Surface functionalization of virus-like particles for bio- conjugation applications

Abstract

Embodiments of the present disclosure generally relate to a platform for vaccine delivery. Specifically, providing a virus-like particle (VLP) as a platform for vaccine delivery. The VLP platform includes a bacteriophage with a plurality of coat proteins, the plurality of coat proteins operable to assemble into a VLP, and a protein anchor system including a first portion inserted in at least one coat protein of the plurality of coat proteins and a second portion, wherein the first portion and the second portion are operable to link together.

Description

TXTU-0009PCSURFACE FUNCTIONALIZATION OF VIRUS-LIKE PARTICLES FOR BIOCONJUGATION APPLICATIONS BACKGROUNDField

[0001] Embodiments of the present disclosure generally relate to a platform for vaccine delivery. Specifically, providing a virus-like particle as a platform for vaccine delivery.Description of the Related Art

[0002] Peptide and protein antigens are alternatives to traditional vaccines such as live-attenuated vaccines and inactivated vaccines. The antigens exhibit excellent safety profiles. Peptides and some protein vaccines are however less immunogenic and require conjugation or display on larger immunogens such as Keyhole limpet hemocyanin to enhance their immunogenicity. Within the last decade, virus-like particles (VLPs) have gained attention as a display platform for peptide and some protein antigens. VLPs are self-assembling structural proteins (cage-like structures derived from viruses) that look like viruses except that they lack viral genome; they are highly immunogenic. Given their immunogenic properties, VLPs have been used as display platforms for less immunogenic antigens; less immunogenic antigens are displayed multivalently on the surface of VLPs. Peptides or protein antigens can be displayed on VLPs by genetic insertion or by chemical conjugation. The chemical conjugation approach is expensive and complicated. The peptides in question typically have to be first synthesized in a commercial setting with a cysteine residue at either the N- or C-terminus of the peptide for conjugation to VLPs. For example, the synthesis of only 4 mg of a 22 amino acid peptide can cost about $425. Moreover, peptides with cysteine residues cannot be conjugated because they form disulfide bonds with cysteine residue added at the N- or C-terminus of a peptide for conjugation. This conjugation approach cannot even be used for proteins due to the limitation on the size of protein that can be chemically synthesized (<165 amino acids). On the other hand, the genetic insertion approach is more cost effective and can be scaled up for large scale production; for example, 10 - 1000 mg of a protein antigen of 450 amino acids can be developed, expressed, and purified from a 1 L culture at < $1,000. However, the genetic insertion approach can be very challenging with some antigens.TXTU-0009PCGenetic insertion of some antigens on the coat proteins can sometimes prevent the coat proteins from assembling to form VLPs.

[0003] Accordingly, what is needed in the art is a new bio-conjugation system that allows for the conjugation of a protein on VLPs, such that VLPs may be used as a platform for a variety of applications.SUMMARY

[0004] In a first embodiment, a virus-like particle (VLP) platform is disclosed. The VLP platform includes a bacteriophage with a plurality of coat proteins, the plurality of coat proteins operable to assemble into a VLP, and a protein anchor system including a first portion inserted in at least one coat protein of the plurality of coat proteins and a second portion, wherein the first portion and the second portion are operable to link together.

[0005] In another embodiment, a virus-like particle (VLP) platform is disclosed. The VLP platform includes a bacteriophage with a plurality of coat proteins, the plurality of coat proteins operable to assemble into a VLP and a protein anchor system including a first portion inserted into a N-terminus or an AB-loop of at least one coat protein of the plurality of coat proteins and a second portion coupled to at least a foreign protein of interest, wherein the first portion and the second portion are operable to link together.

[0006] In another embodiment, a method of forming a virus-like particle (VLP) platform is disclosed. The method includes expressing a first portion of a protein anchor system in a plurality of coat proteinsof a bacteriophage, forming a recombinant coat protein such that the plurality of coat proteins form the VLP and the first portion of the protein anchor system is expressed along an exterior of the VLP, fusing a target protein with a second portion of the protein anchor system; and conjugating the VLP portion with the second portion of the protein anchor system to form a VLP platform such that the first portion of the anchor protein system forms a bond with the second portion of the anchor protein system.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure,TXTU-0009PCbriefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments of the disclosure and are therefore not to be considered limiting of scope of the disclosure, may admit to other equally effective embodiments.

[0008] Figure 1 shows a virus-like particle (VLP) platform, according to one or more embodiments.

[0009] Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, and 2J show insertion of Spytag or Spytag003 into a coat protein, according to one or more embodiments.

[0010] Figures 3A, 3B, and 3C show the SDS-PAGE results of small-scale expression of the recombinant coat proteins of Qp-Spytag003 and PP7-Spytag003 in C41 (DE3), Rosetta (DE3) pLysS, and BL21 Star (DE3) pLysS. Figure 3A shows expression of Q -N-SpytagOO3 and Qp-C-Spytag003 coat proteins in C41 cells. Figure 3B shows expression of PP7-N-SpyTag003 and PP7-C-Spytag003 coat proteins in C41. Figure 3C shows expression of PP7-N-Spytag003 and PP7-C-Spytag003 coat proteins in Rosetta cells and BL21 Star cells. Four hours after induction, bacteria are pelleted, lysed with 8M urea and lysates are run on SDS PAGE gels. In all cases, recombinant coat protein is induced with 0.5 mM IPTG at 37 °C for 4 hours. Induced-1, Induced-2, and Induced-3 represent independent bacterial colonies collected from the agar plate transformed with the plasmids expressing the coat proteins above. Arrows indicate expected proteins sizes (-16.78 KDa for Qp-N-Spytag003 and -30.59 KDa for PP7-C-Spytag003).

[0011] Figure 4A and 4B show a solubility assessment of the coat proteins of Qp-Spytag003 coat protein and PP7-Spytag003 coat protein. Figure 4A shows C41 bacterial pellet expressing Qp-N-Spytag003 coat protein is lysed with BugBuster reagent supplemented with 0.1% triton-X-100, centrifuged and the supernatant and lysate analyzed on SDS PAGE gel. Figure 4B shows the bacterial pellet expressing PP7-C-Spytag003 coat protein is lysed similar to Figure 4A and supernatant and lysates analyzed on SDS PAGE gel. Arrows indicate expected proteins sizes (-16.78 KDa for Qp-N-Spytag003 and -30.59 KDa for PP7-C-Spytag003).TXTU-0009PC

[0012] Figure 5A, 5B, 5C, and 5D show the purification of the coat proteins of Qp-N-Spytag003 coat protein and PP7-C-Spytag003 coat protein. Bacterial pellet expressing Qp-N-Spytag003 coat protein (Figure 5A) and PP7-C-Spytag003 coat protein (Figure 5B) are sequential lysed with SCB buffer, Borax, and BugBuster reagent followed by lysis with 0.5M, 1M, and 8M urea. Supernatant after each lysis is run on SDS PAGE gel. Qp-N-Spytag003 coat proteins (lysed with 0.5 and 1 M urea) are combined and are dialyzed (buffer exchanged with PBS) using Amicon Ultra Centrifugal Filter, 3 KDa molecular weight cutoff. Samples are run on SDS PAGE (as shown in the right of panel Figure 5A). Positive controls represent the lysates of Qp-N-Spytag003 coat protein and PP7-C-Spytag003 coat protein lysed with 8M urea. Figures 5C and 5D are TEM of negatively stained sample of concentrated Qp-N-Spytag003 from Figure 5A and PP7-C-Spytag003 samples from Figure 5B, respectively. TEM are taken at 50,000 X and 70.000X, respectively.

