Oral fish vaccines and compositions comprising the same

Abstract

Disclosed herein is an oral fish vaccine comprising a recombinant cell comprising virus-like particles, wherein the recombinant cell is intact, inactivated, and does not exhibit antibiotic resistance. In one preferred embodiment, the inactivation is performed using sodium hypochlorite. In another preferred embodiment, the recombinant cell is Escherichia coli (E. coli) or Lactococcus lactis (L lactis). In a further preferred embodiment, the virus-like particles comprise nervous necrosis virus capsid proteins and the vaccine is used in treating or preventing viral nervous necrosis.

Description

ORAL FISH VACCINES AND COMPOSITIONS COMPRISING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore provisional application no.10202500093S, filed 10 January 2025, as well as Singapore provisional application no.10202503439Q, filed 20 November 2025, the contents of both applications being hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of molecular biology and biotechnology. In particular, the present invention relates to the use of recombinant cells containing overexpressed proteins as vaccines.BACKGROUND OF THE INVENTION

[0003] Nervous necrosis virus (NNV) is one of the major fish pathogens, causing viral encephalopathy and retinopathy disease in marine and freshwater fish species.

[0004] Most vaccine formulations, including the commercial products, rely on injection. Injection is an efficient way to deliver antigens (usually obtained from inactivated viruses) to the immune system. However, this method can only be administered to fish of fairly large size, is stressful to the fish, causes damage at the injection site, and is labour intensive. Injections are challenging for fingerlings smaller than 5 g per fish. Although automated injection methods have been developed to reduce labour costs, the methods tend to increase injection-induced injuries Moreover, injection vaccination is not feasible for early juvenile fish because of their small size. Virus infection may therefore occur in unvaccinated fish, from the larval stage to just before these fish can be vaccinated by injection. In addition, mortality caused by NNV in hatchery-reared larvae and juveniles can reach up to 100% of the population.

[0005] Thus, there is an unmet need for a non-invasive method of inoculating or vaccinating fish.SUMMARY

[0006] In one aspect, the present disclosure refers to a recombinant cell comprising virus-like particles, wherein the recombinant cell is intact, inactivated, and does not exhibit antibiotic resistance.

[0007] In another aspect, the present disclosure refers to an oral vaccine comprising the recombinant cell as described herein.

[0008] In yet another aspect, the present disclosure refers to a fish feed additive comprising the recombinant cell as described herein.

[0009] In a further aspect, the present disclosure refers to a method of inoculating a subject against nervous necrosis virus, the method comprising administering the recombinant cell as described herein.

[0010] In another aspect, the present disclosure refers to a method of treating or preventing viral nervous necrosis in a subject, the method comprising administering the recombinant cell as described herein.

[0011] In yet another aspect, the present disclosure refers to the use of the recombinant cell as disclosed herein in the manufacture of a medicament for eliciting an immune response against nervous necrosis virus (NNV).

[0012] In a further aspect, the present disclosure refers to the use of the recombinant cell as disclosed herein in the manufacture of a medicament for preventing or treating a nervous necrosis virus (NNV) infection.

[0013] In another aspect, the present disclosure refers to a method of obtaining a recombinant cell comprising virus-like particles, wherein the recombinant cell is intact, inactivated, and does not exhibit antibiotic resistance; the method comprising: a. removing antibiotic resistance genes from a plasmid to obtain an antibiotic resistance free plasmid; b. inserting the gene encoding a viral capsid protein into the antibiotic resistance free plasmid of step a. to obtain an overexpression plasmid; c. transforming the overexpression plasmid of step b. into competent cells; d. growing and selecting the transformed competent cells of step c. under conditions that allow overexpression of the gene encoding the viral capsid protein and self-assembly of the virus-like particles; e. inactivating the cells of step d., wherein the inactivation leaves the recombinant cell intact; thereby obtaining intact recombinant cell comprising virus-like particles.

[0014] In a further aspect, the present disclosure refers to a recombinant cell obtained using the method disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0016] FIG. 1 shows SDS-PAGE results of samples from the E. col cells containing NNV capsid protein without inactivation (A) and with inactivation by using 2000mg / L sodium hypochlorite (B) WC = whole cell lysate after sonication; S = soluble fraction after sonication.

[0017] FIG. 2 shows SDS-PAGE results of E. coli samples induced at 18°C and 37°C for 4 hours and 20 hours, respectively.

[0018] FIG. 3 shows SDS-PAGE results of E. coli samples containing nervous necrosis virus (NNV) capsid proteins before (left panel) and after (right panel) passing through a Capto core 400 column.

[0019] FIG. 4 shows transmission electron microscopy (TEM) images (A) and hydrodynamic diameter (B) of NNV capsid protein purified using a Capto core 400 column. The average diameter of about 33nm indicates the self-assembly of NNV capsid protein into VLPs inside the E. coli cells.

[0020] FIG. 5 shows a graph depicting the hydrodynamic diameter of NNV capsid protein extracted from E. coli cells treated with sodium hypochlorite by using a Capto core 400 column. The average diameter of about 34 nm indicates the self-assembly of NNV capsid protein into VLPs inside the treated E. coli cells.

