Baculovirus expression systems and methods thereof

Abstract

An improved baculovirus expression system for enhanced heterologous protein expression in hosts selected from Bombyx mori larvae, pupae, primary cells, or established insect cell lines is disclosed. The expression system is derived from a wild-type strain of Bombyx mori nucleopolyhedrovirus (BmNPV) by deletion or inactivation of non-essential genes, of the BmNPV genome. The system includes novel transfer vectors designed for targeted gene modification and incorporation of a bacterial origin of replication and an insertion site for a gene of interest (GOI). This engineered bacmid maintains infectivity while offering enhanced genetic stability and significantly improved heterologous protein expression in insect cells, larvae, or pupae. Methods for constructing this bacmid via homologous recombination and using it for high-level protein production are also described.

Description

[0001] BACULO VIRUS EXPRESSION SYSTEMS AND METHODS THEREOF

[0002] PRIORITY CLAIM

[0003] The instant patent application is related to and claims priority from the India provisional application entitled, “BACULOVIRUS EXPRESSION SYSTEMS AND METHODS THEREOF”, Application No.: 202441052521, Filed: 09 July 2024, which is incorporated in its entirety herewith to the extent not inconsistent with the description herein.

[0004] TECHNICAL FIELD OF INVENTION

[0005] Embodiments of the present disclosure relate generally to baculovirus expression systems and methods of construction for recombinant expression of heterologous genes.

[0006] BACKGROUND

[0007] Expression systems involving mammalian cells offer distinct advantages due to their ability to facilitate essential processes such as proper protein folding, post-translational modifications, and product assembly. However, these systems often encounter challenges in achieving satisfactory yields for intracellular proteins. While advancements have been made in multiprotein expression technologies for mammalian cells, they are still in the early stages of development (Tepap, Cromwel Zemnou, Anissi, Jaouad, and Bounou, Salim. ‘Recent Strategies to Achieve High Production Yield of Recombinant Protein: A Review’. 1 Jan. 2023: 25-37).

[0008] As an alternative to mammalian systems, heterologous gene expression using recombinant baculoviruses to infect insect cells has emerged in recent years. Insects such as Drosophila melanogaster, Bombyx mori (Bm, silkworm), Anopheles stephensi (mosquito), Periplaneta americana (the American cockroach), Tribolium castaneum (red flour beetle), Trichoplusia ni (T. ni, cabbage looper), and Spodoptera frugiperda (fall army worm) serve as promising models in biomedical research due to the conservation of various biological pathways and structural similarities with humans. Since insects reproduce rapidly and can be easily maintained, these models offer an economical solution when compared to other systems such as mammalian or yeastbased platforms.

[0009] Silkworms are included among the insect species suitable for baculovirus vector applications. In recent years, silkworms are being explored for use as living biofactories for protein production, as silkworm rearing is a traditional practice and an economically significant industry, especially in India. In contrast, T. ni and Spodoptera frugiperda are agricultural pests that cause significant damage to a wide range of crops, including vegetables, grains, and cotton. Given their domestication, silkworms are preferred as expression hosts.

[0010] Nucleopolyhedroviruses (NPVs) have emerged as powerful tools for both insect pest management and biotechnological applications. These viruses infect and replicate within insect hosts, primarily in the order Lepidoptera (moths and butterflies). They are highly specific to their host insects and exhibit little to no harmful effects on non-target organisms, making them ideal candidates for biological control agents in agriculture. Beyond pest control, NPVs have gained importance in biotechnology due to their ability to serve as vectors for recombinant protein expression. Baculovirus expression vectors based on NPVs are widely used in insect cell systems for the production of recombinant proteins, vaccines, and viral vectors for gene therapy.

[0011] The baculovirus expression system (BES) offers several advantages for protein expression, including high-level expression of heterologous proteins, post-translational modification capabilities similar to those of mammalian cells, scalability for large-scale production, and safety due to the inability of baculoviruses to replicate in mammalian cells. Most expression systems developed to date harbour the strong and highly active polyhedrin promoter (PolH or pPolH), capable of driving high-level expression of recombinant proteins during the late phase of viral infection. These promoters are derived from the polyhedrin gene of baculoviruses, such as Autographa califomica nucleopolyhedrovirus (AcNPV), and are among the most commonly used in baculovirus expression vectors. However, this promoter is active only during the late phase of baculovirus infection, resulting in a delayed onset of recombinant protein expression. Further, it lacks defined regulatory elements, such as tissue-specific enhancers or inducible elements, which may limit its versatility for specialized applications requiring precise control over gene expression.

[0012] Host specificity is another defining feature of baculovirus systems. Among the two widely studied baculoviruses, AcNPV infects Spodoptera frugiperda and Trichoplusia ni, while BmNPV infects Bombyx mori. Silkworms are preferred hosts for baculovirus expression systems because they support high levels of viral replication and maintain sufficient larval viability to allow efficient protein production, with minimal interference in silk gland function when timed appropriately. This tolerance allows for efficient expression of foreign genes in silkworm cells using baculovirus vectors. While Bombyx morz'-derived cell lines have been used, direct protein expression in silkworm larvae or pupae yields 10- to 100-fold higher expression levels.

[0013] Baculoviruses, members of the family Baculoviridae , are a type of DNA virus that infect insects.

[0014] NPVs are part of this family and represent one of the largest DNA viruses, characterized by a circular, double- stranded, supercoiled genome typically ranging from 80 to 180 kilobases in size. They harbour a genome comprising over 100 genes that are orchestrated in a stage-dependent manner, including immediate -early, delayed-early, late, and very-late phases. Approximately half of the NPV genes are deemed essential for viral propagation, encompassing roles in gene expression, DNA replication, and virion assembly. Despite gene knock-out (KO) studies on baculoviruses like AcMNPV and BmNPV, the functions of more than one-third of viral genes remain elusive. The two most studied baculoviruses include AcMNPV and BmNPV.

[0015] While US 2015 / 0361403 Al describes improved baculoviral expression systems by manipulating non-essential genes, primarily focusing on Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), it does not describe the specific features that can improve the stability of the hetereologous gene and its expression.

[0016] Although BmNPV is a widely used baculovirus for insect cell expression systems, existing BmNPV vectors are not optimized for the unique characteristics of endogenous strains of Bombyx mori. The characteristics of different Bombyx mori strains, particularly those from different geographic regions such as India, can vary significantly. The long history of silkworm domestication and the ease of rearing and maintenance in laboratory conditions, compared to other insect species, make silkworms a convenient and cost-effective platform for biotechnological applications such as recombinant protein expression. Thus, there is a need in the art to develop recombinant baculoviruses specifically tailored to silkworms and their pupae for large-scale protein production.

[0017] However, the inherent DNA instability of currently used baculovirus genomes presents a challenge. As the bacmid replicates, it can progressively accumulate deletions or rearrangements, especially in regions encoding non-essential genes or within inserted heterologous gene cassettes. This issue is exacerbated by the presence of repeated sequences, such as homologous regions (hrs), and mutational hotspots including the Tn7 insertion site, which is widely used for transgene delivery. These features promote recombination events that compromise the integrity of the recombinant bacmid, reduce plasmid yield, and ultimately affect the fidelity and efficiency of downstream protein expression in insect hosts. Therefore, there is a need for a BmNPV expression system with enhanced genomic stability during bacterial propagation, increased packaging efficiency, and improved capacity for accommodating large heterologous inserts without compromising virus assembly or infectivity. Aspects of the present disclosure are directed to baculovirus expression systems with improved packaging, enhanced stability, and increased expression of heterologous genes, including those encoding multiple and / or complex proteins.

[0018] BRIEF DESCRIPTION OF DRAWINGS

[0019] Example embodiments of the present disclosure will be described with reference to the accompanying drawings briefly described below.

[0020] Figure 1 is a diagram depicting the process flow for R&D Loopsynth.

[0021] Figure 2 is a diagram depicting the process involved in the construction of BmNPV bacmid devoid of some non-essential genes.

[0022] Figure 3A is a diagram depicting a vector map of transfer vector, TF-01 with a mini-F replicon backbone and Homology arms that will target the genes Chitinase and cathepsin for knockout and knock-in of mini-F replicon.

[0023] Figure 3B is a diagram depicting a vector map of transfer vector TF-02, comprising a pUC-19 backbone and PolH homology arms for knockout of the PolH gene and knock-in of the LacZ gene. Figure 4 is a schematic representation of the vector used for bacmid generation and the engineered Loopbacl construct.

[0024] Figure 5 is an image depicting colony growth on LB agar containing chloramphenicol, showing functional validation of Loopbacl bacmid replication in E. coli DH10B cells.

[0025] Figure 6 is an agarose gel image depicting PCR-based confirmation of the inhibition of copy control or inCC region within the mini-F backbone, where Bad and Bac2 represent two individual clones used for the experiment.

[0026] Figure 7 is a schematic diagram depicting the TF2_HDR fragment used for PolH gene knockout and LacZa insertion.

[0027] Figure 8 is a genomic map depicting the TF2_HDR integration site within the baculoviral genome. Figure 9 is an image depicting blue-white screening of E. coli Loopbac2 colonies containing the Mini-F origin and LacZa cassette.

[0028] Figure 10 is an agarose gel image depicting PCR confirmation of LacZa gene insertion in the Bacl construct.

[0029] Figure 11A are images depicting blue-white screening of E. coli colonies harboring the LoopBac2 bacmid and pHelper plasmid.

[0030] Figure 12A is a fluorescence microscopy image depicting GFP expression in Sf21 infected cells at 72 hours post-infection.

[0031] Figure 12B is a fluorescence microscopy image depicting GFP expression in Sf21 infected cells at 96 hours post-infection. Figure 12C is a fluorescence microscopy image depicting GFP expression in Sf21 infected cells at 120 hours post-infection.

[0032] Figure 12D is a bar graph depicting quantitative analysis of fluorescent cell counts at 72 h, 96 h, and 120 h post-infection, corresponding to Figures 12A-12C.

