VLP, VLP-RNA complex and recombinant expression platform for expression thereof

Abstract

The present invention provides a versatile recombinant expression platform for expression of multi-antigen VLPs and VLP-RNA complexes particularly, VLP-mRNA complexes. The present invention provides a VLP-mRNA complex encapsulating mRNA inside VLP with increased thermostability and protection from ribonucleases, hence, have a shelf life up to 2 years, increased template copy number with enhanced therapeutic outcome. The multivalent VLPs and complexes of the present invention have great potential applications against one or more respiratory, bacteriological and oncological diseases.

Description

[0001] VLP, VLP-RNA COMPLEX AND RECOMBINANT EXPRESSION PLATFORM FOR EXPRESSION THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention generally relates to the field of medical sciences, vaccine technology. Particularly, the invention provides Virus-like Particles (VLPs) and VLP / RNA complexes and a recombinant expression platform for their expression.

[0004] BACKGROUND OF INVENTION

[0005] VLPs are nanostructures assembling viral proteins which mimic viruses but do not contain any infectious viral genetic material. VLP vaccines thus characterized by their non-infectious nature and highly immunogenic properties, represent a promising avenue against diverse pathogens. Furthermore, VLPs can serve as platforms for various epitopes, displaying high immunogenicity even for poorly immunogenic targets (Rourke et al., 2015).

[0006] In addition, VLPs have also been explored as nanocarriers for cargo delivery (RNA or protein) as they combine the key advantages of viral and non-viral vectors. Said particles protect cargo molecules from degradation, have good cell penetrating property to mediate cargo passing the cell membrane and release into cells, making them an ideal tool for intracellular delivery of biomolecules and drugs. Chu and Quan (2023) summarize the most recent progress in pre-clinical and clinical VLP vaccine development and outline various strategies that contributed to improving the efficacy of VLP based vaccines.

[0007] Production of complex encapsulated and enveloped VLP based vaccines present various challenges such as expression of each protein in stoichiometric amounts for efficient production, poor characterization at the biophysical level due to non-uniformity in structure, storage and transportation etc. Furthermore, conventional vaccine development approaches also suffer from challenges in providing comprehensive protection against multiple pathogens, necessitating innovative strategies to address these limitations (Gupta et al., 2023). Thus, it is critical to maintain their structural integrity and stability for optimum immune recognition, and pathogenicity as well as target multiple pathogens. The existing expression systems may prove inefficient for expression of multiple proteins and the yield of the protein may also be compromised.

[0008] Therefore, the present invention seeks to address these challenges by employing bidirectional promoters, which offers an efficient approach to gene regulation and expression in genetic circuits.

[0009] By utilizing bidirectional promoters, the present invention aims to improve the assembly and regulation of transcriptional units, thereby creating a more robust and efficient genetic circuit.

[0010] Moreover, bidirectional promoters facilitate better stoichiometric balance between the transcribed and expressed genes, which is crucial in applications requiring precise ratios of gene products. This improved stoichiometry can significantly enhance the overall functionality and performance of genetic circuits, making them more suitable for complex biotechnological applications, such as synthetic biology, metabolic engineering, and therapeutic gene delivery.

[0011] Besides this, RNA based therapeutics have also emerged as promising candidates for the prevention and treatment of various infectious diseases. However, they too, suffer from various limitations due to high susceptibility to degradation by RNases due to its intrinsic biochemical nature. Thus, there is a need for improved approaches for increasing stability, immunogenicity, translation efficiency, and safe delivery of mRNA as vaccines.

[0012] The present invention utilizes the unique properties of bidirectional promoters to overcome existing limitations in genetic circuit design and expression systems, offering an efficient solution that enhances transcriptional coordination, reduces expression variability, and achieves optimal gene product ratios for expression of VLP and VLP-RNA complex.

[0013] OBJECTIVES OF THE INVENTION

[0014] An important object of the present invention is to provide a recombinant expression platform for the expression of VLP and VLP-RNA complex.

[0015] Another object of the present invention is to provide a novel approach for the development of a multi-antigen VLP with improved immunogenicity against respiratory viruses and having enhanced stability. A further object of the present invention is to provide a VLP-RNA complex comprising viral mRNA encapsulated within VLP with increased thermostability and efficacy.

[0016] Another object of the present invention is to develop a proof-of-concept for in-vivo recombinant expression of dual-target VLP-RNA complexes.

[0017] Another important object of the present invention is to provide a methodology for circular RNA encapsulation within VLPs, ensuring stability and protection.

[0018] Another object of the present invention is to provide the recombinant VLP approach as a potential plug-and-play platform for in-vivo encapsulation of RNA antigens.

[0019] A further object of the present invention is to provide a multivalent vaccine against one or more antigenic targets.

[0020] Another object of the present invention is to provide a VLP / VLP-RNA based vaccine with enhanced thermostability and shelf-life for preventing or treating one or more respiratory, bacterial or oncological diseases.

[0021] A further object of the present invention is to provide a method of preventing or treating one or more respiratory, bacterial or oncological diseases by administering a therapeutically effective amount of the VLP / VLP-RNA complex.

[0022] Additional objects, advantages, and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention.

[0023] SUMMARY OF THE INVENTION The following summary is provided to facilitate an understanding of some of the innovative features unique to the disclosed embodiments and is not intended to be the full description of the invention.

[0024] The present invention provides a novel approach for efficient recombinant expression of Virus- Like Particle (VLP) and VLP-mRNA complexes, targeting one or more antigenic targets. Particularly, one or more antigenic targets may be associated with respiratory infectious diseases selected from but not limited to influenza, RSV, SARS-Cov2, hMPV, MERS, NIP AH or bacterial or oncological diseases.

[0025] Particularly, the VLP-mRNA complex of the present invention comprises a VLP encapsulating an mRNA expressed using a recombinant expression platform based on a protease deficient yeast host system.

[0026] Particularly, the present invention provides a recombinant expression platform comprising a high copy episomal expression vector (pYRE400) operably linked with a bidirectional promoter Gall / Gal 10 and a CYCT1 terminator, an integration vector (pYRHOO) operably linked with a promoter Gall and a CYCT1 terminator, and an engineered protease deficient yeast host cell. This platform is designed for the expression of a VLP / VLP-mRNA complex. These vectors collectively provided a robust framework for gene expression regulation. Furthermore, an essential element of the recombinant expression platform involves the utilization of an engineered protease-deficient S. cerevisiae yeast host cell. This modification ensures the stability and longevity of the expressed genes, preventing unwanted degradation. The combination of the high copy episomal expression vector, integration vector, and the engineered host cell enables efficient and controlled gene expression.

[0027] In addition, the expression system of the present invention employs bidirectional promoters, to drive the transcription of two genes simultaneously, but in opposite directions. This characteristic enables the coordinated regulation of multiple genes, ensuring synchronized expression and transcription of both protein and RNA candidate with aligned functional interplay within a genetic system. One key advantage of this approach is the potential to minimize noise in gene expression, which often arises due to variability in transcriptional activity. Reduced noise ensures that both transcription and gene expression levels are more predictable and consistent, enhancing the reliability of the system.

[0028] The recombinant expression platform is highly versatile owing to its ability to express one or more immunogenic antigen sequences against multiple viruses, making it a promising solution for broad-spectrum vaccine development. The encapsulated mRNA within the VLP-mRNA complex enhances the thermostability of the generated complex, resembling that of a native virus.

[0029] In an embodiment, the present invention provides a recombinant expression platform comprising: i. a high copy episomal expression vector; ii. an integration vector; and iii. engineered protease deficient yeast host cell, wherein said vectors are operably linked with a bidirectional promoter Gall / GallO and comprise a CYCT1 terminator, a selection marker selected from Ura3c or Leu2; an Ampicillin resistance marker; an origin of replication site pUC ori; a 2 micron origin; and a sequence region comprising of multiple cloning sites; wherein said platform enables insertion of full length or truncated polynucleotide sequence for expression of desired antigenic polypeptides into the host cell.

[0030] In some embodiments of the present invention, the recombinant expression platform comprises an episomal vector selected from pYRE400 or pYRElOO.

[0031] In some embodiments of the present invention, the recombinant expression platform comprises a pYRHOO integration vector.

[0032] In some embodiments of the present invention, the recombinant expression platform is capable of expressing a VLP and / or a VLP-RNA complex.

[0033] In some embodiments of the present invention, the protease deficient yeast host is Saccharomyces cerevisiae.

[0034] In some embodiments of the present invention, the recombinant expression platform of the present invention is capable of expressing two or more immunogenic antigen sequences against two or more viruses.

[0035] In some embodiments of the present invention, the episomal vector expresses VLP antigens and regulatory sequences for circularization and packaging of mRNA. In some embodiments of the present invention, the integration vector is integrated into the genome comprising the assembly and packaging recognition fusion protein and expresses VLP antigens.

[0036] In some embodiments of the present invention, the episomal and integration vector enables assembly of VLP and packaging of the mRNA.

[0037] In some embodiments of the present invention, the episomal and integration vector enables parallel assembly of VLP and packaging of the mRNA.

[0038] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 1 for expression of Influenza VLP assembly protein matrix protein (Ml) wherein said construct comprises a nucleic acid sequence encoding the influenza membrane protein (Ml) of H1N1, and an integration expression vector comprising Leu2 selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal promoter.

[0039] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 2 for expression of Influenza VLP assembly Hemagglutinin (HA) and Neuraminidase (NA) protein of H1N1, wherein said construct comprises nucleic acid sequences encoding the Hemagglutinin (HA) and Neuraminidase (NA) protein of H1N1, and an episomal expression vector comprising Ura3C selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal promoter.

[0040] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 3 for expression of Influenza VLP assembly protein matrix protein (Ml) wherein said construct comprises a nucleic acid sequence encoding the influenza membrane protein (Ml) of H5N1, and an integration expression vector comprising Leu2 selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal promoter.

[0041] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 4 for expression of Influenza VLP assembly Hemagglutinin (HA) and Neuraminidase (NA) protein of H5N1, wherein said construct comprises nucleic acid sequences encoding the Hemagglutinin (HA) and Neuraminidase (NA) protein of H5N1, and an episomal expression vector comprising Ura3C selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal promoter.

[0042] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 5 for expression of Influenza VLP assembly protein named matrix protein (Ml) in fusion with assembly cum packaging recognition fusion protein (CP) wherein said construct comprises nucleic acid sequences encoding the matrix protein (Ml) and assembly cum packaging recognition fusion protein (CP) of H1N1, and an integration expression vector comprising Leu2 selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal promoter.

[0043] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 6 for transcription of VLP prefusion conformation-stabilized RSV fusion protein (preF) in fusion with assembly cum packaging recognition fusion protein (CP) wherein said construct comprises a nucleic acid sequence encoding the preF of RSV and assembly cum packaging recognition fusion protein (CP), and an episomal expression vector comprising Ura3C selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with bidirectional Gall / GallO promoter.

[0044] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 7 for transcription of prefusion conformation-stabilized RSV fusion protein (preF) and expression of Neuraminidase (NA) wherein said construct comprises nucleic acid sequences encoding the preF protein of RSV with assembly cum packaging recognition fusion protein (CP) and Neuraminidase (NA) protein, and an episomal expression vector comprising Ura3C selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with bidirectional Gall / GallO promoter.

[0045] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 8 for expression of Influenza VLP assembly protein named matrix protein (Ml) in fusion with assembly cum packaging recognition fusion protein (CP) and Hemagglutinin (HA) wherein said construct comprises nucleic acid sequences encoding matrix protein (Ml), assembly cum packaging recognition fusion protein (CP) and Hemagglutinin (HA), and an integration expression vector comprising Leu2 selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with promoter.

[0046] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 9 for transcription of Hemagglutinin (HA)H1N1 wherein said construct comprises nucleic acid sequences encoding assembly cum packaging recognition fusion protein (CP) and Hemagglutinin (HA) of H1N1, and an episomal expression vector comprising Ura3c selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with bidirectional Gall / GallO promoter.

