u003cem\\u003eλD\\u003c/em\\u003e gene is modified for generating \\u003cem\\u003eλD\\u003c/em\\u003e gene fusions. The counter-selectable \\u003cem\\u003etetA-sacB\\u003c/em\\u003e marker cassette is inserted just upstream or downstream of the \\u003cem\\u003eD\\u003c/em\\u003e gene in the prophage by selecting for tetracycline resistance. These two Tet\\u003csup\\u003eR\\u003c/sup\\u003e prophage strains are respectively, XTL1059 and XTL1048. Both become sucrose sensitive because of the \\u003cem\\u003esacB\\u003c/em\\u003e gene function linked to the \\u003cem\\u003etetA\\u003c/em\\u003e marker, which can then be replaced with the sequence of the fusion peptide. Strain XTL1048 was used to create the C-terminal SARS-CoV-2 spike protein λD fusion phage described in the results.\\u003c/p\\u003e \\u003c/p\\u003e\\u003cp\\u003e \\u003ch2\\u003eXTL1439\\u003c/h2\\u003e \\u003cp\\u003eThis strain completes the truncation of Stf without placing a fusion peptide at the Stf location. Strain XTL1226 is recombineered with a gene block to replace the \\u003cem\\u003estf386-tetA-sacB\\u003c/em\\u003e cassette with a stop codon, truncating Stf. A new \\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB\\u003c/em\\u003e cassette is then inserted at the C or N terminus of this strain\\u0026rsquo;s \\u003cem\\u003eλD\\u003c/em\\u003e gene to create strains XTL1441 and XTL1442. These two strains were used to then make melanoma epitope λD display phages in a strain with a truncated Stf to create an identical genetic background for comparison with the Stf melanoma epitope display phages.\\u003c/p\\u003e \\u003c/p\\u003e\\u003cp\\u003e \\u003ch2\\u003eProphage induction procedure\\u003c/h2\\u003e \\u003cp\\u003eOnce foreign proteins have been genetically fused to the \\u003cem\\u003eD\\u003c/em\\u003e or \\u003cem\\u003estf\\u003c/em\\u003e gene of the λ lysogen, the prophage can be induced to create a bacterial lysate which may be further purified and used in animal studies. To induce prophage, the \\u003cem\\u003eE. coli\\u003c/em\\u003e strain containing the fusion prophage is grown at 32\\u0026deg; C in LB broth to 2x10\\u003csup\\u003e8\\u003c/sup\\u003e cell/ml before quickly shifting to 42\\u0026deg; C for 30 min and back to 39\\u0026deg; C for 3 hrs (Fig.\\u0026nbsp;7). Additionally, 0.2% total arabinose can be added to induce host chromosome wild-type λD to mitigate potential phage particle instability caused by the fusion (Fig.\\u0026nbsp;6). Due to the \\u003cem\\u003eSam7\\u003c/em\\u003e mutation that inactivates the phage holin protein, a lysate of the induced phage can be prepared by at least two methods. The first is the addition of chloroform, and the second is spinning the cells down into a pellet, resuspending in a small volume, and freezing overnight at -80\\u0026deg; C. After freezing, the resuspended pellets are thawed to lyse the cells. The resulting crude lysate is then spun down to form a pellet and the supernatant filtered through 0.45 and 0.2 micron filters to collect the clear phage lysate. Freeze/thaw was the method used for lysis in the studies described here. Whether made through freeze/thaw or chloroform addition, the lysate is then titered on a \\u003cem\\u003esupF\\u003c/em\\u003e strain (XTL1212) which suppresses the \\u003cem\\u003eSam7\\u003c/em\\u003e mutation to make gpS and allow lysis and plaque formation. Strain XTL1212 also contains the plasmid pLXT42, which produces λD protein to complement D-fusion phage particles that may be somewhat defective in D function and cannot form plaques efficiently on their own. The λD fusion identity can then be confirmed by western blot and DNA sequencing before the phage lysates are purified by tangential flow filtration.\\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eTangential flow filtration\\u003c/strong\\u003e \\u003cp\\u003eEngineered phage lysates were filtered and their titer concentrated by passage through a Pellicon\\u0026reg; Mini Cassette 0.1 mm Composite Regenerated Cellulose filter by Millipore. The filter retains the phage particles and allows bacterial waste to pass through and be discarded. Filtration occurs by running 5\\u0026ndash;7 liters of 10 mM Tris and 50mM MgCl₂ buffer\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e through the filter system to purify phage. Phage are resuspended after filtration in a small volume (5\\u0026ndash;15 ml) of the same buffer. The MgCl₂ stabilizes phage particles and allows for long-term storage. This buffer can be diluted in PBS before injection into mice to reduce MgCl₂. Endotoxin readings were taken after filtration using the Endosafe\\u0026reg; Nextgen-PTS\\u0026trade; (Portable Test System) by Charles River to ensure that the lysate was below an acceptable level of toxin for animal injection.\\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eMouse study protocol\\u003c/strong\\u003e \\u003cp\\u003eThe mouse study conducted to test the immunogenicity of XTL1382 display phage utilized six- to eight-week-old Balb/c female mice. Groups of five mice were injected with antigen fusion phage, vector phage, and 10 mM Tris and 10mM MgCl₂ buffer. Mice were injected intraperitoneally with 500 \\u0026micro;l doses of 2x10⁹ pfu three times over the course of the 49-day study, on days 0, 13, and 34. Bleeds were taken at five time points on days 0, 13, 27, 34, and terminally on day 49. This study was performed under Integrated Biotherapeutics\\u0026rsquo; (Rockville, MD) approved IACUC protocol #160805.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eAcknowledgment\\u003c/h2\\u003e \\u003cp\\u003eThis research was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute (NCI), and the Center for Cancer Research. (CCR). The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.\\u003c/p\\u003e \\u003cp\\u003eWe would like to thank Hong Zhou for performing ELISA assays and collecting data found in supplemental Fig.\\u0026nbsp;1. We also thank all other members of our lab for their helpful assistance.\\u003c/p\\u003e\\u003ch2\\u003eData Availability Statement:\\u003c/h2\\u003e \\u003cp\\u003eRelevant data are included in the manuscript.