[0033] Figure 13 is a Western blot image depicting GFP protein expression in insect cells at multiple time intervals (24h, 48h, 72h and 96h).

[0034] Figure 14A is an image depicting silkworm larvae under UV illumination, showing wild- type and GFP-expressing specimens.

[0035] Figure 14B is an image depicting silkworm pupae under UV illumination, showing wild- type and GFP-expressing specimens.

[0036] Figure 14C is an image depicting silkworm hemolymph under UV illumination, showing wildtype and GFP-expressing samples.

[0037] Figure 15 is a Western blot image depicting GFP expression in silkworm pupae at 72 hours postinfection.

[0038] Figure 16 is a vector map depicting the BmNPV genomic DNA with non-essential genes identified for knockout.

[0039] In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number. In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements.

[0040] DETAILED DESCRIPTION

[0041] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

[0042] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

[0043] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0044] The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a nonexclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more compositions or elements or structures or components preceded by “comprises... a” does not, without more constraints, preclude the existence of other compositions or elements or other structures or other components or additional compositions or additional elements or additional structures or additional components.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The compositions, methods, and examples provided herein are illustrative only and not intended to be limiting.

[0046] Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.

[0047] 1. Overview

[0048] Aspects of the present disclosure are directed improved baculovirus expression system (BEV), for expression of heterologous proteins, in particular, a BmNPV shuttle vector, a “Bacmid” wherein the non-essential genes have been deleted and / or mutated. The present disclosure provides a recombinant baculovirus, and methods thereof.

[0049] According to an aspect of the present disclosure, a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid is provided for enhanced heterologous protein expression in hosts selected from Bombyx mori larvae, pupae, primary cells, or established insect cell lines. The bacmid includes a BmNPV genome derived from a wild-type strain, wherein the BmNPV genome is reduced in size relative to the wild-type BmNPV genome by deletion of at least one base pair of at least one non-essential gene, or at least one non-essential gene is inactivated by mutation pr a combination of both. The bacmid also includes a bacterial origin of replication enabling stable propagation in Escherichia coli, and an insertion site for at least one gene of interest (GOI) comprised within the BmNPV genome, wherein the insertion site is positioned at a distance from the bacterial origin of replication to enhance stability of the GOI during bacmid maintenance or viral passage.

[0050] In an embodiment, the wild-type strain is an Indian strain.

[0051] In an embodiment, at least one non-essential gene is inactivated by mutation to reduce metabolic burden or suppress undesired protein expression that interferes with heterologous gene expression.

[0052] In another embodiment, the non-essential gene is selected from polyhedrin (PolH) gene, chitinase gene, and cathepsin gene.

[0053] In yet another embodiment, the non-essential gene is further selected from the group comprising of plO, p26, 39k, pif, p74, AcMNPV orfll, AcMNPV orfl3, egt, acl8, arifl, ac22 (pif-2), ac23 (gp64 homolog), AcMNPV orf29, ac30, fibroblast growth factor (fgf), p43, p47, gta, orf34, orf35 (ubiquitin-like), ac57, ac58 / ac59, ac60, fp25k (25k), orf51, gp37, p30 (arif-1), orf74, BroB, He65, gcn-2, AC122, pp34, orfl lOa, acl50, acl49, BroE, and pl5..

[0054] In yet another embodiment, the bacterial origin of replication is selected from pUC, Fl, colEl, or mini-F origin of replication.

[0055] In yet another embodiment, the origin of replication is mini-F.

[0056] In yet another embodiment, the insertion site further includes at least one synthetic site-specific transposon insertion sequence or recombinase recognition sequence, and the GOI is integrated using a corresponding transposase or recombinase.

[0057] In yet another embodiment, the transposon insertion sequence is an attTn7 transposition sequence.

[0058] In an embodiment, the transposition sequence is further selected from piggyBac inverted terminal repeats (ITRs), mariner transposon inverted repeats, Sleeping Beauty inverted or direct repeats, Tol2 inverted repeats, or equivalents thereof.

[0059] In an embodiment, the recombinase recognition sequence is selected from loxP, attP, attB, FRT, or equivalents thereof. In yet another embodiment, the BmNPV bacmid is capable of assembling into infectious baculovirus particles upon transfection into hosts selected from Bombyx mori larvae, pupae, primary cells, or established insect cell lines.

[0060] In yet another embodiment, the heterologous protein expressed is selected from a therapeutic protein, vaccine antigen, monoclonal antibody, or industrial enzyme.

[0061] In another aspect of the invention, at least one recombinant transfer vector for generating a Bombyx mori nucleopolyhedrovirus (BmNPV) have been disclosed. The vectors comprise at least one pair of homologous recombination arms, wherein each arm is derived from a region of the BmNPV genome derived from a wild-type strain. The genomic region may comprise essential or non- essential gene. The vector further comprises atleast one functional element positioned between the homologous recombination arms. The functional element may be selected from a bacterial origin of replication, a selectable marker, or a transgene insertion site. The vector comprises a bacterial plasmid backbone, enabling propagation of the vector in a E.coli. The vector is configured to enable targeted replacement of BmNPV non-essential gene regions with elements that improve recombinant bacmid stability or heterologous gene expression.

[0062] In one embodiment, least one pair of homologous recombination arms derived from a region of the BmNPV genome derived from a wild-type Indian strain. The genomic region may comprise essential or non-essential gene.

[0063] In another embodiment, non-essential gene region may be selected from a polyhedrin (PolH) gene, a chitinase gene, or a cathepsin gene. The non essential gene may further be selected from plO, p26, 39k, pif, p74, AcMNPV orfll, AcMNPV orfl3, egt, acl8, arifl, ac22 (pif-2), ac23 (gp64 homolog), AcMNPV orf29, ac30, fibroblast growth factor (fgf), p43, p47, gta, orf34, orf35 (ubiquitin-like), ac57, ac58 / ac59, ac60, fp25k (25k), orf51, gp37, p30 (arif-1), orf74, BroB, He65, gcn-2, AC122, pp34, orfl lOa, acl50, acl49, BroE, and pl5..

[0064] In an embodiment, the bacterial origin of replication may selected from pUC, Fl, colEl, R6kGamma, or mini-F origin of replication, in particular the bacterial origin of replication is a mini-F replicon.

[0065] In another embodiment, the functional element may be a selectable marker selected from green fluorescent protein (GFP), luciferase, chloramphenicol acetyltransferase (CAT), beta- glucuronidase (GUS), in particular the selectable marker is lacZa.

[0066] In an embodiment, the functional element is a transgene insertion site, selected from an attTn7 transposition sequence or a recombinase recognition sequence. The attTn7 transposition sequence is selected from piggyBac inverted terminal repeats (ITRs), mariner transposon inverted repeats, Sleeping Beauty inverted / direct repeats, or Tol2 inverted repeats. The recombinase recognition sequence may be selected from loxP, attP, attB, or FRT.

[0067] According to another aspect of the present disclosure, a method for producing a bacmid includes isolating genomic DNA from a wild-type strain of BmNPV; genetically modifying the wild-type strain of BmNPV genome by deleting at least one base pair of at least one non-essential gene to reduce the genome size relative to the wild-type BmNPV genome, or by inactivating at least one non-essential gene or a combination thereof; introducing a bacterial origin of replication; introducing an insertion site for at least one gene of interest (GOI) into the BmNPV genome positioned at a distance from the bacterial origin of replication to enhance stability of the GOI during bacmid maintenance or viral passage; introducing the genetically modified BmNPV genomic DNA into E. coli', and isolating the resulting bacmid DNA.

[0068] In an embodiment, the strain of BmNPV is a wild-type Indian strain.

[0069] In another embodiment, the at least one non-essential gene is inactivated by mutation to reduce metabolic burden or suppress undesired protein expression that interferes with heterologous gene expression.

[0070] In yet another embodiment, the non-essential gene is selected from polyhedrin (PolH) gene, chitinase gene, and cathepsin gene.

[0071] In an embodiment, the non-essential gene is further selected from the group consisting of plO, p26, 39k, pif, p74, AcMNPV orfl l, AcMNPV orfl3, egt, acl8, arifl, ac22 (pif-2), ac23 (gp64 homolog), AcMNPV orf29, ac30, fibroblast growth factor (fgf), p43, p47, gta, orf34, orf35 (ubiquitin-like), ac57, ac58 / ac59, ac60, fp25k (25k), orf51, gp37, p30 (arif-1), orf74, BroB, He65, gcn-2, AC122, pp34, orfl lOa, acl50, acl49, BroE, and pl5.

[0072] In yet another embodiment, the bacterial origin of replication is selected from pUC, Fl, colEl, or mini-F origin of replication. In yet another embodiment, the bacterial origin of replication is mini-F.

[0073] In yet another embodiment, the insertion site further includes at least one synthetic site-specific transposon insertion sequence or recombinase recognition sequence, and the GOI is integrated using a transposase or recombinase.

[0074] In yet another embodiment, the transposon insertion sequence is an attTn7 transposition sequence.

[0075] In yet another embodiment, the transposition sequence is further selected from piggyBac inverted terminal repeats (ITRs), mariner transposon inverted repeats, Sleeping Beauty inverted or direct repeats, Tol2 inverted repeats, or equivalents thereof.

[0076] In yet another embodiment, the recombinase recognition sequences are selected from loxP, attP, attB, FRT, or equivalents thereof.

[0077] In yet another embodiment, the deletion of the non-essential gene is performed using a technique selected from the group consisting of recombineering, Cre-LoxP recombination, CRISPR-Cas9 gene editing, FLP-FRT recombination, TALEN-mediated gene editing, zinc finger nucleases, restriction enzyme -based deletion and ligation, or transposon-mediated mutagenesis.

[0078] In an embodiment, the bacmid is competent for transfection into hosts selected from Bombyx mori larvae, pupae, primary cells, or insect cell lines derived therefrom for expression of the gene of interest.