[0047] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 10 for transcription of Influenza VLP assembly cum packaging recognition fusion protein (CP) in fusion with Hemagglutinin (HA) and expression of Neuraminidase (NA) wherein said construct comprises nucleic acid sequences encoding assembly cum packaging recognition fusion protein (CP), with Hemagglutinin (HA) and Neuraminidase (NA) of H1N1, and an episomal expression vector comprising Ura3c selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with bidirectional Gall / GallO promoter.

[0048] In some embodiments, the present invention provides a nucleic acid construct with SEQ ID NO. 11 for transcription of Influenza VLP assembly cum packaging recognition fusion protein (CP) in fusion with Hemagglutinin (HA) wherein said construct comprises nucleic acid sequences encoding assembly cum packaging recognition fusion protein (CP), with Hemagglutinin (HA) of H5N1, and an episomal expression vector comprising Ura3c selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with bidirectional Gall / GallO promoter.

[0049] In some embodiments, the present invention provides a recombinant VLP-mRNA complex comprising mRNA transcript encapsulated within VLP particles wherein, said complex is capable of expressing two or more immunogenic antigen sequences against one or more viruses.

[0050] In some embodiments of the present invention, the VLP-mRNA complex is expressed using protease deficient recombinant expression platform, comprising one or more antigenic sequences expressed through Virus like particles (VLPs) and one or more antigenic sequences expressed using mRNA transcript.

[0051] In some embodiments of the present invention, the virus is a respiratory virus selected from H1N1, H5N1, RSV, SARS CoV2, hMPV, MERS and NIP AH.

[0052] In some embodiments of the present invention, the virus is a respiratory virus of the family Orthomyxoviridae.

[0053] In some embodiments of the present invention, the virus is an Influenza virus.

[0054] In some embodiments of the present invention, the virus is selected from, but not limited to H1N1, H5N1.

[0055] In some embodiments of the present invention, the mRNA is a positive or a negative strand of RNA virus.

[0056] In some embodiments of the present invention, the mRNA encodes an anti-tumor protein.

[0057] In some embodiments of the present invention, the mRNA encodes an anti-bacterial protein.

[0058] In some embodiments of the present invention, the viral mRNA is from a virus selected from but not limited to RSV, H1N1, H5N1, SARS CoV, hMPV, MERS, NIP AH. In some embodiments of the present invention, the viral mRNA is a virus from the family

[0059] Pneumoviridae and is a PreFusion (PreF), RSV mRNA.

[0060] In some embodiments of the present invention, the viral mRNA is a virus from the family

[0061] Orthomyxoviridae and is a Hemagglutinin (HA), H1N1 mRNA.

[0062] In some embodiments of the present invention, the viral mRNA is a virus from the family

[0063] Orthomyxoviridae and is a HA, H5N 1 mRNA.

[0064] In some embodiments of the present invention, the complex comprises Influenza VLP encapsulating RS V or HINI or H5Nl mRNA.

[0065] In some embodiments of the present invention, the complex comprises H1N1 VLP encapsulating PreF RSV, HA H1N1, HA H5N1 mRNA.

[0066] In some embodiments of the present invention, the mRNA is encapsulated in circular or linear form.

[0067] In some embodiments, the present invention provides a recombinant virus like particle (VLP) comprising at least one or more antigenic polypeptides of a respiratory virus selected from but not limited to H1N1, H5N1, RSV, SARS CoV2, hMPV, MERS, NIP AH.

[0068] In some embodiments of the present invention, the virus is an Influenza virus selected from, but not limited to H1N1, H5N1.

[0069] In some embodiments of the present invention, the VLP comprises one, two or three antigenic polypeptides of H1N1 virus.

[0070] In some embodiments of the present invention, the antigenic polypeptides are selected from but not limited to the group of Hemagglutinin (HA), Neuraminidase (NA), and Matrix protein (Ml).

[0071] In some embodiments of the present invention, the VLPs maintain a size range and stoichiometry range of the antigenic peptides.

[0072] In some embodiments of the present invention, the antigenic polypeptide is a full-length sequence or fragment thereof.

[0073] In some embodiments of the present invention, the VLP is cloned and expressed using episomal and integrated expression vectors in a protease deficient yeast host system. In some embodiments, the present invention provides a method of producing recombinant VLP using the recombinant expression platform as claimed in claim 1, comprising the steps of: a. cloning one or more antigenic polypeptides using one or more combination of episomal expression vectors and integration vectors; b. codon optimizing the antigenic polypeptide sequences for expression; and c. transforming and expressing in a protease deficient yeast host.

[0074] In some embodiments, the present invention provides a method of producing a recombinant VLP-mRNA complex using the recombinant expression platform as claimed in claim 1, said method comprising: i. cloning one or more antigenic polypeptides using one or more combination of episomal expression vectors and integration vectors; ii. codon optimizing the antigenic polypeptide sequences for expression; iii. transformation and integration of integrating expression vector in genome of protease deficient yeast host; iv. transformation of episomal vector in engineered yeast host of step iii; v. co-expression of VLP proteins and in parallel transcription of mRNA, regulatory and packaging sequences in yeast host of step iv from episomal and integration vector; and vi. parallel assembly of VLP proteins and encapsulation of mRNA to form recombinant virus like particle (VLP)-mRNA complex.

[0075] In some embodiments of the present invention, the antigenic polypeptides are selected from but not limited to Hemagglutinin, Neuraminidase, and Matrix protein, PreF, CP.

[0076] In some embodiments of the present invention, the episomal expression vector is pYRE400 or pYRElOO and the integration vector is pYRHOO.

[0077] In some embodiments of the present invention, the episomal expression vector comprises a bidirectional Gall / Gal 10 promoter and a CYCT1 terminator.

[0078] In some embodiments of the present invention, the integration vector comprises a Gall promoter and a CYCT1 terminator.

[0079] In some embodiments of the present invention, the codon optimization includes modifying the codon sequences of antigenic polypeptides to improve expression in protease deficient yeast host.

[0080] In some embodiments of the present invention, the protease-deficient yeast host is Saccharomyces cerevisiae, deficient in pep4 and prbl proteases. In some embodiments of the present invention, the VLP-mRNA complex encapsulates circular RNA for enhanced stability and protection against RNases.

[0081] In some embodiments of the present invention, the mRNA includes regulatory sequences for circularization and packaging into the VLP.

[0082] In some embodiments of the present invention, the VLP-mRNA complex targets one or more respiratory viruses selected from Influenza H1N1, H5N1, RSV, SARS-CoV-2, NIP AH, hMPV and MERS.

[0083] In some embodiments of the present invention, the transcription of mRNA is driven by a galactose- inducible promoter system.

[0084] In some embodiments of the present invention, the co-expression step involves simultaneous expression of VLP proteins and transcription of mRNA, the stoichiometric assembly of VLP- mRNA complexes.

[0085] In some embodiments, the present invention provides a method for production of a scalable amount of recombinant VLP-mRNA complex comprising the steps of: i. overexpressing recombinant VLPs-mRNA co expression complex in a transformed yeast host cell culture; ii. growing the transformed yeast cell culture to a stage of log phase growth; iii. inoculating and inducing the culture in a suitable media; and iv. expression of VLP proteins, transcription of mRNA and purification of the VLP encapsulating mRNA transcript.

[0086] In some embodiments, the present invention provides a vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition comprising the recombinant VLP or recombinant VLP-mRNA complex and a pharmaceutically acceptable carrier or excipient.

[0087] In some embodiments of the present invention, the vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition is thermostable and has a shelf life of up to 2 years.

[0088] In some embodiments of the present invention, the vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition can be used preventing or treating one or more respiratory infections or diseases. In some embodiments of the present invention, the vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition can be used for preventing or treating one or more bacterial or oncological diseases.

[0089] In some embodiments, the present invention provides a method of preventing or treating one or more respiratory infections or diseases comprising administering a therapeutically effective amount of recombinant VLP or recombinant VLP-mRNA complex or vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition.

[0090] In some embodiments, the mRNA is encapsulated inside a VLP and wherein the recombinant VLP- mRNA complex has potential as a dual vaccine target for use in preventing or treating one or more respiratory infections or diseases.

[0091] In some embodiments, the vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition can be used for preventing or treating one or more bacterial or oncological diseases by engineering the VLP to carry an antigen that elicits an immune response against the bacterial cell or the cancerous cell.

[0092] In another important embodiment, the recombinant virus like particle (VLP) -mRNA complex comprises mRNA encapsulated inside a VLP and wherein the recombinant VLP-mRNA complex has potential as a dual vaccine target for use in preventing or treating one or more respiratory infections or diseases.

[0093] Further areas of applicability of the present invention will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and specific examples, while indicating the important embodiments of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

[0094] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0095] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations and are not intended to limit the scope of the present disclosure. The invention itself, however, both as to organization and method of operation, may best be understood by reference to the detailed description which follows taken in conjunction with the accompanying drawings in which: Figure 1 shows A: the scheme of genetic constructs for bidirectional and unidirectional expression of VLP and VLP-RNA complex generation in Yeast; B: restriction map of pYRE400 and C: restriction map of pYRHOO.

[0096] Figure 2 shows the restriction maps of A: pYRHOO Ml and B: pYRE100_HA+NA

[0097] Figure 3 shows results of immunoblot analysis of structural proteins- HA, NA and Ml expression from S. cerevisiae -based expression platform at small scale- A: HA immunoblot; B: NA immunoblot; C: Ml Immunoblot.

[0098] Figure 4 demonstrates the results of 48th hr Post induction expression analysis from scale up culture- A: Immunoblot with HA specific polyclonal antibody; B: Immunoblot with NA specific polyclonal antibody; C: Immunoblot with Ml specific polyclonal antibody.

[0099] Figure 5 shows A: the particle size distribution and B: the co-relation curve of purified H1N1 VLP.

[0100] Figure 6 shows the electron microscopy analysis of H1N1VLP.

[0101] Figure 7 shows the absorbance data for purified standard proteins of single antigen for the measurement of each antigen in co expressed VLP and stoichiometry.

[0102] Figure 8 shows the bar graph depicting the total IgG response results.

[0103] Figure 9 shows the bar graph depicting the individual Antigen IgG response results.

[0104] Figure 10 shows the bar graph depicting the MNT assay results.

[0105] Figure 11 shows the bar graph depicting the HI assay results.

[0106] Figure 12 shows the body weight trends of mice over time across different dose groups (0 pg, 2.5 pg, 5 pg, 20 pg, and 52 pg) for VLP_HA+NA+M1 (without adjuvant) and VLP_HA+NA+Ml+AddaVax adjuvant formulations.

[0107] Figure 13 shows a H5N1 VLP_HA + NA Episomal, Ml Integrated, showing a small-scale culture at 15ml culture: 48thhr Post Induction Expression Analysis- A: Immunoblot with HA specific polyclonal antibody; B: Immunoblot with NA specific polyclonal antibody; C: Immunoblot with Ml specific polyclonal antibody; D: SDS-PAGE. Figure 14 shows a H5N1 VLP_HA + NA Episomal, Ml Integrated, showing a Scale up culture at 3L flask level: 48thhr Post Induction Expression Analysis- A: Immunoblot with HA specific polyclonal antibody; B: Immunoblot with NA specific polyclonal antibody; C: Immunoblot with Ml specific polyclonal antibody; D: SDS-PAGE.

[0108] Figure 15 shows the construct map of plasmid pYRI100_Ml (H5N1), demonstrating its structural organization.

[0109] Figure 16 shows the construct map of plasmid pYRElOO HA+NA (H5N1 ), demonstrating its structural arrangement.

[0110] Figure 17 shows the particle size distribution as a function of particle diameter (in nm) with a peak at the measured size range.