[0021] FIG.6 shows images showing the stability of feed pellets immersed in water with slight agitation for 0 (A) and 10 (B) minutes

[0022] FIG. 7 shows column graphs depicting (A) NNV neutralization titres against 100 TC D50 of GGNNV virus on day 28. Comparison of NNV neutralizing titres in the sera of control, intraperitoneally (IP) and orally vaccinated ABS. Fig. 7 (B) shows NNV neutralizing titres in sera of control and orally vaccinated ASB. Each point represents the geometric mean neutralizing titres in vaccinated fish (n=10 fish / group) ± standard Error (SE). #The lowest serum dilution analysed was 1:10, with <1:10 set as 0. ***, P < 0.001; ns, non-significant.

[0023] FIG. 8 shows a column graph showing the comparison of brain NNV viral titres by q-PCR after 7 days post-challenge with NNV in ASB. Each point represents the arithmetic mean of viral copies in the brain (n=5 fish / group) ± standard Deviation (SD).

[0024] FIG. 9 shows an image of an SDS PAGE of lysate of L. lactis cells expressing NNV capsid protein (middle lane), flow-through fraction after the lysate passed through a Capto Core 400 column (right lane), and protein markers (left lane).

[0025] FIG. 10 shows transmission electron microscopy (TEM) images of NNV capsid proteins extracted from L. lactis cells without (A) and with (B) treatment using 2000 mg / L hypochlorite.

[0026] FIG. 11 shows images of the results of a SDS-PAGE analysis of control and heat-inactivated L. Lachs cells. WC, whole-cell lysate, S, soluble fraction. The band between 35 kDa and 40 kDa corresponds to theNNV capsid protein (molecular weight (MW): 37 kDa), as indicated by a black arrow. Heat-inactivated cells were washed with PBS to remove leaked proteins before sonication for lysate preparation. The sample amounts loaded in the middle panel were half of those in the right panel.

[0027] FIG. 12 shows the results of an SDS-PAGE (A) and a Western Blot with NNV-specific antibody (B) of L. Lactis cells inactivated by sodium hypochlorite. WC, whole-cell lysate; S, soluble fraction. Inactivated cells were washed with PBS to remove leaked proteins before sonication for lysate preparation. Tn panel B, “No induction” refers to cells before protein overexpression, while “Control” denotes the NNV VLPs purified from E. coli expression.

[0028] FIG. 13 shows graphs showing the results of NNV VLP-specific IgM and neutralization antibody titres induced by orally immunizing ABS fingerlings with live L. lactis cells expressing VLPs (A and B) and heat-inactivated L. lactis (C and D). For comparison, purified VLPs (from E. coli) were administered via injection (IP VLP) or orally (Oral VLP). Oral Lactisl-VLP: live L. lactis expressing VLPs; Oral lactis2:lactis expressing VLPs inactivated at 37°C. Results are expressed as mean value (n=8), with whiskers representing the standard deviation (SD) (***p<0.001).

[0029] FIG. 14. shows column graphs depicting NNV VLP-specific IgM titres (A) and neutralization antibody titres (B) induced by orally immunizing ABS fingerlings with hypochlorite-inactivated L. lactis cells expressing VLPs, and viral copies post-challenge (C). For comparison, purified VLPs (from E. coli) were administered via injection (IP VLP) or orally (Oral VLP). Oral Lactis- VLP: L. lactis expressing VLPs inactivated by 2000 mg / L sodium hypochlorite. (A, B) Each point represents the mean value (n=8) ± SD (***p<0.001). (C) Each point represents the mean value (n=5) ± standard error (**p<0.01; ***p<0.001).DETAILED DESCRIPTION

[0030] In the fish farming industry, viral infections are a significant issue, with reduced production yields and significant economic losses, resulting from the lack of simple and effective procedures to treat infected fish. Among these, viral nervous necrosis (VNN), which is caused by nervous necrosis virus (NNV), is one of the major devastating diseases affecting economically important fish species such as grouper, European seabass, and Asian seabass (ASB). Fish are particularly vulnerable in the larval and juvenile stages, with the disease having a near 100% mortality rate in the larval stage. Moreover, the growth is reduced even if some fish survive. Atpresent, there are no simple and effective treatments available forNNV infection, with vaccination playing an increasingly important role in controlling and preventing infectious fish diseases.

[0031] Described herein is the use of recombinant cells, such as, but not limited to, Escherichia coh (E. coli) and Laclococcus lactis (L. lactis) cells, as systems for expressing and encapsulating viral capsid proteins in a virus-like particle (VLP) form. Also disclosed herein are methods for inactivating the cells without changing the properties of such capsid proteins. Thus, inactivated recombinant cells containing VLPs can be incorporated into compositions, such as, but not limited to, fish feed as oral vaccines in aquaculture. The recombinant cells disclosed herein do not contain antibiotics resistant vectors and are inactivated, causing no biosafety or environment issues when used in aquaculture

[0032] Thus, in one example, the present disclosure refers to a recombinant cell comprising virus-like particles, wherein the recombinant cell is intact, inactivated, and does not exhibit antibiotic resistance. In another example, the virus-like particles comprise nervous necrosis virus (NNV) capsid proteins.

[0033] In another example, the recombinant cell comprises virus-like particles. In another example, the recombinant cell is intact, inactivated, and does not exhibit antibiotic resistance. In yet another example, the viral capsid proteins are soluble viral capsid proteins.

[0034] As used herein, the term “inactivated” refers to a cell (recombinant or otherwise) that has been modified or manipulated, either physically or chemically, so that it is no longer able to replicate or multiply.