[0079] According to another aspect of the present disclosure, a method is provided for expressing a heterologous protein. The method includes introducing a recombinant bacmid as described above into a host selected from Bombyx mori larvae, pupae, primary cells, or established insect cell lines; allowing viral infection and expression of the heterologous gene; and harvesting the protein product from an infected host.

[0080] In an embodiment, the heterologous protein is selected from growth factors, antigens, enzymes, antibodies, virus-like particles, antimicrobial peptides, or diagnostic or therapeutic proteins.

[0081] In an embodiment, the larvae, pupae, or primary cells used are of the same lineage as the BmNPV isolate used to derive the bacmid. In another embodiment, the protein is recovered from Bombyx mori larvae or pupae by lysis, clarification, and chromatographic purification.

[0082] According to another aspect of the present disclosure, a heterologous protein is provided, obtained from the method described above, wherein the protein is selected from therapeutic proteins, vaccine antigens, monoclonal antibodies, virus-like particles, diagnostic proteins, antimicrobial peptides, or industrial enzymes.

[0083] 3. Embodiments

[0084] The invention discloses improved baculovirus expression systems (BEV), wherein 'baculovirus' refers to members of the Baculoviridae family of enveloped, double-stranded DNA viruses that infect insects, including Bombyx mori nucleopolyhedrovirus (BmNPV). As used herein, the term “BmNPV” refers specifically to the nucleopolyhedrovirus strain that naturally infects the silkworm Bombyx mori. BmNPV is widely used in insect expression systems and serves as the viral backbone of the modified bacmids described in the present disclosure. According to an aspect, the BmNPV shuttle vector, Bacmid wherein the non-essential genes have been mutated and / or deleted and recombinant baculovirus, and methods thereof. As used herein, the term “shuttle vector” refers to a vector capable of replication in two or more different host species. The recombinant bacmid comprising a BmNPV genome derived from a wild-type strain, in particular an Indian strain, engineered to replicate stably in Escherichia coli via a bacterial origin of replication (e.g., mini-F replicon), while also being competent for transfection into insect cells or Bombyx mori larvae / pupae, where it assembles into infectious baculovirus particles and enables expression of one or more heterologous genes of interest (GOIs). The invention discloses a BmNPV-based expression system as a modular, platform suitable for scalable recombinant protein production.

[0085] As used herein, the term “non-essential gene” refers to a gene that is not required for the survival or viability of a virus, cell, or organism under defined environmental or culture conditions. Disruption, deletion, or modification of non-essential genes do not result in lethality or loss of fundamental viral functions. These non-essential genes can be removed to reduce genome size and improve stability or heterologous gene expression. Non-essential genes may also express proteins that interfere with heterologous protein folding, glycosylation, or stability. These genes may be inactivated by, for example, point mutations, frame shifts, or early stop codons to inactivate these interfering genes without physically removing large segments of DNA — preserving regulatory spacing or structural integrity of the genome. For example, mutational inactivation of genes such as ieO, iel, plO, or 39k, without deleting them entirely, maintains compatibility with native promoters or enhances future editing flexibility.

[0086] The genes comprised in the bacmid of the invention may be rendered inactive by point mutations / modifications or deletion of at least one base or insertion of at least one base or substituting at least one base for another. The inactivation may be by mutation techniques known in the art including site-directed mutagenesis, CRISPR-cas9 genome editing, insertional mutagenesis, and targeted mutagenesis using nucleases (Zinc Finger Nucleases (ZFN), Transcription Activator-Like Effector Nucleases (TALENs).

[0087] In one aspect, the invention discloses a bacmid of reduced genome size compared to its wild-type strain for improved packaging, enhanced stability, and increased expression of the heterologous genes.

[0088] The BmNPV genome size may be decreased by at least 5%, more preferably 10-20%. The modified genome is capable of assembling into an infectious baculovirus. The assembled baculovirus can express heterologous proteins upon infecting a suitable host cell. The host cell may be prokaryotic or eukaryotic and may be selected from Bombyx mori larvae, pupae, primary cells, or established insect cell lines In particular, the host cell may be an insect cell. In an embodiment, the insect cell is derived from Bombyx mori, including primary cultures from larval or pupal tissues, or established insect cell lines derived from Bombyx mori.

[0089] “Primary culture” as used herein refers to the initial, in vitro cultivation of cells directly isolated from Bombyx mori tissues or organs (such as those from larvae or pupae). These cells are immediately placed into a controlled artificial environment (culture medium) where they can survive, attach, and proliferate for a limited period.

[0090] “Established insect cell lines”, as used herein, refer to continuously passaged and genetically stable cell cultures derived from Bombyx mori tissues that are capable of sustained propagation under defined in vitro conditions. Examples of such cell lines include BmN cells, Bm5 cells, and BmS cells.

[0091] Unlike "established cell lines" (which are immortalized and can be continuously passaged), primary cells have a limited lifespan and typically only divide a finite number of times before undergoing senescence (aging) or apoptosis (programmed cell death). They tend to retain more of the morphological and functional characteristics of the original tissue from which they were derived, making them valuable for studying in vzvo-like physiological responses.

[0092] In addition to the non-essential genes explicitly exemplified herein, selected from polyhedrin (PolH) gene, chitinase gene, and cathepsin gene, the present invention further contemplates the deletion or inactivation of other non-essential genes within the BmNPV genome to further reduce genome size, enhance stability, and / or improve heterologous protein expression. Such additional non-essential genes, or their functional orthologs in BmNPV, may include, but are not limited to, plO, p26, p74, 39k, pif, AcMNPV orfl 1, AcMNPV orfl3, egt, acl8, arifl, ac22 (pif-2), ac23 (gp64 homolog), AcMNPV orf29, ac30, fibroblast growth factor (fgf), p43, p47, gta, orf34, orf35 (ubiquitin-like), ac57, ac58 / ac59, ac60, fp25k (25k), orf51, gp37, p30 (arif-1), orf74, BroB, He65, gcn-2, AC122, pp34, orfl 10a, acl50, acl49, BroE, and pl5. These genes are considered non- essential based on extensive literature review and comparative genomic analyses with known baculovirus strains.

[0093] In one aspect, the invention relates to improved BmNPV vectors and recombinant baculovirus. As used herein, the term “recombinant baculovirus” refers to a baculovirus whose genome has been modified by insertion, deletion, substitution, or rearrangement of one or more nucleotide sequences such that it comprises at least one heterologous gene not present in the wild-type genome. It may retain infectivity and be used to express the inserted gene in insect cells.

[0094] BmNPV baculovirus is well known in the art for the expression of recombinant proteins in insect cells as it is compatible with a wide range of insect cell lines. The present invention encompasses novel BmNPV vectors and recombinant baculovirus constructs from strains of wild-type BmNPV isolated from India. In a specific aspect of the invention, the wild-type BmNPV has been genetically manipulated to generate a recombinant baculovirus comprising genetic modifications or improved BmNPV vectors. The improved baculovirus expression system of the invention offers improved and efficient protein production capabilities.

[0095] “Baculovirus Expression System” or BEV as used herein refers to a recombinant DNA-based system that uses baculoviruses — particularly Bombyx mori nucleopolyhedrovirus (BmNPV) — to express heterologous genes of interest (GOIs) in insect hosts, such as Bombyx mori larvae, pupae, primary cells, or established insect cell lines.

[0096] “BmNPV” or “wild-type BmNPV” refers to a naturally occurring Bombyx mori nucleopolyhedrovirus isolated from infected Bombyx mori larvae or environmental sources in India, which serves as the genomic backbone for recombinant manipulation. The terms “Recombinant bacmid” or “BmNPV bacmid” or “modified BmNPV bacmid” are used interchangeably to refer to a shuttle vector comprising a modified BmNPV genome engineered for propagation in Escherichia coli (via a bacterial origin of replication such as the mini-F replicon), and for transfection into insect cells for expression of one or more heterologous genes. In this context, the term “bacmid” may refer to a circularized DNA molecule, typically over 100 kb, that includes essential baculoviral replication elements and optionally non-essential gene deletions or insertions for heterologous gene expression. “Loopbacl,” “Loopbac2” refer to specific recombinant bacmid constructs developed in the course of this invention. These vectors differ in the non-essential genes deleted and the location / structure of the GOI insertion site.

[0097] The wild-type strain may be isolated from infected Bombyx mori larvae, pupae, hemolymph of infected larvae, excreta, or silk glands, dead or silk worm waste, leaves, mulberry beds, or soil, by methods known in the art. Particularly, the genomic DNA may be isolated from the wild-type strain and subjected to sequencing to determine its complete nucleotide sequence. It is known in the art that genomes of NPV exhibit a high degree of conservation and the similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several algorithms known in the art, preferably BLAST, and CLUSTALW. The inventors were able to identify 40 genes as non- essential and dispensable for virus replication through sequence alignment with the BmNPV T3 genome (NCBI Accession Number: NC_001962). Table 1 shows the list of these 40 non-essential genes.

[0098] Through extensive literature searches and comparative genomic analyses, including sequence alignments of the isolated BmNPV wild- type genome with established reference baculovirus genomes (e.g., the well-characterized BmNPV T3 genome, NCBI Accession Number: NC_001962), the inventors were able to identify 40 genes as non-essential and dispensable for virus replication. This identification was based on their established functional characterization as non-essential in various baculovirus strains, including their orthologs in BmNPV. These identified genes, upon deletion or inactivation, are specifically contemplated for use in reducing the bacmid genome size while maintaining infectivity and are particularly advantageous for improving genetic stability and / or enhancing heterologous protein expression. Table 1 below provides a comprehensive list of these 40 identified non-essential genes. " TABLE 1

[0099]

[0100] Table 1: A list of 40 non-essential genes identified in the BmNPV genome

[0101] The list of non-essential genes identified in Table 1 is provided for illustrative purposes. Additional non-essential genes may be determined through functional genomics studies, sequence alignments, or empirical screening, and may likewise be deleted or modified without departing from the scope of the invention.