[0013] Figure 6A and 6B show SDS-PAGE results of small-scale expression of the recombinant coat proteins of PP7-Spytag. Expression of PP7-N-Spytag (Figure 6A) and PP7-AB-loop- Spytag (Figure 6B) in C41 cells. In all cases, recombinant coat protein is induced with 0.5 mM IPTG at 37 °C for 4 hours. Four hours after induction, bacteria are pelleted, lysed with 8M urea and lysates are run on SDS PAGE gels. Lanes labeled 1-6 correspond to independent colonies that are picked after transformation of the plasmid into competent cells. Arrows indicate expected protein size -30.59 KDa).

[0014] Figures 7A, 7B, 7C, and 7D show purification and TEM analysis of PP7-N-Spytag and PP7-AB-loop-Spytag. Figure 7A shows SDS-PAGE analysis of purified fractions (by SEC using a Sepharose CL-4B column) of expressed PP7-N-Spytag coat protein. Figure 7B shows a TEM image of purified PP7-N-Spytag VLPs. Figure 7C shows a SDS-PAGE analysis of purified fractions (by SEC using a Sepharose CL-4B column) of expressed PP7-AB-loop-Spytag coat protein. Figure 7D shows a TEM analysis of purified PP7-AB-loop-Spytag. Arrows indicate expected size (-30.59 KDa) of recombinant coat protein. TEM images are taken at 70.000X. Sizes of LVPs are -27-30 nm.

[0015] Figures 8A, 8B, and 8C show SDS-PAGE analysis of expressed AP205 in C41 cells (Figure 8A), and MS2 coat proteins with Spytag003 insertion in C41 cellsTXTU-0009PC(Figure 8B) and Rosetta cells (Figure 8C). A) Vectors expressing AP205-N-Spytag003 and AP205-C-Spytag003 are transformed into C41 cells. Cells are induced at 37 °C for 3 hours with 0.5 mM IPTG, lysed with 8M urea and are run on SDS PAGE followed by staining with Coomassie Blue. Vectors expressing MS2-N-Spytag003 and MS2-C-Spytag003 are transformed into C41 cells (Figure 8B) or Rosetta cells (Figure 8C). Proteins are induced and analyzed as described in panel A. M = protein marker.

[0016] Figures 9A, 9B, 9C, 9D, and 9E show the solubility of AP205-Spytag003 and MS2-Spytag003 coat proteins. Figure 9A shows AP205-N-Spytag003 and AP205-C-Spytag003 or Figure 9B shows MS2-N-Spytag003 and MS2-C-Spytag003 are induced at 37 °C for 3 hours with 0.5 mM IPTG. Figure 9C shows AP205-N-Spytag003 and AP205-C-Spytag003 or Figure 9D shows MS2-N-Spytag003 and MS2-C-Spytag003 are induced at 20 °C for 16 hours with 0.5 mM IPTG. Figure 9E shows C41 bacterial cells expressing two chaperone proteins (groES and groEL) are transformed with expression vector expressing MS2-C-Spytag003 and expression done at 16 °C and room temperature (RT) for 16 hours or at 37 °C for 4 hours. groES and groEL chaperone proteins are induced as described in text with 0.5 mg / ml of arabinose. In all cases, cells are pelleted and lysed with both BugBuster protein extraction reagent and with 2% lysozyme solution; in panel E, only lysis with BugBuster is used. Lysates are spun down (10,000 rpm) and supernatants are run on SDS PAGE followed by staining with Coomassie Blue. M = protein marker.

[0017] Figures 10A and 10B show purification data, SDS-PAGE and TEM images of AP205-N-Spytag003 VLPs (Figure 10A) and MS2-N-Spytag003 VLPs (Figure 10B). Bacterial supernatant expressing: AP205-N-Spytag003 VLPs (Figure 10A) and MS2-N-Spytag003 VLPs (Figure 10B) are spun on 23%, 29%, and 35% Optiprep gradient and layers (numbered 1-7; left image) are analyzed by SDS PAGE (middle) and TEM (right). TEM images are taken at 50,000 X or 70,000 X, respectively.

[0018] Figures 11A and 11 B show the conjugation of Spycatcher003 protein on AP205-N-Spytag003 VLPs analyzed by SDS-PAGE (Figure 11 A) and TEM (Figure 11 B). Figure 11A shows Spycatcher003 is conjugated to AP205-N-Spytag003 VLPs at 1.5:1 molar ratio and mixture is incubated at room temperature for up to 3 hours. Following conjugation, samples are run on SDS PAGE gel. M = protein marker. Band intensities are quantified by densitometry using Imaged. Conjugation efficiency isTXTU-0009PCcalculated as follows: total coat protein before conjugation minus unconjugated coat protein, divided by the total coat protein before conjugation.

[0019] Figure 12 shows the conjugation of PP7-N-Spytag VLPs with Spycatcher003. SDS-PAGE data showing conjugation of PP7-N-Spytag with Spycatcher003 at a molar ratio of 1 :3.25 (VLPs:Spycatcher003). PP7-N-Spytag alone (-30.59 KDa); Spycatcher003 alone (-15.15 KDa); conjugation reaction, showing the up-shifted PP7-Spytag-Spycatcher003 complex (-45.6 KDa). The conjugation reactions are done at room temperature for 3 hours. Conjugation efficiency is calculated by subtracting unconjugated coat protein from total coat protein before conjugation, divided by the total coat protein before conjugation.

[0020] Figures 13A and 13B show analysis by SDS-PAGE (Figures 13A and 13B) and TEM (Figures 13C and 13D). Spycatcher003-Target Antigen is conjugated to AP205-N-Spytag003 VLPs (Figure 13B) and to MS2-N-Spytag003 VLPs (Figure 13C) at 1:8 molar ratio and 1.7:1 molar ratio, respectively. Following conjugation, samples are run on SDS PAGE gel. Figure 13C shows TEM images of samples. Figure 13D shows TEM images of samples. Band intensities are quantified by densitometry using Imaged. Conjugation efficiency is calculated as follows: total coat protein before conjugation minus unconjugated coat protein, divided by the total coat protein before conjugation.

[0021] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.DETAILED DESCRIPTION

[0022] Embodiments of the present disclosure generally relate to a platform for vaccine delivery. Specifically, providing a virus-like particle as a platform for vaccine delivery. Currently, what is needed in the art is a bio-conjugation platform that enables robust conjugation / display of heterologous peptides and proteins on VLPs, while minimizing interference with VLP assemblies, reducing impact of pre-existing immunity, and facilitating rapid, high-efficiency conjugation under mild conditions. Existing systems fail to address one or more of these simultaneously as manyTXTU-0009PCheterologous proteins are too large to be inserted into VLPs and existing platforms are inhibited by the human immune system. As disclosed herein, Spycatcher, Spytag, Spycatcher003, Spytag003 have been tested and compared for use as a protein anchor system. In some embodiments, the human population includes pre-existing antibodies against the Spycatcher and Spytag pairing. The pre-existing antibodies led to the exploration of Spycatcher003 and Spycatcher003 pairing as the protein anchor system. Both systems show potential for use as part of a VLP platform.Definitions

[0023] The term “virus-like particle” or “VLP” refers to one or several recombinantly expressed viral capsid proteins, which spontaneously assemble into macromolecular particulate structures mimicking the morphology of a virus coat, but lacking infectious genetic material.

[0024] The term self-assemble refers to the process in which a system of preexisting components, under specific conditions, adopts a more organized structure through interactions between the components themselves. As described herein, the term self-assemble refers to the coat proteins to self-assemble into a VLP.

[0025] The term protein anchor system refers to a first portion and a second portion where each protein is a peptide or protein and the first portion and the second portion are operable to couple together through a chemical bond.