[0111] Figure 18 shows the correlation function (g2) against delay time (in seconds), demonstrating the dynamic light scattering (DLS) data for particle analysis.

[0112] Figure 19 provides an overview for preparation of H1N1 VLP (Ml) / cRNA_PreF (RSV).

[0113] Figure 20 shows the 48thhr Post induction expression analysis at small scale- A: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; B: SDS-PAGE.

[0114] Figure 21 shows a 48thhr Post induction expression analysis of selected clone at 2L scale- A: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; B: SDS- PAGE.

[0115] Figure 22 shows the construct map of plasmid pYRIJMl(HlNl) + CP construct, demonstrating its structural arrangement.

[0116] Figure 23 shows the construct map of plasmid pYRE400_PreFCP construct, demonstrating its structural arrangement.

[0117] Figure 24 shows DLS and homogeneity curve.

[0118] Figure 25 shows PCR confirmation of Encapsulated mRNA (PreF) using PreF specific primers.

[0119] Figure 26 provides an overview for preparation of H1N1 VLP (NA-Ml) / cRNA_PreF (RSV). Figure 27 shows a 48thhr Post induction expression analysis at small scale- A: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; B: Immunoblot using NA antibody; C: SDS-PAGE.

[0120] Figure 28 shows a 48th hr Post induction expression analysis at 2L scale- A: Immunoblot using NA antibody; B: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; C: SDS-PAGE.

[0121] Figure 29 shows the construct map of plasmid pYRE400_PreFCP+NA construct, demonstrating its structural arrangement.

[0122] Figure 30 shows PCR confirmation as large length encapsulated mRNA (PreF) was conducted using PreF-specific primers.

[0123] Figure 31 provides an overview for preparation of H1N1 VLP (HA-NA-Ml) / cRNA_PreF (RSV).

[0124] Figure 32 shows a 48thhr Post induction expression analysis at small scale- A: Immunoblot using HA antibody; B: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; C: Immunoblot using NA antibody; D: SDS-PAGE.

[0125] Figure 33 shows a 48thhr Post induction expression analysis of selected clone at 2L scale- A: Immunoblot using HA antibody; B: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; C: Immunoblot using NA antibody; D: SDS-PAGE.

[0126] Figure 34 shows an encapsulation of PreF (RSV) mRNA inside VLP (HA-NA-M1) by PCR.

[0127] Figure 35 shows H1N1 VLP (HA-NA-M1) / cRNA_PreF (RSV)- pYRHOO -Integration vector components for the expression of Ml in fusion with packaging recognition protein and HA in separate cassette.

[0128] Figure 36 shows H1N1 VLP (HA-NA-M1) / cRNA_PreF (RSV)-pYRE400 -Episomal vector components for the transcription of circularization, packaging unit and IRES and expression of NA protein.

[0129] Figure 37 shows the antigens were verified by immunoblot using HA, Ml, and NA-specific antibodies in purified VLP Figure 38 shows the encapsulated mRNA (PreF) was confirmed by PCR.

[0130] Figure 39 shows the results of dynamic light scattering (DLS) analysis.

[0131] Figure 40 shows the results obtained through homogeneity curve.

[0132] Figure 41 shows the results of mouse serum analysis for Total IgG-Coated VLP.

[0133] Figure 42 shows the results of mouse serum analysis for Total IgG-Coated Pre-F.

[0134] Figure 43 shows the results of mice intramuscularly injected with the VLP liquid formulation safety study at doses of 10 pg, 20 pg, 50 pg, and 100 pg, with no adjuvant in multiple cohorts.

[0135] Figure 44 provides an overview for preparation of VLP(Ml) / cRNA_HA (H1N1).

[0136] Figure 45 shows a 48thhr Post induction expression analysis at small scale- A: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; B: SDS-PAGE.

[0137] Figure 46 shows a 48thhr Post induction expression analysis of selected clone at 2L scale- A: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; B: SDS- PAGE.

[0138] Figure 47 shows an encapsulation of HA mRNA inside VLP(Ml) by PCR.

[0139] Figure 48 shows the construct map of plasmid pYRE400_HA(HlNl) CP construct, demonstrating its structural arrangement.

[0140] Figure 49 shows encapsulation m VIP, capturing a large sequence.

[0141] Figure 50 shows Dynamic Light Scattering (DLS) analysis indicated a particle size of 149.86 nm.

[0142] Figure 51 shows the results demonstrated by the homogeneity curve.

[0143] Figure 52 provides an overview for preparation of VLP(HA-NA-Ml) / cRNA_HA (H1N1).

[0144] Figure 53 shows a 48thhr Post induction expression analysis at small scale- A: Immunoblot using HA antibody; B: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; C: Immunoblot using NA antibody; D: SDS-PAGE. Figure 54 shows a 48thhr Post induction expression analysis of selected clone at 2L scale- A: Immunoblot using HA antibody; B: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; C: SDS-PAGE.

[0145] Figure 55 shows an encapsulation of HA (H1N1) mRNA inside VLP(HA-NA-Ml) by PCR.

[0146] Figure56 shows the construct map of plasmid pYRE400_HA(HlNl) CP+NA construct, demonstrating its structural arrangement.

[0147] Fig re 57 shows the results demonstrated by DLS and homogeneity curve.

[0148] Figure 58 shows the large length sequence capture and encapsulation in Virus-Like Particles (VLPs).

[0149] Figure 59 shows the detection of HA RNA transcripts highlights the functionality of the VLP formulations and their potential for targeted delivery and prolonged RNA stability.

[0150] Figure 60 provides an overview for preparation of VLP(Ml) / cRNA_HA (H5N1).

[0151] Figure 61 shows a 48thhr Post induction expression analysis at small scale- A: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; B: SDS-PAGE.

[0152] Figure 62 shows a 48thhr Post induction expression analysis of selected clone at 2L scale- A: Immunoblot with packaging recognition fusion protein specific polyclonal antibody; B: Immunoblot with packaging recognition fusion protein specific polyclonal antibody.

[0153] Figure 63 shows an encapsulation of HA (H5N1) mRNA inside VLP(Ml) by PCR.

[0154] Figure 64 shows the construct map of plasmid p YRE400 HACP (H5N 1 ) construct, demonstrating its structural arrangement.

[0155] Figure 65 shows the result of the large length sequence capture and encapsulation in Virus-Like Particles (VLPs).

[0156] Figure 66 shows the results of thermostability studies of liquid and revived lyophilized different day time point samples in purified VLP-mRNA from H1N1 capsid protein Ml fused with packaging recognition fusion protein encapsulating cPreF RSV mRNA. Figure 67 shows the result of an experiment focused on verifying the stability of the VLPs surface antigens by immunoblot analysis.

[0157] Figure 68 shows analysis focused on verifying the stability of surface antigens of VLP in liquid formulations stored under different conditions: 120 days at 25±2°C and 240 days at 5±3°C.

[0158] Figure 69 shows analysis performed focused on verifying the stability of surface antigens of VLP in lyophilized formulations stored under different conditions: 120 days at 25±2°C and 240 days at 5±3°C.

[0159] Figure 70 shows the results of lyophilization enhanced RNA stability, as indicated by consistent RT-PCR amplification of the target RNA.

[0160] DETAILED DESCRIPTION OF THE INVENTION

[0161] The details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.

[0162] ABBREVIATIONS USED

[0163] VLP - Virus like particle mRNA - Messenger RNA

[0164] NA- Neuraminidase

[0165] HA- Hemagglutinin hMPV- Human metapneumovirus

[0166] Ml - Matrix protein

[0167] MERS-CoV- Middle East respiratory syndrome coronavirus

[0168] ORF- Open Reading Frame

[0169] RS V - Respiratory syncytial virus PreF - Pre-fusion antigen RNA

[0170] IRES- Internal ribosome entry site

[0171] The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.

[0172] Unless otherwise defined, scientific and technical terms used herein shall have meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

[0173] Definitions

[0174] As used herein, the term "expression vector" refers to a recombinant vector capable of expressing a target protein in a suitable host cell and referred to a gene construct containing essential regulatory factors operably linked to express a DNA insert.

[0175] As used herein, the term “VLPs” used herein refers to virus-derived structures made up of one or more different antigens with the ability to self-assemble, mimicking the form and size of a virus particle but lacking the genetic material so they are not capable of infecting the host cell.

[0176] As used herein, the term “Influenza” used herein refers to one or more viruses belonging to the family Orthomyxoviridae selected from but not limited to strain serotypes H1N1, H5N1 etc.

[0177] As used herein, the term “RSV” used herein refers to a common respiratory virus. It usually causes mild, cold-like symptoms. But it can cause serious lung infections, especially in infants, older adults, and people with serious medical problems.

[0178] The foregoing broadly outlines the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying the disclosed methods or for carrying out the same purposes of the present disclosure. The present invention provides an improved recombinant expression platform for efficient expression of VLP / VLP-RNA complex, particularly VLP / VLP-mRN A complex. Particularly, the present invention provides a multi-antigen VLP expressed in a protease deficient yeast (Saccharomyces cerevisiae) host system. The expressed VLP exhibits good immunogenicity and great thermostability with or without use of an adjuvant. Thus, the resultant VLP has a broader and long-lasting immune response and reduced toxicity as well as improved safety owing to lack of an adjuvant.

[0179] Further, by leveraging the inherent advantages of VLPs, such as stability and protection for encapsulated mRNA, the present invention aims to provide an efficient dual target vaccine with a prolonged shelf life, increased template availability, and enhanced therapeutic efficacy. The encapsulation of linear and circular mRNA within VLPs represents a pivotal aspect of the present invention, conferring protection against ribonuclease degradation and offering logistical advantages through lyophilization and thermostability.

[0180] The present invention meets the need for more effective and versatile recombinant expression platform for expression of VLP, VLP-RNA complexes. VLP / VLP-RNA complex expressed using said recombinant expression platform can be used against one or more antigenic targets associated with various respiratory infectious diseases, such as influenza and Respiratory Syncytial Virus (RSV) as well as several bacterial or oncological diseases. More particularly, the present invention provides a novel approach for vaccine designing through the utilization of Virus-Like Particles (VLPs) and VLP-mRNA complexes for increased stability and efficacy.

[0181] The present invention also provides a recombinant expression platform comprising a high copy episomal expression vector operably linked with a bidirectional promoter Gall / Gal 10 and a CYCT1 terminator, an integration vector (pYRHOO) operably linked with a promoter Gall and a CYCT1 terminator, and an engineered protease deficient yeast host cell. This platform is designed for the expression of a VLP-RN A complex. The encapsulated RN A within the VLP-RN A complex enhances the thermostability of the generated VLP-RN A complex, resembling that of a native virus. Thermostability' of produced vaccine candidate also have an added advantage to benefit logistics limits in maintaining cold chain, transportation to have further cost-effective productions, lead affordable and niche area reach to the mass population, especially in low- and middle-income countries (LIMCs). The recombinant expression platform's versatility is highlighted by its ability to express two or more immunogenic antigen sequences, making it a promising solution for broadspectrum vaccine development. This integrated approach enables the parallel assembly of VLP and packaging of RNA, contributing to the efficiency and scalability of vaccine production and further cost-effective solutions as two or multiple disease targets in one formulation.

[0182] More particularly, the present invention involves the encapsulation of linear or circular mRNA within VLPs, providing stability, protection from ribonucleases, and a longer shelflife. The present approach also provides for efficient and cost-effective, scalable production and purification, ensuring consistent high yields. The recombinant VLP approach involves in-vivo encapsulation of mRNA antigens, offering efficiency compared to in-vitro packaging. The emphasis on translation in the human host aims to achieve human glycosylation, enhancing vaccine efficacy.

[0183] The present invention provides an efficient methodology for the generation of circular RNA, incorporating sequence elements responsible for circularization, packaging and expression from internal ribosomal entry site signals in an episomal vector. An integration vector is used for VLP assembly and an independent expression cassette for the high copy antigen protein expression. The integration of these components in two vectors results in a complete unit of encapsulated VLP- mRNA complexes, showcasing a comprehensive and innovative approach to vaccine development.