[0035] As used herein, the term “intact” refers to the characteristic of a recombinant cell disclosed herein which has been inactivated but is still a whole cell. In other words, the term “intact” refers to the status of the cell membrane, indicating that the cell membrane of the recombinant cell has not been disrupted or lysed while deactivating the recombinant cell using the methods disclosed herein.

[0036] Also disclosed herein is the use of nervous necrosis virus (NNV) capsid proteins which were expressed in either Lactococcus lactis (L. lactis) or Escherichia colt (E. coli), and are evaluated as a vaccine in Asian seabass (Lates calcarifer) fingerlings. As shown herein, the capsid protein assembled into virus-like particles (VLPs), structurally similar to those produced in E. coli In one example, nervous necrosis virus capsid VLPs disclosed herein are produced in Laclococcus lactis or Escherichia coli. In another example, the virus-like particles self-assemble from viral capsid proteins.

[0037] Intraperitoneal injection of purified virus-like particles (VLPs) elicited strong humoral immunity, with NNV-specific IgM titres four-fold higher than oral delivery, despite a tenfoldlower antigen dose. In contrast, encapsulation of VLPs in live or heat -inactivated L. lactis cells failed to induce protective immunity, likely due to poor antigen release. Sodium hypochlorite-inactivated L. lactis according to the present disclosure preserved VLP encapsulation, solubility, and structural integrity. Oral vaccination with these cells induced approximately two-fold higher antibody and neutralizing titres than the purified VLPs. Challenge studies demonstrated a roughly 2.5-log reduction in brain viral load 7 days post-challenge. In other words, oral vaccination with hypochlorite-inactivated cells was shown to reduce brain viral load. Taken together, the findings disclosed herein highlight the use of hypochlorite-inactivated cells as an oral vaccine platform.

[0038] Thus, in one example, hypochlorite-inactivated cells are as described herein. As disclosed herein, such hypochlorite-inactivated cells preserved VLPs and enhanced immunity. In another example, the hypochlorite-inactivated cells can be L. lactis or E. coli.

[0039] Such recombinant, modified cells can be obtained using methods known in the art. In one example, the vector used in the methods, and the recombinant cells disclosed herein, is a modified pET32a(+) vector, which had been modified by replacing the ampicillin gene with a FabI gene and deleting unnecessary tags and cleavage sites. In addition, the modified pET32a(+) vector does not contain any antibiotics resistance genes. Clone selection was performed using triclosan, and the competent cells used were BL21 DE.

[0040] For L. lactis, vector pNZ8149 (MobiTec GmbH) was used, which is free of antibiotics resistance genes. In other words, when generating the vector for L. lactis, the vector pNZ8149 was used, which does not comprise antibiotics resistance genes.

[0041] Thus, in one example, the recombinant cells disclosed herein do not contain any antibiotic resistance genes. In other words, the recombinant cells disclosed herein can be, but are not limited to, inactivated cells which are free of or lack antibiotic resistance. Such cells can be, but are not limited to, E. coli and A. lactis cells. Tn one example, the cell is a recombinant, inactivated, antibiotic resistance-free E. coli cell. In another example, the cell is a recombinant, inactivated, antibiotic resistance-free L. lactis cell.

[0042] In another example, the recombinant cells disclosed herein comprise virus-like particles. As used herein, the term “virus-like particles” (VLPs) refers to virus-derived structures made up of one or more different viral molecules (for example, but not limited to, nucleic acids, proteins, peptides, DNA, RNA) via self-assembly. Examples of viral molecules that make up virus-like particles are, but are not limited to, structural proteins, capsid proteins, envelope proteins, core (functional) proteins, receptor binding proteins, and combinations thereof. In one example, the virus-like particles can be formed using proteins or molecules obtained from one type of virus. In another example, the virus-like particles can be formed from proteins obtained from differentviruses (so-called chimeric virus-like particles). These virus-like particles mimic the form and size of a virus particle but lack the genetic material usually enclosed within the virus. Because of the lack of genetic payload within the virus-like particles, these particles are not capable of infecting the host cell. Expression and self-assembly of the viral structural proteins can take place within a living cell expression system or within a cell-free expression system, after which the viral structures can be assembled and reconstructed. Thus, in one example, the virus-like particles comprise capsid proteins.

[0043] In another example, the capsid proteins expressed by the recombinant cells disclosed herein are shown to assemble into virus-like particles within the recombinant cells. This negates the need to purify any expressed viral proteins, and results in the generation of a low-cost vaccine The recombinant cells disclosed herein were shown to be inactivated but were shown to contain functional and assembled virus-like particles.

[0044] Oral vaccination is suitable for fish at any developmental stage. It does not cause stress in the fish and is low cost. It is also simple to administer and does not require handling of individual fish. Oral vaccines can be administered repeatedly throughout the life of cultured fish. Having said that, the efficacy of oral vaccination remains lower compared to injection vaccination. To achieve similar survival rates for grouper larvae, immunization via the oral route required about 80 times more nervous necrosis virus (NNV) virus-like particles (VLPs) compared to the amount required to be administered via intramuscular injection. This is mainly due to antigen (e.g., capsid protein) denaturation and degradation in the acidic gastric environment, which maintains a low pH and a high concentration of digestive enzymes. To reduce the antigen denaturation and degradation, live artemia (a genus of aquatic crustaceans, also known as brine shrimp), often used as fish feed, have been used to encapsulate E. coli cells which had been modified to express NNV capsid proteins or variants thereof, as described herein, as well as being used as a vehicle to orally deliver the viral antigen. The NNV antigen delivered in this manner is shown to have an immunoprotective effect against NNV infection in grouper larvae. It has also been shown herein that E. coli cells can be used as a vaccine vehicle to encapsulate and deliver NNV capsid protein. European seabass vaccinated with the feed coated with E. coli cells expressing NNV capsid protein were shown to be protected from NNV infection.