[0026] The term Spytag or Spytag003 refers to a part of the protein anchor system optimized to bind to the Spycatcher or Spycatcher003. The Spytag includes, but is not limited to a Seq. ID No. 1. The Spytag includes at least the following amino acids: AHIVMVDAYKPTK. Spytag003 includes, but is not limited to Seq. ID No. 2. The Spytag003 includes, at least the follow amino acids: RGVPHIVMVDAYKRYK.

[0027] The term Spycatcher or Spycatcher003 refers to a part of the protein anchor system optimized to bind to Spytag or Spytag003. The Spycatcher includes, but is not limited to the sequence: Seq. ID No. 3 includes at least the following amino acids: VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTIST WISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA HI. Spycatcher003 includes, but is not limited to the sequence: Seq. ID No. 4. TheTXTU-0009PCSeq. ID No. 4 includes at least the following amino acids: VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIST WISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDA HT.

[0028] The term “biologically active” means the ability to form a VLP.

[0029] The term “sequence variant” refers to a polypeptide (e.g., amino acid) sequence with at least at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99.5%, such as 100% sequence identity to the said polypeptide sequence.

[0030] VLPs are non-infectious scaffolds and are highly immunogenic. Virus-like particles (VLPs) are protein-based nanoscale assemblies derived from structural proteins of viruses. Thus, VLPs are widely used as scaffolds to display and enhance the immunogenicity of less immunogenic foreign antigens. The VLPs disclosed herein encapsidate endogenous adjuvant, single-stranded RNA (ssRNA), which is a ligand for toll-like receptor 7 / 8 and activate T-helper 1 responses. ssRNA induces efficient co-stimulation of antigen presenting cells by engaging TLR7 / 8 and is associated with strong T-helper 1 responses. Different approaches, including genetic insertions, can be used to display foreign antigens on VLPs. However, the conventional approaches have limitations; such as the inability of viral coat proteins to tolerate a foreign insertion. As disclosed herein, the tolerability of bacteriophages Qp, PP7, AP205, and MS2, to coat proteins to insertions of a Spytag003 peptide or a Spytag peptide (hereinafter the peptides or the anchor system) are shown. The coat proteins, the peptides, and a foreign protein of interest form a bio-conjugation system. In one or more embodiments, the bio-conjugation system is referred to as a VLP platform.

[0031] Specifically, some conventional bio-conjugation systems consist of a 13 amino acid peptide (Spytag; AHIVMVDAYKPTK) and its binding partner, 113 amino acids protein (Spycatcher). In one or more embodiments, Spycatcher is preceded by a 25 amino acid sequence: SYYHHHHHHDYDIPTTENLYFQGAM, which includes the histidine tag and a TEV protease cleavage site, forming SEQ No. 5. Spytag is genetically inserted on the VLPs while Spycatcher (with a histidine tag and a tobacco etch virus (TEV) protease cleavage site) is expressed as a recombinant protein linked to a protein of interest. Mixing of the recombinant VLPs (displaying Spytag) with aTXTU-0009PCSpycatcher linked to a protein of interest leads to the conjugation of the protein on the VLPs. The referenced approach has been used to bio-conjugate foreign proteins on the surfaces of VLPs derived from bacteriophage AP205, norovirus, hepatitis B, and / or porcine circovirus type 2. However, the original SpyTag / SpyCatcher system is derived from Streptococcus pyogenes, and, therefore, antibodies against SpyCatcher are frequently found in humans due to natural exposure to this bacterium. The high level of pre-existing immunity limits the usefulness for vaccine applications and can attenuate the efficacity of the system and consequently any protein conjugated using this system. To obviate the limitation, a new bio-conjugation system (Spytag003 / Spycatcher003) is utilized. Spytag003 (RGVPHI MVDAYKRYK) is three amino acids longer than the prototype Spytag (AHIVMVDAYKPTK) and differs from prototype Spytag by 6 amino acids (highlighted in bold text, the substituted amino acids are underlined). Spycatcher003, the binding partner of Spytag003, differs from the prototype Spycatcher by 13 amino acids. Unlike the prototype Spytag / Spycatcher system, Spytag003 / Spycatcher003 reaction is 400 times faster and has less preexisting antibodies in the population. The disclosed bio-conjugation system has not been explored to display the foreign protein of interest on any VLP platform previously. The insertion of Spytag003 on the coat proteins of four bacteriophages (PP7, Qp, AP205, and MS2) is disclosed herein such that a VLP platform may be used to bioconjugate foreign proteins.

[0032] The surface functionalization capabilities of VLPs allow them to be optimal platforms for vaccine delivery of large foreign proteins or peptides as the proteins or peptides can be displayed on the surface of the VLPs, which increases the size as well as the valency or the repetitiveness of the displayed target protein. VLPs are empty viral shells derived from the expression of viral structural proteins such as the capsid or envelope proteins; expressed coat proteins spontaneously self-assemble into VLPs that resemble (structurally and immunogenetically) an authentic virus except that they do not have the viral genome. Thus, the VLPs are safe for humans regardless of their immune status. Studies have shown that nanoparticles, such as VLPs, that are about 20 nanometers (nm) to about 200 nm in diameter can be easily taken up by antigen presenting cells (APCs) for presentation to the immune system. Soluble proteins or peptides that are greater than 10 nm cannot be easily taken up by APCs. VLPs are used as a display platform to enhance the immunogenicity of lessTXTU-0009PCimmunogenic peptides or proteins from other infectious agents. VLPs displaying heterologous peptides or proteins are called chimeric VLPs. The goal of chimeric VLPs is to elicit immune responses against the foreign protein on the VLP and not necessarily against the VLP in question.

[0033] Foreign proteins that have highly dense multivalent / repetitive structures, such as VLPs, can activate B cells (by cross-linking B-cell receptors) and induce antibody responses at much lower concentrations (e.g., about 5 pg) when compared to a monomeric protein; our VLPs encapsidate endogenous adjuvant, single-stranded RNA (ssRNA), which is a ligand for toll-like receptor 7 / 8 and activate T-helper 1 responses. ssRNA induces efficient co-stimulation of antigen presenting cells by engaging TLR7 / 8 and is associated with strong T-helper 1 responses. VLPs can enter the lymphatic system efficiently by direct diffusion. VLPs can be cross-presented in association with major histocompatibility complex (MHC) class I; immunization with VLPs displaying T cell epitopes elicit robust protective T cell responses (both CD4 and CD8) against cancer, bacterial, and viral infections. The referenced features have allowed VLPs to be used as stand-alone vaccines. VLP-based vaccines have been approved against human papillomaviruses (HPVs) and hepatitis B virus. Unlike live-attenuated vaccines, VLP vaccines are very safe and are used in individuals with a depressed cell mediated immunity. Accordingly, VLPs provide a platform for vaccine delivery. In one or more embodiments, the VLP platform are suitable for use in diagnostic assays, targeted delivery of therapeutic proteins, enzyme display, immune modulation, or combinations thereof.

[0034] Figure 1 is a representative illustration of a virus-like particle (VLP) platform 100. The VLP platform 100 includes a virus-like particle (VLP) 102 and a target protein 112 coupled to the exterior surface 110 of the VLP 102 with a protein anchor system 108. Each VLP includes a plurality of coat proteins which are operable to selfassemble to form the VLP. As shown in Figure 1, the protein anchor system 108 includes a first portion 104 and a second portion 106. In one or more embodiments, the protein anchor system 108 is the aforementioned peptides (e.g., Spytag003, Spycatcher003, Spytag, or Spycatcher). Mixing the first portion 104 and the second portion 106 allows a covalent bond to form with a strong affinity of 0.2 |j.M. The protein anchor system 108 allows for foreign proteins (e.g., the target protein 112) to be displayed on the exterior of VLPs (e.g., VLP 102). In one or more embodiments, eachTXTU-0009PCVLP includes the target protein 112, which is the same protein. In one or more embodiments, each VLP may include a variety of target proteins.

