A novel virus-like particle platform based on intein-mediated bioconjugation with target antigen and encapsulated with immunomodulatory factors

Abstract

The present invention deals with a novel virus-like particle (VLP) platform for developing enhanced vaccines. This novel VLP-platform consists of two unique attributes: i) intein-mediated protein bioconjugation, a method that allows for precise and efficient attachment of target antigens to the VLP surface without leaving undesirable residual tags or scars; and ii) encapsulation of different small molecule immunomodulators (including cytokines, adjuvants, and small molecule metabolites) for specific priming of tailored immune response through influencing the outcome of interactions between the antigen-presenting cells (APCs) and the T-cells towards eliciting a specific type of immune response (Th1: humoral, Th1: cell mediated, Tfh, Th2, Th17, or Treg). This intein-mediated bioconjugation based VLP platform facilitates a "plug-and-display" approach, significantly reducing the time and resources required for development of vaccines suitable for a wide range of applications, including vaccines against infectious diseases, cancer immunotherapies, and treatments for autoimmune disorders. Additionally, encapsulation of immunomodulatory factors within the VLP core, enables delivery to APCs thereby generating robust and specific type of immune response for enhanced vaccine efficacy. This invention provides a versatile, scalable, and cost-effective solution for rapid development of vaccines that induce comprehensive and tailored immune responses, including both humoral and cellular immunity with long-term memory.

Description

A NOVEL VIRUS-LIKE PARTICLE PLATFORM BASED ON INTEIN-MEDIATED BIOCONJUGATION WITH TARGET ANTIGEN AND ENCAPSULATED WITH IMMUNOMODULATORY FACTORS

[0001] The present invention relates to the field of recombinant vaccines, specifically to the design and development of a novel Virus-Like Particle (VLP) platform capable of encapsulating various immunomodulatory factors within its core and the VLP surface can be efficiently bioconjugated with different antigens of choice (target antigens) through specific intein-mediated bioconjugation, and enables site-specific delivery of encapsulated immunomodulatory factors to generate tailored immune response against target antigens.

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome, rendering them non-infectious. Due to their structural similarity to viruses, VLPs are highly immunogenic, making them ideal platforms for vaccine development. VLPs can present antigens in a repetitive and multivalent manner, which enhances immune recognition and activation, leading to robust antibody responses. As a result, VLP-based vaccines have become an attractive strategy for combating various infectious diseases, including hepatitis B, human papillomavirus (HPV), and others. Despite their promise, current VLP technologies face significant challenges in terms of flexibility of different antigen integration, scalability, and the induction of a comprehensive and desired type of immune response, including both humoral and cellular immunity.

[0004] The majority of VLP-based vaccines rely on genetic fusion or chemical conjugation techniques to display the target antigens on the VLP surface. Genetic fusion methods involve inserting the gene encoding the target antigen into the capsid protein sequence of the VLP. This approach often disrupts the capsid protein’s ability to self-assemble into VLPs due to structural alterations, necessitating extensive optimization for each specific antigen. For example, GSK’s RTS,S / AS01 (Mosquirix), the first licensed malaria vaccine, took over 20 years of development. A significant portion of this time was spent optimizing the production of chimeric VLPs, as inserting the malaria antigen into the hepatitis B surface antigen capsid protein backbone required complex adjustments to maintain the VLP structure. Such lengthy development timelines and the need for extensive optimization pose substantial barriers to the rapid deployment of vaccines, especially during outbreaks of emerging infectious diseases besides adding to the costs.

[0005] Chemical conjugation techniques, another commonly used approach, involve chemically linking antigens to the VLP surface. While this method avoids the structural disruptions associated with genetic fusion, it often lacks site specificity, which can result in heterogeneous products with variable immunogenicity. Moreover, chemical conjugation may leave behind undesirable chemical residues, and the harsh conditions required for the reactions can damage sensitive antigens, reducing their immunogenicity. The unpredictability and complexity of these processes increase the cost and time required for vaccine development.

[0006] Additionally, current VLP-based vaccines primarily focus on eliciting antibody responses. However, for many infectious diseases, including those caused by intracellular pathogens like viruses and certain bacteria, effective protection also requires a robust cellular immune response, particularly the activation of T-cells. Existing VLP platforms often struggle to induce strong T-cell responses, limiting their efficacy against such pathogens. Moreover, while some VLPs have shown promise in presenting antigens for cancer immunotherapy, the capacity to modulate immune responses specifically to target tumors or autoimmune conditions remains limited.

[0007] Several companies and research institutions are developing VLP-based vaccines using alternative technologies. For example, SpyBiotech employs the SpyCatcher-SpyTag system for antigen conjugation. This technology utilizes a peptide-protein pair that spontaneously forms a covalent bond, allowing antigens to be "plugged" onto the VLP surface. While this method provides a rapid and flexible way to display antigens, it leaves behind a significant residual scar—approximately 120 amino acids—from the conjugation tags. This residual sequence can be immunogenic itself, potentially interfering with the immune response against the target antigen and complicating the vaccine formulation.

[0008] Other approaches involve using chemical cross-linkers or coupling agents that enable antigen attachment through covalent bonds. However, these methods often face issues of reproducibility and scalability. The need for stringent reaction conditions can also pose compatibility challenges with the antigen or VLP components. Furthermore, as chemical conjugation typically lacks site specificity, it may result in a mixture of product variants, complicating downstream purification and quality control processes.

[0009] The insertion of antigens into the capsid protein backbone is another commonly employed strategy but requires meticulous optimization to ensure that the VLPs retain their structural integrity and immunogenic properties. For instance, in the development of tetravalent dengue vaccines, extensive optimization was necessary to successfully insert multiple dengue virus antigens into the VLP capsid proteins. This time-intensive process significantly lengthened the pre-clinical phase, delaying the availability of effective vaccines.

[0010] The principal drawback of these existing technologies lies in their limited flexibility and scalability, particularly when targeting a wide variety of diseases. Each antigen requires a tailored approach to ensure proper integration into the VLP structure, a process that is both time-consuming and resource-intensive. This limitation is especially problematic when rapid vaccine development is needed, such as in response to emerging pathogens or in developing personalized vaccines for cancer immunotherapy.

[0011] Given the challenges associated with current VLP-based vaccine technologies, there is a pressing need for a versatile and efficient platform that can rapidly accommodate a wide array of antigens with minimal optimization. Such an improved VLP platform would significantly reduce the time and cost associated with vaccine development and enable the production of vaccines that elicit comprehensive immune responses, including both humoral and cellular immunity. Furthermore, a VLP platform that allows for the targeted delivery of encapsulated immunomodulatory factors could specifically modulate priming of the immune response through influencing the cross-talk between the Antigen Presenting Cells (APCs) and the T-cells to generate a tailored immune response towards the target antigen, is highly desirable, and could potentially improve the vaccine efficacy against both infectious and non-communicable diseases.