[0102] Towards the improvement of the BmNPV expression vector system, the ~130 kb genome of the BmNPV has been mapped, and the 40 non-essential and dispensable genes were selected from the BmNPV genome. The improved baculovirus genome generated by deleting and / or inactivating at least one non-essential gene, increased the stability of the bacmids. As used herein, the term “bacmid” refers to a baculovirus genome maintained as a bacterial artificial chromosome (BAC) in Escherichia coli, allowing stable propagation and manipulation of the viral genome in bacterial hosts. Such engineered viruses further reduce the likelihood of genetic rearrangements and deletions during propagation in bacterial hosts, and offer stability during passages and infection. This leads to more consistent and reliable expression of heterologous genes over time. The increased ability to accommodate larger foreign gene insertions without compromising the ease of handling. This allows for the efficient packaging of larger amounts of foreign DNA into the viral capsids, for higher expression levels of the desired gene. Smaller bacmids with reduced size and complexity simplify the production process by allowing for faster growth and higher yields of recombinant virus in bacterial hosts. The inventors have developed a modular, iterative synthetic biology workflow, Loopsynth for the systematic construction, testing, and optimization of customized recombinant BmNPV bacmids. Figure 1 illustrates a schematic overview of the R&D Loopsynth platform, which underpins the strategic design and development of engineered baculovirus expression systems. The platform is designed to facilitate “design-build-test-learn” cycles for rapid prototyping of viral vectors and expression constructs. Within Loopsynth, synthetic DNA modules — including bacterial replicons, recombination cassettes, and gene expression elements — are computationally assembled, validated, and experimentally integrated into the native BmNPV genome using molecular cloning and homologous recombination. The expression system of the invention has superior protein production properties without affecting the ability of the recombinant baculovirus to infect and propagate effectively in cells. This is achieved by knocking out or mutating specific regions of the baculovirus genome that are known to interfere with heterologous protein expression. Overall construction process of recombinant BmNPV bacmids derived from Indian wild-type strains, in particular Indian strains comprises deletion of one or more non-essential genes, as depicted in Figure 2. The deletion of non-essential genes such as chitinase, cathepsin, or polyhedrin (PolH) enables improved genetic stability and higher expression of heterologous genes of interest (GOIs). The process comprises (i) identifying and validating non-essential genes; (ii) constructing transfer vectors with appropriate homology arms; (iii) integrating bacterial propagation elements (e.g., mini-F replicon) into the BmNPV genome via homologous recombination; and (iv) confirming functional bacmid assembly and expression in E. coli and insect hosts.

[0103] The invention further provides a method for introducing a gene of interest (GOI) into BmNPV geneome, for example, between open reading frames (ORF) ORF2 and ORF1629 of the BmNPV genome by homologous recombination. A schematic overview of the generation of the recombinant BmNPV bacmid is shown in Figure 3A. The insertion of the gene of interest (GOI) at a genomic locus distant from the bacterial replicon is depicted in Figure 3B, which demonstrates the modularity of the vector architecture

[0104] An embodiment of the invention disclosed, TF-01 and TF-02 (also referred to as Transfer Vector 1 and Transfer Vector 2) plasmid constructs used for homologous recombination with the BmNPV genome, wherein TF-01 targets the chitinase and cathepsin genes and introduces the mini-F replication origin into the bacmid genome and TF-02 targets the polyhedrin (PolH) gene locus and enables the knock-in of a lacZa gene. The vector map of the transfer vectors (e.g., TF-01 and TF- 02) are depicted in Figure 3 A and 3B. Figure 3A depicts the TF-01, which utilizes a mini-F replicon backbone. The vector includes homology arms that flank the chitinase and cathepsin genes in the native BmNPV genome. This design enables site-specific recombination, wherein these proteolysis-related non-essential genes are knocked out and replaced with bacterial propagation elements, allowing the modified viral genome to be stably maintained and replicated in Escherichia coli as a bacmid. Vector map of TF-02 is depicted in Figure 3B, a transfer plasmid with a pUC-19 backbone. This vector carries homology arms targeting the PolH gene locus of BmNPV, facilitating its knockout and the concurrent insertion of a lacZa reporter gene. The lacZa cassette includes an attTn.7 insertion site, enabling future modular insertion of one or more GOIs via Tn7-mediated transposition. This setup allows blue-white screening of recombinant colonies in E. coli and provides a flexible and traceable platform for high-throughput gene insertion.

[0105] “Vector,” “Expression Vector,” “Transfer Vector,” and “Donor Vector” refer to plasmid-based constructs used in the generation of recombinant bacmids. While overlapping in function, Transfer vector (e.g., TF-01, TF-02) contain regions of homology for site-specific recombination into the BmNPV genome (e.g., chitinase, cathepsin, or polyhedrin loci), and may include selection markers, attTn7 sites, and reporter genes like lacZa and donor vector carries the gene of interest (GOI) flanked by recombination or transposition sites. “Expression vector” includes promoter (e.g., PolH) and regulatory elements enabling transcription of the GOI in insect cells. Where context permits, “vector” may refer generally to either a plasmid used in E. coli or a recombinant bacmid used in insect cells.

[0106] A “homologous recombination arm” as used herein refers to a DNA sequence that is identical or very similar (homologous) to a specific region of a target DNA molecule, such as a viral genome or a chromosome. In genetic engineering, these arms are designed to flank a desired DNA sequence (e.g., a gene to be inserted, a selectable marker, or a bacterial origin of replication) within a transfer vector. When this transfer vector is introduced into a cell, the homologous recombination arms facilitate the precise exchange of genetic material between the vector and the target DNA, allowing the desired sequence to be integrated into the specific genomic location. For example, the homologous recombination arm enable the targeted deletion of non-essential genes and the insertion of new elements (like the mini-F replicon or the LacZ / Tn7 cassette) into the BmNPV genome.

[0107] As illustrated generally in Figure 4, the overall process involves precisely modifying the BmNPV genome. Key modifications include the deletion of genes expressing proteases, such as polyhedrin (PolH) gene, and chitinase and cathepsin genes. These genes when knocked out show decrease in protein degradation when knocked out increasing the yield of the desired recombinant protein. Additionally, deletion of non-essential genes such as plO, p24, p26, and p74 has been shown to improve protein expression. A comprehensive list of such non-essential genes identified for knockout is presented, Table 1, and their locations on the BmNPV genome are depicted in an accompanying Figure 16. Key functional elements are annotated in Figure 4, including regions responsible for replication and selection in E. coli, as well as viral elements required for infection and propagation in insect hosts. The proprietary Loopbacl backbone is specifically optimized for high-efficiency transduction in Indian silkworm strains, improving overall recombinant protein yield. This figure also shows the inclusion of "A miniF origin of replication, facilitating stable maintenance and amplification in E. coli; An antibiotic resistance marker, enabling straightforward selection of positive clones; LoxP sites, allowing Cre-lox mediated recombination to support multigene expression. The site designated TF-1 "serves as the miniF region insertion site (where Cathepsin and chitinase genes were present). Figure 5 depicts the colony growth on LB agar containing chloramphenicol, following transformation with the engineered BmNPV bacmid construct (Loopbacl). The observed colony growth confirms successful replication and maintenance of the bacmid, validating the functional integration of the miniF origin. Figure 6 is an agarose gel image depicting PCR-based confirmation of the inCC region within the mini-F backbone, where Bad and Bac2 represent two individual clones obtained during transformation and selection in E. coli. The agarose gel image showing PCR-based confirmation of successful modification of the mini-F backbone in recombinant bacmid clones is presented in Figure 6 and validated the presence and structural integrity of the inCC (inhibition of copy control) region, a key regulatory sequence in the mini-F origin of replication. Clear amplification bands corresponding to the expected product size in both Bad and Bac2 confirmed correct insertion and stable propagation of the mini-F replicon in the Escherichia coli host.

[0108] The expression of other non-essential genes such as anti-apoptotic gene p35 may enhance both protein expression and cell viability by maintaining the host cells in a viable state for extended periods. Further silencing of other genes like ODV E-26 and orf34 may improve protein production. The recombinant baculovirus of the invention provides a genome of a BmNPV, wherein the genome size is reduced by deletion of non-essential genes described in this disclosure achieving a reduction in the genome size relative to the wild type by at least 10%, preferably by at least 20% wherein the baculovirus is capable of assembling into an infectious particle.

[0109] In another aspect, the invention provides for genetically configurable baculovirus expression system, wherein the bacmid vector and associated viral components may be subjected to a variety of genetic manipulations to optimize performance, broaden host range, or enhance protein expression capabilities. Such genetic modifications may include, but are not limited to deletion of additional non-essential genes, insertion of regulatory elements, codon optimization of GOI, fusion with tags (e.g, reporter, affinity purification etc.), integration of recombinase-based genes (e.g., attP / attB, loxP, FRT, or piggyBac inverted terminal repeats). The manipulated vector s may be propagated in both Escherichia coli and insect cells. These engineered vectors are capable of replicating in both E. coli and insect cells and can be used for cloning, genetic manipulation, and propagation of recombinant baculovirus DNA in both host systems. The methods include the use of at least one donor or transfer vector, helper vector, and recombinant bacmid vector described earlier for genetic manipulation as depicted in Figures 3 A and 3B, the donor or transfer vectors (e.g., TF-1 and TF-2) of the invention are designed to comprise modified BmNPV DNA in which one or more non-essential genes have been targeted for deletion, and include flanking regions to enable recombination with the wild-type baculovirus genome. These and other customizable features render the system highly adaptable for diverse industrial, pharmaceutical, and research applications, including but not limited to antibody production, vaccine development, enzyme synthesis, and metabolic engineering. Figure 7 illustrates one example of such genetic flexibility, depicting the design and use of the TF2_HDR fragment for targeted gene knockout (e.g., PolH) and insertion of a ZocZa-tagged cassette containing an attTn7 insertion site, enabling future modular transgene integration through recombinase or transposase-based methods. The terms TF_HDR1 and TF_HDR2 are used interchangeably with TF-01 and TF-02, respectively, and represent the homology-directed recombination (HDR) versions of these vectors. Figure 8 illustrates the genomic integration site of the TF2_HDR fragment within the BmNPV genome. The figure maps the precise insertion of the LacZa-attTn7 cassette into the PolH locus. This engineered site serves as a stable and traceable landing pad for subsequent GOI insertions, enabling controlled expression under known regulatory elements. The map also confirms that no unintended genomic rearrangements have occurred during recombination.