[0035] In one or more embodiments, the VLP platform 100 includes at least one of a bacteriophage PP7, bacteriophage Qp, bacteriophage AP205, bacteriophage, or bacteriophage MS2. In one or more embodiments, the VLP platform 100 may include RR1, AIN000, AIN002, AIN003, AIN010, AVE017, AVE019, AVE021, Beihai16, Beihai17, Beihai18, Beihai19, Beihai21, Beihai23, Beihai26, Beihai27, Beihai28, Beihai30, Beihai32, Beihai33, Beihai34, EMS014, EOC000, EOC005, ESE005, ESE006, ESE007, ESE009, ESE010, ESE012, ESE019, ESE021, ESE024, ESE025, ESE029, ESE030, ESE037, ESE046, ESE058, ES0010, Hubei2, Hubei3, Hubei6, Hubei8, HubeilO, Hubei14, NFYT01000214, NFZC01009824, Shahe3, Wenling2, Wenling3, Wenzhou4, AVE004, AVE006, AVE007, AVE022, AVE024, AVE039, GALT01000492, GALT01093879, AVE000, AVE005.AVE015, AVE016, AVE018, AVE020, AVE023, Beihai12, GALQ01044112, AVE002, GALQ01040378, Beihai13, ESE017, GALQ01034907, Beihai20, Cb5, EMS002, EMS011, EMS017, ESE016, ES0001, Beihai3, Beihai6, MB, Beihail, EMS007, ESE008, ESE015, ESE022, ESE026, ESOOOO, Wenzhou2, Beihai9, Wenlingl, Changjiangl, EMS005, Wenzhoul, EMM000, EMS001, ESE020, ESE041, AC, NFYT01000391 , NFZC01007443, EMS000, EMS003, AVE001 , AVE003, Beihai14, ESE001, ES0003, AIN001, Human and animal papillomaviruses, hepatitis B virus, parvoviruses, norovirus, HIV, influenza viruses, hepatitis E, Zika, chikungunya virus, Nipah virus, Hendra virus, rotavirus, coronaviruses, dengue viruses, Japanese encephalitis virus, Ebola viruses, Infectious bursal disease virus, rabbit hemorrhagic disease virus, Newcastle disease virus, porcine circoviruses, blue tongue virus, African swine fever virus, poliovirus, goose hemorrhagic polyomavirus, rabies virus, foot and mouth disease virus, Piscine myocarditis virus, Atlantic cod nervous necrosis virus, Cowpea chlorotic mottle virus, Cucumber mosaic virus, Papaya mosaic virus, Tobacco mosaic virus, herpes viruses, bovine viral diarrhea virus, vesicular stomatitis virus, pseudorabies virus, poxviruses, herpesviruses, or combinations thereof. Although bacteriophage PP7, bacteriophage Qp, bacteriophage AP205, bacteriophage, or bacteriophage MS2 are used as exemplary options, it should be understood that any of the listed options may be part of the VLP platform 100.TXTU-0009PC

[0036] The VLP platform further includes Spytag003 or Spytag inserted into at least one coat protein of the bacteriophage where Spytag003 or Spytag is the first portion 104 of the protein anchor system. The coat proteins assemble to form a VLP that displays at least the Spytag003 or the Spytag on the exterior of the VLP. In one or more embodiments, the Spytag003 and the Spytag are operable to couple to the Spycatcher003 and the Spycatcher, which may be conjugated with a target protein. In one or more embodiments, the VLP platform may be freeze dried and stored at room temperature of up to 6 months. In one or more embodiments, the VLP platform may be freeze dried and stored at 37 °C for about 2 months.

[0037] For example, the VLP platform including the coat proteins of PP7 and Spytag003 includes SEQ. ID NO. 6 or a biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99.5%, such as 100% sequence identity to the amino acid sequence of SEQ. ID No. 6. For example, the VLP platform including the coat proteins of PP7 and Spytag003 includes the following amino acid sequence: MARGVPHIVMVDAYKRYKGSGGSGGSGKTIVLSVGEATRTLTEIQSTADRQIFEEK VGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADWDCSTSVCGELPKVRYTQVWS HDVTIVANSTEASRKSLYDLTKSLVATSQVEDLWNLVPLGRYGSKTIVLSVGTATR TLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDCST SVCGELPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLWNLVPL GR. As disclosed herein, Spytag003 is operable to be inserted at the N-terminus or the C-terminus of the coat proteins of PP7.

[0038] For example, the VLP platform including the coat proteins of PP7 and Spytag includes SEQ. ID NO.: 7 or a biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99.5%, such as 100% sequence identity to the amino acid sequence of Seq. ID No. 7. For example, the VLP platform including the coat proteins of PP7 and Spytag includes the following amino acid sequence: MAHIVMVDAYKPTKGGGSAKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLR LTASLRQNGAKTAYRVNLKLDQADWDCSTSVCGELPKVRYTQVWSHDVTIVANS TEASRKSLYDLTKSLVATSQVEDLWNLVPLGRYGSKTIVLSVGTATRTLTEIQSTATXTU-0009PC DRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDCSTSVCGELPK VRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLWNLVPLGR. As disclosed herein, Spytag is operable to be inserted at the N-terminus or the C-terminus of the coat proteins of PP7.

[0039] For example, the VLP platform including the coat proteins of Qp and Spytag003 includes Seq. ID No. 8 or a biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99.5%, such as 100% sequence identity to the amino acid sequence of Seq. ID No. 8. For example, the VLP platform including the coat proteins of Qp and Spytag003 includes the following amino acid sequence: MARGVPHIVMVDAYKRYKGSGGSGGSGKLETVTLGNIGKDGKQTLVLNPRGVNPT NGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSV TRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY. As disclosed herein, Spytag003 is operable to be inserted at the N-terminus or the C-terminus of the coat proteins of Qp.

[0040] For example, the VLP platform including the coat proteins of Qp and Spytag includes Seq. ID No. 9 or a biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99.5%, such as 100% sequence identity to the amino acid sequence of Seq ID No. 9. For example, the VLP platform including the coat proteins of Qp and Spytag includes the following amino acid sequence:MAHIVMVDAYKPTKGGGSKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQA GAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVT FSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY. As disclosed herein, Spytag is operable to be inserted at the N-terminus or the C-terminus of the coat proteins of Qp.

[0041] For example, the VLP platform including the coat proteins of AP205 and Spytag003 includes Seq. ID No. 10 or a biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99.5%, such asTXTU-0009PC100% sequence identity to the amino acid sequence of Seq. ID No. 10. For example, the VLP platform including the coat proteins of AP205 and Spytag003 includes the following amino acid sequence: MARGVPHIVMVDAYKRYKGSGGSGGSGNKPMQPITSTANKIVWSDPTRLSTTFSA SLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGS AENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA. As disclosed herein, Spytag003 is operable to be inserted at the N-terminus or the C-terminus of the coat proteins of AP205.

[0042] For example, the VLP platform including the coat proteins of AP205 and Spytag includes Seq ID No. 11 or a biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99.5%, such as 100% sequence identity to the amino acid sequence of Seq ID No. 11. For example, the VLP platform including the coat proteins of AP205 and Spytag includes the following amino acid sequence: MAHIVMVDAYKPTKGGGSNKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVG IAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAE WETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTTA. As disclosed herein, Spytag is operable to be inserted at the N-terminus or the C-terminus of the coat proteins of AP205.