[0012] To address these needs, the present invention aims to provide a novel VLP platform that leverages intein-mediated bioconjugation of the target antigen as a promising solution. Inteins are protein segments that can excise themselves from a host protein sequence and facilitate the ligation of the flanking protein sequences, effectively "splicing" them together. This intein-mediated bioconjugation allows for site-specific and efficient linkage of antigens to VLPs under mild physiological conditions, preserving antigen integrity and VLP assembly. The result is a "plug-and-display" approach that requires minimal optimization for each new antigen, drastically reducing the pre-clinical development phase. Additionally, the encapsulation of immunomodulatory factors within the VLP core could serve as a novel strategy for modulating the immune response. By delivering these factors directly to the APCs, such VLP platform can prime and enhance the desired immune response against the target antigen. This capability is particularly advantageous for designing vaccines against pathogens that require a balanced immune response, including strong T-cell activation with a possibility of generating long term immune memory against the target antigen, also applicable for conditions like cancer and autoimmune disorders, where modulating immune responses is crucial for therapeutic success.

[0013] The intein-mediated VLP platform introduces several key advantages over existing technologies:Near trace-less Conjugation: Intein-mediated bioconjugation leaves minimal (few amino acids) to absolutely no residual tag remnants, avoiding the immune interference issues seen with other technologies like SpyCatcher-SpyTag.Modularity and Flexibility: The VLP platform’s modular design allows for the rapid bioconjugation with various antigens without extensive optimization, significantly accelerating the pre-clinical development process.Enhanced Immune Modulation: By encapsulating immunomodulatory factors within the VLP core, this platform can deliver these agents directly to APCs, enhancing the specificity and strength of the desired type of immune response against the target antigen.Broad Applicability: Such VLP platform is versatile enough to be used for both infectious diseases (caused by various pathogens) and non-communicable conditions like cancer and autoimmune disorders, expanding its potential applications beyond traditional vaccine targets.Scalability and Cost-Effectiveness: The use of intein-mediated conjugation under mild physiological conditions simplifies the production process, making it more scalable and cost-effective compared to current VLP technologies that require harsh conditions or extensive adjustments.The novel VLP platform of the present disclosure represents a significant advancement in the field of vaccine technology. By addressing the limitations of current VLP-based approaches, this intein-mediated bioconjugation enabled novel VLP-platform offers a versatile, efficient, and scalable solution for developing vaccines against a wide range of diseases. Further, its ability to modulate immune responses through site-specific delivery of encapsulated immunomodulatory factors further enhances its potential as a next-generation vaccine platform, capable of addressing the growing global need for more effective and rapidly deployable vaccines.

[0014] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.OBJECTS OF THE INVENTION

[0015] The principal object of the present invention is to provide a VLP platform that utilizes intein-mediated bioconjugation for the efficient, site-specific, and near trace-less attachment of antigens.

[0016] Another object of the present invention is to enhance the immunogenicity of the VLP-surface bioconjugated antigens, facilitating the rapid and scalable production of vaccines.

[0017] Another object of the present invention is to provide a VLP platform capable of specific and efficient bioconjugation with a wide variety of antigens, including viral, bacterial, fungal, parasitic, cancer, and autoimmune disease-related antigens, without compromising the structural integrity of the VLP or the antigen.

[0018] Another object of the present invention is to significantly shorten the pre-clinical development phase of VLP-based vaccines by employing a “plug-and-display” approach using intein-mediated bioconjugation with various antigens of interest (AOI). This reduces the extensive optimization typically required in existing technologies, thus saving time and resources.

[0019] Yet another object of the present invention is to establish a bioconjugation method that leaves minimal to no residual tags or scars on the final VLP-antigen product, ensuring that the conjugation process does not interfere with the immunogenic properties of the antigen or the functionality of the VLP.

[0020] Yet another object of the present invention is to encapsulate the VLP core with immunomodulatory (small molecules: metabolites, adjuvants / cytokines) factors capable of modulating the priming and outcome of the adaptive immune response of a specific type (Th1: humoral, Th1: cell-mediated, Tfh, Th17, Th2 or Treg) against the target antigen through influencing the cross-talk between the APCs and the T-cells. This ensures obtaining tailored robust immune response against the target antigen thereby enhancing the vaccine efficacy.SUMMARY

[0021] This invention relates to the field of recombinant vaccines, particularly the development of a novel and versatile VLP platform designed to improve the immunogenicity of a wide range of antigens. This innovative VLP platform employs intein-mediated bioconjugation to efficiently and site-specifically bioconjugate with antigens of choice to the surface of pre-assembled and purified VLPs. The approach enables a “plug-and-display” mode, facilitating the rapid screening and prioritization of vaccine candidates, thereby accelerating the pre-clinical development of vaccines.

[0022] The VLP platform addresses critical limitations in current VLP-based vaccine technologies, such as the extensive optimization required for antigen insertion within capsid proteins, which often disrupts the VLP structure and significantly extends development timelines. Traditional methods, like those used in GSK's RTS,S / AS01 malaria vaccine and other dengue vaccines, involve complex processes to ensure proper VLP assembly, often necessitating dilution with wild-type backbones or employing chemical conjugation techniques that leave undesirable remnants on the final product.

[0023] In contrast, VLP platform of the present disclosure utilizes intein-based protein bioconjugation, which is highly efficient and nearly trace-less, leaving minimal or no residual amino acids from the conjugation tags. This not only preserves the structural integrity and immunogenicity of the VLP-antigen complex but also significantly reduces the time and cost involved in pre-clinical development. Additionally, this novel VLP platform can be employed for the encapsulation of various immunomodulatory factors, including cytokines, adjuvants, and small molecule metabolites, within the VLP core. This feature allows for precise site-specific delivery of these factors to the APCs, enhancing generation of tailored immune response against the target antigen and thereby improving vaccine efficacy.

[0024] The versatility of this novel VLP platform extends to its application across a broad spectrum of infectious diseases where antibodies play a primary protective role. Furthermore, it can be adapted for use in non-infectious diseases, such as cancer and autoimmune disorders, by priming effective modulation of immune response through delivery of the encapsulated immunomodulators for generating specific T-cell responses (Th1: humoral, Th1: cell-mediated, Tfh, Th2, Th17 or Treg). This adaptability makes the platform a powerful tool for modulating immune responses, either by enhancing or selectively abrogating specific signaling pathways, for obtaining a tailored immune response effective against the target antigen and as per the therapeutic needs of various cancers and autoimmune disorders.

[0025] Compared to existing technologies, VLP platform of the present disclosure offers several distinct advantages. For instance, the SpyCatcher-SpyTag system used by some competitors, while similar in its plug-and-display approach, leaves a substantial scar of about 120 amino acids from the conjugation tags, which can potentially interfere with the desired immune response against the target antigen or the functionality of the VLP. The ntein-based method of the present disclosure overcomes this limitation by providing a near scar-free bioconjugation process with trace-less or minimal (few amino acids) carryover from the conjugation-tag sequences, allowing for a cleaner and more efficient conjugation of target antigen with the VLP for vaccine development.

[0026] Another significant advantage of the novel VLP platform is its ability to encapsulate and deliver immunomodulatory factors (including cytokines, adjuvants, and small molecule metabolites) alongside priming the immune system against the target antigen. This capability provides a dual mechanism of action—one that primes APCs and another that modulates the ensuing immune response towards promoting a tailored, robust and specific immune response against the target antigen for enhancing the vaccine efficacy. This approach not only improves the initial priming of specific immune response but could also support the induction of strong memory of immune responses, crucial for long-term protection.