[0110] Targeted deletions of non-essential genes may be achieved using recombineering strategies, reducing genome size and potentially redirecting cellular machinery toward heterologous gene expression. The overall bacmid construction scheme shown in Figure 4. A transfer vector specifically designed to contain the GOI expression cassette flanked by recombination arms corresponding to desired insertion site (e.g., 0RF2 and ORF1629) may be introduced into insect cells, larvae, or pupae together with a linearised bacmid. Introduction may be carried out using chemical transfection reagents such as Cellfectin, Lipofectamine, or XtremeGene, or by microinjection using a 10 pL Hamilton syringe. Homologous recombination between the transfer vector and the bacmid at the designated ORF sites results in the insertion of the GOI expression cassette into the bacmid genome. This method reduces the time required to generate recombinant bacmids, particularly in cases where rapid expression in pupae is necessary or where the protein of interest does not express optimally in insect cell lines.

[0111] In an embodiment to further simplify the process and avoid the need for blue-white screening in E.coli, the recombinant bacmid vector contains a homing endonuclease site enabling linearization by a homing endonuclease selected from Pl-Pful, I-Ceul, I-SceII, I-TevIII, or I-Cfal, which is used to linearize the bacmid. The linearized bacmid and the transfer vector, (containing recombination regions corresponding to 0RF2 and ORF1629 on either side of the GOI expression cassette), may be introduced into insect cells, larvae, or pupae. Recombination in the host generates a functionally replicating bacmid containing the GOI expression cassette.

[0112] The gene of interest (GOI) may encode any biologically active or industrially relevant protein, including but not limited to enzymes, structural proteins, immune modulators, therapeutic proteins, diagnostic markers, or synthetic constructs. Expression cassettes may vary in design depending on the application, or regulatory requirements

[0113] Examples of donor vectors include utilized in the present invention include commercially available systems such as pFastBac™ vectors (e.g., pFastBacl, pFastBacDual) and Bac-to-Bac® Baculovirus Expression System vectors. In an embodiment of this disclosure, Transfer Vector-01 (TF-01) is designed as a homologous recombination vector that specifically targets Chitinase and cathepsin gene for knock-out and facilitates the replacement with the mini-F replicon region as depicted in Figure 3 A. The essential components of TF-01 vector include the homologous recombination arm of Gp64 and chitinase gene with a miniF replicon backbone. Similarly, Transfer Vector-02 (TF-02) functions as a homologous recombination vector that targets PolH gene and replaces it with LacZ gene. The components of TF-02 vector include the Polyhedrin gene-Left and Right homologous recombination arms with LacZ gene having a pUC-19 backbone (Figure 3B). Figure 9 is an image depicting blue- white screening of E. coli Loopbac2 colonies containing the Mini-F origin and LacZa cassette. Figure 10 is an agarose gel image depicting PCR confirmation of LacZa gene insertion.

[0114] As used herein the specification, Loopbacl and Loopbac2 refer to the first and second versions, respectively, of the recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid constructs engineered during the course of the invention. These are derived from wild-type Indian strains of BmNPV and contain defined deletions of non-essential genes, insertion of a bacterial mini-F replicon, and engineered insertion sites for genes of interest (GOI). According to an aspect of the invention, these transfer or donor vectors may be developed with BmNPV regions that are necessary for expression such as promoter, enhancer, other regulatory genetic elements and the origin of replication in E coli such as ColEl, R6Gamma, or F origin as shown in Figures 3 A and 3B. The transfer or donor vectors may also contain antibiotic markers for selection in E.coli. These vectors may contain antibiotic markers for selection in E. coli and may include specific recombination sites such as Tn7 recombination sites, Cre-Fox site or other recombination sites for Lamda recombination. Furthermore, the said vectors may also contain a multiple cloning sites wherein a GOI may be introduced into the vectors via restriction enzymebased cloning. In another embodiment, these vectors may contain rare cutting endonucleases that may be used to introduce large or whole gene expression cassettes from other transfer vectors via Cre-Lox recombination, standard cloning methods or other methods of integration. As used herein, the term “gene expression cassette” refers more specifically to a DNA segment containing a gene along with regulatory elements such as promoters, enhancers, and terminators that enable and control the gene’s expression in host cells.

[0115] In another embodiment, these transfer or donor vectors may contain single promoters or dual promoters along with their regulatory elements or combinations thereof for enhancing protein expression. The size of the plasmid will be kept at minimal sizes of 2-3 kb to enhance cloning of larger fragments and stability of the plasmid. Multiple expression cassettes could be combined via Cre-Lox or endonuclease-based cloning hence creating a large transfer or donor vector containing multiple genes as different cassettes in one single large plasmid. As part of the overall strategy outlined in Figure 4, this can be used to transfer all such gene clones into the bacmid via transposition or Cre-Lox recombination in E.coli, insect cells, insect larvae or pupae.

[0116] The expression system may further comprise a promoter for driving the expression of the foreign gene(s) and viral sequences necessary for recombination with the baculovirus genome. In one embodiment, the vector may comprise promoters such as polyhedrin, plO or unique promoters of the invention, flanking regions of homology, and baculovirus-specific transcriptional and translational signals. The transfer vectors may be introduced into host cells (e.g., E. coli or insect cells) along with a helper vector for recombination to generate recombinant baculoviruses. Donor or transfer vectors may also carry bacterial selection markers (e.g., antibiotic resistance genes as depicted in Figures 3A and 3B) for selection in E. coli. These vectors may be generated by standard cloning methods or In-fusion PCR reactions. After transforming into cloning E. coli strains, these vectors may be selected, sequenced and verified for functional regions. In an embodiment, the vector comprises miniF amplicon, kanamycin-resistant gene (Kan) between gp64 and chitinase overhang. The overhangs as well as miniF and Kan are PCR amplified using primer sequences of Table 2 and ligated for generation of TF-01.

[0117] Table 2: A non- exhaustive list of primers used for the assembly of transfer / donor plasmids TF01 and TF02

[0118] TF_HDR1 and TF_HDR2 are used interchangeably with TF-01 and TF-02, respectively, and represent the homology-directed recombination (HDR) versions of these vectors. This is used for introducing miniF region into BmNPV virus for generation of a functional Bacmid. In another embodiment, for the generation of TF-02 (as depicted in Figure 3B), the E.coli origin of replication (ori) and Ampicillin resistant gene (Amp) are amplified from pUC 19 plasmid, LacZ reporter gene is amplified from AcMNPV bacmid placed between PolH left and right overhang and ligated for generation of TF-02. TF-02 is used for transferring lacZ gene along with Tn7 attachment sites into the bacmid. The Tn7 attachment sites are crucial for introducing GOI containing expression cassette into the bacmid from Donor vectors explained in the section above. All sequences required for the generation of vectors may be PCR amplified using primers of Table 2 and may be ligated through methods known in the art including traditional methods using restriction enzymes and T4 DNA ligase, TA cloning, Gibson assembly, ligation independent cloning, and / or golden gate assembly. It is to be understood that the generation of the vectors and the methods disclosed herein are only representative and does not restrict to the sequences disclosed herein. Helper vectors, or helper bacmid vectors, may also be used in E. coli to provide the necessary baculovirus genes in trans for generating recombinant baculoviruses. Helper vectors may also carry bacterial selection markers for selection in E. coli. They also carry the genes for making Transposase complex which will help in transferring GOI expression cassette into bacmid attachment site as part of the overall process illustrated in Figure 4.1n one embodiment the donor and helper vectors are simultaneously introduced into E. coli cells along with bacmid vector derived from the BmNPV genome. The invention takes advantage of recombination-based methods to replace the non-essential gene(s) in the wild-type BmNPV genome with the modified DNA from the donor plasmids. Subsequently, E. coli clones containing the desired recombinant bacmids are selected based on antibiotic resistance and / or reporter gene screening (e.g., blue -white screening, visually represented by colonies in Figure 9). The selected clones contain recombinant bacmids with the desired genetic modifications.

[0119] In another aspect genetic elements of AcMNPV or other baculovirus may be included to produce hybrid BmNPV bacmids that have the capacity to infect various lepidopteran insect cells or larvae for expression of recombinant proteins. Included herein are genetic elements such as coat protein elements, viral capsid elements, enhancers, or gene regulatory elements that make the recombinant baculovirus compatible with a larger set of hosts.

[0120] Another aspect of the invention provides for the generation of recombinant viruses. The method may comprise transfection of insect cells recombinant bacmids of the invention. The insect cells may comprise any insect cell lines such as Spodopterafrugiperda (Sf9) and Trichoplusia ni (High Five) cells known in the art and / or Bombyx mori derived cells such as BmN, Bm5, Bml2, and BmE. The Bombyx mori cells may be primary cells or novel cell lines may be generated. Homologous recombination between the recombinant bacmid DNA and the endogenous BmNPV genome results in recombinant BmNPV viruses with the desired gene deletions. As used herein, the term “homologous recombination” refers to a molecular mechanism by which nucleic acid sequences are exchanged between two similar or identical molecules of DNA. In this disclosure, it is used to introduce heterologous DNA into specific regions of the baculovirus genome using flanking regions of homology.

[0121] The vectors of the present disclosure may comprise at least one nucleic acid sequence encoding core viral genome, foreign genes, expression cassette comprising regulatory elements and nucleic acid sequence operably linked to a gene encoding at least one protein, and selectable marker or a combination thereof. The baculovirus may be comprised in a cell, insect or insect larvae. Thus the recombinant baculovirus strains of the invention can be used for the efficient production of recombinant proteins in insect cell culture systems, including but not limited to, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five) cells and BmN ,Bm5, BmE, Bml2 or primary cells isolated from host Bombyx mori larvae. The establishment of newer insect cell lines, specifically Bombyx mori cell lines allows for convenient screening of recombinant viruses and recombinant proteins can be expressed with efficiency comparable to or higher than current baculovirus expression systems.