[0045] Thus, in one example, an oral vaccine against nervous necrosis virus (NNV) using edible bacteria, such as but not limited to, Lactococcus lactis (L. lactis), has been developed, as described herein, as an antigen expression system and as a carrier / vehicle system.

[0046] In one example, the recombinant cells disclosed herein can be Escherichia coli (E. coli) cells or Lactococcus lactis cells, or combinations thereof. These cells have the advantage ofresulting in a high protein overexpression yield, as well as having a fast growth rate and being low cost in cell culture cultivation requirements.

[0047] Recombinant L. lactis and E. coll cells, which had been engineered to express NNV capsid proteins in the cytoplasm, were generated by DNA cloning using the method disclosed herein. The expressed capsid proteins were then shown to self-assemble into VLPs inside the recombinant cells. The capsid proteins (also referred to herein as antigens) were delivered orally by incorporation of the cells into fish feed. Both live, recombinant L. lactis cells and L. lactis cells inactivated by heat did not appear to elicit neutralizing antibodies in Asian seabass. On the other hand, L. lactis cells inactivated by sodium hypochlorite (in accordance with the methods disclosed herein) was shown to induce a higher titre of neutralizing antibodies compared to purified VLPs when administered at equivalent antigen doses. Moreover, the seabass fingerlings vaccinated orally with hypochlorite-inactivated L. lactis cells containing NNV VLPs showed a reduction in brain viral titres of about 2.5-logs on the seventh day after NNV challenge.

[0048] Thus, in one example, the recombinant cell is Escherichia coli (E. coll) or Lactococcus lactis (L. lactis'). In one example, the Escherichia, coli cell is BL21DE3. In another example, the Lactococcus lactis cell is NZ3910.Plasmid devoid of antibiotic resistance gene

[0049] The fabl gene containing 789 bp was amplified from the genomic DNA of E. coli BL21(DE3) using forward primer (5 ’ -GAGTCC ATGGGTTTTCTTTCCGGT A-3 ’ : SEQ ID NO: 1) and reverse primer (5 ’ -C AGCCCGGGTTATTTC AGTTCGAGTTCG-3 ’ ; SEQ ID NO: 2), where the underlined sequences correspond to the restriction enzyme sites Ncol and Smal, respectively. pET32a(+) was modified to obtain a smaller vector pET32a(+)s by deleting TrxA, S-tag, enterokinase site, and Ncol and EcoRV restriction enzyme sites. To remove the ampicillin resistance gene from pET32a(+)s, whole-plasmid PCR amplification was performed using forward primer (5’-GTAACCCGGGCTGTCAGACCAAGTTTACTCA-3’; SEQ ID NO: 3) and reverse primer (5’-GGACCCATGGACTCTTCCTTTTTCAATATTATTG-3’; SEQ ID NO: 4), respectively. The PCR amplified vector fragment contained restriction enzyme sites Smal (CCCGGG; SEQ ID NO: 5) and Ncol (CCATGG; SEQ ID NO: 6) at its 5’ and 3’ ends, respectively, but contained no ampicillin resistance gene The antibiotics-free vector was generated by ligating the insert (fabl gene) with the PCR amplified vector fragment, which is denoted as pET32a(+)sf. The sequence of the vector was confirmed by DNA sequencing.Recombinant E. coli cells expressing viral antigen in a virus-like particle (I .Pj form

[0050] The nervous necrosis virus (NNV) capsid protein DNA sequence was cloned into the new vector pET32a(+)sf using Ndel (CATATG; SEQ ID NO: 7) and Xhol (CTCGAG; SEQ IDNO: 8) restriction sites. No extra amino acids other than the capsid protein were introduced. The construct was transformed into A'. coJi BL21 (DE3) competent cells. Transformants were selected on 0.25 - 0.5 pM triclosan-containing LB agar plates after 18 hours incubation at 37°C. When following the concentration of 1.0 pM used in known methods for using triclosan as a selection agent, no colonies were found. As the triclosan concentration used here was low, most colonies on the agar plate were false-positive. Positive colonies containing the NNV capsid protein gene were identified by colony PCR using NNV primers. The identified positive clones were further confirmed by DNA sequencing The recombinant cell obtained here is different from the previously published one (TOP 10 E. coli). Moreover, the previous recombinant cell contained an antibiotic-resistant gene and a His-tag in the plasmid and was selected by antibiotic ampicillin.

[0051] Three selected clones with the correct DNA sequence were cultured in 10 mL LB broth supplemented with 1 mM CaCh, but without antibiotics or triclosan, at 37°C until ODeoo reached about 0.6. Subsequently, the cell culture was induced with 0.5mM IPTG and incubated for 20 hours at 18°C for NNV expression. The expression level of NNV capsid protein was assessed by SDS-PAGE. The clone with the highest yield was selected for large scale (1 L culture) expression.