[0027] Furthermore, this design of novel and versatile VLP platform of the present disclosure supports the development of combination multivalent vaccines by enabling the presentation of multiple distinct antigens on the VLP surfaces. Batches of VLPs each could be easily bioconjugated with target antigens from multiple strains of a pathogen or a range of related pathogens. The desired combinations of such VLPs could then be co-administered, as well as for creating combination vaccines that can protect against multiple diseases simultaneously.

[0028] The commercial potential of this invention is significant, given the estimated market size of over USD 20 billion by 2030 for vaccines targeting both infectious and non-infectious diseases. The scalability and versatility of the novel VLP platform makes it suitable for large-scale commercial production, meeting the high demand for effective vaccines in global markets. Additionally, the modularity of this novel VLP platform allows for rapid adaptation and customization, which is particularly valuable in responding to emerging infectious threats or tailoring vaccines to individual patient needs in personalized medicine.

[0029] Overall, this invention offers a transformative approach to VLP-based vaccine development. By leveraging intein-mediated bioconjugation and the encapsulation of immunomodulatory factors, the novel VLP platform provides a powerful, flexible, and efficient tool for enhancing vaccine efficacy across a wide range of applications. This innovative technology not only addresses the shortcomings of existing methods but also opens new avenues for the development of next-generation vaccines that are faster, more effective, and more adaptable to diverse medical challenges.

[0030] These and other features will become apparent from the following detailed description of illustrative embodiments thereof, which is to be read in connection with the accompanying drawings. While the invention has been described and shown with reference to the preferred embodiment, it will be apparent that variations might be possible that would fall within the scope of the present invention.

[0031] So that the manner in which the above-recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may have been referred by embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

[0032] These and other features, benefits, and advantages of the present invention will become apparent by reference to the following text figure, with like reference numbers referring to like structures across the views, wherein:

[0033] shows a schematic representation of the novel VLP platform along with all steps and major components involved therein.

[0034] shows AP-205-VLP bioconjugated with AOI (R0.6C, a chimericPlasmodium falciparumantigen with subdomains from GLURP and Pfs48 / 45) and encapsulated with Resiquimod.

[0035] shows split HBcAg-VLP bioconjugated with AOI (JEV-EDIII, a subdomain from Japanese Encephalitis Envelop protein) and encapsulated with Kynurenine.

[0036] Figure 4 shows immunomodulator encapsulation efficiency within the VLP core as a function of the various concentrations of the immunodulator used during the encapsulation process.DETAILED DESCRIPTION OF THE INVENTION

[0037] While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and the detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claim.

[0038] As used throughout this description, the word "may" is used in a permissive sense (i.e. meaning having the potential to), rather than the mandatory sense, (i.e. meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein are solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers, or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents acts, materials, devices, articles, and the like are included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention.

[0039] In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase “comprising”, it is understood that we also contemplate the same composition, element, or group of elements with transitional phrases “consisting of”, “consisting”, “selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.

[0040] The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only and are not intended to limit the scope of the claims. In addition, several materials are identified as suitable for various facets of the implementations.

[0041] The present invention pertains to a novel and versatile VLP platform designed to enhance the immunogenicity of various antigens for vaccine development. This VLP platform leverages intein-mediated bioconjugation to achieve efficient, site-specific, and trace-less conjugation of target antigens to the surface of pre-assembled and purified VLPs. Additionally, the VLP core encapsulated with immunomodulatory factors (including cytokines, adjuvants, and small molecule metabolites) could stimulate generation of tailored, robust and specific type of immune response against the target antigens for enhancing the vaccine efficacy. This invention thus, represents a significant advancement in the field of recombinant vaccines, offering a streamlined, adaptable and scalable approach to developing vaccines with enhanced efficacy against a wide range of infectious and non-infectious diseases.

[0042] The VLP platform of the present disclosure consists of pre-assembled virus-like particles that serve as a scaffold for antigen presentation and encapsulated immunomodulatory factors within its core. VLPs are non-infectious, virus-mimicking structures that retain the immunogenic properties of viruses, making them ideal candidates for vaccine development. The novel aspect of this platform consists of the following two attributes: i) use of intein-mediated protein conjugation, a bioconjugation method that allows for the precise and efficient attachment of antigens to the VLP surface without leaving undesirable residual tags or scars; and ii) encapsulation of different small molecule immunomodulators (including cytokines, adjuvants, and small molecule metabolites) for specific priming of tailored immune response through influencing the outcome of interactions between the APCs and the T-cells towards eliciting a specific type of immune response (Th1: humoral, Th1: cell mediated, Tfh, Th2, Th17, or Treg).

[0043] In an embodiment, the disclosure relates to a VLP platform for vaccine development, comprising a VLP structure capable of encapsulating immunomodulatory factors within its core and presenting antigens on its surface; and an intein-mediated bioconjugation system for the site-specific attachment of a target antigen to the VLP structure. The intein-mediated bioconjugation occurs under mild physiological conditions, preserving the structural integrity of the VLP-backbone and the target antigen.

[0044] The intein-mediated bioconjugation system comprises of short intein sequences, flanked by the VLP capsid protein, and its cognate intein-partner with the target antigen sequence, enabling self-excision of the intein-tag and its cognate partner, and surface ligation of the VLP capsid protein to the target antigen sequence.

[0045] Intein-mediated bioconjugation is a protein splicing mechanism that enables the covalent linkage of proteins or peptides under mild physiological conditions. In this platform, short cognate tags are engineered on both the backbone capsid protein of the VLP and the target antigen. Upon mixing, these tags undergo intein-mediated splicing, resulting in a covalent and site-specific bioconjugation of the target antigen to the VLP. This process is highly efficient and nearly traceless, leaving minimal (a few amino acids) or no residual amino acids from the tag sequences, thereby maintaining the integrity and functionality of both the VLP and the target antigen.

[0046] is schematic representation of the novel and versatile VLP platform of the present disclosure, capable of enhancing vaccine efficacy. This novel VLP platform consists of a unique combination of two key capabilities: 1. Encapsulation of different immunomodulators within the VLP core capable of generating tailored immune response against the antigen of interest (AOI); and 2. Short split intein (In) fusion tag designed to be exposed on the VLP-surface capable of trans-splicing mediated bioconjugation with cognate intein (cIn) fusion tag expressed with AOI. This novel VLP platform could be customized to display variety of antigens from different infectious diseases, cancer and autoimmune disorders, as well as specific priming and generation of tailored immune response against the AOI which expedites pre-clinical vaccine development and rapid selection of best combination of target antigen and immunomodulators with improved vaccine efficacy outcomes.

[0047] In an embodiment, there is provided a method of producing a VLP-based vaccine, comprising:

[0048] (a) providing a VLP structure capable of encapsulation of immunomodulatory factors within its core and presenting antigens on its surface;

[0049] (b) providing an antigen of interest capable of specific bioconjugation with the VLP surface; and

[0050] (c) utilizing intein-mediated bioconjugation to attach the antigen to the VLP surface (encapsulated with immunomodulatory factors) in a site-specific manner under mild physiological conditions.