[0122] In some embodiments, the invention may provide methods to infect insect larvae, hemolymph or pupae using recombinant baculoviruses. Vectors of the invention are capable of replicating efficiently in various cell types, including insect larvae and pupae. These vectors are designed to facilitate quick replication within cell lines and establish a commercialization system for infecting larvae and pupae of silkworms. To enhance the versatility of the baculovirus, additional genes may be incorporated into its genome. These genes enable the baculovirus to replicate in a wider range of host cell lines, thereby expanding its application potential.

[0123] According to a specific aspect of the invention, novel expression systems have been engineered for optimized protein production by deleting or inactivating genes. The methods described comprise eliminating non-essential genes by homologous recombination, CRISPR / cas9 genome editing, transposase mediated integration, transposon mutagenesis, recombineering, Cre-Lox mutagenesis, or a combination thereof to decrease the size of the BmNPV vector or to inactivate genes. In another embodiment, heterologous GOI may be introduced into the BmNPV bacmid of the invention. The genes may be introduced by methods known in the art comprising Tandem Recombineering (TR), involving sequence-and-ligation-independent cloning (SLIC) and Cre- LoxP recombination, transfer vectors created in this way are introduced into the MultiBac baculovirus genome by the Tn7 transposon, in E. coli strains modified for this purpose. In one embodiment, the present disclosure exemplifies the knockout of genes that code for proteins such as cathepsin and chitinase, which are late -expression genes. (Example 3).

[0124] The vectors and constructs may be designed to incorporate genetic modifications, such as the insertion of additional promoter elements, enhancers, or regulatory sequences, to enhance protein expression levels, optimize protein folding and secretion, or facilitate downstream purification processes.

[0125] The vectors of the invention are designed to exhibit high efficiency and specificity in infecting Indian Bombyx mori strains and cells isolated from these strains, thereby facilitating the production of recombinant proteins for various applications. The BmNPV vectors were tested for their ability to infect Indian Bombyx mori cells and express foreign genes. Expression levels, viral titres, and other relevant parameters were evaluated to assess the efficiency and functionality of the vectors (Figures 13-15).

[0126] In one another aspect, the invention provides an improved BmNPV vector incorporating a heterologous nucleotide sequence. The heterologous nucleotide sequence may be a GOI encoding for a protein of interest, such as green fluorescent protein, used in the present invention as a reporter to evaluate vector performance. Figures 10 through 12 illustrate the validation of the system as seen by GFP expression, Figure 10 presents fluorescence microscopy images at 72, 96, and 120 hours post-infection, showing progressive increase in fluorescence intensity, correlating with robust GFP expression over time. Similarly, Figure 11 shows Western blot analyses confirming the presence of GFP at different time intervals in both insect cells and pupae, with a consistent 27.9 kDa band corresponding to GFP. Figure 12 demonstrates systemic expression of GFP in Bombyx mori larvae, pupae, and haemolymph, visually confirming successful infection and expression following bacmid delivery.

[0127] The invention also provides genetic elements — including miniF replicons, attTn7 insertion sites, and modular recombination cassettes — facilitating the construction and propagation of BmNPV bacmid vectors for heterologous protein expression. The method includes infecting insect cells, silkworm larvae, or pupae with the recombinant baculovirus comprising at least one GOI.

[0128] Figures 13-15 demonstrate expression of GFP in the insect cells and the Figure 15 is a Western blot image depicting GFP expression in silkworm pupae at 72 hours post-infection. The improved expression system of the invention may provide for large-scale protein synthesis and the method of production of protein. In particular, the invention discloses DNA elements to facilitate the construction and propagation of bacmid BmNPV vectors for the expression of GOI in insect cells. The method may comprise infecting insect cells, insects or insect larvae with the recombinant baculovirus comprising at least one GOI, and monitoring expression using functional assays, as exemplified by GFP expression results.

[0129] "Heterologous" refers to sequences not inherent to a baculovirus, preferably eukaryotic genes or cDNA. Consequently, a native promoter of the baculovirus rearranged within the genome is also considered heterologous. The expression of genes within the heterologous nucleotide sequence encompasses promoters originating from prokaryotic, viral, mammalian, or insect cellular sources, either individually or in combination. Some promoters include IE1, polyhedrin, or plO promoters for driving the expression of GOI.

[0130] The GOI may be introduced by methods known in the art including homologous recombination, Cre-Lox recombination, gateway recombination, CRISPR / cas9 genome editing, Tn transposition system or a combination thereof. In particular, the invention provides Tn7 transposition system wherein the attTn7 insertion site is comprised in a reporter gene. As used herein, the term “Tn7 transposition system” refers to a site-specific DNA insertion system that utilizes the Tn7 transposase complex to insert DNA into an attTn7 site within a bacmid or plasmid. This allows targeted and stable integration of an expression cassette. The reporter gene may include GFP, luciferase, beta-gal, alkaline phosphatase, lacZ, but not limited to these. The insertion of the GOI results in loss of function of the reporter gene. The improved BmNPV vector may further comprise DNA sequence responsible for replication (ori) of the bacmid vector in Escherichia coli (E. coli) cells such as pUC, Fl, colEl, preferably MiniF.

[0131] The mini-F origin is a low-copy number bacterial replicon known for its stability and reduced recombination during propagation in Escherichia coli. This low-copy number origin supports stable maintenance making it well-suited for large viral genomes. As a result, mini-F-based bacmids exhibit structural rearrangement during cloning and storage. In an embodiment of the present invention, the GOI that is incorporated into the baculovirus genome, may include enzymes, cytokines, transcription factors, transmembrane proteins, hormones, antibodies, antigen proteins, antibody fragments, growth factors, virus-like particles, virus structural proteins, antimicrobial peptides, fusion proteins, synthetic peptides, polymers, diagnostic molecules, secreted proteins, complex proteins supramolecular proteins or a combination thereof.

[0132] In another aspect, the invention provides for identifying new promoter sequences from isolated Indian strains of BmNPV for optimum expression of GOI. The method involves, whole -genome sequencing to obtain the complete sequence of the BmNPV genome, including potential promoter regions followed by analysis on bioinformatic tools to analyze the BmNPV genome and identify potential promoter sequences. The analysis may be on promoter prediction algorithms known in the art to identify regions with characteristics typical of promoter sequences, such as TATA boxes, transcription start sites, and regulatory elements. The method may further involve selecting potential promoter candidates for experimental validation based on sequence conservation, proximity to known genes, and predicted activity.

[0133] In one embodiment, the putative promoter sequences are cloned upstream of reporter genes such as lacZ, GFP, or luciferase to create promoter-reporter gene fusion constructs and transfected into insect cells or insect larvae, and reporter gene expression levels were measured in beta gal assays, fluorescent microscopy or luminometer, or qRT-PCR, western blots etc and compared with well- characterized strong promoters, such as the polyhedrin promoter.

[0134] The present disclosure will be further described below with reference to specific examples, so that a person of ordinary skill in the art can understand the present disclosure more comprehensively, but does not limit the present disclosure in any way. Unless otherwise specified, the test methods involved in the examples are all conventional methods or manufacturer-suggested methods; and the reagents, materials and the like used can be obtained through commercial routes, unless otherwise specified.

[0135] EXAMPLES

[0136] Example 1: Obtaining Indian strains of BmNPV

[0137] The wild-type BmNPV virus strain was isolated from lyophilized PDVs purchased from Shimoga University or isolated from the silkworm rearing facility in GKVK, Bangalore, followed by sequencing. The strains obtained from Shimoga University were named after their geographical locations as BmNPV KNK strain, BmNPV HSR strain, BmNPV VKK strain, and BmNPV MSR strain. The scope of this disclosure encompasses not only these specified strains but also extends to other strains isolated from diverse geographic regions throughout the country. Genomic DNA (gDNA) from the said BmNPV strains was isolated and sequenced. Researchers in the art can also separate the viruses from silkworm pupae in rearing that are naturally infected by BmNPV, or pupae that are commercially available.

[0138] The genomes of NPV exhibit a high degree of conservation. In this disclosure, references to coding segments (CDS), spacers or repeat sequences (br) are made with regard to the genomes of BmNPV. Using standard alignment tools, skilled practitioners can identify corresponding sequences in other NPV alpha baculoviruses. Accordingly, a comparative analysis was conducted on the effects of knocking out various genes from both AcMNPV and BmNPV. A total of 40 genes were identified as non-essential and dispensable for virus replication through sequence alignment of obtained sequences with the NCBI BmNPV T3 genome (Accession Number: NC_001962). Table 1 shows the list of these 40 non-essential genes.

[0139] Example 2: A Method for Constructing Compact BmNPV Bacmid:

[0140] In an aspect, the present disclosure describes a method for constructing the BmNPV (Bombyx mori nuclear polyhedrosis virus) bacmid that involves the following steps. The procedure begins with collecting multiple BmNPV strains from silkworm larvae and isolating polyhedron-derived virions (PDVs). Genomic DNA is extracted from these PDVs, sequenced, and analyzed to pinpoint non-essential genes suitable for knockout. Two transfer vectors are then prepared. TF-1 contains a miniF replicon, homologous regions for Gp64 and chitinase, and a kanamycin-resistance marker, all amplified from either a selected BmNPV strain or the bMON 14272 vector. TF-2 uses a pUC- 19 backbone and carries left and right polyhedrin overhangs flanking a LacZ reporter cassette. Insect cells are co-transfected with TF-1, TF-2, and the parental bacmid, allowing homologous recombination that deletes chitinase, cathepsin, and polyhedrin from the bacmid. Recombinant clones are identified and validated by blue-white screening based on LacZ activity. Additional dispensable genes can subsequently be excised from the engineered bacmid using genome-editing methods such as CRISPR-Cas9.