[0052] The NNV capsid protein was expressed mainly in a soluble form at high expression level (FIG. 1A). The yield of the soluble capsid protein was estimated to be about 100 mg in 1 L of cell culture by using western blot. The protein molecules were shown to assemble into VLPs as described below. The condition used for cell culture is different from method previously described, as shown in Table 1.Table 1. Comparison of protein expression conditions used here and previous study

[0053] Under the culture conditions used herein, VLPs formed more efficiently inside the cells. When the cells were cultured at 37°C for 4 hours or 18 hours, the NNV capsid protein was expressed mainly in an insoluble form or as inclusion bodies. However, the protein was expressed mainly in a water-soluble form under the condition used here (FIG. 2).

[0054] Thus, in one example, there is disclosed a method of obtaining recombinant cell comprising virus-like particles, wherein the recombinant cell is intact, inactivated, and does notexhibit antibiotic resistance; the method comprising a. removing antibiotic resistance genes from a plasmid to obtain an antibiotic resistance-free plasmid, b. inserting the gene encoding a viral capsid protein into the antibiotic resistance free plasmid of step a. to obtain an overexpression plasmid; c. transforming the overexpression plasmid of step b. into competent cells; d. growing and selecting the transformed competent cells of step c. under conditions that allow overexpression of the gene encoding the viral capsid protein and self-assembly of the virus-like particles; e. inactivating the cells of step d., wherein the inactivation leaves the recombinant cell intact; thereby obtaining intact recombinant cell comprising virus-like particles. In another example, the inactivation in step e. is performed using sodium hypochlorite.

[0055] As can be appreciated by a person skilled in the art, if the plasmid used does not contain any antibiotic resistance genes, then step a. of the method disclosed herein may not be required. Therefore, in one example, there is described a method of obtaining recombinant cell comprising virus-like particles, wherein the recombinant cell is intact, inactivated, and does not exhibit antibiotic resistance; the method comprising inserting the gene encoding a viral capsid protein into an antibiotic resistance free plasmid to obtain an overexpression plasmid; transforming the overexpression plasmid of the preceding step into competent cells; growing and selecting the transformed competent cells of the preceding step under conditions that allow overexpression of the gene encoding the viral capsid protein and self-assembly of the virus-like particles; inactivating the cells obtained from the preceding step, wherein the inactivation leaves the recombinant cell intact; thereby obtaining intact recombinant cell comprising virus-like particles. In another example, step a. of the method disclosed herein is optional.

[0056] In another example, the inactivation in step e of the method disclosed herein is performed for up to 3 hours. In a further example, the inactivation in step e. is performed for at least 0.25 hour (about 15 minutes). In other words, the inactivation in the method disclosed herein can be performed for between 0.25 to 3 hours, or for about 0.25 hour (about 15 minutes), about 0.5 hour (about 30 minutes), about 0.75 hour (about 45 minutes), about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours or about 2.75 hours.

[0057] In another example, there is disclosed a recombinant cell obtained using the methods disclosed herein.Characterization of VLP formation and VLP quantification

[0058] To examine if nervous necrosis virus (NNV) capsid protein molecules assemble to form virus-like particles (VLPs) inside E. coli cells under the culture condition used herein, cells were resuspended in PBS buffer and disrupted by sonication. Nervous necrosis virus (NNV) VLPs werepurified from the soluble fraction via size exclusion chromatography using a Capto core 400 column (Cytiva). This column traps smaller protein molecules (<400 kDa) and allows larger molecules (<400 kDa) to pass through the column

[0059] According to the SDS-PAGE of the samples before and after passing through the column (FIG. 3), the capsid protein was not trapped inside the column, indicating that the capsid protein forms VLPs and exists in a multimer with a total molecular weight larger than 400 kDa. It is of note that one NNV capsid protein consists of 338 amino acids and has a molecular weight of about 37 kDa. The diameters of the molecules in the sample after flowing through the column was found to be a range of 31 to 35 nm by electron microscopy (EM; FIG. 4A). In aqueous solution, the average diameter of the purified capsid protein was shown to be about 33 nm by dynamic light scattering (DLS; FIG. 4B). This size is similar to that of in vitro assembled VLPs.Inactivation ofE. coli cells without affecting encapsulation and property of VLPs

[0060] The cell culture was incubated with 1000 to 2000 mg / L sodium hypochlorite for 1 to 2 hours at room temperature. Subsequently, the cells were centrifuged down and washed several times with PBS buffer to remove excess sodium hypochlorite. After the last wash, cells were collected as oral vaccine material by centrifugation. The residual sodium hypochlorite in these samples was measured using a modified colorimetry method. To improve the detection limit, the absorbance was measured at 350 nm, rather than at 470 nm. The lower limit to detect hypochlorite was reduced by 22 times, compared to know methods. The residual sodium hypochlorite in the cells from 1 L culture was found to be 7 pg, which is much lower than the allowed hypochlorite concentration of 5 mg / L in drinking water, thereby showing that the oral vaccine material obtained using the method disclosed herein is safe to fish and having a negligible effect on water.

[0061] To examine if there are any residual live cells after inactivation, the collected cells were resuspended in PBS buffer, plated on LB agar plates, and incubated the plates at 37°C overnight. After the incubation, colony counting was performed.