[0051] The method further comprises a step of encapsulating immunomodulatory factors (metabolites, small molecules, pharmacologically active compounds, peptides, proteins, cytokines, hormones, signalling molecules, or adjuvants) within the VLP core to enhance the immune response.

[0052] The intein-mediated bioconjugation is performed such that minimal to no residual amino acids (near trace-less) from the intein-tags remain on the final VLP-antigen bioconjugated product.

[0053] The target antigen or antigen of interest can be selected from viral proteins, bacterial proteins, cancer antigens, and autoimmune disease-related antigens.

[0054] Table 1 shows the list of antigens of interest for infectious diseases.

[0055] Table 1:Infectious DiseasesAntigen(s) of Interest (AOI)Human Papillomavirus (HPV)L1, L2, E1, E2, E3, E4, E5, E6, E7, and subdomains thereofInfluenzaHemagglutinin (HA), Neuramimidase (NA), Nucleoprotein (NP), Matrix proteins (M1& M2), NS1, and strain transcendent protective antigenic subdomains from HA, NA and M2 individually or any combinations thereofCoronaviruses Beta-CoV (SARS-CoV, SARS-CoV2, MERS-CoV)Spike protein (S1 and S2) and subdomains thereof including the RBD, Nucleocapsid protein (NP) from each of the respective coronaviruses, and subdomains thereofHuman Immunodeficiency Virus (HIV)Envelop proteins gp120, gp41, gp160, and subdomains thereofHuman adenovirus (HAdv)Hexon protein, penton base protein, fiber-knob (FK) protein from all HAdv serotypes, and subdomains thereofVaricella-zoster virus (Chickenpox) & Herpes zoster (Shingles)Glycoprotein E (gE / ZoE) and subdomains thereofEpstein-Barr Virus (EBV)Glycoprotein gp350 / 220, gH, gL, gp42, EBNA-1, EBNA-2, EBNA-3C, EBV-poly, and subdomains thereofHuman Parvovirus B19Capsid protein VP1, VP2, and subdomains thereofHuman Papilloma Virus (HPV)Capsid proteins L1, L2, oncoproteins: E1, E2, E3, E4, E5, E6, E7, and subdomains thereofMpox VirusMPXV antigens A29, M1, A35, B6, and subdomains thereofHuman Polyomavirus (HPyV) 6 / 7 & JC Polyomavirus (JCPyV)Structural capsid protein VP1, VP2, VP3, oncoproteins: large T antigen (Lag), small T antigen (sTAg), and subdomains thereofAfrican swine fever virus (ASFV)Structural proteins p30, p54, p72, pp62, CD2v, and subdomains thereofLassa virusGlycoprotein (GPC, GP1, GP2), Nucleoprotein (NP), viral matrix protein (Z), and subdomains thereofHuman Astrovirus (MLB1)Capsid protein (CP / VP1), and subdomains thereofRotavirusVP2, VP4, VP6, VP7, VP8, and subdomains threofHepatitis E virusHEV Capsid protein (ORF2: S, M and P domains), p239, p179, TrpE-C2, and subdomains thereofRift Valley Fever (RVF) VirusRVFV NSs protein, NSm1, NSm2, Gn, Gc, and subdomains thereofCrimean-Congo hemorrhagic fever virusCCHFV nucleoprotein (NP), GPC (GP38), and subdomains thereofNorwalk virusNoV Capsid proteins VP1, VP2, and subdomains thereofEbola hemorrhagic fever (EHF) virusEHFV Nucleoprotein (NP), Glycoprotein (GP), VP40, and subdomains thereofMarburg hemorrhagic fever (MHF) virusMHFV Nucleoprotein (NP), Glycoprotein (GP), VP40, and subdomains thereofWest Nile virus (WNV)WNV envelop protein (E), and subdomains thereofYellow Fever virus (YFV)YFV envelop protein (E), membrane protein (M), non-structural proteins (NS1, NS2, NS3, NS4, NS5), and subdomains thereofDengue virusEnvelop protein (E) from four serotypes (DENV1-4), non-structural proteins (NS1, NS2A, NS2B,NS3, NS4A, NS4B, NS5), membrane protein (prM), and subdomains thereofJapanese Encephalitis virus (JEV)JEV envelop protein (E) from all prevailing serotypes, non-structural proteins (NS1, NS2A, NS2B,NS3, NS4A, NS4B, NS5), membrane protein (prM), and subdomains thereofZika virusZIKV envelop protein (E), non-structural proteins ((NS1, NS2A, NS2B,NS3, NS4A, NS4B, NS5), membrane protein (prM), and subdomains thereofTick-borne Encephalitis (Kyasanur Forest disease virus, Alkhurma virus, Omsk hemorrhagic fever virus, Powassan virus, Langat virus)Envelop protein (E) from all prevailing subtypes, non-structural proteins (NS1, NS2A, NS2B,NS3, NS4A, NS4B, NS5), membrane protein (prM), and subdomains thereofMeasles virus (MV)MV Fusion Protein (F), MV Haemagglutinin Protein (H), MV Neuraminidase Protein (N), and subdomains thereofHuman parainfluenza virus (PIV) types 1, 2, 3, 4PIV glycoproteins: Haemagglutinin-Neurminidase (HN) and fusion (F) from all prevailing subtypes, and subdomains thereofRespiratory Syncytial Virus (RSV) subtypes A & BRSV Fusion protein (F) in various conformation (pre-fusion and post-fusion) from all prevailing subtypes, and subdomains thereofMumps virus (MuV)subtypes A, G and HMuV Fusion protein (F) in various conformation (pre-fusion and post-fusion), Haemagglutinin-Neurminidase (HN) from all prevailing subtypes, and subdomains thereofPicornaviruses (Encephalomyocarditis virus, Foot-Hand and-mouth disease virus, human coxsackieviruses A & types, human echovirues, Reovirus, Rhinovirus, human enterovirus, vilyuish virus)Capsid proteins VP1, 2, 3, and 4, from the respective virus types, and subdomains thereofRabies Virus (RABV)RABV Glycoprotein (G), Matrix protein (M), Phosphoprotein (P),Nucleoprotein (N), large polymerase protein (L), and subdomains thereofHenipaviruses: Hendra virus (HeV) & Nipah virus (NiV)Soluble G glycoprotein of HeV / NiV (subtypes M and B), and subdomains thereofRubella virusStructural proteins (E1, E2, C), non-structural proteins (p90 and p150), and subdomains thereofChikungunya virus (CHIKV)CHIKV capsid protein (C), Envelop proteins (E1, E2, E3, 6K, TF), non-structural proteins (nsP1, nsP2, nsP3, nsP4), and subdomains thereofAcinetobacter baumanniiOmpA, and subdomains thereofPseudomonas aeruginosaOprF, exotoxin A (EPA), and subdomains thereofEscherichia coli (ETEC) Virulence Factors (SinH), adhesins: FimH, FdeC, PapDG; Siderophore receptor lutA, IreA, FyuA, IroN, Ybt, Aer; Toxoids derived from LTK63, HlyA, CNF1, LTB enterotoxin; Protein conjugates of O and K capsular antigens; and subdomains thereofStreptococcus mutansSurface adhesins AgI / II, GlnH and PstS proteins, and subdomains thereofVibrio choleraeCholera toxin B (CTXB), OmpW, OmpU, TcpA, TcpF, flagellin, AcfA, and subdomains thereofSalmonella typhiProtein conjugates of Vi capsular