[0184] The approach of the present invention ensures efficient large-scale production and purification with consistently high yields.

[0185] Without limiting the scope of the present invention as described above in any way, the present invention has been further explained through the examples provided below.

[0186] Examples

[0187] Example 1: Construction of Recombinant Expression Platform

[0188] The construction of the recombinant expression platform involves the assembly of key components designed to facilitate efficient gene expression. The platform comprises a high copy episomal expression vector, specifically pYRE400, which is operably linked with a bidirectional promoter Gall / Gal 10 and a CYCT1 terminator. Additionally, an integration vector, denoted as pYRHOO, is incorporated into the platform, being operably linked with a promoter Gall and a CYCT1 terminator. VLP was expressed using a combination of pYRElOO and pYRHOO and VLP-mRNA complex was expressed using pYRE400 and pYRHOO. Figure 1A shows the scheme of genetic constructs for bidirectional and unidirectional expression of VLP and VLP-RNA complex generation in Yeast. Figure IB shows restriction map of pYRE400and Figure 1C shows restriction maps of PYRHOO.

[0189] Table 1 lists different constructs developed in accordance with the present invention with their sequence IDs.

[0190] Example 2: Cloning of HA, NA, Ml of H1N1 influenza strain for co-expression construct development

[0191] Heterologous co-expression of VLP structural protein was carried out, produced recombinantly from combination of episomal expression vector named pYRElOO with Gall promoter and CYCT1 terminator cassette expressing Hemagglutinin (HA) and Neuraminidase (NA) protein from independent expression cassettes and integration vector named pYRHOO with GallP and CYCT1 terminator expressing Influenza VLP assembly protein matrix protein (Ml). The structural protein sequence HA, NA and Ml protein were codon biased and optimized for expression in PEP4, Prbl protease deficient . cerevisiae yeast host strain (as disclosed in W02010134095A2; WO2021156890A2; and WO2021176397A2.). The genes were cloned by cloning methodologies well known in art and each clone was analyzed through restriction digestion. The constructs were transformed into protease deficient host for expression studies. Figure 2 shows the restriction maps of pYRHOO Ml and pYRE100_HA+NA

[0192] Example 3: Recombinant cell line generation for H1N1 VLP generation

[0193] 3.1 Transformation in A. cerevisiae protease deficient host strain

[0194] The integrating vector containing influenza membrane protein (Ml) of H INI was transformed first in protease deficient host by acetate / SS-DNA / PEG mediated protocol well known in art. Colonies were obtained by incubating the plates at 28°C for 2-4 days on LEU2 minus amino acid plates. Subsequently, episomal vector containing HA and NA was transformed and colonies were selected on LEU2 and URA3 minus plates.

[0195] 3.2 Expression at small scale

[0196] Isolated healthy colonies were inoculated in 10 ml of YNB Glucose - LEU and -URA media and were cultured at 28°C, 250rpm as was a control of protease deficient host strain transformed with vector backbone. Expression was analysed up to 48thhr post induced time point. Induction was performed at late log phase A600-6.5 OD / ml; with final concentration of 2% galactose and 1% galactose every 24hr. Samples were analysed for expression by Immuno-blot analysis using HA, NA and Ml protein specific primary antibody followed by incubation in HRP conjugated goat anti-rabbit secondary antibody.

[0197] Immunoblot analysis using commercially available antibodies for each of HA, NA and Ml proteins confirmed the expression of all three proteins (HA (~71 to 91kda), NA (-54-71 kDa) and Ml (~ 29 to 35kda)) (Figure 3). The size aberration is due to glycosylated nature of expressed proteins. The studies confirm that the S. cerevisiae- as . expression platform is able to express the three antigens. The process was scaled up 50 times for co-expression of three antigens and VLP purification and analysis. Example 4: Scale up culture at shake flask.

[0198] Scale up culture was performed at shake flask level at 500ml (X4) scale & was analysed for expression. Inoculum of 100ml was prepared for 25 ml inoculum in respective flask in YNB Glucose -URA / LEU) for 2L scale up culture. Late log phase culture of the healthy grown preculture was inoculated into YPD broth. The cultures were induced at a final concentration of 2% galactose for 48hrs with supplemented 1% galactose in 24hr. VLPs were purified from pooled scaleup culture and expression was analysed by Immuno-blot analysis using HA, NA and Ml protein specific primary antibody followed by incubation in HRP conjugated goat anti-rabbit secondary antibody.

[0199] Immunoblot analysis using commercially available HA, NA and Ml antibodies confirmed the expression of three proteins in scale up culture; HA ( ~71 to 91kda), NA (-54-71 kDa) and Ml (~ 29 to 35kda). The studies confirm that the S. cerevisiae -based expression platform is able to coexpress the three antigens which co-assemble to form a VLP. Figure 4 shows the results of 48th hr post induction expression analysis from scale up culture.

[0200] Example 5: Purification of VLP

[0201] The recombinant VLP was purified through a series of purification steps comprising of microfiltration, diafiltration and chromatography steps, mixed with an array of different buffer compositions (Potassium phosphate buffer (pH6.8 to 7.8) , 200-2M potassium chloride along with sugar and detergent) and formulating bulking agents for preparing a stable formulation for the multiple antigen VLP molecule to be lyophilized.

[0202] The purified VLP was formulated in different buffer systems and formulating bulking agents to provide best environment for stability, handling, conformational and physical consistency. The formulations that were tried in a combination, were selected from Potassium phosphate buffer, Potassium chloride and phosphate buffer saline (PBS); with and without use of stabilizers / cryoprotectants i.e., polysorbate 80 (0.05%-0.0001%) and with and without use of a bulking agent being a disaccharide sugar as Mannitol, Sucrose and Trehalose and different percentages or combinations of the same (2 to 20%).

[0203] Example 6: Measurement of VLP size, morphology and aggregation properties The size of the VLP was measured by Dynamic Light Scattering (DLS) and co-relation curve to show solution homogeneity.

[0204] Post filtration sample was subject to particle size analysis using Anton Paar Litesizer 500 at 25°C. Correlation curve was monitored to understand formation of aggregated species, if any. Obtained results described the VLP size of + / -150 to 200nm size and co-relation curve defines the assembled VLP is homogeneous in solution. Figure 5 shows the particle size distribution and the co-relation curve of purified H1N1 VLP.

[0205] Example 7: Electron microscopy analysis of VLP

[0206] Sample was applied on the grid after dilution. Uniform uranyl acetate staining was performed. After drying, sample was checked under light microscope to check formation of visible aggregate, if any and image was captured using Talos F200C TEM (120-200kV) instrument. Figure 6 shows the electron microscopy analysis of H1N1VLP.

[0207] Electron microscopy of the H1N1 VLP showed it to be pleomorphic with a size range distribution size of 181nm in comparison to literature-based size of 120nm for H1N1 VLP and varies from strain to strain. The resultant VLP showed close structure of H1N1 VLP in electron microscopy with high copy representation of NA immunogen as confirmed through stoichiometry ratios in comparison to literature based native virus ratio of HA to NA of 10: 1. The obtained size variation further supports the high copy presentation of NA copies. Close structure proximity to native virus is further confirmed from their functional ability to produce high IgGtire and neutralization nature.

[0208] Tables 2 and 3 below show results for VLP coating of about 250ng and 500ng respectively.

[0209] Table 2

[0210] Table 3

[0211] Example 8: Stoichiometric ratios calculation for HA, NA and Ml protein in the produced VLP from co expression

[0212] ELISA: Purified proteins were coated on ELISA plate. VLP was coated at 250ng per well. After overnight coating, 1% BSA solution was used for blocking. Specific antibody for the detection of VLPs was used independently. After washing, respective secondary antibodies were applied. Finally, absorbance was measured with TMB solution at 450nm, and data was plotted accordingly. Ratio determined as HA: NA: Ml was 10: 420±10: 2600±250. Basis literature the abundance of each protein varies among virus subtypes, with the HA-NA ratio of influenza virus A / WSN / 33 (H1N1) being approximately 10 to 1 (doi: 1.0. n.28 / ivi.7 .1.:1601.5-60^ Further, the experiment predicted, presence of 10 copies of HA (present as trimer on virus) and ~410 NA per particle which exists as a tetramer on virus. The ratio describes the presence of most immunogenic HA molecules, as per the native virus while the NA expression over the surface of VLP is 400 times of the molecules present in native virus. The present invention design further aims at the organization of these molecules’ expression from expression vector in order to obtain NA in high copy number, an important antigen to provide broad protection along with HA the main protection antigen. This further, defines the present VLP candidate as potential effective vaccine candidate for Influenza H1N1 infections. Further in literature, Ml, (less immunogenic as compared to HA or NA) is demonstrated as probable immunogen to elicit both humoral and cellular immune response (h tips: / ' / doi. org / 10. 1093 / infdis / i iab269) which also been presented in effective copy number in obtained VLP from present yeast host system. Figure 7 shows the absorbance data for purified standard proteins of single antigen for the measurement of each antigen in co expressed VLP and stoichiometry.

[0213] Example 9: In Vivo Studies to measure Immunogenicity

[0214] In vivo studies in mice model were carried out to measure the ability of the triple antigen VLP to surmount an immune response with high titres in mice. Also, the antibodies were seen to be neutralizing in nature.

[0215] 9.1 Humoral immune responses and Immunization in mice

[0216] BALB / c mice were immunized with VLP formulation with & without adjuvant Addavax (AV) & Squalene (SQ). VLP amount of 2.5 pg, 5 pg, 20 pg and 52 pg were injected. Three control groups were set with buffer alone and with adjuvant only (AD (Addavax) and SQ (Squaline) injection. The animals were injected at 28 days interval (Day 0, 28) with one primary and two booster doses.

[0217] 9.2 Serum Antibody measurements

[0218] Immunized animal sera at 0, 28, 42, 56 and 84 days, was collected. IgG mediated antibody response and titre was determined by enzyme-linked immunosorbent assay (ELISA) in 96 well plate coated with 0.25 pg of VLP at 2-8 °C overnight and blocked with 2% BSA for 1 h at RT. Serum dilutions were applied to the well and incubated for at 37 °C for 2 h, followed by incubation with HRP conjugated goat anti-mouse antibodies at 37 °C for Ih. The plate was developed using TMB, following 2 M H2SO4 addition to stop the reaction, and read at 450 nm using an ELISA plate reader.

[0219] 9.3 Antigen Specific ELISA Antigen-specific IgG response was assessed under similar conditions as applied in example 8.2. Diluted sera (1 : 1000) was used and incubated for 2 hours at 37 °C. Plate was developed as per method explained in example 8.2.

[0220] 9.4 Influenza MINI Microneutralization assays

[0221] The advanced-MEM (Gibco) with 10% FBS and lx penicillin-streptomycin grown MDCK- London cells were overlayed in 96 well plate one day before the experiment, and cells with confluency of 70-80% were used. 56°C heat inactivated serum samples (30 min.) were used. Different concentration of serum (1:100 to 2-fold dilutions up to five dilutions) with 100 TCIDso of virus was incubated for 1 hr in virus growth medium in U-bottom 96 well plates at 37°C with positive serum, virus control. After incubation the mixture was overlayed on 96-well MDCK plates for 48 h of treatment and the cells were fixed in 4% PF A and stained with 0.5% crystal violet for the cytopathic effect (CPE), based upon the CPE. The NT50 was calculated, and the cell control, virus control and Cal / 09 positive serum, was taken as a positive control in each experiment. The analysis was conducted in triplicates.