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eSternberg, N., Hoess, R.H.: Display of peptides and proteins on the surface of bacteriophage lambda. Proc. Natl. Acad. Sci. U S A. \\u003cb\\u003e92\\u003c/b\\u003e, 1609\\u0026ndash;1613 (1995)\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSmith, G.P., Petrenko, V.A.: Phage Display. Chem. Rev. \\u003cb\\u003e97\\u003c/b\\u003e, 391\\u0026ndash;410 (1997)\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang, C., Zeng, J., Wang, J.: Structural basis of bacteriophage lambda capsid maturation. 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Biol. \\u003cb\\u003e4\\u003c/b\\u003e, 11 (2003)\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eTable 1A \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; Table 1B \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Table 1C\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eStf strain name\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAntigen\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTiter\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026lambda;D C-terminal strain name\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAntigen\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTiter\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026lambda;D N-terminal strain name\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAntigen\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTiter\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1429\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAtr\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e4x10\\u003c/strong\\u003e\\u003csup\\u003e12\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1443\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAtr\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e1.2x10\\u003c/strong\\u003e\\u003csup\\u003e12\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1453\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAtr\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e6x10\\u003c/strong\\u003e\\u003csup\\u003e9\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1430\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eDennd6b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2x10\\u003c/strong\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1444\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eDennd6b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2x10\\u003c/strong\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1454\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eDennd6b\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e8x10\\u003c/strong\\u003e\\u003csup\\u003e10\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1431\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGnaq\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e8x10\\u003c/strong\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1445\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGnaq\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e6x10\\u003c/strong\\u003e\\u003csup\\u003e10\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1455\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGnaq\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e1x10\\u003c/strong\\u003e\\u003csup\\u003e10\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1432\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eKras\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2x10\\u003c/strong\\u003e\\u003csup\\u003e12\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1446\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eKras\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2x10\\u003c/strong\\u003e\\u003csup\\u003e9\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1456\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eKras\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e1.4x10\\u003c/strong\\u003e\\u003csup\\u003e9\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1433\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMctp1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e1.6x10\\u003c/strong\\u003e\\u003csup\\u003e12\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1447\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMctp1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e9x10\\u003c/strong\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1457\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMctp1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e1.4x10\\u003c/strong\\u003e\\u003csup\\u003e9\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1434\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMfn1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e1.2x10\\u003c/strong\\u003e\\u003csup\\u003e12\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1448\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMfn1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e4x10\\u003c/strong\\u003e\\u003csup\\u003e10\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1458\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMfn1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2.4x10\\u003c/strong\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1435\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ent5dc1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e1.2x10\\u003c/strong\\u003e\\u003csup\\u003e12\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1449\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ent5dc1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e4x10\\u003c/strong\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1459\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ent5dc1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2.8x10\\u003c/strong\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1436\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrpf38a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e1.8x10\\u003c/strong\\u003e\\u003csup\\u003e12\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1450\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrpf38a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2x10\\u003c/strong\