antigenShigella flexneriProtein conjugates of O-polysaccharide (OPS), virulence protein VirG, Invasin proteins IpaB and IpaC, and subdomains thereofStaphylococcus aureusClumping Factor A (ClfA), Manganese Transporter Subunit C (MntC), Protein conjugates of Capsular polysaccharides (CP5 and CP8), surface adhesins SpA, IsdB, AdsA, toxoids from Staphylococcal Enterotoxin B (SEB), TSST-1 (Toxic Shock Syndrome Toxin-1), and subdomains thereofChlamydia trachomatisMajor Outer Membrane Protein (MOMP), PMPs, Hsp60, CPAF, Pgp3, and subdomains thereofNeisseria gonorrhoeaePorin outer membrane protein: PorB; Phospholipase PldA, PLD; Pilin: PilE, PilC, PilQ,; Porin; Other outer membrane proteins: Opa, OpcA, OmpA, Nhba; Siderophore recpetor: TbpA, TbpB, LbpA, LbpB, FetA, FetB, TdfJ, TdfH; AniA, Lst, NspA, LgtG, Iga, Mip, MsrA / B, MtrE, Ngophi6, Acp, Iga2, BamA, BamE, CsgG, LolB, Lprl, LptD, LptE, MetQ, NGO0416, NGO0690, NGO0948, NGO1043, NGO1215, NGO1251, NGO1701, NGO2054, OmpU, SliC, TamA, and subdomains thereofTreponema pallidumMembers of outer membrane protein (Tpr) family: Tp0751, Tp0136, Tp0326, Tp0633, Tp086, Tp0897, and subdomains thereofBorrelia burgdorferi Variants of Outer surface proteins OspA, OspB, OspC and OspS, CspZ, DbpA, Bbk32 (p35), Subolesin, TROSPA, Salp25D, and subdomains thereofFrancisella tularensisOuter membrane protein OmpA, FTT0814, FTT0438, FTT1043, GltA, Mip, IgIC,PilA, DnaK, Tul4, FopA, SucB; Protein conjugates of O-antigen capsular polysaccharide from FTT, and subdomains thereofRickettsia rickettsia(RMSF)Outer membrane proteins Adr2, RickA, OmpA (Sca0), OmpB (Sca5), Sca2, Sca4, and subdomains thereofRickettsia prowazekii(epidemic typhus)Outer membrane proteins Sca0 (OmpA), Sca1, Sca2, Sca3, Sca4, Sca4 (OmpB), Adr1, Adr2, TolC, OmpW, Porin-4, YbgF, GroEL, PrsA, RplY, RpsB, SurA, RP403, RP598, RP739, RP778, RP884, and subdomains thereofRickettsia typhiOuter membrane protein B (OmpB), OmpA, and subdomains thereofOrientia tsutsugamushiTSA56 (Sta47), TSA47 (Sta22), Sta56, ScaA, ScaC, ScaD, ScaE, and subdomains thereofAnaplasma phagocytophilum&Anaplasma marginaleMSP1a, MSP2, VirB10, OMP1, OMP11, subdomains thereofCoxiella burnetii(Q fever)CBU1910, CBU0307, CBU0311, CBU0952, CBU0630, P1-HspB, and subdomains thereofYersinia pestisMajor capsule protein F1, LcrV, V307, combinations and subdomain thereofEhrlichia canis&E. chaffeensisp28, TRP19, TRP36, TRP140, Ank200, and subdomains thereofCorynebacterium diphtheriaeDiphtheria Toxin A or B subunit, CRM197, and subdomains thereof.Bordetella pertussis Pertusis exotoxin (S1, S2, S3, S4 and S5 subunits), filamentous haemaglutinin (FHA), fimbrial antigens (FIM2 / 3), pertactin (PRN), and subdomains thereofLegionella pneumophilaLpg0127 (AcsB), Lpg0199 (CydA), Lpg0533 (SucB), Lpg0534 (SucC), Lpg1594 (LdsB), Lpg1595, Lpg1596 (YfcX), Lpg2271, Lpg2311, Lpg2312, Lpg2272, Lpg2273 (UgpB), Lpg2217, Lpg0688, Lpg2025, and subdomains thereofMycobacterium tuberculosisAg85A / B, ESAT-6, CFP10, TB10.4, MPT64, MPT70, MPT83, PPE18, PPE68, RpfA, RpfB, RpfC, RpfD, RpfE, CysD, Rv2875, Rv3044, Rv2073c, Rv0577, Rv2608, Rv3619, Rv3620, Rv1813, Rv2660c, EspA, EspD, EspC, EspE, EspR, EspI, H74, Mtb 32A, Mtb 39A, and subdomains thereofStreptococcus pyogenes(Strep A)M protein (CRR domains), SpyCEP, SpyAD, SLO, Spy7, C5a peptidase, AD1, TF, Spy0762, Spy0651, Obp, pullulanase, Nbp, Sortase A, all variants of HVR (M protein), J8 / J14 / p145, Protein conjugates of Group A carbohydrate (GAC), and subdomains thereofMeningococcal meningitisProtein conjugates of capsular antigens from serogroups A,B, C, W, X & YStreptococcus pneumoniaeSurface proteins PspA, PspC, choline-binding protein (CbpA), PcsB, StkP, PcpA, PlyD1, PhtD, virulence factors: Pneumolysin (Ply), LytA, PsaA, PiaA and NanA, Protein conjugates of variants of pneumococcal capsular antigens, and subdomains thereofKlebsiella pneumoniaeOuter membrane proteins OmpA, OmpW, OmpK36, FepA, OmpK17, GlnH, FimA, MrkA, TonB, Kleb-SRP, Klebsiella cytotoxins (KCT-1, 2 & 3), Protein conjugates of variants of the capsular polysaccharide (CPS), O polysaccharide (OPS), and subdomains thereofClostridium tetaniTetanus toxoid (TT), THc, and subdomains thereofClostridium perfringens&Clostridium septicumAlpha-toxin, perfringolysin O, epsilon toxin, Flagellar biosynthetic protein (FliR), and subdomains thereofToxoplasma gondiiTgHSP70, TgCDPK6, TgGRA1, TgGRA2, TgGRA4, TgGRA5, TgGRA6, TgGRA7, TgGRA24, TgMIC2, TgMIC3, TgMIC4, TgMIC8, TgMIC11, TgMIC13, TgSAG1(P30), TgSAG2(P22), TgSAG3(P43), TgROP1, TgROP8, TgROP16, TgROP18, TgROP54,TgNTPase-II, and subdomains thereofEntamoeba histolyticaGalactose-adherence lectin, virulence factors: amebapores, arginase, alcohol dehydrogenase, peroxidoxin, lipopeptidophosphoglycan, Serine-richE. histolyticaprotein (SREHP), alkyl hydroperoxide reductase, MLIF, Ehcp112, Ehadh112, EhCBP30, HSBP, Eh29, EhCPADH, and subdomains thereofTrichomonas vaginalisTvAP65, TpAP33, alpha-actinin, and subdomain thereofTrypanosoma cruzi Flagellar Tc24, Tc52, Tans-sialidase (TSA1, ASP1 / 2, ASP9, TS, SAPA, TSf), Amastigote Surface proteins: MASP1 / 2, GP63, TcG1, TcG2, TcG4, Lyt1, GP72, Ech1 / 2, Complement regulatory protein (CRP), cruzipain (SCz), GP82, KMP11, paraflagellar rod proteins (PFR), and subdomains thereofTrypanosoma bruceiDifferent variants of the variable surface glycoprotein (VSG), enolase, Invariant Surface Glycoprotein (ISG65, ISG75), transferrin receptors ESAG6 / 7, TbTubulin, TbActin, congopain, sialidase, cation ATPase, and subdomains thereofGiardia lambliaUridine phosphorylase-like protein-1 (UPL-1), protein 21.1, alpha1-giardin, alpha11-giardin, beta-giardin, gamma-giardin, protein disulphide isomerase 5, cyst wall protein (CWP2), different variants of the VSPs, and subdomains thereofLeihmania donovani, L. infantum, L. major, L. braziliensis, L. panamensis, L. Mexicana,Homologs of H2A, H3, H4, A2, KMP11, HSP70, LiP2a, LiP2b, LiP0, TSA, LmSTI1, LeIF, NH36, SMT, KMP-11, HASPB, Leish-F1 / Leish-111f, Leish-F2, Leish-F3, CPA, CPB, PSA-50S, A2, LACK, gp63, and subdomains thereofPlasmodium falciparumCSP, TRAP, LSA1, LSA2, LSA3, STARP, SALSA, Pf11-1, Var2CSA, PIESP2, AMA1, MSP1, MSP2, MSP3, MSP3-family of