[0141] Example 3: Method for knockout of non-essential genes such as cathepsin and chitinase from BmNPV vector

[0142] In an embodiment, PCR amplification of overhang regions of chitinase and GP64 genes of the BmNPV gDNA was done to enable the knockout of chitinase and cathepsin genes, respectively. Specific primers were designed to amplify the regions upstream and downstream of the chitinase and GP64 genes. These DNA fragments serve as homology arms or regions that facilitate the precise insertion or deletion of the target genes through homologous recombination. The bacmid DNA from AcMNPV sourced from DHIOBac E. coli cells (Thermo Fischer Scientific) was isolated. This DNA served as the backbone for constructing the BmNPV bacmid. Kanamycin resistance (Kan R) cassette and a 6.5 kb miniF replicon (without LacZ) of the AcMNPV gDNA were amplified by PCR. These components were incorporated into the final BmNPV bacmid construct. Fig. 3 A shows a vector map of TF-01, that comprises 650-900 bases homology regions (hrs) of chitinase and gp64 genes, along with a miniF replicon, excluding LacZ. This assembly can be done by Gibson cloning, restriction digestion or In-Fusion cloning method. In-Fusion cloning is a DNA assembly method that allows for the seamless joining of DNA fragments with overlapping ends. It eliminates the need for restriction enzymes or ligases traditionally used in cloning processes. By designing primers with homologous ends between inserts and vectors, the fragments can anneal and ligate in a single step with In-Fusion cloning enzymes.

[0143] In another embodiment of the present disclosure, a vector TF-02 was constructed by amplifying different parts of the pUC-19, AmMNPV and BmNPV gDNA. First, the Ori and AmpR regions from pUC-19 vector and upstream and downstream sequences of polyhedrin overhang regions from BmNPV gDNA were amplified. The backbone vector used here is not limited to pUC19, but other vectors such as pBluescript, pET, pGEM, pBAD can also be used. The LacZ region from AcMNPV bacmid is also PCR amplified. Assembly of TF-02 vector is done by stitching together PolH-L-overhang, LacZ, PolH-R-overhang, and PUC-19 regions using In-fusion assembly. Alternatively, restriction digestion or Gibson cloning may also be used. Fig. 3B depicts the vector map of TF-02. Table 2 gives a non-exhaustive list of primers that can be used for In-Fusion assembly of TF-01 and TF-02.

[0144] In another embodiment of the present invention, TF-01 and TF-02 can be synthesised using a restriction digestion and ligation standard cloning protocol. Through overlap PCR with primers mentioned in Table 3, two or more fragments can be fused by ligation after using restriction enzymes such as EcoRI, Hindlll, BamHI, Mini, Avril, or Notl.

[0145] Table 3: A non-exhaustive list of primers used in restriction digestion and ligation standard cloning of TF 01 and TF 02.

[0146] Fig.2 shows a scheme of the process involved in the construction of BmNPV bacmid devoid of some non-essential genes. It provides an overview of the various steps involved and introduces the prospects of this invention.

[0147] The compact BmNPV genome enables the expression of various recombinant proteins with potential applications in animal feed. These proteins may include enzymes like phytases, xylanases, proteases, and carbohydrases, aimed at improving nutrient utilization and digestion. Additionally, growth factors such as insulin-like growth factor (IGF) can be expressed to promote animal growth and development. Certain proteins rich in essential nutrients, such as amino acids or vitamins, may also be produced as feed supplements. Moreover, insect cell-expressed recombinant proteins like subunit vaccines, monoclonal antibodies, and hormones like insulin, insulin-like growth factor 1 (IGF-1), and human growth hormone (HGH) have diverse applications in both human and animal health.

[0148] Example 4: Method to knockout non-essential genes sequentially using CRISPR-cas9 system In an embodiment, the CRISPR-cas9 system is employed to excise non-essential parts of the genome and introduce new elements to create an engineered plasmid. Initially, a CRISPR plasmid will be linearized at the specified loci within the AcMNPV or BmNPV genome, namely the Orfl l(BmNPV orf4) and Orf36 (BmNPV orf 27) regions. Subsequently, engineered plasmids will be designed encompassing 700 to 1000 base pairs upstream of AcMNPV Orfl l and downstream of Orf36, containing essential genes along with their regulatory regions and a reporter Yellow Fluorescent Protein (YFP). Other reporter genes such as Luciferase, Green Fluorescent Protein (GFP) or P-glucuronidase (GUS) may also be used in place of YFP. The engineered plasmids will also include sequences for homologous recombination with the viral genome. Utilizing homologous recombination techniques, these engineered plasmids will be integrated into the AcMNPV or BmNPV viral genome at the targeted loci (Orfl 1 and Orf36). This process involves introducing the engineered plasmids into AcMNPV / BmNPV-infected insect cells, where they will undergo recombination with the viral DNA, resulting in the insertion of the desired genes and regulatory elements. Selection of clones with the desired gene insertions and removals will be facilitated through the expression of reporter genes, agarose gel electrophoresis, and PCR analysis.

[0149] In another embodiment, the CRISPR plasmid will be linearized at other loci within the AcMNPV / BmNPV genome, such as odve-66 and Gp37 loci. Subsequently, engineered plasmids will be constructed utilizing regions upstream and downstream of these loci. This engineered plasmid will contain a selection of specific genes, their regulatory elements, and LoxP sites. This process aims to reduce the size of the plasmid to approximately 113 kilobases, thereby enhancing its efficiency in gene editing experiments.

[0150] In an aspect of the present disclosure, other non-essential genes identified in Table 1, can be targeted for removal from the obtained BmNPV bacmid using CRISPR technology, thereby optimizing the size and functionality of the bacmid. Fig. 16 shows a vector map of BmNPV gDNA isolated and sequenced by Loopworm, representing non-essential genes (grey) for knockout considerations. In accordance with the disclosed method, once the non-essential genes from Table 1 are identified and mapped, the CRISPR-Cas9 system may be employed to excise or modify the genes annotated in grey, as shown in the figure.

[0151] Additional preferred heterologous nucleotide sequences that can be incorporated into the genome include but are not limited to TnT sites; sequences encoding resistance genes; homing endonuclease sites; mutated fp genes; loxP sites; various promoters such as IE1, polyhedrin, and plO; reporter genes and fluorescent markers such as green, red, or yellow fluorescent proteins; enhancer elements; epitope tags; secretion signals; or regulatory elements from different organisms.

[0152] In an embodiment, repeated burst sequences (BSs) are used as transcription enhancers for improving polyhedrin promoter activity. BSs are located 50 bps downstream of the polyhedrin promoter and facilitate effective expression of very late viral promoters during the late phase of infection (Manoher et.al., 2010).

[0153] “Burst sequences”, as used herein are also known as homologous repeat regions or homologous repeats, are specific sequences found within the genome of Bombyx mori nucleopolyhedrovirus (BmNPV) and play crucial roles in the replication and propagation of the virus within host cells. In another embodiment, homologous regions (hrs) are used in addition to the BSs as transcription enhancers. The hrs are repetitive sequences naturally found in baculovirus DNA, ranging in size from 150 to 800 base pairs. The BmNPV T3 strain harbours 7 hrs, including hrl, hr2L, hr2R, hr3, hr4L, hr4R, and hr5. Among these, hr3 serves as a cis-regulatory element (CRE) in overexpression vector constructs due to its strong affinity for transcription factors, thereby enhancing the transcriptional efficiency of several promoters such as gp64, 39 K, and vp39.

[0154] In an embodiment, homing endonuclease sites, also known as homing endonuclease recognition sites or HE sites are incorporated into the expression vector to enable targeted DNA cleavage and insertion of GOI into the genome. Homing endonucleases are enzymes that cleave DNA at specific sites, facilitating gene editing and genetic engineering applications.

[0155] In an aspect of the present disclosure, a method for producing recombinant proteins using the enhanced BEVS system is included which comprises: i) Transfecting insect cells with the modified BmNPV bacmid lacking non-essential genes; ii) Cultivating the transfected insect cells under appropriate conditions to induce recombinant protein expression; iii) Harvesting and purifying the expressed recombinant proteins for various applications in biotechnology and pharmaceutical industries.

[0156] In an aspect of the present disclosure, to improve the recombinant protein purity and yield, epitope tags are genetically fused to the target protein of interest. Epitope tags include FLAG (DYKDDDDK, recognised by anti-FLAG antibodies), HA tag (YPYDVPDYA, recognized by anti-HA antibodies), Myc tag (EQKLISEEDL, recognized by anti-Myc antibodies), His tag (Polyhistidine stretch, allowing purification using immobilized metal affinity chromatography), GST tag (Glutathione S -transferase, facilitating purification using glutathione affinity chromatography), V5 tag (GKPIPNPLLGLDST, recognized by anti-V5 antibodies), or T7 tag ( MASMTGGQQMG, recognized by anti-T7 antibodies). They are not typically involved in the protein's function but serve as molecular handles for various applications. Epitope tags are especially useful for protein detection and purification because they allow for specific and efficient isolation of the tagged protein from complex mixtures. These tags can enhance protein production indirectly by aiding in protein purification, which ensures higher protein yields and purity. Additionally, some tags may stabilize the protein of interest, leading to improved expression levels. In another embodiment, signal peptides are fused to the N-terminus of recombinant protein to facilitate their secretion from insect cells infected with BEVS. Some examples of signal peptides that can be used include Honeybee melittin signal peptide, Bombyx mori fibroin signal peptide, AcMNPV GP64 signal peptide or Nuclear localization signals (NLS). This embodiment is not limited to the above signal peptides, it can also include signal peptides from other baculovirus species, such as Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) or Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV). The embodiments described above represent examples of the present disclosure, and it is important to note that individuals skilled in the relevant field may introduce various enhancements and modifications without deviating from the fundamental technical aspects of the present disclosure. Such modifications and enhancements are also encompassed within the scope of protection of the present disclosure.