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1460\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrpf38a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e6x10\\u003c/strong\\u003e\\u003csup\\u003e9\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1437\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTop2a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2x10\\u003c/strong\\u003e\\u003csup\\u003e12\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1451\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTop2a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e4x10\\u003c/strong\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1461\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTop2a\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2x10\\u003c/strong\\u003e\\u003csup\\u003e8\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1438\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMtmr10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2.2x10\\u003c/strong\\u003e\\u003csup\\u003e12\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1452\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMtmr10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2.4x10\\u003c/strong\\u003e\\u003csup\\u003e11\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1462\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMtmr10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e3x10\\u003c/strong\\u003e\\u003csup\\u003e10\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 1:\\u003c/strong\\u003e \\u003cstrong\\u003eTiters of \\u0026lambda; phage lysates displaying melanoma antigens in three engineering constructions: Stf, \\u0026lambda;D C-terminal, and \\u0026lambda;D N-terminal.\\u003c/strong\\u003e Phage titers from each of thirty phage lysates prepared by induction and freeze/thaw lysis. Each phage displays one of ten antigens found in mouse melanoma cell line B2905 in three differing fusion constructions: A: Stf, B: C-terminal \\u0026lambda;D, and C: N-terminal \\u0026lambda;D. Prophage genotypes are listed in Table 2. Titers were determined by plating serial phage lysate dilutions on \\u003cem\\u003esupF\\u003c/em\\u003e strain XTL1212 and counting resulting plaques after overnight incubation.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cbr\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 2\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"619\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eProphage Strain Name\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eStrain Genotype\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eMelanoma Neo-Epitope\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1429\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1226 (stf386~Melanoma Atr-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAtr - RMKEAYTHAQIAKNNELKDTLILTT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1430\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1226 (stf386~Melanoma Dennd6b-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eDennd6b - PPNVVLGVTNPFIIKTLQHWPHVLR\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1431\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1226 (stf386~Melanoma Gnaq-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGnaq - QSVIFRMVDVGGLRSERRKWIHCFE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1432\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1226 (stf386~Melanoma Kras-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eKras - MTEYKLVVVGADGVGKSALTIQLI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1433\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1226 (stf386~Melanoma Mctp1-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMctp1 - MYQLDITLRRGQGLAARDRGGTSDP\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1434\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1226 (stf386~Melanoma Mfn1-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMfn1 - CSDFQEDIVFRFFLGWSSLVHRFLG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1435\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1226 (stf386~Melanoma Nt5dc1-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNt5dc1 - DKPGWYSQGNAALLYELLKKMTSKP\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1436\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1226 (stf386~Melanoma Prpf38a-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrpf38a - GLTAELVVDKAMKLKFVGGVYGGNI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1437\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1226 (stf386~Melanoma Top2a-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTop2a - IAGSTKDVKVFLSGNSLPVKGFRSY\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1438\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1226 (stf386~Melanoma Mtmr10-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMtmr10 - DPYFRTITGFQSLIQKEWVWRAISS (containing single amino acid deletion)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1443\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1441 (\\u0026lambda;D~Melanoma Atr-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAtr - RMKEAYTHAQIAKNNELKDTLILTT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1444\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1441 (\\u0026lambda;D~Melanoma Dennd6b-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eDennd6b - PPNVVLGVTNPFIIKTLQHWPHVLR\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1445\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1441 (\\u0026lambda;D~Melanoma Gnaq-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGnaq - QSVIFRMVDVGGLRSERRKWIHCFE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1446\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1441 (\\u0026lambda;D~Melanoma Kras-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eKras - MTEYKLVVVGADGVGKSALTIQLI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1447\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1441 (\\u0026lambda;D~Melanoma Mctp1-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMctp1 - MYQLDITLRRGQGLAARDRGGTSDP\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1448\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1441 (\\u0026lambda;D~Melanoma Mfn1-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMfn1 - CSDFQEDIVFRFFLGWSSLVHRFLG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1449\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1441 (\\u0026lambda;D~Melanoma Nt5dc1-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNt5dc1 - DKPGWYSQGNAALLYELLKKMTSKP\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1450\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1441 (\\u0026lambda;D~Melanoma Prpf38a-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrpf38a - GLTAELVVDKAMKLKFVGGVYGGNI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1451\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1441 (\\u0026lambda;D~Melanoma Top2a-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTop2a - IAGSTKDVKVFLSGNSLPVKGFRSY\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1452\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1441 (\\u0026lambda;D~Melanoma Mtmr10-neo-epitope)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMtmr10 - DPYFRTITGFQSLIQKEWVWRAISS (containing single amino acid deletion)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1453\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1442 (Melanoma Atr-neo-epitope~\\u0026lambda;D)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAtr - RMKEAYTHAQIAKNNELKDTLILTT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1454\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1442 (Melanoma Dennd6b-neo-epitope~\\u0026lambda;D)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eDennd6b - PPNVVLGVTNPFIIKTLQHWPHVLR\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1455\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1442 (Melanoma Gnaq-neo-epitope~\\u0026lambda;D)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGnaq - QSVIFRMVDVGGLRSERRKWIHCFE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1456\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1442 (Melanoma Kras-neo-epitope~\\u0026lambda;D)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eKras - MTEYKLVVVGADGVGKSALTIQLI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1457\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1442 (Melanoma Mctp1-neo-epitope~\\u0026lambda;D)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMctp1 - MYQLDITLRRGQGLAARDRGGTSDP\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1458\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1442 (Melanoma Mfn1-neo-epitope~\\u0026lambda;D)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMfn1 - CSDFQEDIVFRFFLGWSSLVHRFLG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1459\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1442 (Melanoma Nt5dc1-neo-epitope~\\u0026lambda;D)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNt5dc1 - DKPGWYSQGNAALLYELLKKMTSKP\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1460\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1442 (Melanoma Prpf38a-neo-epitope~\\u0026lambda;D)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrpf38a - GLTAELVVDKAMKLKFVGGVYGGNI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1461\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1442 (Melanoma Top2a-neo-epitope~\\u0026lambda;D)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTop2a - IAGSTKDVKVFLSGNSLPVKGFRSY\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eYXTL1462\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXTL1442 (Melanoma Mtmr10-neo-epitope~\\u0026lambda;D)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMtmr10 - DPYFRTITGFQSLIQKEWVWRAISS (containing single amino acid deletion)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eRed letter in neo-epitope sequence denotes single amino acid mutation found in mouse melanoma cell line B2905\\u003csup\\u003e36\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e* ~ symbol refers to a genetic fusion\\u003c/p\\u003e\\n\\u003cp\\u003eY= temperature sensitive \\u003cem\\u003ecI857\\u003c/em\\u003e mutation\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 2:\\u003c/strong\\u003e \\u003cstrong\\u003eMelanoma prophage strains\\u003c/strong\\u003e. Prophage strains developed to generate phage displaying each of ten melanoma neo-epitopes at three fusion locations: C terminus of Stf, C terminus of \\u0026lambda;D, and the N terminus of \\u0026lambda;D. Parental strains and melanoma epitopes listed for each prophage developed.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cbr\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 3\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"658\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eProphage Strain\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eStrain Genotype\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSpike Protein Antigen Sequence\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eLength (aa)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSpike Protein Domain Boundary\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1312\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1048 \\u003cem\\u003eD~linker~spike epitope\\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eLinker and antigen are inserted at \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene TAA stop.