proteins, MSP4, MSP5, MSP7, MSP8, MSP9, MSP10, GLURP, RH2a / b, RH4, RH5, EBA175, EBA181, EBA140, MAEBL, PfMA, GAMA, CyRPA, Ripr, SERA5, SERA8, RESA, RAP1, GARP, PfCTMAG, AARP, RALP1, variants of the Var-gene (PfEMP1) family, variants of STEVOR, variants of RIFIN, PfP0, PfP2, Pfs25, Pfs28, Pfs48 / 45, Pfs47, Pfs230, and subdomains thereofPlasmodium vivaxHomologs of allPlasmodium falciparumantigens listed above, PvCelTOS, PvP01_0534300, PvP01_0948700, Pv50, PvEBP family of proteins (PvDBP, PvEBP2), PvTRAgs family of antigens including PvTRAg26, PvPH, PvSOP26, and subdomains thereofPlsmodium ovale, P. malariae,P. knowlesiHomologs of allPlasmodium falciparumandPlasmodium vivaxantigens listed above, and subdomains thereofCryptococcus neoformans&Cryptococcus gattiiProtein conjugates of cell wall glucans (beta-glucans), chitin, chitosan (Cda1, Cda2), galactoxylomannan (GalXM), glucosylceramide (GlcCer), glucuronoxylomannan (GXM), virulence factor ZNF2, Heat-Shock proteins (HSPs), mannoprotein (MP), melanin, sterylglucosidase-1, sterylglucosides, and subdomains thereofHistoplasma capsulatumProtein conjugates of cell wall glucans (1,3 beta-glucans), Hsp60 / HIS62, H-antigen, F-box protein, enolase, HSC82, Histone2B, beta-1,3-glucanosyltrasferase and subdomains thereofAspergillus fumigatus, A. flavus,A. niger, A. terreus, A. nidulansProtein conjugates of cell wall glucans (beta-1, 3-D-glucan), glycans, mannans, NXT-2, Asp f3, Crf1 / Asp f9, Gel1, Pep1, and subdomains thereofCandida albicans, C. tropicalis,C. parapsilosis, C. glabrata, C. krusei, C. aurisProtein conjugates of cell wall glucans (beta-1,3 glucan), Als3 / NDV3, Saps (Sap2), Hyr1, Hsp90, and subdomains thereofCoccidioides posadasii, C. immitisAg2 / Pra, Pmp1, Cs-Ag, Cpa1, and subdomains thereofSporothrix schenckii, S. brasiliensis,S. globose, S. lurieiGlycoprotein Gp70, Gp60, importin (ZR8, ZR3), enolase, ssCWP47, ssCWP71, ssCWP100, and subdomains thereofPneumocystis carinii, P. murina,P. jirovecii,Gsc1, p55, Pca1, KEX1, and subdomains thereofMucor circinelloide, Rhizopus delemar, R. azygosporus, R. stolonifer,FTR1, Cell wall proteins, and subdomains thereofCancersCervical, Anal & OropharyngealHuman papilloma virus L1 and L2 proteins from different HPV types, E1, E2, E3, E4, E5, E6, E7, and subdomains thereofHepatocellular CarcinomaHepatitis B surface antigen, Hepatitis B core antigen, and subdomains thereofNon-Small Cell Lung CancerEpidermal growth factor (EGF), tumor-associated antigens (TAAs): MAGE-A3, NY-ESO1, CEA, WT1, MUC1; Neoantigens: KRAS G12C, Tp53 mutations / variants, and subdomains thereofMelanomaTAAs: MART1, gp100 (PMEL), Tyrosinase and tyrosinase related proteins (TRP1 / TRP2), NY-ESO1, MAGE-A1, A3, A4, PRAME, melanoma specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofKidney cancer / Renal cell carcinomaTAAs: Carbonic Anhydrase IX (CAIX), 5T4 Oncofetal antigen, WT1, MUC1, heat shock proteins (HSPs), NY-ESO1, EGFR, VEGFR, neoantigens: mutated PDL1, mutated CXCR4, and subdomains thereofPancreatic cancerTAAs: Mesothelin, alpha-1,3-galactosyltransferase, MUC1, CEA, WT1, pancreatic cancer specific neoantigens (mutated antigens) identified through genome sequencing such as KRAS mutations, Survivin mutations, hTERT mutations, and subdomains thereofGlioblastomaEGFR variants, IDH1 variants, Survivin variants, WT1, HER2 / neu, CMV(pp65 antigen), HSP96 / HSPPC-96, glioblastoma specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofProstate cancerSerine protease, PSMA, PAP, STEAP1, Prostein (SLA45A3), PSCA, MUC1, Androgen receptor variants, NY-ESO1, MAGE-A3, prostate cancer specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofHead and Neck cancersE6 and E7 oncoproteins of HPV, MAGE-A3, MAGE-A4, NY-ESO1, HSP70 variants, HSP90 variants, p53 variants, EGFR variants, IDO1 variants, mutated PDL1, hTERT variants, head and neck tumor specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofColorectal cancerTAAs: CEA, MUC1, HER2 / neu, hTERT variants, Survivin variants, MSI-H variants, KRAS mutations, APC mutations, Tp53 mutations, colorectal cancer specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofBreast cancer &Triple negative Breast cancerHER2, MUC1, CEA, Mammaglobin-A, NYESO1, MAGE-A3, WT1, Folate receptor-alpha, hTERT variants, p53 variants, breast cancer specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofOvarian cancerCA125 / MUC16, HER2 / neu, Folate receptor-alpha, NY-ESO1, WT1, Mesothelin, Human epididymis protein-4 (HE4), ovarian cancer specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofAutoimmune DisordersType 1 Diabetes (T1D)Insulin / Proinsulin, GAD65, Islet Antigen-2, Zinc transporter-8, IGRP, Tyrosine phosphatase-like protein (IA2), Islet amyloid polypeptide precursor protein, Coxsackievirus B proteins, T1D specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofMultiple Sclerosis (MS)Myelin basic protein (MBP), Myelin oligodendrocyte glycoprotein (MOG), Proteolipid protein (PLP), Citrullinated proteins, Epstein-Barr virus antigens, HSP60 and HSP70, MS specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofRheumatoid Arthritis (RA)Citrullinated proteins (CCP, vimentin, fibrinogen, alpha-enolase), Proteoglycan, Filaggrin, HSPs, Type-2 Collagen, Rheumatoid factor antigens, RA specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofSystemic Lupus Erythematosus (SLE)Smith (Sm) antigen, U1 snRNP, histone (H4), nucleosome proteins, Ro / SSA, La / SSB, CD22, CD20 / CD19, HSPs, BAFF antagonists, SLE specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofCeliac DiseaseGluten peptides, Glutenin, alpha-gliadin, gamma-gliadin, tissue Transglutaminase (tTG), Celiac disease specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofMyasthenia Gravis (MG)Acetylcholine receptor (AChR), muscle-specific kinase (MuSK), Low-density lipoprotein receptor-related protein4 (LRP4), Agrin, cortactin, MG specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereofInflammatory Bowel Disease (IBD)Flagellin (FliC) and OMPs ofE. coli,Clostridium difficilederived proteins / virulence factors,Mycobacterium aviumsubsp.paratuberculosis(MAP), HSPs, IBD specific neoantigens (mutated antigens) identified through genome sequencing, and subdomains thereof