[0062] No live cells were found after the cell culture was incubated for 2 hours at a sodium hypochlorite concentration of 2000 mg / L.

[0063] To investigate leakage of NNV capsid protein during the treatment by sodium hypochlorite, the protein amounts in the treated and untreated cells were compared. If the cells were lysed during the inactivation, NNV capsid protein and other A. coli proteins would be washed out during the wash process, and protein contents in the sample treated with sodium hypochlorite would be much lower than those in the untreated sample. The SDS-PAGE (FIG. 1) shows that the NNV capsid protein amount in the treated sample was similar to that in the untreated sample, indicating that the treatment disclosed herein does not cause significant cell lysis.

[0064] Conventional inactivation methods, such as using formaldehyde and heat, were also tested. Using these methods, the cells were killed effectively without significant leakage of proteins. However, no soluble NNV capsid protein could be detected after cell lysis by sonication, indicating that the capsid protein was crosslinked with other biomolecules by formaldehyde or denatured to form large aggregates by heat. When the cells were treated with sodium hypochlorite, it was found that the capsid protein still remained soluble (FIG. IB). The protein extracted from the treated cells could also be purified using the Capto Core 400 column. The diameter of the purified protein from treated cells had a similar size to that of the protein from the untreated cells (FIG. 5), i.e. the protein molecules still assembled to form VLPs after the cells were killed using sodium hypochlorite. The result shows that the property of the NNV capsid protein is not affected by the treatment.

[0065] Thus, in one example, the cells disclosed herein are inactivated using sodium hypochlorite (NaOCl). In another example, the concentration of the sodium hypochlorite used in the methods disclosed herein is at least 10 mg / 1. In another example, the concentration between 10 mg / l to 5500 mg / 1 (which may also be written as 0.1 g / 1 to 5.5 g / 1). That is to say, the concentration of sodium hypochlorite is between 10 mg / 1 to 2500 mg / 1, between 10 mg / 1 to 50 mg / 1, between 20 mg / 1 to 100 mg / 1, between 50 mg / 1 to 200 mg / 1, between 100 mg / 1 to 250 mg / 1, between 150 mg / 1 to 300 mg / 1, between 250 mg / 1 to 400 mg / 1, between 300 mg / 1 to 600 mg / 1, between 500 mg / 1 to 1500 mg / 1, between 800 mg / 1 to 1000 mg / 1, between 750 mg / 1 to 1750 mg / 1, between 1250 mg / 1 to 2250 mg / 1, or between 1900 mg / 1 to 2100 mg / 1. In one example, the concentration of sodium hypochlorite is between 10 mg / 1 to 200 mg / 1. In another example, the concentration of sodium hypochlorite is about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 110 mg / ml, about 120 mg / ml, about 1 0 mg / ml, about 140 mg / ml, about 150 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, about 200 mg / ml, about 210 mg / ml, about 220 mg / ml, about 230 mg / ml, about 240 mg / ml, about 250 mg / 1, about 260 mg / 1, about 270 mg / 1, about 280 mg / 1, about 290 mg / 1, about 300 mg / 1, about 350 mg / 1, about 400 mg / 1, about 450 mg / 1, 500mg / l, about 550 mg / 1, about 600 mg / 1, about 650 mg / 1, about 700 mg / 1, 750 mg / 1, about 800 mg / 1, about 850 mg / 1, about 900 mg / 1, about 950 mg / 1, 1000 mg / 1, 1250 mg / 1, 1500 mg / 1, 1750 mg / 1, 1800 mg / 1, 1950 mg / 1, 2000 mg / 1, 2150 mg / 1, 2250 mg / 1, 2500 mg / 1, about 3000 mg / 1, about 3500 mg / 1, about 4000 mg / 1, about 4500 mg / 1, or about 5000 mg / 1. In one example, the concentration of sodium hypochlorite used is about 2000 mg / 1. In another example, the concentration of sodium hypochlorite used is about 200 mg / 1.

[0066] In another example, the inactivation of the cells described herein takes place in cell culture.

[0067] The method as disclosed herein can also include further steps of, such as, but not limited to, resuspending cells in PBS buffer after harvesting and then performing the inactivation step. Thus, in another example, the cells disclosed herein are harvested and resuspended in PBS buffer, and subsequently inactivated using sodium hypochlorite (NaOCl). In this example, the concentration of sodium hypochlorite in this method can be between 10 mg / 1 to 200 mg / 1.Formulation of oral vaccine for fish

[0068] Inactivated whole cells containing NNV VLPs were resuspended in PBS buffer. Cell suspension was mixed with finely ground fish feed powder, along with 1 % fish oil and 10% starch. The mixture was extruded through a pellet maker, and the resulting feed pellets were dried overnight at 37°C. Higher temperatures were avoided in order to ensure functional VLPs were not affected. The dried feed pellets retained their shape for 10 minutes without obvious disintegration in water (FIG. 6). Since the cells are incorporated into the feed, they will not fall off during feeding, unlike in feed coated with the cells which were used in in the art. These feed pellets can be used directly as oral vaccine for fish.