[0222] 9.5 Hemagglutination (HI) Assay

[0223] Virus HA titer was determined by Hemagglutination Assay first by incubating two-fold dilutions of virus with 0.5% chicken RBCs (either U-bottom or V-bottom plates). The highest dilution showing agglutination was taken as the HA titre of virus. For hemagglutination inhibition assay, two-fold dilution of heat inactivated and RDE-treated sera starting from 1 :40 dilution was incubated with 25pr 4HAU virus for 1 hour and 0.5% RBCs were added and plate was read for teardrop formation as a sign of protective antibody response and inhibition. Highest dilution showing tear drop formation was taken as HI titre of the serum sample. Mouse sera raised against lnfA / California / 07 / 2009 was used as positive control for Hl Assay. One-way ANOVA was used for analysis. Each group had 5 experimental repeats' [p<0'0001].

[0224] Results

[0225] The total IgG binding endpoint titers (EPTs) from all the immunized mice groups were measured. Results showed that all the VLP vaccinated groups elicited IgG-mediated response as compared to controls. A dose-dependent increase in titre was observed. Interestingly, the VLP was seen to be immunogenic even without the adjuvant, suggesting its potential to generate an immune response. Further, binding of specific antibodies of HA, NA and Ml were observed during ELISA as compared to control which highlights the potential of VLP in inducing a strong and potent immune response by eliciting effective antibody responses for all three antigens and, therefore, may serve as an effective vaccine candidate for Influenza H1N1 infections. Figure 8 shows the bar graph depicting the total IgG response results. Figure 9 shows the bar graph depicting the individual Antigen IgG response results.

[0226] In the microneutralization inhibition assay, sera from all the VLP vaccinated groups showed significant neutralization potential as compared to control groups (placebo sera).

[0227] The MNT50 is the microneutralization assay test performed with the test serum with the virus for the complete or >50% neutralization at a specific dilution of the serum. However, maximum neutralization was observed at the highest (52 pig) dose VLP as compared to control and Addavax groups. The present experiment showed a titre in the range of 1 :500 to 1:3200 in applied dose range which is considered to be highly effective titre. Figure 10 shows the bar graph depicting the MNT assay results.

[0228] Hemagglutination (Hl) Assay was performed to assess the anti-influenza HA antibody titers against IAV / California / 07 / 2009 influenza virus strains. The gold standard test for quantifying anti- HA antibodies was performed by using a Hemagglutination Inhibition assay. Historically HAI titer of 1:40 or greater has been co related with protection. The VLPs of the present invention showed mouse sera protected against tested homologous virus i.e., IAV / California / 07 / 2009 influenza strain with a high HI titer of >500 at 52 pg dose as compared to the control groups.

[0229] The results thus comprehensively show that VLP (without adjuvant) was able to surmount an immune response which has neutralization potential. Figure 11 shows the bar graph depicting the HI assay results.

[0230] Example 10: Safety of the vaccine formulation over large range of dose formulation

[0231] Extensive studies were performed to demonstrate that the enveloped VLP for Influenza H1N1 virus, composed of three antigens, HA, NA and Ml and produced using the yeast expression platform has considerable safety even in multiple doses. In vivo studies were performed in mice to demonstrate the safety of this formulation. 10.1 Studies in mice (Mus musculus)

[0232] Multiple cohorts of Mice (Mus musculus sp.), Strain Balb / c, gender female, age 6-8 weeks, were procured from Palamur Biosciences Private Limited; Telangana (India) and under all applicable guidelines, injected intramuscularly with liquid formulation of the VLP in dose of 2.5 pg, 5 pg, 20 pg and 52 pg. Three vaccine formulations were studied simultaneously: a) with commercially available adjuvant Addavax (AD) and squalene (SQ) and b) VLP alone with no adjuvant. In each cohort, six animals were included to maintain statistical significance of the data observed.

[0233] The mice were given two doses, on day 0, and day 28 and observed for any signs / symptoms of morbidity or other changes. No mortality was documented through the course of study. All animals were observed to maintain a healthy condition post injection, and all through the course of study, there were no visible changes in their motility, no behavioral changes inside the cage were observed or fighting wounds or any skin infections were observed, no lack of shininess of hair was observed. Food habits were observed to be normal for all the groups. Body weight was normal across the study in all the groups of animals. The obtained data showed the safety of the vaccine formulation over large range of dose without any health issues.

[0234] The animals were humanely sacrificed by carbon dioxide asphyxiation at termination of the analysis.

[0235] Figure 12 shows the body weight trends of mice over time across different dose groups (0 pg, 2.5 pg, 5 pg, 20 pg, and 52 pg) for VLP HA+NA+M1 (without adjuvant) and VT.P_HA+NA+Ml+AddaVax adjuvant formulations.

[0236] Example 11: Preparation of H5N1 VLP HA + NA +M1

[0237] Triple antigen VLP of H5N1 was also prepared following similar protocol as set out below:

[0238] The H5N1 Virus-Like Particle (VLP) expressing HA + NA Episomal+Ml Integrated was characterized through predicted molecular weights and immunoblot analysis at both small and large-scale cultures. The predicted molecular weights for the components were 64.08 kDa for HA, 51.59 kDa for NA, and 27.89 kDa for Ml protein. Higher size bands were also obtained due to glycosylated nature of proteins. Immunobloting was conducted with specific polyclonal antibodies for HA, NA and Ml and the results were corroborated with SDS-PAGE analysis. In the 48th-hour post-induction expression analysis at the 3L flask level (Figures 13 and 14), HA expression was observed between approximately 64 to 71 kDa, while NA expression was detected at around between 51 to 70 kDa in the pellet samples. Importantly, no immunoblot signal was detected in the control sample (protease deficient yeast host--- Saccharomyces cerevisiae) against the expected bands of HA and NA proteins, indicating specificity in the expression constructs. The HA + NA Episomal and Ml Integrated construct in the large-scale culture, specifically in the HA +NA (episomal)- l integrated-pro tease deficient yeast clone, successfully expressed both HA and NA proteins. This comprehensive analysis provided valuable insights into the accurate expression of the targeted components in the designed H5N1 VLP construct. Figure 15 shows the construct map of plasmid pYRIl 00__Ml (H5N1), demonstrating its structural organization. Figure 16 shows the construct map of plasmid pYRElOO HA+NA (H5N1), demonstrating its structural arrangement. Figure 17 shows the particle size distribution as a function of particle diameter (in nm) with a peak at the measured size range. Figure 18 shows the correlation function (g2) against delay time (in seconds), demonstrating the dynamic light scattering (DLS) data for particle analysis.

[0239] Example 12: Cloning and co-expression construct development for VLP-mRNA complex

[0240] Heterologous co-expression of VLP structural proteins and mRNA transcription were produced recombinantly from combination of episomal expression vector named pYRE400 which transcribes RSV PreF antigen mRNA. The vector also carries sequences for the mRNA circularization as well as its packaging. The vector also carries expression cassette for structural protein expression of recombinant Neuraminidase (NA). Both functions are transcribed from Galactose based induced promoter and CYCT1 terminator.

[0241] Further, integration vector named pYRHOO was used for the cloning of Influenza VLP assembly protein named matrix protein (Ml) in 3’ fusion with assembly cum packaging recognition fusion protein (CP) recognized by packaging signal from transcribed RNA from episomal vector. The integrating expression vector also contained an independent expression cassette for the expression of HA ORF Influenza structural antigen protein. Both cassettes are under Gall promoter and with CYCT1 terminator. The complete unit of episomal and integration vector resulted in an encapsulated VLP-mRNA complex.

[0242] Codon biased and optimized structural sequences of HA, NA and Ml protein used for the generation of H1N1 VLP, has also been used for the generation H1N1VLP in VLP-mRNA complex of HINIVLP / c RNA PreF (RSV for expression in PEP4, Prbl protease deficient 5. cerevisiae yeast host).

[0243] The genes were cloned by cloning methodologies well known in art into respective episomal and integration expression vectors. The cloned gene was analyzed through restriction digestion. The construct was transformed into protease deficient host for expression studies.

[0244] Example 13: Recombinant cell line development for VLP-mRNA complex generation

[0245] Various single, dual and multi-antigen VLPs were prepared in accordance with the present invention as per the protocol set out in examples 2-8.

[0246] 13.1 Encapsulated RNA Analysis

[0247] RNA was isolated from 500 pg of VLP using known protocols in the art using a mixture of Trizol and chloroform, solution was mixed and was centrifuged for 15 minutes at 16 300 x g. The RNA retained in the aqueous phase was precipitated by Isopropanol followed by 15 minutes centrifugation at 16 300 x g. RNA pellet was washed three times with 80% ethanol. RNA was dissolved in nuclease free water and stored at -80 °C. This was followed by PCR analysis using gene specific primers using methods known in art. cDNA was prepared from 1 pg of the RNA by using iScript cDNA Synthesis kit (Bio-Rad, 1708891) and has been used as a template for PCT or qPCR for encapsulated RNA analysis.

[0248] 13.2 Preparation of H1N1 VLP (Ml) / cRNA PreF (RSV) Complex

[0249] The preparation of expression constructs involved the generation and analysis of two key constructs: the integration construct (pYRHOO) and the episomal construct (pYRE400). Figure 19 provides an overview for preparation of H1N1 VLP (Ml) / cRNA_PreF (RSV). In the integration construct, the Ml gene in fusion with assembly protein (CP) recognizing packaging signal was expressed. The confirmation of expression was validated through immunoblotting, with the absence of signals in the control sample affirming specificity. The episomal construct, consisting of PreF and regulatory elements for circular RNA generation and encapsulation sequence elements, was transformed into the integrated clone. At the 48thhour post-induction, expression analysis was performed both at a small scale (Figure 20) and the 2L scale (Figure 21). Immunoblotting using SDS-PAGE and specific antibodies for assembly protein in fusion with Ml protein was employed for validation. The results confirmed the expression assembly protein in fusion with Ml protein (~43 kDa) during small-scale analysis. Importantly, no immunoblot signal was detected in the control sample (protease deficient yeast host Saccharomyces cerevisiae with transformed backbone plasmid), providing evidence of construct specificity. Purification of VLP was performed from scale up culture. The encapsulation ofRNA within these VLPs was confirmed through PCR, with the detection of PreF-RSV mRNA using specific primers. RNA isolation was performed from purified VLPs followed by cDNA conversion which was used as template for PCR analysis. Further, the product's sequencing validated the successful encapsulation and expression of PreF RSV mRNA (Figure 25). Further analysis included studying mRNA stability after lyophilization and storage at 4°C, and DLS revealed a particle size of 197.40 nm (Figure 24). Real-time PCR corroborated the encapsulation, with a CT mean of 22.7. This comprehensive process ensured the successful generation, expression, and encapsulation of the desired constructs for potential applications in vaccine development or therapeutic purposes. Figure 22 shows the construct map of plasmid pYRI Ml(HlNl) +CP construct, demonstrating its structural arrangement, and Figure 23 shows the construct map of plasmid pYRE400_PreFCP construct, demonstrating its structural arrangement.

[0250] 13.3 Preparation of H1N1 VLP (NA-Ml) / c RNA PreF (RSV) Complex

[0251] The process of preparing expression constructs involved two critical components: the integration construct (pYRHOO) and the episomal construct (pYRE400). Figure 26 provides an overview for preparation of H1N1 VLP (NA-Ml) / cRNA_PreF (RSV). In the integration construct, the Ml gene in fusion with assembly protein (CP) recognizing packaging signal was expressed. The episomal construct, on the other hand, incorporated PreF and NA and regulatory elements for circular RNA generation and encapsulation. The transformation of the episomal expression construct into the integrated clone marked a pivotal step in the procedure. Following this, small-scale expression analysis was conducted, and the expression was subsequently scaled up to a 2L culture. The purification of Virus-Like Particles (VLPs) was undertaken to ensure the isolation of the desired constructs. At the 48thhour post-induction, expression analysis was performed both at a small scale (Figure 27) and the 2L scale (Figure 28). Immunoblotting using SDS-PAGE and specific antibodies for assembly protein in fusion with Ml protein and NA proteins was employed for validation. The results confirmed the expression of NA (-54-71 kDa) and assembly protein in the fusion protein (-43 kDa) during small-scale analysis. Importantly, no immunoblot signal was detected in the control sample (PYPD - Saccharomyces cerevisiae transformed with vector backbone), providing evidence of construct specificity. The expression was further confirmed at the 2L culture scale, and the resulting culture pellet was subjected to purification for downstream applications. The meticulous observation and validation steps ensured the successful generation of the intended expression constructs and encapsulation of PreF RSV in VLPs. Figure 29 shows pYRE400_PreFCP+NA construct.