[0043] For example, the VLP platform including the coat proteins of MS2 and Spytag003 includes Seq. ID No.: 12 ora biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99.5%, such as 100% sequence identity to the amino acid sequence of Seq. ID No. 12. For example, the VLP platform including the coat proteins of MS2 and Spytag003 includes the following amino acid sequence: MARGVPHIVMVDAYKRYKGSGGSGGSGSNFTQFVLVDNGGTGDVTVAPSNFANG VAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWR SYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGLYGNFTQFVLVDNG GTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVA TQTVGGVE LPVAAWRS YLN M E LTI P I FATNS DCE LI VKAMQG LLKDG N PI PS Al AANTXTU-0009PCSGIY. As disclosed herein, Spytag003 is operable to be inserted at the N-terminus or the C-terminus of the coat proteins of MS2.

[0044] For example, the VLP platform including the coat proteins of MS2 and Spytag includes Seq. ID No. 13 or a biologically active sequence variant that has at least 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 96%, such as, 97%, such as 98%, such as 99%, such as 99.5%, such as 100% sequence identity to the amino acid sequence of Seq. ID No. 13. For example, the VLP platform including the coat proteins of MS2 and Spytag includes the following amino acid sequence: MAHIVMVDAYKPTKGGGSSNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNS RSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTI PIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGLYGNFTQFVLVDNGGTGDVTVA PSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVE LPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY. As disclosed herein, Spytag is operable to be inserted at the N-terminus or the C-terminus of the coat proteins of MS2.Materials and MethodsInsertions of Spytag003 or Spytag into the Coat Proteins

[0045] As shown in Figures 2A-2H the DNA sequence encoding Spytag003 peptide (RGVPHIVMVDAYKRYK) is inserted at either the N-terminus or the C-terminus of the coat proteins of bacteriophage Qp (GenBank: ACY07236.1), the coat proteins of the single-chain dimer of bacteriophage PP7, the coat proteins of AP205, and the single-chain dimer of MS2. In one or more embodiments, a flexible linker is incorporated between each Spytag003 or Spytag and the coat protein. The flexible linker improves the ability for the coat proteins to self assemble into a VLP as well as assure that Spytag003 or Spytag are exposed on the surface of the VLP for conjugation. Typically, the flexible linker includes a plurality of amino acids such as G or S. In one or more embodiments, a nine amino acid flexible linker, Seq. ID No.14 (GSGGSGGSG) is incorporated in-between each of Spytag003 sequence and coat protein to promote flexibility of the tag on the coat proteins and to reduce steric hindrance. The coat protein sequences with the insertions are codon-optimized for E. coli expression and are synthesized and cloned into a pET28a expression vector byTXTU-0009PCEpoch Life Sciences. In one or more embodiments, in addition to the insertions of Spytag003 peptide to the coat protein of PP7, the prototype Spytag (AHIVMVDAYKPTK) is separately inserted at either the N-terminus or the AB loop of the coat protein as shown in Figure 21 and 2J. For N-terminal insertion, a four amino acid flexible linker, Seq. ID No. 15 (GGGS) is included in-between Spytag and the coat protein. For the AB-loop insertion, Spytag (flanked by the four amino acid flexible linker and Kpnl site on both sides) is inserted between amino acids 138 and 139. For PP7, the insertion is done in pDSP7K expression vector, which expresses the singlechain dimer of PP7 coat protein.

[0046] Figures 2A-2F show a schematic design of Qp-Spytag003, PP7-Spytag003, and PP7-Spytag expression constructs. Schematics of pET28a-Qp-N-Spytag003 (Figure 2A), pET28a-Qp-C-Spytag003 (Figure 2B), pDSP7K-PP7-N-Spytag003 (Figure 2C), and pDSP7K-PP7-C-Spytag003 (Figure 2D) vectors show the insertions of Spytag003 peptide (RGVPHIVMVDAYKRYK) at the N-terminus or C-termini of the coat proteins of Qp and single-chain dimer PP7 (pDSP7k). Linker sequence, GSGGSGGSG, is highlighted. Insertions are done using Ncol and BamHI restriction sites. Schematics of pDSP7K-PP7-N-Spytag (Figure 2E) and pDSP7K-PP7-Spytag AB-loop (Figure 2F) vectors showing the insertions of Spytag peptide (AHIVMVDAYKPTK) in the N-terminus and in the AB-loop (in-between amino acids 138 and 139) of the coat protein of the single-chain dimer of PP7. Linker sequences (GGGS) used for insertion are within a rectangle, as shown in Figures 2A-2J. For AB-loop insertion, a Kpnl site (italicized, as shown in Figure 2F) flanking the linker sequences is included for cloning. The six amino acids that makes Spytag003 different from Spytag are in bold text in Figures 2A-2D while the three amino acids that makes Spytag different from Spytag003 are highlighted in bold text in Figures 2E and 2F.

[0047] Figures 2G-2J show a schematic design of the insertion of Spytag003 at the N-terminus (Figure 2G) and on the C-terminus (Figure 2H) of AP205 coat protein, and the insertion of Spytag003 at the N-terminus (Figure 2I) and on the C-terminus (Figure 2J) of the single chain dimer of MS2 coat protein. Nine amino acid linker sequence between Spytag003 and coat protein is are within a rectangle, as shown in Figures 2G-2J.TXTU-0009PC

[0048] In one or more embodiments, for protein expression in PP7 and Qp the plasmids are transformed into different strains of E. coli with different features: For protein expression, the plasmids are separately transformed into E. coli C41(DE3), BL21 Star pLysS, or Rosetta pLysS. Single colonies are inoculated into Luria-Bertani (LB) broth and grown at 37 °C until an OD600of about 0.6 is reached. Protein expression is induced with 0.5 mM isopropyl p-D-1 -thiogalactopyranoside (IPTG) at 37 °C for about 4 hours. Cells are harvested by centrifugation (5,000 x g, 10 min, 4 °C), lysed and lysates are run on SDS PAGE to confirm expression. Bacterial cells that showed the highest level of protein expression are screened for solubility as follows: bacterial pellets expressing PP7 and Qp coat proteins with Spytag003 or Spytag insertions are lysed with BugBuster® (Millipore Sigma; Cat 70584-M) supplemented with 0.1% Triton X-100, and soluble (supernatant) and insoluble (pellet dissolved in 8M urea) fractions are analyzed by SDS PAGE.

[0049] Insoluble bacterial pellets expressing recombinant coat proteins of PP7 and Qp are subjected to sequential lysis as follows. Cells are first lysed with Sepharose column buffer (SCB; 20 mM Tris-HCI pH 7.4, 100 mM NaCI, 0.1 mM MgS04), and supernatants are collected. The remaining pellets are subsequently re-lysed with Borax buffer (10mM sodium tetraborate decahydrate, and 10mM NaCI), followed by BugBuster reagent. Residual insoluble material is then solubilized using increasing concentrations of urea (0.5 M, 1 M, and 8 M, sequentially). Supernatants from each step are analyzed for the presence of target proteins by SDS PAGE.