[0069] As will be appreciated by a person skilled in the art, the amount of recombinant cells mixed into or present in the feed has to be large enough in order to have the effect as described herein. In other words, the amount of virus-like particles comprised within the fish feed would need to be in an amount that can elicit an immune response. Such an amount of virus-like particles can be, but is not limited to, between 300 pg to 6000 pg of VLPs in one gram feed; or about 300 pg, about 400 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, about 1000 pg, about 1200 pg, about 1400 pg, about 1600 pg, about 1800 pg, about 2000 pg, , about 2200 pg, , about 2400 pg, about 2600 pg, about 2800 pg, about 3000 pg, about 3200 pg, about 3400 pg, about 3600 pg, about 3800 pg, about 4000 pg, about 43000 pg, about 4600 pg, about 5000 pg, about 5500 pg or about 6000 pg.

[0070] Thus, in one example, the fish feed as disclosed herein comprises between 2xl09CFU to 4xlO10CFU of recombinant cells in one gram feed disclosed herein. In another example, the amount of recombinant cells used in the fish feed can be about 2xl09CFU, about 2.5xl09CFU, about 3.0xl09CFU, about 3.5xl09CFU, about 4xl09CFU, about4.5xl09CFU, about 5xl09CFU, about 5.5xl09CFU, about 6xl09CFU, about 6.5xl09CFU, about 7xl09CFU, about 8xl09CFU, about 9xl09CFU, about IxlO10CFU, about L5xlO10CFU, about 2xlO10CFU, about 3xl010CFU, or about 4x1010CFU. In one example, recombinant L lactis, as described herein, is used in anamount of about 1.4xlO10CFU. In another example, recombinant E. coli, as described herein, is used in an amount of about 5xl09CFU.Immune response of encapsulated and non-encapsulated NNV VLPs

[0071] 400 Asian seabass (ASB) fingerlings (average fish body weight of 1.0 g) were obtained from the Marine Aquaculture Center, Singapore. After a 1-week acclimatization period, fish weighing 1.5 ± 0.2 g were selected and randomly divided into groups of 50 fingerlings each. Two intraperitoneal injection control groups (n=50 fingerlings / group) were set up, the first group was immunized with 15 pg of purified nervous necrosis virus (NNV) virus-like particles (VLP) in 20 pl of PBS. The second IP control group was immunized with 10 pl of lysed A. coli cells containing 15 pgNNV-VLP. For the oral vaccination, fish fingerlings (n=50 fingerlings / group) were fed with feed incorporated with either purified NNV VLP, whole E. coli cells expressing NNV VLP or lysed £ coli cells expressing NNV VLP. One gram of each feed contained 666 pg NNV VLP. A control group was fed regular commercial feed for comparison. The feed was administered over the course of three days and consisted of 5% of the body weight of the experimental fish. Two weeks following the initial vaccination, all groups of fish were vaccinated in an identical manner to the primary vaccination. On day 27, ten (10) fish per experimental group were euthanized for the collection of plasma, intestine and gill for evaluation of anti-NNV specific immunoglobulins (Igs) for ELISA or virus microneutralization (VMN). On day 28, thirty (30) fish from each experimental group were challenged with 20 pl of 106TCID50 grease grouper nervous necrosis virus (GGNNV).

[0072] Virus neutralization activity of sera from groups of vaccinated fish was assessed against live, grease grouper nervous necrosis virus (GGNNV) using an in vitro assay. Briefly, Asian Seabass fibroblast (SB) cells were seeded at 1.5 x 104cells / well 1 day prior to infection in a 96-well plate. Sera from ASB were then serially diluted from 1 : 10 to 1 : 1280 with minimum essential medium (MEM; Gibco) and incubated with 100 times the 50% Tissue Culture Infectious Dose (100 TCID50) of GGNNV for 2 hours in a separate 96-well plate for neutralization. Following neutralization, the virus-antibody mixture was added to wells containing the monolayer of SB cells for infection for two hours at 28°C. The virus-antibody mixture was then removed, and fresh MEM media was added to the ASB cells prior to incubation at 28°C for 4 days. After 3 to 4 days, the cytopathic effect (CPE) was observed under an inverted microscope (Olympus 1X71), and the neutralizing titre was assessed as the highest dilution of vaccinated sera or positive and negative controls that provided complete protection. For further confirmation, the cells were fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature (RT). The fixed cells were tested by indirect immunofluorescent assay (IF A) with mouse anti-NNV polyclonal sera for 1 hour atroom temperature, followed by incubation with a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated rabbit anti -mouse immunoglobulin (Dako, Denmark) for 1 hour at room temperature. The fluorescence signal was evaluated by wide-field epi-fluorescence microscopy (Olympus 1X71).

[0073] ASB immunized twice intraperitoneally with lysed E. colt expressing NNV-VLP or NNV-VLP alone produced a higher virus neutralization titre compared to various orally vaccinated groups (FIG. 7A). Among the oral vaccination groups, fish immunized twice with whole E. coli expressing NNV VLP exhibited about 2.5 times more virus-neutralizing titre, compared to the group immunized with lysed E. coli expressing NNV VLP or NNV-VLP alone (FIG. 7B).

[0074] Viral titres in the brains of post challenge ASB were assessed using RT-qPCR. Brains of 5 fish were collected on day 7 post-challenge, and viral RNA was extracted using Trizol. The viral RNA was then reverse transcribed into cDNA, and lOOng of cDNA was used as a template for amplification of the NNV capsid RNA2. A standard curve was generated using NNV standards serially diluted 10-fold from 10 ' to 10°. Viral titres in the NNV VLP IP group, E. coli lysed and E. coli whole oral groups, were compared to the control group.