[0252] Figure 30 shows PCR confirmation of encapsulated mRNA (PreF) was conducted using PreF- specific primers, with the amplification of a large-length gene of interest (GOI) sequence (2889 bp) to confirm the presence of intact RNA and efficient encapsulation in VLP. The samples tested included the Gene Ruler 1 Kb ladder and cDNA from RNA isolated from purified VLP, using both F and reverse primers.

[0253] Table: 4

[0254] 13.4 Preparation of H1N1 VLP (HA-NA-Ml) / cRNA PreF (RSV) complex

[0255] The preparation of H1N1 Virus-Like Particles (VLP) expressing HA-NA-M1 and cirRSV involved the generation of integration and episomal constructs, followed by expression analysis and purification. Figure 31 provides an overview for preparation of H1N1 VLP (HA-NA- Ml) / cRNA_PreF (RSV). The integration construct, contains Ml protein fused with assembly cum packaging recognition fusion protein +HA, expressed as another independent expression cassette contains Gall promoter and CYCT1. Simultaneously, the episomal construct, including PreF and NA and regulatory elements for circular RNA generation and encapsulation, was transformed into the integrated clone (Figures 35 and 36) cell line. Expression analysis began with a small scale, progressed to a 2L scale, followed by purification for further study. At the 48thhour post-induction, expression analysis was conducted at both scales. Observations from the analysis included the detection of HA expression between 71kDa and 91kDa using an HA-specific antibody and NA expression between 54kDa and 71kDa with an NA-specific polyclonal antibody in the pellet samples. Expression of Ml protein fused with assembly cum packaging recognition fusion protein (~43 kDa) was confirmed using a packaging recognition fusion protein specific antibody at a small scale (Figure 32). Importantly, the control sample (protease deficient yeast host- Saccharomyces cerevisiae transformed with vector backbone) showed no immunoblot signal, affirming construct specificity. Expression confirmation at the 2L culture scale (Figure 33) preceded purification. Encapsulation of mRNA was assessed through PCR (Figure 34), using isolated encapsulated RNA converted to cDNA which was used as template by Pref specific PCR primers revealing the study of PreF RSV mRNA from purified VLP. The expected product size of 214 was detected. Further, the purified preparation of H1N1 VLP (HA-NA-Ml) / cRNA_PreF (RSV) was obtained from a 2L scale batch. Antigens were verified by immunoblot using HA, M, and NA-specific antibodies Figure 37. Encapsulated mRNA (PreF) in purified VLP was confirmed by PCR (Figure 38). and dynamic light scattering (DLS) analysis was performed, and results are shown under Figure 39. The homogeneity curve was also generated (Figure 40). This comprehensive process ensured the successful preparation and characterization of the H1N1 VLP expressing HA-NA-M1 and cPreF RSV constructs.

[0256] 13.5 In Vivo Studies to Measure Immunogenicity

[0257] In vivo studies were conducted to measure the immunogenicity of the dual vaccine candidate Hl N1 VLP (HA-NA-Ml) / cRNA_PreF (RSV) complex. The immunogenicity study involved 6-8-week- old Balb / c mice (N=6 / group), with a dosing volume of 100 pl via intramuscular administration. Doses of 10 pg, 20 pg, 50 pg, and 100 pg were administered, with a placebo group included. Serum samples were collected on Day 0, Day 28, and Day 42 to analyze total IgG levels (Figure 41) against VLP and Pre-F. Immunogenicity was observed at 42 days post-immunization across all doses, with significant immune responses at 50 and 100 pg. The 50 pg dose elicited a more effective immune response against Pre-F (RSV) (Figure 42) at both Day 28 and Day 42. The response against individual antigens was also captured. 13.6 Safety of the vaccine formulation over large range of dose formulation

[0258] Extensive studies were performed to demonstrate that the enveloped VLP for Influenza H1N1 virus, composed of three antigens, HA, NA and Ml encapsulating PreF (RSV) mRNA and produced using the yeast expression platform has considerable safety even in multiple doses. In vivo studies were performed in mice to demonstrate the safety of this formulation.

[0259] 13.7 Studies in mice (Mus musculus)

[0260] In vivo studies were carried out in mice (Mus musculus, Balb / c strain, 6-8 weeks old, female) under applicable guidelines. The mice were intramuscularly injected with the VLP encapsulating PreF mRNA liquid formulation at doses of 10 pg, 20 pg, 50 pg, and 100 pg, with no adjuvant Figure 43 in multiple cohorts (six animals per cohort to ensure statistical significance). The mice were administered two doses, on Day 0 and Day 28, and monitored for any signs of morbidity or adverse effects. No mortality was observed throughout the study. The animals remained healthy, with no changes in motility, behavior, or signs of skin infections, and their food intake remained normal. The study demonstrated no abnormalities or adverse reactions post-immunization, with body weight remaining stable across all groups. Mice were humanely sacrificed by carbon dioxide asphyxiation at the end of the study. Observations confirmed the safety of the vaccine formulation across the tested doses.

[0261] 13.8 Preparation of (Ml) / cRNA_HA(HlNl) complex

[0262] The preparation of the VLP(Ml) / cRNA_HA(HlNl) expression constructs involved two main components: an integration construct and an episomal construct. Figure 44 provides an overview for preparation of VLP(Ml) / cRNA_HA (H1N1). The integration construct, designated pYRHOO Ml+assembly cum packaging recognition fusion protein, was designed for the expression of the Ml protein fused with an assembly and packaging recognition fusion protein. On the other hand, the episomal construct, named pYRE400-HA+regulatory elements for circular RNA generation & encapsulation, was engineered with regulatory elements to enable circular RNA generation and encapsulation of the desired HA. The transformation of the episomal expression construct into an integrated clone cell line was followed by small-scale expression analysis, and subsequently, the expression was scaled up to 2 liters. Purification of the Virus-Like Particles (VLPs) was carried out to isolate the VLP-mRNA complex. At the 48thhour post-induction, expression analysis was performed at both small scale and 2L scale (Figures 45 and 46). Immunoblotting using a specific polyclonal antibody against the packaging recognition fusion protein confirmed the expression of Ml in fusion with the assembly and packaging recognition fusion protein (~43 kDa) at the small scale. No immunoblot signal was detected in the control sample (protease deficient yeast host - Saccharomyces cerevisiae transformed with backbone plasmid). The expression of the selected clone at the 2L culture scale was also confirmed, and the culture pellet was further processed for purification. Sequencing of the expression construct plasmid revealed the presence of the HA and matrix protein gene in the expression vector.

[0263] Subsequent analysis focused on encapsulation of mRNA using PCR, studying circularization, and amplifying large-length mRNA. Purified VLPs were subjected to RNA isolation followed by cDNA conversion and PCR using HA-specific primer PCR, revealing an expected product size of 229 bp (Figure 47). Amplification of a large-length DNA fragment was also performed to demonstrate efficient encapsulation in VLP, capturing a large sequence Figure 49. The samples tested included the Gene Ruler 1 Kb ladder and cDNA from RNA isolated from purified VLP, using F and reverse primers, with an expected product size of 3115 bp.. Dynamic Light Scattering (DLS) analysis indicated a particle size of 149.86 nm Figure 50, and the homogeneity curve Figure 51. was also generated to assess the uniformity of the VLP preparation.

[0264] Table: 5

[0265] Further, sequencing of the encapsulated amplified HA gene after conversion to cDNA was performed and confirmed the presence of the HA gene. Figure 48 shows the construct of pYRE400_HA(HlNl) CP. 13.9 Preparation of VLP (HA-NA-Ml) / cRNA HA (H1N1) complex

[0266] The VLP (HA-NA-Ml) / cRNA_HA (H1N1) expression system was prepared using a combination of an integration construct (pYRHOO Ml+assembly & packaging recognition fusion protein+HA) and an episomal construct (pYRE400-HA-NA+regulatory elements for circular RNA generation and encapsulation). Figure 52 provides an overview for preparation of VLP(HA-NA- Ml) / cRNA_HA (H1N1) complex. The episomal construct was transformed into the integrated clone having integrated Ml protein fused with assembly cum packaging recognition fusion protein +HA protein, followed by small-scale expression analysis and subsequent scale-up to 2 liters. Purification of the VLP was carried out, and analytical studies were conducted at the 48thhour post-induction for large scales (Figures 53 and 54). The expected sizes of HA, NA, and Ml protein fused with assembly cum packaging recognition fusion protein were 63.1 kDa, 51.59 kDa, and 43 kDa (with deviated size of HA ~71-91kda, NA-54-71 kDa due to glycosylation), respectively. Expression of HA, NA, and Ml+assembly cum packaging recognition fusion protein was confirmed using specific antibodies, with no signal detected in the control sample (protease deficient yeast host - Saccharomyces cerevisiae transformed with vector backbone plasmid). Sequencing of the expression construct plasmid confirmed the presence of the HA NA and Ml+CP gene. Further analysis involved transcription and encapsulation of mRNA through PCR. The observations indicated successful HA mRNA encapsulation, with the expected product size of 256 bp using HA specific primers (Figure 55). RT-PCR analysis showed the HA gene was detected in the PCR, with a real-time PCR Ct value of 18.8±4 (~30 to 70 ng encapsulated mRNA). Further, sequencing of the encapsulated amplified HA gene after conversion to cDNA was performed and confirmed the presence of the HA gene Figure 57 shows the DLS and homogeneity curve.

[0267] 13.10 Amplification of Large Length DNA Fragment

[0268] The study also focuses on the amplification of a large-length DNA sequence to demonstrate efficient large length insert sequence capture and encapsulation in Virus-Like Particles (VLPs) Figure 58. The experimental design included the use of cDNA obtained from RNA isolated from purified VLPs. Specific forward and reverse primers were employed to amplify the DNA, with the expected product size being 3115 bp. As a reference, a Gene Ruler 1 Kb ladder was utilized to verify the amplification. Analytical techniques such as RT-PCR confirmed the successful detection of the HA gene, with the mean cycle threshold (Ct) value being 12.54, which further indicates a significant presence of the target encapsulated RNA sequence within the VLPs.

[0269] Table: 6

[0270] Figure 56 shows the construct of pYRE400_HA(HlNl) CP+NA.

[0271] 13.11 MDCK Cell Binding Assay for Functional Analysis of VLP (HA-NA-Ml) / cRNA_HA (H1N1) Complex

[0272] A functional assay was performed using Madin-Darby Canine Kidney (MDCK) cells to evaluate the binding and internalization of VLPs containing HA-NA-M1 proteins and circular RNA (cRNA) of the HA gene. MDCK cells express sialic acid residues on their glycoconjugates through a2-3 or a2-6 glycosidic bonds. The surface antigen “NA” in the VLPs facilitates binding to a2-6 sialic acid, aiding endocytosis. After incubating MDCK cells with the VLP formulations for 30 minutes, total RNA was isolated and converted to cDNA. PCR amplification using HA-specific primers confirmed the presence of encapsulated HA RNA, indicating successful internalization. The Ct value obtained from RT-PCR analysis was approximately 25, confirming the presence of HA RNA transcripts.