[0056] Advantages Over Existing Technologies

[0057] Traditional VLP-based vaccine technologies rely on either direct insertion of antigens into the capsid protein backbone or chemical conjugation methods, such as those employed by GSK for their RTS,S / AS01 malaria vaccine or similar approach used for dengue vaccine by other competitors. These approaches often require extensive optimization to ensure proper VLP assembly, as any insertion within the backbone protein can disrupt the overall three-dimensional structure, leading to prolonged pre-clinical development times and increased costs.

[0058] Additionally, the SpyCatcher-SpyTag system, another plug-and-display technology used by some competitors, results in a large residual scar of approximately 120 amino acids, which can negatively impact or interfere with the target antigen's immunogenicity or may even interfere with the VLP's structure and stability. In contrast, the intein-based bioconjugation process of the present disclosure is nearly scar-free, providing a cleaner and more efficient alternative that preserves the desired properties of the VLP-antigen complex.

[0059] Encapsulation of Immunomodulatory Factors

[0060] A unique feature of the novel VLP platform of the present disclosure is its ability to encapsulate various immunomodulatory factors within the VLP core. These factors can include metabolites, small molecules, pharmacologically active compounds, peptides, proteins, cytokines, hormones, signaling molecules, or adjuvants. Encapsulation allows for the precise delivery of these immunomodulatory agents to APCs, which could influence the priming of T-cell for generating a tailored and robust specific type of immune response against the conjugated antigen and thereby enhancing the vaccine efficacy.

[0061] This dual-functionality—where the VLP serves both as an antigen presentation scaffold and as a delivery vehicle for immunomodulatory factors—enables the modulation of immune responses in a controlled manner capable of generating tailored, robust and specific immune response against the target antigen for enhancing the vaccine efficacy. For example, encapsulated immunomodulators can boost the activation of APCs, while cytokines can steer the immune response towards a desired pathway, such as enhancing T-cell activation or promoting antibody production or cell mediated immunity, as well as development of desired long-term immune memory. This capability is particularly valuable for developing vaccines against complex diseases, including cancer and autoimmune disorders, where tailored immune modulation is crucial.

[0062] is schematic representation of different steps involved in AP205 VLP platform encapsulated with Resiquimod and bioconjugated with AOI (R0.6C, a chimericPlasmodium falciparumantigen with subdomains from GLURP and Pfs48 / 45). Recombinant AP205 VLP backbone (SEQ ID 3) with N-terminal intein (In) tag (NpuDnaEC) (SEQ ID 1) was expressed and purified under denaturing conditions fromE.coliextracts. The In-tag AP205 is dialysed extensively through 3.5kDa membrane to remove denaturing urea in presence of Resiquimod to enable encapsulation within the assembling AP205-VLP core. Excess Resiquimod (which was not encapsulated) was removed by extensive dialysis through 300 kDa membrane. Recombinant AOI (SEQ ID 4) with C-terminal cognate intein (cIn) tag (NpuDnaEN) (SEQ ID 2) was expressed and purified fromE. coliextract, and mixed with In-tag AP205-VLP (SEQ ID 3) under physiological conditions (PBS, pH 7.4). The gel inset picture shows >98% bioconjugation of AOI with AP205-VLP. A representative scanning electron microscopy image shown below shows stable AP205-VLP encapsulated with Resiquimod and bioconjugated with AOI (R0.6C).

[0063] shows split HBcAg-VLP bioconjugated with another AOI (JEV-EDIII, a subdomain from Japanese Encephalitis Envelop protein) and encapsulated with Kynurenine. A schematic representation of different steps involved in HBcAg split VLP platform encapsulated with Kynurenine and bioconjugated with AOI (JEV-EDIII domain). Recombinant HBcAg VLP backbone (SEQ ID 5) is expressed as split VLP encoding a N-term fragment (Nf) with a stop codon and internal ribosomal binding site (IRBS) followed by N-terminal intein (In) tag (NpuDnaEC) (SEQ ID 1) in fusion with the C-term VLP fragment (Cf). Both Nf and Cf VLP fragments were co-purified under denaturing conditions fromE.coliextracts. In-tag HBcAg fragments were dialysed extensively through 3.5kDa membrane to remove denaturing urea in presence of Kynurenine to enable encapsulation within the assembling AP205-VLP core. Excess Kynurenine (which was not encapsulated) was removed by extensive dialysis through 300 kDa membrane. Recombinant AOI (SEQ ID 6) with C-terminal cognate intein (cIn) tag (NpuDnaEN) (SEQ ID 2) was expressed and purified fromE. coliextract, and mixed with In-tag HBcAg-VLP under physiological conditions (PBS, pH 7.4). The gel inset picture shows >98% bioconjugation of AOI with HBcAg-VLP. A representative scanning electron microscopy image shown below shows stable HBcAg-VLP encapsulated with Kynurenine and bioconjugated with AOI (JEV-EDIII domain).