[0050] In one or more embodiments, for protein expression in MS2 and AP205 the plasmids are transformed into different strains of E. coli with different features: C41(DE3) which promote the expression of toxic or membrane associated proteins, Rosetta 2(DE3)pLysS which promotes the expression of heterologous proteins with rare codons in bacteria, BL21 Star(DE3) that enhances RNA stability and consequently high levels of protein expression, or ArcticExpress (DE3) which promotes the yield of soluble proteins in E. coli. Individual bacterial colonies transformed with plasmids are picked from agar plates and are screened for protein expression as follows: colonies are resuspended in Luria-Bertani (LB) broth, and the cultures are grown for about 3 hours to about 4 hours after which the cultures are induced with 0.5 mM Isopropyl p-d-1 -thiogalactopyranoside (IPTG) for 3 hours. For induction of chaperone proteins in C41 cells (C41-pGro7 cells have a plasmid, pGro7,TXTU-0009PCexpressing chaperone proteins, groES and groEL), 0.5 mg / ml of arabinose is added to the growth media at the same time the media is inoculated with bacterial culture. The cultures are grown until an ODeoo of 0.6. Protein expressions are induced with 0.5 mM IPTG at 16 °C and room temperature for 16 hours or 37 °C for 4 hours. For protein expression in ArcticExpress cells, bacteria are grown until an ODeoo of 0.6 and protein is induced with same concentration of IPTG at 16 °C for 16 hours. After the indicated time of induction, all cultures are spun down, lysed with 8M urea and lysates are run on SDS PAGE gels. Bacterial cells that showed the highest level of protein expression are screened for solubility studies.Purification of Recombinant Coat Proteins and Assessment of Assembly into Chimeric VLPs

[0051] The recombinant coat proteins (e.g., the recombinant coat proteins described above of the PP7, Qp, AP205, MS2 or combinations thereof) are purified from a large-scale culture by lysing the bacterial pellet with the BugBuster protein extraction reagent. About 0.1% triton X-100 is added to bacterial lysates, the lysates are centrifuged at 10,000 rpm for 10 minutes and the supernatant are run on 23%, 29%, and 35% Optiprep gradient (Millipore Sigma; Cat D1556). The gradient is run at 50,300 rpm at 16 °C for 3.5 hours. For the purification of PP7 coat proteins with Spytag insertions, bacterial pellets are lysed with 0.2% lysozyme solution supplemented 0.05% with deoxycholate. DNase I (0.01 mg / mL) and MgCI2(0.2 M) are added to lysates and the mixtures are incubated at 37 °C for about 1 hour. Lysates are clarified by centrifugation at 3,700 rpm for 30 minutes at 4 °C, and ammonium sulfate (70% saturation) is added to the supernatant. Samples are incubated on ice for 30 min and centrifuged at 10,000 rpm for 10 minutes at 4 °C. Pellets are resuspended in SCB buffer (20 mM Tris-HCI, 100 mM NaCI, 0.1 mM MgS04, pH 7.4) and clarified by centrifugation (10,000 rpm, 10 minutes, 4 °C). Following ammonium sulfate precipitation, supernatants are loaded onto a Sepharose CL-4B size-exclusion column equilibrated with SCB buffer (20 mM Tris-HCI, 100 mM NaCI, 0.1 mM MgS04, pH 7.4) and purified. For example, the samples are purified by flowing the sample into the beads by gravity. Once all the samples are in the beads, SCB buffer is connected to the column and fractions (3 mL) from the column are collected and analyzed on SDS-PAGE gel. In one or more embodiments, the samples are purifiedTXTU-0009PCby gel filtration, fast protein liquid chromatography, or combinations thereof. The method results in the formation of the VLP portion of the VLP platform. The VLP portion of the VLP platform at least includes the recombinant coat proteins where the coat proteins include Spytag or Spytag003.Expression and Purification of Spycatcher0003 and a Foreign Protein of Interest

[0052] In one or more embodiments, the Spycatcher003 is fused with a target protein 112. For example, as shown in Figure 1 , the target protein 112 is bonded to the second portion 106 of the protein anchor system 108. Abacterial pellet expressing a foreign protein linked to Spychatcher-his-tag can be lysed with 0.2% lysozyme solution, BugBuster reagent, and / or with 2-8 M urea buffer (20 mM Nab PC , 20 mM Na2HPO4, supplemented with 400 mM NaCI, 50 mM imidazole, 10% tween 20, 10 mM beta-mercaptoethanol, pH 7.5). Supernatant with foreign protein can then be purified by affinity chromatography, ion-exchange chromatography or size exclusion chromatography.Conjugation of Spycatcher003 to the VLP Portion of the VLP Platform

[0053] For conjugation, for example, the PP7-N-Spytag VLPs are incubated with Spycatcher003 (Bio-Rad; Cat TZC025) at 1:3.25 molar ratio, respectively. To conjugate Spycatcher003-taret protein to the VLPs, purified Spycatcher003-target protein is conjugated to PP7-N-Spytag VLPs at 1:0.43 molar ratio. In one or more embodiments, Qp-N-Spytag VLPs are incubated with Spycatcher003 at a 1:3 molar ratio. The conjugation results in a covalent bond between at least the first portion 104 of the protein anchor system 108 and the second portion 106 of the protein anchor system 108. All reactions are carried out at room temperature for 3 hours. Conjugation products are analyzed by SDS PAGE, and band intensities are compared using Imaged software to assess relative conjugation efficiency.

[0054] For conjugation, in another example, the Spycatcher003-target protein and AP205-N-Spytag003 VLPs are incubated with at a 1.5:1 molar ratio, respectively. To conjugate Spycatcher003-target protein to the VLPs, purified Spycatcher003-target protein is conjugated to AP205-N-Spytag003 VLPs and to MS2-N-Spytag003 VLPs at 1:8 molar ratio and 1.7:1 molar ratio, respectively. All bio-conjugation reactions areTXTU-0009PCincubated at room temperature for about 1.5 hours to about 3 hours after which they are run on an SDS PAGE gel to assess conjugation efficiency.

[0055] In one or more embodiments, conjugation occurs at a ratio of VLP first portion to second portion of the protein anchor system at a molar ratio of 1:1, 1:1.5, 1:2, 1:3, 1:4, 2:2, or 2:1. In one or more embodiments, the molar ratio of VLP to conjugated protein ranges from about 10:1 to about 1:10, including any sub-ranges thereof, depending on antigen size, valency, and desired surface density. The molar ratios disclosed herein may apply to any of the disclosed VLPs (e.g., bacteriophage PP7, bacteriophage Qp, bacteriophage AP205, bacteriophage, or bacteriophage MS2) with a first portion 104 of the anchor system and the second portion 106 of the anchor system. For example, the molar ratio is 1:8, VLP with a first portion 104: second portion 106. In one or more embodiments, the conjugation occurs for up to 12 hours at room temperature.Transmission Electron Microscopy (TEM) analysis

[0056] Purified conjugated VLPs are absorbed onto carbon-coated, glow-discharged copper grids for 2 minutes. The grids are washed with distilled water and negatively stained with 2% uranyl acetate for 2 minutes. Conjugated VLPs are visualized using a Hitachi H-7500, H-7650, or HT7700 transmission electron microscope at varying magnification such as 50,000X-70,000X.Statistical Analysis

[0057] Statistical analysis is performed using GraphPad Prism software. Comparisons between groups are made using Mann-Whitney U tests. P-values < 0.05 are considered statistically significant.ResultsInsertion of Spytag003 and Spytag into the Coat Proteins of Q / 3 and PP7

[0058] Four constructs are designed with Spytag003 inserted at either the N-terminus or C-terminus of the coat proteins of Qp and PP7 (single-chain dimer): pET28a-Qp-N-Spytag003, pET28a-Qp-C-Spytag003, pDSP7K-N-Spytag003 and pDSP7K-C-Spytag003, as shown in Figures 2A-2D. The coat proteins of Qp with N-TXTU-0009PCand C-terminal insertions (Qp-N-Spytag003 and Qp-C-Spytag003) and the coat protein of PP7 with N- and C-terminal insertions (PP7-N-Spytag003 and PP7-C-Spytag003) are successfully expressed in C41 cells. As shown in Figures 3A and 3B, bands of about 16.78 KDa and about 30.59 KDa (which correspond to expected sizes of the coat proteins of Q and PP7 with insertions, respectively) are observed in the induced samples but not in uninduced samples. The expression levels of Qp with Spytag003 inserted at the N-terminus are better than the insertion at the C-terminus. PP7 with the insertion of Spytag003 at the N-terminus of the coat protein did not show any signs of protein expression. To evaluate whether lack of visible levels of protein expression is due to the host cell in question, pDSP7K-N-Spytag is transformed (the expression vector that contained the protein) into other strains of E. coli (Rosetta pLysS and BL21 StarpLysS). Rosetta 2 is a strain of BL21 cells with additional copies of genes that code for rare tRNA, which promotes the expression of heterologous proteins with rare codons in bacteria. BL21 Star, on the other hand, is a strain of BL21 cells with a mutation in rne131 gene which enhances RNA stability and consequently high-levels of protein expression. As shown in Figure 3C, only PP7 with the insertion of Spytag003 at the C-terminus is expressed in both cells.