[0273] Binding and internalization of the H1N1 VLP (HA-NA-Ml) / cRNA_HA complex was assessed by detecting encapsulated HA cRNA within VLPs. The results confirmed the presence of HA RNA in VLP-treated cells, validating the proper assembly, important for internalization and functionality of viral proteins. Notably, the stability of the cRNA inside MDCK cells was significantly higher than that of linear RNA encapsulated in lipid nanoparticles (LNPs). While linear RNA from LNPs typically degrades within five minutes in the bloodstream, the circular HA RNA remained stable inside MDCK cells for at least 30 minutes, highlighting its resistance to nuclease-mediated degradation.

[0274] Protocol and Results

[0275] To test the functionality of the VLP formulations, MDCK cells were incubated with - VLPs encapsulating HA circular RNA. These cells were exposed to the VLPs at a concentration of 200 pg for 30 minutes. Post incubation, total RNA was extracted using the Trizol method, followed by cDNA synthesis and RT-PCR analysis. HA plasmid served as a positive control. Obtained Ct values, for the HA-NA and cRNA HA formulations, are 25.63 and 28.87. The positive control plasmid exhibited a Ct value of 5.38, while no Ct values were detected for untreated MDCK cells, confirming the specificity of the assay.

[0276] The data conclusively demonstrates the successful binding and internalization of VLPs in MDCK cells, indicated by the presence of RNA transcripts of HA. cRNA HA where only Ml is used for VLP generation and ct values (28.87) showed no effective binding due to absence of NA on VLP surface. The higher Ct values observed in comparison to plasmid test control for the HA-NA are attributed to the lower expression of a2-6 sialic acid on MDCK cell line used. The findings underscore the stability of circular RNA, as evidenced by its prolonged presence in MDCK cells for up to 30 minutes. This stability provides a significant advantage over the rapid degradation of linear RNA in LNP formulations. Furthermore, the successful detection of HA RNA transcripts highlights the functionality of the VLP formulations and their potential for targeted delivery and prolonged RNA stability (Figure 59).

[0277] Table: 7 In conclusion, the observed Ct values confirm the efficient encapsulation of HA circular RNA within VLPs, its internalization in MDCK cells, and the stability of cRNA once internalized.

[0278] Further, MDCK cell binding assay was performed for functional Analysis of VLP (HA-NA-M1) / cRNA_PreF RSV complex to check the long-term stability of the encapsulated mRNA till 120hrs.

[0279] The same methodology detailed above in Example 13.7 was applied in the context of the functional assay was performed using Madin-Darby Canine Kidney (MDCK) cells to evaluate the binding and internalization of VLPs comprising (HA-NA-M1) / cRNA_PreF RSV.

[0280] To test the functionality of the VLP formulations, here MDCK cells were incubated with - VLPs encapsulating PreF circular RNA and cells were exposed to the two amounts of VLPs which are 200 pg and 300 pg. Incubation was analyzed for three, time points 30, 60 and 120 minutes to further analyze the VLP-mRNA complex stability for longer time as indicated under Table 8 below.

[0281] Post incubation, total RNA was extracted using the Trizol method, followed by cDNA synthesis and RT-PCR analysis was performed. PreF plasmid served as a positive control.

[0282] Table: 8

[0283] In conclusion, the observed Ct values confirm the efficient encapsulation of PreF circular RNA within VLPs, its internalization in MDCK cells, and the stability of cRNA once internalized. Ct value at different time points was same, defines no more internalization after 30 min but maintaining the internalized circular RNA till 120 min. The study further showed long term stability of encapsulated RNA to help suffice effective drug substance availability.

[0284] This underscores the potential of VLP formulations as robust and stable delivery systems for circular RNA, with promising applications in therapeutics and vaccine development.

[0285] 13.12 Preparation of VLP(M1) / HA cRNA (H5N1) complex

[0286] The expression constructs for the VLP (M1) / HA cRNA (H5) involved a combination of an integration construct, pYRHOO Ml+assembly & packaging recognition fusion protein to facilitate the expression of the fusion protein consisting of Ml and assembly & packaging recognition sequences and an episomal construct, pYRE400-HA- regulatory elements, aimed at circular RNA generation of HA and encapsulation, incorporating regulatory elements. Figure 60 provides an overview for preparation of VLP(Ml) / cRNA_HA (H5N1).

[0287] The transformation of the episomal expression construct into Ml+protein fused with assembly cum packaging recognition fusion protein + integrated finalized clone was followed by small-scale expression analysis and subsequent scale-up expression in a 2 L culture.

[0288] The purification process of the VLP involved various steps, including analytics such as encapsulation of mRNA by PCR at the 48thhour post-induction, in large-scale expressions (Figures 61 and 62). Immunoblotting using a packaging recognition fusion protein-specific polyclonal antibody and SDS-PAGE were carried out for expression analysis. The observations revealed that the Ml expression, fused with the assembly & packaging recognition fusion protein, was confirmed at the small scale, with a molecular weight of approximately 43 kDa. No immunoblot signal was detected in the control sample (protease deficient yeast host - Saccharomyces cerevisiae transformed with backbone plasmid). The expression was further confirmed at a larger scale, and the culture pellet was processed for purification. The RNA transcription and encapsulation from purified VLP was analyzed using HA-specific primers. An expected product size of 217 bp was detected, confirming successful transcription and encapsulation. Sequencing of the encapsulated amplified HA gene after conversion to cDNA was performed and confirmed the presence of the HA gene (Figure 63) in agarose gel.

[0289] 13.13 Amplification of large length DNA fragment

[0290] The study also focuses on the amplification of a large-length DNA fragment to demonstrate efficient sequence capture and encapsulation in Virus-Like Particles (VLPs) Figure 65. The experiment utilized cDNA obtained from RNA isolated from purified VLPs as the template, with specific forward and reverse primers designed for amplification. The expected product size of the amplified DNA fragment was 3068 bp. A Gene Ruler 1 Kb ladder was used as a molecular size marker to verify the successful amplification of the target DNA sequence. This process highlights the efficiency of encapsulating large-length sequences in VLPs for downstream applications. Figure 64 shows the construct of pYRE400_HACP(H5Nl).

[0291] Example 14: Lyophilization and Thermostability analysis

[0292] Purified VLP-mRNA of H1N1 capsid protein Ml protein fused with assembly cum packaging recognition fusion protein encapsulating Circ-RSVpreF mRNA was used as sample here. Different day samples and comparisons were analyzed against day 1, as fresh sample results. One of the samples kept at 2-8°C for 45 days and the other day 1 sample was lyophilized, regenerated and analyzed for thermostability.

[0293] For analysis, encapsulated mRNA was isolated from each of, specified day sample and conditions. cDNA was prepared and PCR was performed by PreF specific primers with PCR conditions of initial denaturation at 95°C for 1 cycle followed by 25 cycles of denaturation 95°C for 30sec annealing 58°C for 30sec, extension at 72°C for 15s and the final extension 1 cycle at 72°C for lOmin was performed.

[0294] Presence of correct amplified band size of 214bp in fresh day 1 sample and further at different time points and confirms the presence of intact mRNA isolated from intact VLP in stability study. Figure 66 shows the results of the thermostability studies of liquid and revived lyophilized different day time point samples in purified VLP-mRNA from H1N1 capsid protein Ml fused with packaging recognition fusion protein, encapsulating cPreF RSV mRNA. 14.1 Long-Term Thermostability of VLP-circRNA Complex

[0295] The long-term thermostability of the H1N1 VLP (HA-NA-Ml) / cRNA_PreF (RSV) complex was analyzed through two key experiments. The first experiment focused on verifying the stability of the VLPs by immunoblot analysis Figure 67 to detect the intactness of the three surface proteins HA, NA, and M protein. This analysis was performed for both lyophilized and liquid formulations stored under different conditions: 120 days at 25±2°C Figure 68 and 240 days at 5±3°C Figure 69. Antigen-specific antibodies targeting HA, NA, and M proteins were used in the immunoblot assays. The stability of the VLPs was initially assessed on Day 0, and further analysis confirmed the presence of intact surface proteins after storage under the specified conditions for both liquid and lyophilized formulations.

[0296] The second experiment aimed to evaluate the stability of the encapsulated RNA within the VLPs. For this purpose, RNA was isolated from purified VLPs that had been incubated under the specified conditions, and RT-PCR was performed using RSV-specific PreF primers. The results demonstrated that lyophilization enhanced RNA stability, as indicated by consistent RT-PCR amplification of the target RNA (Figure 70). The Ct values for both liquid and lyophilized formulations remained within the range of 19.8±5 even after 210 days depicts ~30ng to 70 ng mRNA encapsulation in VLP, confirming the stability of the encapsulated RNA. These findings highlight the effectiveness of lyophilization in preserving the VLP-circRNA complex, ensuring the integrity of both the structural proteins and the encapsulated RNA over extended storage periods.

[0297] It is to be noted that the present invention is susceptible to modifications, changes, and adaptations by those skilled in the art. Such modifications, changes adaptations are intended to be within the scope of the present invention. Some of the key advantages of the present invention are listed below:

[0298] Advantages

[0299] • Robust and versatile expression platform for efficient expression of VLP and VLP-mRNA complex.

[0300] • Multivalent VLP / VLP-mRNA complexes with broad spectrum applicability against one or more antigenic targets.

[0301] • Multivalent VLPs have improved immunogenicity with / without use of adjuvants. • mRNA encapsulation in VLPs ensures thermostability, protection from ribonucleases, and a longer shelf life.

[0302] • Platform adaptability for linear and circular mRNA encapsulation.

[0303] • Efficient VLP assembly and mRNA packaging integrates vectors for streamlined vaccine production.

[0304] • Enable simultaneous VLP assembly and mRNA packaging for increased efficiency.

Claims

THE CLAIM:

1. A recombinant expression platform comprising: i. a high copy episomal expression vector; ii. an integration vector; and iii. engineered protease deficient yeast host cell, wherein said vectors are operably linked with a bidirectional promoter Gall / Gal 10 and comprise a CYCT1 terminator, a selection marker selected from Ura3c or Leu2; an Ampicillin resistance marker; an origin of replication site pUC ori; a 2 micron origin; and a sequence region comprising of multiple cloning sites; wherein said platform enables insertion of full length or truncated polynucleotide sequence for expression of desired antigenic polypeptides into the host cell.

2. The recombinant expression platform as claimed in claim 1 , wherein the episomal vector is selected from pYRE400 or pYRElOO.

3. The recombinant expression platform as claimed in claim 1 , wherein the integration vector is pYRHOO.

4. The recombinant expression platform as claimed in claim 1, wherein said platform is capable of expressing a VLP and / or a VLP-RNA complex.

5. The recombinant expression platform as claimed in claim 1 , wherein said protease deficient yeast host is Saccharomyces cerevisiae.

6. The recombinant expression platform as claimed in claim 4, expressing two or more immunogenic antigen sequences against two or more viruses.

7. The recombinant expression platform as claimed in claim 4, wherein the episomal vector expresses VLP antigens and regulatory sequences for circularization and packaging of mRNA.

8. The recombinant expression platform as claimed in claim 4, wherein the integration vector is integrated into the genome comprising the assembly and packaging recognition fusion protein and expresses VLP antigens.

9. The recombinant expression platform as claimed in claim 4, wherein the episomal and integration vector enables assembly of VLP and packaging of the mRNA.

10. The recombinant expression platform as claimed in claim 4, wherein the episomal and integration vector enables parallel assembly of VLP and packaging of the mRNA.

11. A nucleic acid construct with SEQ ID NO. 1 for expression of Influenza VLP assembly protein matrix protein (Ml) wherein said construct comprises a nucleic acid sequence encoding the influenza membrane protein (Ml) of H1N1, and an integration expression vector comprising Leu2 selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal promoter.

12. A nucleic acid construct with SEQ ID NO. 2 for expression of Influenza VLP assembly Hemagglutinin (HA) and Neuraminidase (NA) protein of H1N1, wherein said construct comprises nucleic acid sequences encoding the Hemagglutinin (HA) and Neuraminidase (NA) protein of H1N1, and an episomal expression vector comprising Ura3C selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal promoter.