\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e47\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e602-648\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1313\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSSSGWTAGAAAYYVGYLQPRTFLL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e254-277\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1314\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATN\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e125\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1-125\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1315\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDS\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e133\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e122-254\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1316\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e133\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e253-385\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1317\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e133\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e384-516\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1318\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e133\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e515-647\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1319\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e133\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e646-778\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1320\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e133\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e777-909\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1321\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKR\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e133\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e907-1039\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1322\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDIS\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e133\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1038-1170\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1323\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e108\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1166-1273\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1375\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e33\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1038-1070\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1376\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPRE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e33\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1060-1092\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1377\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e33\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;1082-1114\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1378\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e33\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1104-1136\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1379\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKN\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e33\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1126-1158\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1380\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eFKEELDKYFKNHTSPDVDLGDIS\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e23\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1148-1170\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1381\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIV\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e84\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1049-1133\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1382\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSame as above\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYDPLQPELDSFKEELDKYFKNHTSPDVDLGD\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e31\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1138-1168\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e* ~ symbol refers to a genetic fusion\\u003c/p\\u003e\\n\\u003cp\\u003eY= temperature sensitive\\u003cem\\u003e\\u0026nbsp;cI857\\u003c/em\\u003e mutation\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 3:\\u003c/strong\\u003e \\u003cstrong\\u003eSARS-CoV-2 spike protein prophage strains and their displayed epitopes.\\u0026nbsp;\\u003c/strong\\u003eProphage strains developed for use in detecting a suitable SARS-CoV-2 spike protein antigen. Each strain is a modification of YXTL1048, containing a partial sequence of the spike protein fused to the C-terminal of the prophage\\u0026rsquo;s \\u003cem\\u003e\\u0026lambda;\\u003c/em\\u003e\\u003cem\\u003eD\\u003c/em\\u003e gene with a single unit linker in the sequence: GGGGS. Highlighted sequences are those displayed antigens which produced a strong response against COVID+ donor serum.\\u003cstrong\\u003e\\u003cbr\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 4A\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"624\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBacterial Strains\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eRelevant Genetic Markers\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSource\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eProphage\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLE392\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003etyrT(supF58) glnV(supE44) hsdR514(r-m+) lacY1 galK2 galT22 metB1 trpR55\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLynn Enquist\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e_\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1212\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLE392 p\\u0026Delta;\\u003cem\\u003e\\u003csub\\u003eBAD\\u003c/sub\\u003e\\u003c/em\\u003e::\\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e* /pLXT42**\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e_\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSJ_XTL175\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMG1655, \\u003cem\\u003elacY\\u003csub\\u003eA177C\\u0026nbsp;\\u003c/sub\\u003e\\u003c/em\\u003e, \\u003cem\\u003e\\u0026Delta;araFGH::spec\\u003csup\\u003eR\\u003c/sup\\u003e\\u003c/em\\u003e, \\u0026Delta;\\u003cem\\u003elacI,\\u003c/em\\u003e \\u0026Delta;\\u003cem\\u003earaE\\u003c/em\\u003e,\\u003c/p\\u003e\\n \\u003cp\\u003e\\u0026Delta;araD::\\u003cem\\u003etetA-sacB-amp\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLi, Xt., Jun, Y., Erickstad, M. et al. tCRISPRi: tunable and reversible, one-step control of gene expression. Sci Rep 6, 39076 (2016).