[0059] To determine if expressed proteins are soluble for purification purposes and for the purpose of evaluating assembly to VLPs, solubility studies are conducted. Bacterial pellets expressing Qp-N-Spytag003, Qp-C-Spytag003 and PP7-C-Spytag003 coat proteins are lysed with BugBuster reagent supplemented with 0.2% Triton-X 100. Only Qp-N-Spytag003 and PP7-C-Spytag003 are partially soluble after lysis with the reagent, as shown in Figure 4A and Figure 4B.

[0060] Attempts to purify the samples by ultracentrifugation using 23%, 29%, and 35% Opti Prep density gradients or 29%, 35%, and 41% Opti Prep density gradients supplemented with 0.1% Triton-X 100 are unsuccessful. In most of the layers of samples collected after ultracentrifugation, Qp-N-Spytag003 and PP7-C-Spytag003 coat proteins co-purified with contaminating bacterial proteins. Sequential lysis of bacterial pellet (expressing the recombinant coat proteins) with SCB, Borax, and BugBuster, followed by lysis with increasing concentrations of urea (0.5-8 M), removed some of the contaminating bacterial proteins and improved solubility of the proteins (Figure 5A and 5B). To assess whether some of the solubilized proteins with low concentration of urea (0.5 and 1 M) are VLPs, a buffer-exchange (of urea) isTXTU-0009PCcompleted with PBS buffer and concentrated the samples and analyzed them under the TEM. Small oval to irregular-shaped protein structures (particles), -5 nm for QP-N-Spytag003 and -11-14 nm for PP7-C-Spytag003, are observed under TEM, shown in Figure 5C and 5D. These results suggest that Qp-N-Spytag003 (coat proteins) and PP7-C-Spytag003 (small nanoparticles) can be successfully purified from bacterial lysates. In one or more embodiments, the potential of the purified Qp-N-Spytag003 coat protein to (re-)assemble into VLPs can be explored in vitro (re-assembly). In one or more embodiments, the purification process can be explored using different lysis buffer to prevent denaturation of the protein.

[0061] Insertion of Spytag into the N-terminus or the AB-loop (between residues 138-139) of the coat protein of the single-chain dimer of PP7 allowed for the coat proteins to assemble into VLPs. As mentioned above, Spytag is three amino acids shorter than Spytag003 and differs from Spytag003 by 6 amino acids. Similar to the insertion of Spytag003, the insertion of Spytag on the coat protein of PP7 (N-terminus or AB-loop) did not affect the expression of the coat proteins, as shown in Figure 6A and 6B. The insertion of Spytag did not affect the assembly of the coat proteins into VLPs. As shown in Figure 7A, 7B, 7C, and 7D, purified PP7 coat proteins (expected size of -30.59 KDa) with Spytag insertion at either the N-terminus or the AB-loop assembled into VLPs with diameters of about 27 nm to about 30 nm. Given the fact that the insertion of Spytag (unlike Spytag003 above) into the N-terminus of PP7 did not affect the solubility of the recombinant protein nor its ability to assemble into VLPs, the insertion of Spytag on the N-terminus of Qp is expected to not affect the solubility nor the ability of Qp to assemble into VLPs.

[0062] Spycatcher003 conjugated to Spytag on the VLPs even though the former differs from the prototype Spycatcher (the binding partner of Spytag) by 13 amino acids. Given the fact that Spycatcher003 can bind covalently to the prototype Spytag, foreign proteins of interest can be expressed as recombinant proteins linked to Spytag003 and conjugated to the PP7-N-Spytag VLPs or any other bacteriophage (e.g., Qp).Insertion of Spytag003 and Spytag into the Coat Proteins of AP205 and MS2

[0063] Bacteriophage AP205 coat protein with Spytag003 insertions at both the N-and C-termini (AP205-N-Spytag003 and AP205-C-Spytag003, respectively) areTXTU-0009PCsuccessfully expressed at high levels in C41 cells, as shown in Figure 8A. For bacteriophage MS2, the coat protein with insertion at the N-terminus (MS2-N-Spytag003) is expressed in these cells, as shown in Figure 8B. MS2-C-Spytag003 (with a Spytag003 insertion at the C-terminus) is not expressed in C41 cells, expressing the protein in BL21 Star and Rosetta cells is attempted. The protein is expressed in Rosetta cells (at least 4 colonies after transformation), as shown in Figure 8C. However, no colonies are found on agar plates of BL21 Star cells transformed with plasmid expressing MS2-C-Spytag003. The cells are transformed with the vector, pDSP-MS2-N-Spytag003, that is expressible in C41 cells. There is no expression of MS2-N-Spytag003.

[0064] To assess if the expressed proteins are soluble, for purification downstream, bacterial cells expressing the proteins above (AP205-N-Spytag003, AP205-C-Spytag003, MS2-N-Spytag003, MS2-C-Spytag003) are induced at 37 °C for 4 hours; the cells are lysed with BugBuster® protein extraction reagent, or 2% lysozyme solution and supernatants are run on SDS PAGE gel. As shown in Figures 9A and 9B, the proteins are not soluble when expressed at 37 °C regardless of the lysis buffer used. When the proteins are expressed at 20 °C, only the coat proteins with N-terminal insertions (AP205-N-Spytag003 and MS2-N-Spytag003) are soluble in BugBuster® protein extraction reagent, as shown in Figures 9C and 9D. To assess if the proteins with C-terminal insertions (AP205-C-Spytag003 and MS2-C-Spytag003) would be soluble if expressed in different bacterial host, the protein expression vectors are transformed into C41 cells (C41-pGro7 cells have a plasmid, pGro7, that expresses two chaperone proteins, groES and groEL) and ArcticExpress cells. AP205-C-Spytag003 is not soluble in both cells when expressed at 16 °C. MS2-C-Spytag003 is expressed at very low levels and is soluble in C41 cells (with groES and groEL) induced at 16 °C and room temperature for 16 hours or 37 °C for 4 hours (Figure 9E).

[0065] Purification is completed using Optiprep with the addition of 0.2% Triton-X 100 to lysate and buffer led to separation of the proteins, after centrifugation, into layer 7 for AP205 N-Spytag003 (as shown in Figure 10A, left and middle images) and layer 6 for MS2-N-Spytag003 (as shown in Figure 10B, left and middle images). Analysis of the purified coat proteins showed that the coat proteins assembled into VLPs;TXTU-0009PCAP205-N-Spytag003 VLPs (Figure 10A, right image) and MS2-N-Spytag003 VLPs (Figure 10B, right image). To assess whether Spytag003 is exposed on the surface of the VLPs (for bio-conjugation applications), conjugation of Spytag003’s partner protein, Spycatcher003 to AP205-N-Spytag003 VLPs is attempted. As shown in Figure 11A and Figure 11B, Spycatcher003 (about 15.2 KDa) is successfully bioconjugated on the VLPs. Spycatcher003 conjugated to AP205-spytag003 (i.e. Spycatcher003-AP205-spytag003 platform) migrated on SDS PAGE gel at about 34 KDa, which is close to the sum of about 15.2 KDa (Spycatcher003) and 16.5 KDa (AP205-spytag003); Figure 11 A. The result is further confirmed by TEM (Figure 11 B).Chimeric VLPs Conjugated with Spycatcher003 and Foreign Protein of Interest

[0066] To evaluate whether Spytag is displayed on the VLPs, the conjugation of Spytag binding partner-related protein (Spycatcher003) on PP7-N-Spytag VLPs is tested. Spycatcher003 differs from the prototype Spytag binding partner (Spycatcher) by only 13 amino acids. Spycatcher is available commercially and is used to assess the display of Spytag on PP7 VLPs. In one or more embodiments, Spycatcher003 is operable to bind with Spytag and Spytag003 and Spycatcher is operable to bind with Spytag and Spytag003. The molar ratio of 1:3.25 (VLP:Spycatcher003) had the highest rate of conjugated protein with the conjugation efficiency of 84%. As shown in Figure 12, Spycatcher003 is successfully conjugated on PP7-N-Spytag VLPs. The successful conjugation of Spycatcher003 on PP7-N-Spytag VLPs indicates the VLP platform including at least PP7 is operable to display a foreign protein of interest (e.g., a target protein). In one or more embodiments, the conjugation success of PP7 may translate to Q0, where Spycatcher003 is conjugated on Q0-N-Spytag VLPs.