13. A nucleic acid construct with SEQ ID NO. 3 for expression of Influenza VLP assembly protein matrix protein (Ml) wherein said construct comprises a nucleic acid sequence encoding the influenza membrane protein (Ml) of H5N1, and an integration expression vector comprising Leu2 selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal promoter.

14. A nucleic acid construct with SEQ ID NO. 4 for expression of Influenza VLP assembly Hemagglutinin (HA) and Neuraminidase (NA) protein of H5N1, wherein said construct comprises nucleic acid sequences encoding the Hemagglutinin (HA) and Neuraminidase (NA) protein of H5N1, and an episomal expression vector comprising Ura3C selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal promoter.

15. A nucleic acid construct with SEQ ID NO. 5 for expression of Influenza VLP assembly protein named matrix protein (Ml) in fusion with assembly cum packaging recognition fusion protein (CP) wherein said construct comprises nucleic acid sequences encoding the matrix protein (Ml) and assembly cum packaging recognition fusion protein (CP) of H1N1, and an integration expression vector comprising Leu2 selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal promoter.

16. A nucleic acid construct with SEQ ID NO. 6 for transcription of VLP prefusion conformation-stabilized RS V fusion protein (preF) in fusion with assembly cum packaging recognition fusion protein (CP) wherein said construct comprises a nucleic acid sequence encoding the preF of RS V and assembly cum packaging recognition fusion protein (CP), and an episomal expression vector comprising Ura3C selection marker, CYCT1terminator, an Ampicillin resistance marker, pUC ori along with bidirectional Gall / GallO promoter.

17. A nucleic acid construct with SEQ ID NO. 7 for transcription of prefusion conformation- stabilized RSV fusion protein (preF) and expression of Neuraminidase (NA) wherein said construct comprises nucleic acid sequences encoding the preF protein of RSV with assembly cum packaging recognition fusion protein (CP) and Neuraminidase (NA) protein, and an episomal expression vector comprising Ura3C selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with bidirectional Gall / GallO promoter.

18. A nucleic acid construct with SEQ ID NO. 8 for expression of Influenza VLP assembly protein named matrix protein (Ml) in fusion with assembly cum packaging recognition fusion protein (CP) and Hemagglutinin (HA) wherein said construct comprises nucleic acid sequences encoding matrix protein (Ml), assembly cum packaging recognition fusion protein (CP) and Hemagglutinin (HA), and an integration expression vector comprising Leu2 selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with promoter.

19. A nucleic acid construct with SEQ ID NO. 9 for transcription of Hemagglutinin (HA)H1N1 wherein said construct comprises nucleic acid sequences encoding assembly cum packaging recognition fusion protein (CP) and Hemagglutinin (HA) of H1N1, and an episomal expression vector comprising Ura3c selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with bidirectional Gall / GallO promoter.

20. A nucleic acid construct with SEQ ID NO. 10 for transcription of Influenza VLP assembly cum packaging recognition fusion protein (CP) in fusion with Hemagglutinin (HA) and expression of Neuraminidase (NA) wherein said construct comprises nucleic acid sequences encoding assembly cum packaging recognition fusion protein (CP), with Hemagglutinin (HA) and Neuraminidase (NA) of H1N1, and an episomal expression vector comprising Ura3c selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with bidirectional Gall / GallO promoter.

21. A nucleic acid construct with SEQ ID NO. 11 for transcription of Influenza VLP assembly cum packaging recognition fusion protein (CP) in fusion with Hemagglutinin (HA) wherein said construct comprises nucleic acid sequences encoding assembly cum packaging recognition fusion protein (CP), with Hemagglutinin (HA) of H5N1, and an episomal expression vector comprising Ura3c selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with bidirectional Gall / GallO promoter.

22. A recombinant VLP-mRNA complex comprising mRNA transcript encapsulated within VLP particles wherein, said complex is capable of expressing two or more immunogenic antigen sequences against one or more viruses.

23. The recombinant VLP-mRNA complex as claimed in claim 22, wherein the VLP-mRNA complex is expressed using protease deficient recombinant expression platform, comprising one or more antigenic sequences expressed through Virus like particles (VLPs) and one or more antigenic sequences expressed using mRNA transcript.

24. The recombinant VLP-mRNA complex as claimed in claims 22-23, wherein the virus is a respiratory virus selected from H1N1, H5N1, RSV, SARS CoV2, hMPV, MERS and NIP AH.

25. The recombinant VLP-mRNA complex as claimed in claim 24, wherein the virus is a respiratory virus of the family Orthomyxoviridae.

26. The recombinant VLP-mRNA complex as claimed in claim 25, wherein the virus is an Influenza virus.

27. The recombinant VLP-mRNA complex as claimed in claim 25, wherein the virus is selected from, but not limited to H1N1, H5N1.

28. The recombinant VLP-mRNA complex as claimed in claim 22, wherein the mRNA is a positive or a negative strand RNA virus.

29. The recombinant VLP-mRNA complex as claimed in claim 22, wherein the mRNA encodes an anti-tumor protein.

30. The recombinant VLP-mRNA complex as claimed in claim 23, wherein the mRNA encodes an anti-bacterial protein.

31. The recombinant VLP-mRNA complex as claimed in claim 23, wherein the viral mRNA is from a virus selected from but not limited to RSV, H1N1, H5N1, SARS CoV, hMPV, MERS, NIP AH.

32. The recombinant VLP-mRNA complex as claimed in claim 28, wherein the viral mRNA is a virus from the family Pneumoviridae and is a PreFusion (PreF), RSV mRNA.

33. The recombinant VLP-mRNA complex as claimed in claim 28, wherein the viral mRNA is a virus from the family Orthomyxoviridae and is a Hemagglutinin (HA), H1N1 mRNA.

34. The recombinant VLP-mRNA complex as claimed in claim 28, wherein the viral mRNA is a virus from the family Orthomyxoviridae and is a HA, H5N1 mRNA.

35. The recombinant VLP-mRNA complex as claimed in claims 23-28, wherein the complex comprises Influenza VLP encapsulating RSV or H1N1 or H5N1 mRNA.

36. The recombinant VLP-mRNA complex as claimed in claim 34, wherein the complex comprises H1N1 VLP encapsulating PreF RSV, HA H1N1, HA H5N1 mRNA.

37. The recombinant VLP-mRNA complex as claimed in claim 22-28, wherein the mRNA is encapsulated in circular or linear form.

38. A recombinant virus like particle (VLP) comprising at least one or more antigenic polypeptides of a respiratory virus selected from but not limited to H1N1, H5N1, RSV, SARS CoV2, hMPV, MERS, NIP AH.

39. The recombinant VLP as claimed in claim 38, wherein the virus is a respiratory virus of the family Orthomyxoviridae.

40. The recombinant VLP as claimed in claim 37-38, wherein the virus is an Influenza virus selected from, but not limited to H1N1, H5N1.

41. The recombinant VLP as claimed in claims 38-40, wherein the VLP comprises one, two or three antigenic polypeptides of H1N1 virus.

42. The recombinant VLP as claimed in claim 41, wherein the antigenic polypeptides are selected from but not limited to the group of Hemagglutinin (HA), Neuraminidase (NA), and Matrix protein (Ml).

43. The recombinant VLP as claimed in claim 38, wherein the VLPs maintain a size range and stoichiometry range of the antigenic peptides.

44. The recombinant VLP as claimed in claims 38, wherein the antigenic polypeptide is a full- length sequence or fragment thereof.

45. The recombinant VLP as claimed in claim 38, wherein the VLP is cloned and expressed using episomal and integrated expression vectors in a protease deficient yeast host system.

46. A method of producing recombinant VLP using the recombinant expression platform as claimed in claim 1, comprising the steps of:a. cloning one or more antigenic polypeptides using one or more combination of episomal expression vectors and integration vectors; b. codon optimizing the antigenic polypeptide sequences for expression; and c. transforming and expressing in a protease deficient yeast host.

47. A method of producing a recombinant VLP-mRNA complex using the recombinant expression platform as claimed in claim 1, said method comprising: i. cloning one or more antigenic polypeptides using one or more combination of episomal expression vectors and integration vectors; ii. codon optimizing the antigenic polypeptide sequences for expression; iii. transformation and integration of integrating expression vector in genome of protease deficient yeast host; iv. transformation of episomal vector in engineered yeast host of step iii; v. co-expression of VLP proteins and in parallel transcription of mRNA, regulatory and packaging sequences in yeast host of step iv from episomal and integration vector; and vi. parallel assembly of VLP proteins and encapsulation of mRNA to form recombinant virus like particle (VLP)-mRNA complex.

48. The method as claimed in claim 46 or 47, wherein the antigenic polypeptides are selected from but not limited to Hemagglutinin, Neuraminidase, and Matrix protein, PreF, CP.

49. The method as claimed in claim 46 or 47, wherein the episomal expression vector is pYRE400 or pYRElOO and the integration vector is pYRHOO.

50. The method as claimed in claim 46 or 47, wherein the episomal expression vector comprises a bidirectional Gall / Gal 10 promoter and a CYCT1 terminator.

51. The method as claimed in claim 46 or 47, wherein the integration vector comprises a Gall promoter and a CYCT1 terminator.

52. The method as claimed in claim 46 or 47, wherein the codon optimization includes modifying the codon sequences of antigenic polypeptides to improve expression in protease deficient yeast host.

53. The method as claimed in claims 46 or 47, wherein the protease-deficient yeast host is Saccharomyces cerevisiae, deficient in pep4 and prbl proteases.

54. The method as claimed in claim 47, wherein the VLP-mRNA complex encapsulates circular RNA for enhanced stability and protection against RNases.

55. The method as claimed in claim 47, wherein the mRNA includes regulatory sequences for circularization and packaging into the VLP.

56. The method as claimed in claim 47, wherein the VLP-mRNA complex targets one or more respiratory viruses selected from Influenza H1N1, H5N1, RSV, SARS-CoV-2, NIP AH, hMPV and ME RS.

57. The method as claimed in claim 47, wherein the transcription of mRNA is driven by a galactose-inducible promoter system.

58. The method as claimed in claim 47, wherein the co-expression step involves simultaneous expression of VLP proteins and transcription of mRNA, the stoichiometric assembly of VLP-mRNA complexes.

59. A method for production of a scalable amount of recombinant VLP-mRNA complex comprising the steps of: i. overexpressing recombinant VLPs-mRNA co expression complex in a transformed yeast host cell culture; ii. growing the transformed yeast cell culture to a stage of log phase growth; iii. inoculating and inducing the culture in a suitable media; and iv. expression of VLP proteins, transcription of mRNA and purification of the VLP encapsulating mRNA transcript.

60. A vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition comprising the recombinant VLP as claimed in claims 38-45 or recombinant VLP-mRNA complex as claimed in claims 22-37 and a pharmaceutically acceptable carrier or excipient.

61. The vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition as claimed in claim 60, wherein said composition is thermostable and has a shelf life of up to 2 years.

62. The vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition as claimed in claim 60 for use in preventing or treating one or more respiratory infections or diseases.

63. The vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition as claimed in claim 60 for use in preventing or treating one or more bacterial or oncological diseases.

64. A method of preventing or treating one or more respiratory infections or diseases comprising administering a therapeutically effective amount of recombinant VLP as claimed in claims 38-45 or recombinant VLP-mRNA complex as claimed in claims 22-37 or vaccine composition or pharmaceutical composition or immunogenic composition or immunologically active composition as claimed in claims 60-63.

65. The recombinant virus like particle (VLP)-mRNA complex as claimed in claims 22-37 wherein mRNA is encapsulated inside a VLP and wherein the recombinant VLP-mRNA complex has potential as a dual vaccine target for use in preventing or treating one or more respiratory infections or diseases.

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