\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e_\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL981\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSJ_XTL175 \\u0026nbsp;\\u003cem\\u003earaC\\u003csup\\u003e+\\u003c/sup\\u003e\\u003c/em\\u003e \\u003cem\\u003e\\u0026Delta;araBAD\\u003c/em\\u003e::\\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e_\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1026\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL981 \\u0026Delta;\\u003cem\\u003elamB\\u003c/em\\u003e, \\u0026Delta;\\u003cem\\u003efhuA\\u003c/em\\u003e/pKM208***\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1026\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1048\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1026 \\u003cem\\u003eD\\u003c/em\\u003e~linker::\\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB*,\\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eThe \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette is inserted with a linker coding sequence at \\u003cem\\u003e\\u0026lambda;D\\u0026nbsp;\\u003c/em\\u003egene TAA stop.\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1048\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1059\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1026 \\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB\\u003c/em\\u003e::linker~\\u003cem\\u003eD,\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eThe \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette is inserted with a linker coding sequence at \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene ATG start.\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1059\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1226\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1026\\u0026nbsp;\\u003cem\\u003estf386-tetA-sacB\\u0026nbsp;\\u003c/em\\u003einsertion\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1226\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1439\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1226 \\u003cem\\u003estf386\\u0026nbsp;\\u003c/em\\u003etruncation. \\u003cem\\u003etetA-sacB\\u0026nbsp;\\u003c/em\\u003eis replaced by sequence to truncate \\u003cem\\u003estf.\\u003c/em\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1439\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1441\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1439 \\u003cem\\u003eD\\u003c/em\\u003e~linker::\\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB,\\u0026nbsp;\\u003c/em\\u003ethe \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette is inserted with a linker coding sequence at \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene ATG start. Used to create melanoma C-terminal \\u0026lambda;D fusion phage in a strain that included Stf truncation.\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1441\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1442\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1439 \\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB\\u003c/em\\u003e::linker~\\u003cem\\u003eD,\\u0026nbsp;\\u003c/em\\u003ethe \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette is inserted with a linker coding sequence at \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene ATG start. Used to create melanoma N-terminal \\u0026lambda;D fusion phage in a strain that included Stf truncation.\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1442\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e* :: indicates that the gene preceding the double colon is replaced by the gene following it.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e**pLXT42 is a plasmid that expresses wild-type \\u0026lambda;D under the tunable pBAD promoter, inducible by arabinose. It also contains the following elements: \\u003cem\\u003eAmpR\\u0026nbsp;\\u003c/em\\u003egeneand\\u003cem\\u003e\\u0026nbsp;pMBori.\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e***pKM208 is a pSC101 based plasmid that expresses Red functions \\u003cem\\u003eexo bet\\u0026nbsp;\\u003c/em\\u003eand\\u003cem\\u003e\\u0026nbsp;gam\\u003c/em\\u003e from the \\u003cem\\u003elac\\u003c/em\\u003e promoter for recombineering. Obtained from Ken Murphy.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e* ~ symbol refers to a genetic fusion.\\u003c/p\\u003e\\n\\u003cp\\u003eY= temperature sensitive \\u003cem\\u003ecI857\\u0026nbsp;\\u003c/em\\u003emutation\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 4A:\\u003c/strong\\u003e \\u003cstrong\\u003e\\u003cem\\u003eE. coli\\u003c/em\\u003e strains used and/or generated here.\\u0026nbsp;\\u003c/strong\\u003eBacterial strains generated in the evolution of a \\u0026lambda; display platform. Table includes the initial parental strain, each new strain generated to modify the bacterial host, each strain containing modifications to \\u0026lambda; prophage within the host, and the final strains used to create antigen-displaying phages in this study.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e4B\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"535\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eProphage Name\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eProphage Genotype\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSource\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1026\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026lambda;\\u003cem\\u003ecI857,\\u003c/em\\u003e \\u003cem\\u003eSam7, \\u0026Delta;\\u003c/em\\u003e\\u003cem\\u003efhuA,\\u0026nbsp;\\u003c/em\\u003e\\u003cem\\u003e\\u0026Delta;b2, E\\u003csub\\u003eE158K\\u003c/sub\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1048\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1026 \\u003cem\\u003eD\\u003c/em\\u003e~linker::\\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB*,\\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eThe \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette is inserted with a linker coding sequence at \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene TAA stop\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1059\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1026\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB\\u003c/em\\u003e::linker~\\u003cem\\u003eD,\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eThe \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette is inserted with a linker coding sequence at \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene ATG start\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1226\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1026\\u0026nbsp;\\u003cem\\u003estf386-tetA-sacB\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1439\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1226 \\u003cem\\u003estf386\\u0026nbsp;\\u003c/em\\u003etruncation. \\u003cem\\u003eTetA-sacB\\u0026nbsp;\\u003c/em\\u003eis replaced by sequence to truncate \\u003cem\\u003estf.