[0067] The molar ratios disclosed herein may apply to any of the disclosed VLPs (e.g., bacteriophage PP7, bacteriophage Qp, bacteriophage AP205, bacteriophage, or bacteriophage MS2) with a first portion 104 of the anchor system and the second portion 106 of the anchor system (e.g., Spycatcher003). For example, the molar ratio is 1 :8, VLP with a first portion 104: second portion 106. In one or more embodiments, the conjugation occurs for up to 12 hours at room temperature.

[0068] As shown in Figure 13A and 13B, the Spycatcher003-target protein is successfully conjugated on both AP205-N-Spytag003 and MS2-N-Spytag003 VLPs indicating that Spytag003 is exposed on the surface of both VLPs. AP205-N-TXTU-0009PCSpytag003 VLPs formed a complex with Spycatcher003-target protein that migrated at about 60.0 KDa, which is the expected size (about 16.5 KDa for recombinant VLPs + about 43.5 KDaforSpycatcher003-target protein) (Figure 13A). Similarly, the mixing of Spycatcher003-target protein with MS2-N-Spytag003 VLPs gave rise to a complex that migrated at about 73.8 KDa, which is the expected size (about 30.2 KDa for recombinant VLPs + about 43.5 KDa for Spycatcher-target protein) (Figure 13B). The conjugations only took about 1.5 hours to about 3 hours, and efficiency for AP205-N-Spytag003-Spycatcher003 is 66%, for AP205-N-Spytag003-Spycatcher003-target protein VLPs is 40%, and for MS2-N-Spytag003-Spycatcher003-target protein VLPs is 68%. The result is further confirmed by the TEM data shown in Figures 13C and 13D.

[0069] Overall, embodiments of the present disclosure generally relate to a platform for vaccine delivery. Specifically, providing a VLP as a platform for vaccine delivery. The VLP platform disclosed herein allows for the display of foreign proteins on varying VLP platforms to minimize any potential anti-platform immune responses. Spytag003 is successfully inserted to the N-termini of the coat proteins of AP205 and MS2; insertions did not affect the ability of the coat proteins to assemble into chimeric VLPs. Spytag is successfully inserted to the N-termini of the coat proteins of PP7 and Qp, the insertions did not affect the ability of the coat proteins to assemble into chimeric VLPs. PP7-N-Spytag, Qp-N-Spytag, AP205-N-Spytag003, and MS2-N-Spytag003 disclosed herein can be used to display foreign peptides and proteins for candidate vaccine studies with reduced concern of pre-existing antibodies in population.

[0070] While the foregoing is directed to embodiments of the present disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

TXTU-0009PCWhat is claimed is:

1. A virus-like particle (VLP) platform comprising:a bacteriophage with a plurality of coat proteins, the plurality of coat proteins operable to assemble into a VLP; anda protein anchor system comprising:a first portion inserted in at least one coat protein of the plurality of coat proteins; anda second portion, wherein the first portion and the second portion are operable to link together.

2. The VLP platform of claim 1, wherein the first portion comprises Spytag, Spytag003, or combinations thereof.

3. The VLP platform of claim 1, wherein the second portion comprises Spycatcher, Spycatcher003, or combinations thereof.

4. The VLP platform of claim 1 , wherein the bacteriophage comprises at least one of a bacteriophage PP7, a bacteriophage Qp, a bacteriophage AP205, or a bacteriophage MS2.

5. The VLP platform of claim 1 , wherein the first portion is inserted in a N-terminus or an AB-loop end of the at least one coat protein of the plurality of coat proteins.

6. The VLP platform of claim 1, wherein the VLP platform comprises an amino acid sequence of Seq. ID No. 10 or a biologically active sequence variant that has at least 98% sequence identity to the amino acid sequence of Seq. ID No. 10.

7. The VLP platform of claim 1, wherein the VLP platform comprises an amino acid sequence of Seq. ID No. 12 or a biologically active sequence variant that has at least 98% sequence identity to the amino acid sequence of Seq. ID No. 12.

8. The VLP platform of claim 1 , wherein a diameter of the VLP platform is about 20 nanometers (nm) to about 200 nm.

9. A virus-like particle (VLP) platform comprising:TXTU-0009PCa bacteriophage with a plurality of coat proteins, the plurality of coat proteins operable to assemble into a VLP; anda protein anchor system comprising:a first portion inserted into a N-terminus or an AB-loop at least one coat protein of the plurality of coat proteins; anda second portion coupled to at least a foreign protein of interest, wherein the first portion and the second portion are operable to link together.

10. The VLP platform of claim 9, wherein the first portion comprises Spytag, Spytag003, or combinations thereof.

11. The VLP platform of claim 9, wherein the second portion comprises Spycatcher, Spycatcher003, or combinations thereof.

12. The VLP platform of claim 9, wherein the bacteriophage comprises at least one of a bacteriophage PP7, a bacteriophage Qp, a bacteriophage AP205, or a bacteriophage MS2.

13. The VLP platform of claim 9, wherein the VLP platform comprises an amino acid sequence of Seq. ID No. 10 or a biologically active sequence variant that has at least 98% sequence identity to the amino acid sequence of Seq. ID No. 10.

14. The VLP platform of claim 9, wherein the VLP platform comprises an amino acid sequence of Seq. ID No. 12 or a biologically active sequence variant that has at least 98% sequence identity to the amino acid sequence of Seq. ID No. 12.

15. A method of forming a virus-like particle (VLP) platform comprising:expressing a first portion of a protein anchor system in a plurality of coat proteins of a bacteriophage;forming a recombinant coat protein such that the plurality of coat proteins form the VLP and the first portion of the protein anchor system is expressed along an exterior of the VLP;fusing a target protein with a second portion of the protein anchor system; andTXTU-0009PCconjugating the VLP portion with the second portion of the protein anchor system to form a VLP platform such that the first portion of the anchor protein system forms a bond with the second portion of the anchor protein system.

16. The method of claim 15, further comprising purifying the VLP portion by gel filtration, fast protein liquid chromatography, or combinations thereof.

17. The method of claim 15, wherein the bacteriophage comprises at least one of a bacteriophage PP7, a bacteriophage Qp, a bacteriophage AP205, ora bacteriophage MS2.

18. The method of claim 15, wherein the VLP portion and the second portion of the protein anchor system are mixed at a molar ratio of 1:1, 1:1.5, 1:2, 1 :3, 1:4, 2:2, or 2:1.

19. The method of claim 15, wherein the first portion is inserted in a N-terminus or an AB-loop end of the plurality of coat proteins.

20. The method of claim 15, wherein a diameter of the VLP platform is about 20 nanometers (nm) to about 200 nm.

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