Claims

CLAIMS:

1. A recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for enhanced heterologous protein expression in hosts, selected from Bombyx mori larvae, pupae, primary cells, or established insect cell lines, the bacmid comprising:(a) a BmNPV genome derived from a wild-type strain, wherein the BmNPV genome: i. is reduced in size relative to the wild-type BmNPV genome by deletion of at least one base pair of at least one non-essential gene; or ii. at least one non-essential gene is inactivated by mutation; or iii. combination of (i) and (ii);(b) a bacterial origin of replication enabling stable propagation in Escherichia coli',(c) an insertion site for at least one gene of interest (GOI) comprised within the BmNPV genome, wherein the insertion site is positioned at a distance from the bacterial origin of replication to enhance stability of the GOI during bacmid maintenance or viral passage.

2. The bacmid of claim 1, wherein the wild-type strain is an Indian strain.

3. The bacmid of claim 1, wherein at least one non-essential gene is inactivated by mutation to reduce metabolic burden or suppress undesired protein expression that interferes with heterologous gene expression.

4. The bacmid of claim 1, wherein the non-essential gene is selected from polyhedrin (PolH) gene, chitinase gene, and cathepsin gene.

5. The bacmid of claim 1, wherein the non-essential gene is further selected from the group consisting of plO, p26, 39k, pif, p74, AcMNPV orfl l, AcMNPV orfl3, egt, acl8, arifl, ac22 (pif-2), ac23 (gp64 homolog), AcMNPV orf29, ac30, fibroblast growth factor (fgf), p43, p47, gta, orf34, orf35 (ubiquitin-like), ac57, ac58 / ac59, ac60, fp25k (25k), orf51, gp37, p30 (arif-1), orf74, BroB, He65, gcn-2, AC122, pp34, orfl lOa, acl50, acl49, BroE, and pl5.

6. The bacmid of claim 1, wherein the bacterial origin of replication is selected from pUC, Fl, colEl, R6kGamma, or mini-F origin of replication.

7. The bacmid of claim 1, wherein the origin of replication is mini-F.

8. The bacmid of claim 1, wherein the insertion site further comprises at least one synthetic site-specific transposon insertion sequence or recombinase recognition sequence, and wherein the GOI is integrated using corresponding transposase or recombinase.

9. The bacmid of claim 8, wherein the transposon insertion sequence is an attTn7 transposition sequence.

10. The bacmid of claim 8, wherein the transposon insertion sequence is further selected from piggyBac inverted terminal repeats (ITRs), mariner transposon inverted repeats, Sleeping Beauty inverted / direct repeats or Tol2 or equivalents thereof.

11. The bacmid of claim 8, wherein the recombinase recognition sequence is selected from loxP, attP, attB, FRT, or equivalents thereof.

12. The bacmid of claim 1, wherein the BmNPV bacmid is capable of assembling into infectious baculovirus particles upon transfection into hosts selected from Bombyx mori larvae, pupae, primary cells, or established insect cell lines.

13. The bacmid of claim 1, wherein the heterologous protein expressed is selected from a therapeutic protein, vaccine antigen, monoclonal antibody, or industrial enzyme.

14. A recombinant transfer vector for generating a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid, the vector comprising:(a) at least one pair of homologous recombination arms, wherein each arm is derived from a region of the BmNPV genome derived from a wild-type strain;(b) a functional element positioned between the homologous recombination arms, wherein the functional element is selected from a bacterial origin of replication, a selectable marker, or an insertion site for at least one gene of interest (GOI); and(c) a bacterial plasmid backbone, enabling propagation of the vector in a E.coli, wherein the vector is configured to enable targeted replacement of non-essential gene regions of the BmNPV genome with elements that improve recombinant bacmid stability or heterologous gene expression.

15. The recombinant transfer vector of claim 14, wherein the wild-type strain is an Indianstrain.

16. The recombinant transfer vector of claim 14, wherein the region of BmNPV genome comprises an essential gene or non-essential gene17. The recombinant transfer vector of claim 16, wherein the non-essential gene region is selected from a polyhedrin (PolH) gene, a chitinase gene, or a cathepsin gene.

18. The recombinant transfer vector of claim 16, wherein the non-essential gene region is further selected from plO, p26, 39k, pif, p74, AcMNPV orfl l, AcMNPV orfl3, egt, acl8, arifl, ac22 (pif-2), ac23 (gp64 homolog), AcMNPV orf29, ac30, fibroblast growth factor (fgf), p43, p47, gta, orf34, orf35 (ubiquitin-like), ac57, ac58 / ac59, ac60, fp25k (25k), orf51, gp37, p30 (arif-1), orf74, BroB, He65, gcn-2, AC122, pp34, orfl lOa, acl50, acl49, BroE, and pl5.

19. The recombinant transfer vector of claim 14, wherein the bacterial origin of replication is selected from pUC, Fl, colEl, R6kGamma, or mini-F origin of replication.

20. The recombinant transfer vector of claim 19, wherein the bacterial origin of replication is a mini-F replicon.

21. The recombinant transfer vector of claim 14, wherein the selectable marker is selected from green fluorescent protein (GFP), luciferase, chloramphenicol acetyltransferase (CAT), beta-glucuronidase (GUS) or lacZa.

22. The recombinant transfer vector of claim 21, wherein the selectable marker lacZa.

23. The recombinant transfer vector of claim 14, wherein the insertion site further comprises at least one synthetic site-specific transposon insertion sequence or recombinase recognition sequence, and wherein functional element is integrated using corresponding transposase or recombinase.

24. The recombinant transfer vector of claim 23, wherein the transposon insertion sequence is an attTn7 transposition sequence.

25. The recombinant transfer vector of claim 23, wherein the transposon insertion sequence is further selected from piggyBac inverted terminal repeats (ITRs), mariner transposon inverted repeats, Sleeping Beauty inverted / direct repeats or Tol2 or equivalents thereof.

26. The recombinant transfer vector of claim 23, wherein the recombinase recognition sequence is selected from loxP, attP, attB, FRT, or equivalents thereof.

27. A method of producing a bacmid, comprising:(a) isolating genomic DNA from a wild-type strain of BmNPV ;(b) genetically modifying the wild-type strain of BmNPV genome comprising: i. deleting at least one base pair of at least one non-essential gene to reduce the genome size relative to the wild-type BmNPV genome, or inactivating at least one non-essential gene, or a combination thereof; ii. introducing a bacterial origin of replication; iii. introducing an insertion site for at least one gene of interest (GOI) into the BmNPV genome positioned at a distance from the bacterial origin of replication to enhance stability of the GOI during bacmid maintenance or viral passage;(c) introducing the genetically modified BmNPV genomic DNA into E. coli', and(d) isolating the resulting bacmid DNA.

28. The method of claim 27, wherein the strain of BmNPV is a wild-type Indian strain.

29. The method of claim 27, wherein the at least one non-essential gene is inactivated by mutation to reduce metabolic burden or suppress undesired protein expression that interferes with heterologous gene expression30. The method of claim 27, wherein the non-essential gene is selected from polyhedrin (PolH) gene, chitinase gene, and cathepsin gene.

31. The method of claim 27, wherein the non-essential gene is further selected from the group consisting of plO, p26, 39k, pif, p74, AcMNPV orfl l, AcMNPV orfl3, egt, acl8, arifl, ac22 (pif-2), ac23 (gp64 homolog), AcMNPV orf29, ac30, fibroblast growth factor (fgf), p43, p47, gta, orf34, orf35 (ubiquitin-like), ac57, ac58 / ac59, ac60, fp25k (25k), orf51, gp37, p30 (arif-1), orf74, BroB, He65, gcn-2, AC122, pp34, orfl lOa, acl50, acl49, BroE,and pl5..

32. The method of claim 27, wherein the bacterial origin of replication is selected from pUC, Fl, or colEl, R6kGamma or mini-F origin of replication.

33. The method of claim 32, wherein the bacterial origin of replication is mini-F.

34. The method of claim 27, wherein the insertion site further comprises at least one synthetic site-specific transposon insertion sequence or recombinase recognition sequence, and wherein the GOI is integrated using a transposase or recombinase.

35. The method of claim 34, wherein the transposon insertion sequence is an attTn7 transposition sequence.

36. The method of claim 34, wherein the transposition sequence is further selected from, piggyBac inverted terminal repeats (ITRs), mariner transposon inverted repeats, Sleeping Beauty inverted / direct repeats or Tol2, inverted repeats, or equivalents thereof.

37. The method of claim 34, wherein the recombinase recognition sequences are selected from loxP, attP, attB, FRT, or equivalents thereof.

38. The method of claim 27, wherein the deletion of at least one non-essential gene is performed using a technique selected from the group consisting of recombineering, Cre- LoxP recombination, CRISPR-Cas9 gene editing, FLP-FRT recombination, TALEN- mediated gene editing, zinc finger nucleases, restriction enzyme-based deletion and ligation, or transposon-mediated mutagenesis.

39. The method of claim 27, wherein the bacmid is competent for transfection into hosts selected from Bombyx mori larvae, pupae, primary cells, or insect cell lines derived therefrom for expression of the gene of interest40. A method of expressing a heterologous protein, comprising: a) introducing a recombinant bacmid of claim 1 into a host selected from Bombyx mori larvae, pupae, primary cells, or established insect cell lines; b) allowing viral infection and expression of the heterologous gene; andc) harvesting the protein product from an infected host.

41. The method of claim 40, wherein the heterologous protein is selected from growth factors, antigen, enzymes, antibodies, virus-like particles, antimicrobial peptides, or diagnostic or therapeutic proteins.

42. The method of claim 40, wherein the larvae, pupae, primary cells of the same lineage as the BmNPV isolate are used to derive the bacmid.

43. The method of claim 40, wherein the protein is recovered from Bombyx mori larvae, pupae by lysis, clarification, and chromatographic purification.

44. A heterologous protein obtained from the method of claim 40, wherein the protein is selected from therapeutic proteins, vaccine antigens, monoclonal antibodies, virus-like particles, diagnostic proteins, antimicrobial peptides, or industrial enzymes.

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