\\u003c/em\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1441\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1439 \\u003cem\\u003eD\\u003c/em\\u003e~linker::\\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB,\\u0026nbsp;\\u003c/em\\u003ethe \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette is inserted with a linker coding sequence at \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene ATG start. Used to create melanoma C-terminal \\u0026lambda;D fusion phage in a strain that included Stf truncation.\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eYXTL1442\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXTL1439 \\u003cem\\u003etetA\\u003c/em\\u003e-\\u003cem\\u003esacB\\u003c/em\\u003e::linker~\\u003cem\\u003eD,\\u0026nbsp;\\u003c/em\\u003ethe \\u003cem\\u003etetA-sacB\\u003c/em\\u003e cassette is inserted with a linker coding sequence at \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e gene ATG start. Used to create melanoma N-terminal \\u0026lambda;D fusion phage in a strain that included Stf truncation.\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eThis study\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e* ~ symbol refers to a genetic fusion.\\u003c/p\\u003e\\n\\u003cp\\u003eY= temperature sensitive\\u003cem\\u003e\\u0026nbsp;cI857\\u003c/em\\u003e mutation\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 4B:\\u003c/strong\\u003e \\u003cstrong\\u003e\\u0026lambda; prophage used and/or generated here\\u003c/strong\\u003e. Complementary prophage strains to Table 4A, listing the genetic modifications of each prophage strain that was used to generate display phages in this study. \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 4C\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"651\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOligo name\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOligo sequence\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cu\\u003ePurpose\\u003c/u\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT928\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCGGCGTAAACGCCTTATCCGGCCCAGGTTTTGCTATCTATCTCCTGAGTCATTGCTTTTCTTTTTTCACA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003elamB\\u003c/u\\u003e\\u003c/em\\u003e\\u003cu\\u003e\\u0026nbsp;deletion\\u003c/u\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT924\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAAATGGGCACGGAAATCCGTGCCCCAAAAGAGAAATGGTATATCTCTGATGTAAAGTGAATGATAACGTA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003efhuA\\u003c/u\\u003e\\u003c/em\\u003e\\u003cu\\u003e\\u0026nbsp;deletion\\u003c/u\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT992\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCTGGCTCAACCGTTATTGCTGAATTTGAATCGCTGGGTCAGTGCATTCACCACCACAAGCAGATAATAAC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eatt80\\u0026nbsp;\\u003c/u\\u003e\\u003c/em\\u003e\\u003cu\\u003edeletion\\u003c/u\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT957\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eATTGCGGTTATTTTAATAAAATTAAGGGTTACTATTGAGTCATAGAACTTCCATTATTCTCCTGAAGATA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cu\\u003e\\u0026lambda;\\u003cem\\u003eea59\\u003c/em\\u003e \\u003cem\\u003eea31\\u003c/em\\u003e \\u003cem\\u003eb\\u003c/em\\u003e deletion\\u003c/u\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT934\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eACTCCGTGCCGCCGGACTGCGTGATGTTATTCT\\u003cstrong\\u003eCCT\\u003c/strong\\u003eTACTGCGGCCCATATCCACCTCAACCGGATCGAAGGC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cu\\u003e\\u0026lambda;\\u003c/u\\u003e\\u003cu\\u003egene\\u003cem\\u003e\\u0026nbsp;E\\u0026nbsp;\\u003c/em\\u003emutation\\u003c/u\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT914\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGATAGGAGGATGAAACGatgacgagcaaagaaacctttacc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eara P\\u003csub\\u003eBAD\\u003c/sub\\u003e\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cu\\u003e\\u0026lambda;\\u003c/u\\u003e\\u003cu\\u003egene \\u003cem\\u003eD\\u003c/em\\u003e\\u003c/u\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT915\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTGAGTATAGCCTGGTTTCGTTTGATTGGCTGTGGTTTTATACAGTCATTAaacgatgctgattgccgttccg\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eara P\\u003csub\\u003eBAD\\u003c/sub\\u003e\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cu\\u003e\\u0026lambda;\\u003c/u\\u003e\\u003cu\\u003egen\\u003cem\\u003ee D\\u003c/em\\u003e\\u003c/u\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT1021\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCAGCCAATCAAACGAAACCAGGCTATACTCAAGCCTGGTTTTTTGATGGAgttccggatctgcatcgcag\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eara P\\u003csub\\u003eBAD\\u003c/sub\\u003e \\u0026lambda;D\\u003c/u\\u003e\\u003c/em\\u003e\\u003cu\\u003e\\u0026nbsp;retrieval\\u003c/u\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT1022\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTGCAGAATCCCTGCTTCGTCCATTTGACAGGCACATTATGCAAGCATTGCtgtgataaactaccgcattaaagc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eara P\\u003csub\\u003eBAD\\u003c/sub\\u003e \\u0026lambda;D\\u003c/u\\u003e\\u003c/em\\u003e\\u003cu\\u003e\\u0026nbsp;retrieval\\u003c/u\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT1115 \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGCCGCACAGGCGGCCTTTAGTGATGAAGGGTAAAGCCCTTACACTGGTGTGTTCAGCAAATCGTTAACGG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eHomology arms for \\u003cem\\u003e\\u0026lambda;D\\u0026nbsp;\\u003c/em\\u003edeletion\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT918\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eattcttgcggttgctgctgac\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrimer to check \\u003cem\\u003e\\u0026lambda;D\\u0026nbsp;\\u003c/em\\u003eC-terminal fusion gene.