[0064] Purified AP205 (SEQ ID 3) or HBcAg (Nf + Cf) (SEQ ID 5) proteins under denaturing condition (in presence of 6M urea) were adjusted to a concentration of about 1mg / ml and dialysed extensively through 3.5 kDa membrane to remove urea and favor VLP assembly in presence of varying concentrations of imunomodulator (0.1 to 10 mM). The VLP-suspension with encapsulated immunomodulator was separated from free immunomodulator molecules using extensive dialysis using 300kDa membrane. The amount of encapsulated immunomodulator from about 5 x 1014VLPs was collected upon VLP denaturation, and the amount of encapsulated immunomodulator was estimated using UV-Visible spectrophotometry; Resiquimod (260 nm) and Kynurenine (360 nm). The encapsulation efficiency (%) profile for Resiquimod in AP205 VLP and Kynurenine in HBcAg VLP, respectively is plotted as a function of immunomodulator concentration in Figure 4.

[0065] Applications and Versatility

[0066] The VLP platform's versatility allows it to be adapted for a wide range of applications. It is particularly suited for the development of vaccines against various infectious diseases where T-cell mediated antibody responses are critical for protection, such as viral, bacterial, and parasitic infections, as well as generation of long-term memory (Tfh) of protective humoral immune response. Additionally, this VLP platform can be used to effectively prime specifically tailored T-cell responses, making it applicable to several viral diseases (such as CD8+T-cell mediated immunity) and non-infectious diseases like cancer and autoimmune conditions through modulating the balance between Tconvand Tregmediated immune responses against different target antigens.

[0067] Moreover, this novel VLP platform supports the development of multivalent vaccines, where multiple distinct antigens could be easily bioconjugated with target antigens from multiple strains of a pathogen or a range of related pathogens, to be displayed on surface of different batches of VLPs. The desired combinations of such VLPs could then be co-administered, as well as for creating combination vaccines that provide broad protection against various diseases simultaneously.

[0068] Scalability and Commercial Potential

[0069] This novel VLP platform is designed with scalability in mind, making it suitable for large-scale commercial production. The intein-mediated conjugation process is highly efficient and straightforward not requiring the extensive optimization typical of other VLP technologies, reducing both the time and cost of vaccine development. Additionally, the encapsulation of the VLP core with immunomodulatory factors is a straightforward and scalable process easily achieved during the step of VLP assembly. Such ease of scalability ensures that this novel VLP-platform can meet the demands of high-volume vaccine production, addressing global health needs efficiently.

[0070] Given the growing market for vaccines targeting both infectious and non-infectious diseases, the commercial potential of this novel VLP platform is significant. The estimated market size for such vaccines is projected to exceed USD 20 billion by 2030. The platform's adaptability also makes it an attractive option for personalized medicine, where vaccines can be tailored to individual patient profiles, particularly in oncology and chronic disease management.

[0071] Comparative Analysis with Existing Technologies

[0072] Compared to existing VLP-based vaccine technologies, the novel VLP-platform of the present disclosure offers distinct advantages in terms of efficiency, flexibility, and cost-effectiveness. For instance, the need for extensive structural optimization is eliminated, as the intein-mediated conjugation preserves the VLP’s architecture without requiring antigen insertion into the capsid protein backbone. Additionally, minimal or near trace-less nature of the bioconjugation process ensures that the immunogenicity of the antigen remains unaltered, resulting in a more effective immune response.

[0073] The novel VLP-platform of the present disclosure also has the capacity to encapsulate and deliver immunomodulatory factors alongside target antigen to further differentiate it from competitors, providing a unique mechanism to enhance vaccine efficacy through targeted immune modulation. This feature not only improves initial immune priming but also supports generation of tailored immune response such as the development of robust memory responses, which are essential for long-lasting protection.

[0074] The novel VLP platform of the present disclosure represents a transformative approach to vaccine development. By leveraging intein-mediated bioconjugation and incorporating immunomodulatory encapsulation, this platform addresses critical limitations of existing VLP technologies and offers a highly adaptable, efficient, and scalable solution for enhancing vaccine efficacy. This innovative VLP-technology has the potential to revolutionize the field of recombinant vaccines, providing a powerful tool for combating a wide array of diseases and advancing public health on a global scale.

[0075] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

[0076] Thus, the scope of the present disclosure is defined by the appended claims and includes both combinations and sub-combinations of the various features described hereinabove as well as variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description.

Claims

A virus-like particle (VLP) platform for vaccine development, comprising:a VLP structure capable of presenting antigens on its surface;an intein-mediated bioconjugation system for the site-specific attachment of a target antigen to the VLP structure, wherein the intein-mediated bioconjugation occurs under mild physiological conditions, preserving the structural integrity of the VLP-backbone and the target antigen.The VLP platform of claim 1, wherein the intein-mediated bioconjugation system comprises:an intein sequence flanked by the VLP capsid protein and the antigen sequence, enabling self-excision of the intein-tag and ligation of the VLP capsid protein to the antigen sequence.The VLP platform of claim 1, wherein the target antigens or antigens of interest (AOI) are selected from the group consisting of viral proteins, bacterial proteins, cancer antigens, and autoimmune disease-related antigens.The VLP platform of claim 1, further comprising encapsulated immunomodulatory factors within the VLP core, wherein the encapsulated immunomodulatory factors are designed to modulate the immune response.The VLP platform of claim 4, wherein the immunomodulatory factors include various small molecule metabolites, cytokines, chemokines, adjuvants, or any combination thereof.The VLP platform of claim 1, wherein the VLP structure is derived from a virus selected from the group consisting of infectious diseases causing microorganisms, selected from, human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis E virus (HEV), norovirus, and bacteriophages Qβ and AP205 or any other recombinant protein or combination of recombinant proteins capable of assembling into uniform nanoparticles.The VLP platform of claim 1, wherein the intein-mediated bioconjugation leaves minimal to no residual amino acids from the intein-tags, resulting in a near trace-less final VLP-target antigen bioconjugated product.The VLP platform of claim 1, wherein the VLP platform is adaptable for use in vaccines targeting infectious diseases, cancer, or autoimmune disorders.A method of producing a VLP-based vaccine, comprising:(a) providing a VLP structure capable of encapsulation of immunomodulatory factors within its core and presenting antigens on its surface;(b) providing an antigen of interest capable of specific bioconjugation with the VLP surface; and(c) utilizing intein-mediated bioconjugation to attach the antigen to the VLP surface (encapsulated with immunomodulatory factors) in a site-specific manner under mild physiological conditions.The method of claim 9, further comprising the step of encapsulating immunomodulatory factors (metabolites, small molecules, pharmacologically active compounds, peptides, proteins, cytokines, hormones, signalling molecules, or adjuvants) within the VLP core to be co-delivered to the APCs for modulation and generation of tailored immune response against the target antigens.The method of claim 9, wherein the intein-mediated bioconjugation is performed such that minimal to no residual amino acids (near trace-less) from the intein-tags remain on the final VLP-antigen bioconjugated product.

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