\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT919\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eataacctcaccggaaacaatcg\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrimer to check \\u003cem\\u003e\\u0026lambda;D\\u0026nbsp;\\u003c/em\\u003eC-terminal fusion gene.\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT1039\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ecgccgcattctggccgcagc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrimer to check \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e N-terminal fusion gene.\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXT1040\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003etggtgccatcccacgcaacc\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrimer to check \\u003cem\\u003e\\u0026lambda;D\\u003c/em\\u003e N-terminal fusion gene.\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXT40\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAAGTAATCCAGCGATGCGCC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003ePrimer on \\u003cem\\u003etet\\u003c/em\\u003e gene\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXT1116\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCCAACAGCACGTCTGAAACGCTTGCTGCAACGCCAAAGGCGGTTAAGGTCggtggcggagggagtTCCTAATTTTTGTTGACACTCTATC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003ePrimer used to make XTL1026 \\u003cem\\u003estf386_tetA-sacB\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXT1117\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eATTATAAATTTTTATGGTCCGTGGTTGTTCACTCATTCTGAATGCCATTAATCAAAGGGAAAACTGTCCATATGC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003ePrimer used to make XTL1026 \\u003cem\\u003estf386_tetA-sacB\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXT1118\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGCAATACGTGCAAAAAATTCGGC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003ePrimer used to verify XTL1026 \\u003cem\\u003estf386_tetA-sacB\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXT1119\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGCCGGAATATCTGGCGGTGC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003ePrimer used to verify XTL1026 \\u003cem\\u003estf386_tetA-sacB\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXT1387\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGGGGAGTGCGTCAACGGCAT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003ePrimer used to check \\u003cem\\u003estf\\u0026nbsp;\\u003c/em\\u003efusion genes in strain XTL1226\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003eXT1388\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGGTAACGGACCGAGTTCAGAA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"bottom\\\"\\u003e\\n \\u003cp\\u003ePrimer used to check \\u003cem\\u003estf\\u0026nbsp;\\u003c/em\\u003efusion genes in strain XTL1226\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 4C:\\u003c/strong\\u003e \\u003cstrong\\u003eOligonucleotides used to generate bacterial and prophage strains in this study\\u003c/strong\\u003e. Designed for the deletion, modification, and addition of genes or to check by PCR and Sanger Sequencing the identity of modified sequences.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-portfolio\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Nature Portfolio\",\"twitterHandle\":\"\",\"acdcEnabled\":false,\"dfaEnabled\":false,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8435997/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8435997/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eWe have developed a vector platform for delivery of foreign peptides by genetic modification of the temperate lambda (λ) bacteriophage. This delivery platform is capable of displaying peptides or proteins on either terminus of the structural λ head protein D, present in ~\\u0026thinsp;420 copies per phage particle, and λ side tail fiber (Stf), present at 12 copies per phage particle. Proteins and peptides can be easily fused for display through the low-cost and high-efficiency methods of recombineering and λ prophage induction for recombinant phage preparation described here. To improve this vector technology for use in antigen selection and immunotherapy, we introduced several mutations in the bacterial host and resident prophage λ that improve engineering, induction, phage stability, yield, fusion protein accommodation capacity, and longevity in animal systems. We tested the ability of this λ display system to identify useful antigens and generate antibodies in a mouse model. We report its success as a new technology for both applications: the selection and delivery of therapeutic peptides and proteins.\\u003c/p\\u003e\",\"manuscriptTitle\":\"A λ Phage Platform for Successful Therapeutic Display of Protein Antigens\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-02-27 10:36:46\",\"doi\":\"10.21203/rs.3.rs-8435997/v1\",\"editorialEvents\":[],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"communications-biology\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"commsbio\",\"sideBox\":\"Learn more about [Communications Biology](http://www.nature.com/commsbio/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Communications Biology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Communications Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"d5a45d8c-9d35-431b-8273-f2253e7667ea\",\"owner\":[],\"postedDate\":\"February 27th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":61958658,\"name\":\"Biological sciences/Biotechnology/Peptide delivery\"},{\"id\":61958659,\"name\":\"Biological sciences/Biotechnology/Molecular engineering/Protein design\"}],\"tags\":[],\"updatedAt\":\"2026-02-27T10:36:46+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-02-27 10:36:46\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8435997\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8435997\",\"identity\":\"rs-8435997\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}