[0075] NNV titre in the brains (n=5) of the control group revealed an average of >1045NNV load. In contrast, Asian seabass (ASB) immunized intraperitoneally with either NNV VLP or lysed E. coli expressing NNV VLP showed about a 3 -log reduction in brain viral titre compared to the control group. Similarly, ASB orally vaccinated with either lysed or whole-cell E. coli expressing NNV-VLP also demonstrated a 3-log reduction of brain viral titre load. On the other hand, ASB orally immunized twice with NNV VLP exhibited an approximate 2.5 -log reduction in brain NNV titre compared to the control group (FIG. 8).

[0076] A DNA construct devoid of antibiotic resistance gene and capable of producing NNV capsid protein was invented in this work. A protocol was established to express NNV capsid protein at high yield in E. coli cells and further assemble the protein into VLPs inside the cells. The formation of VLPs inside the cells was demonstrated by different techniques. To prevent the VLPs from denaturation, crosslinking with other molecules, or leakage from the cells, a new protocol for killing E. coli cells was devised. The VLPs were shown to be encapsulated inside the inactivated cells and retain a functional form. An oral vaccine formulation and a production procedure were developed by incorporating the inactivated cells into fish feed. Experiments on fish further demonstrated that fish feed incorporated with inactivated E. coli cells elicited about 2.5 times more neutralizing antibodies than that with the lysed cells. The disease challenge also demonstrated that ASB immunized orally vaccinated with either lysed or whole E. coli cells expressing NNV VLPs resulted in a 3-log reduction in brain viral titre compared to the controlgroup Therefore, using this oral vaccine, functional VLPs can be delivered to the gastrointestinal tract of fish by preventing antigens from denaturation in the harsh stomach environment via encapsulation of the VLPs inside cells.

[0077] Without being bound by theory, it is thought that the E. coh cells described herein are likely lysing rapidly in intestine and release the VLPs to the immune system and thus are capable of delivering antigens orally. Moreover, the feed incorporated with whole cells can effectively induce a protective immune response without the need for downstream protein extraction and purification processes, and thus, the inactivated whole E. coll cells disclosed herein are a suitable oral vaccine for fish.

[0078] The oral vaccine disclosed herein can be applied to fish species which are susceptible to nervous necrosis virus (NNV), such as, but not limited to, Asian seabass Lates calcarifer), European Seabass (Dicentrarchus labrax), Atlantic cod Gadus morhua), Pacific cod (Gadus macrocephalus), haddock (Melanogrammus aeglefinus), greasy grouper (Epinephelus tauvina), yellow tang (Zebrasoma flavescens), three spot damsel (Dascyllus trimaculatus), and guppy (Poecilia reticulata). In one example, the fish species is Asian seabass (Lates calcarifer). In another example, the subject is a fish. In another example, the fish is, but not limited to, Asian seabass (Lates calcarifer , European Seabass (Dicentrarchus labrax), Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), haddock (Melanogrammus aeglefinus), greasy grouper (Epinephelus tauvina), yellow tang (Zebrasoma flavescens), three spot damsel (Dascyllus trimaculatus), and guppy (Poecilia reticulata).

[0079] Replacing the DNA sequence encoding the nervous necrosis virus (NNV) capsid protein with other viral proteins in the construct, oral vaccines against other viruses or pathogens can be produced in a similar way.Expression and characterization of nervous necrosis virus virus-like particles in Lactococcus lactis

[0080] Nervous necrosis virus (NNV) capsid protein expressed in Lactococcus lactis (L. lactis) was found predominantly in the soluble fraction after sonication (FIG. 9). When the soluble fraction was applied to a Capto Core 400 column, the capsid protein was detected exclusively in the flow-through, indicating that the capsid molecules assembled into oligomers larger than 400 EDa. It should be noted that this column permits the passage of molecules with apparent molecular weights larger than 400 kDa.

[0081] To examine the morphology of these oligomers, transmission electron microscopy was performed on the sample purified from the flow-through using a size exclusion chromatography column. The images revealed that the capsid protein formed virus-like particles (VLPs) with anaverage diameter of approximately 39 nm (FIG. 10A), consistent with the size of VLPs produced in / / . coll disclosed herein.Heat inactivation of Lactococcus lactis cells

[0082] Treatment of Lactococcus lactis (L. lactis) cells at 37°C in a water bath for 20 hours reduced viable colonies from 1 x 109cfu / ml to 4 x 106cfu / ml, corresponding to an inactivation efficacy of 99.6% or 2.4-log reduction in viability. Exposure to 40°C or 50°C for 2 hours completely abolished colony growth, indicating 100% inactivation. The total protein content in the lysate of control cells (untreated) was comparable to that in the cells exposed to 37°C (FIG. 11). Similarly, the cells treated at 40 °C and 50 °C showed nearly identical total protein levels, also comparable to the control. This result shows that heat inactivation between 37°C to 50°C does not lead to protein leakage from the cells, nor does it cause significant damage to the cell wall. However, the soluble fraction of NNV capsid proteins within the cells was affected by heat treatment: after incubation at 37°C for 20 hours, about 30% of proteins remained soluble (FIG.11), whereas treatment at 40°C for 2 hours resulted in about a 90% loss of soluble capsid proteins, and at 50°C for 2 hours, soluble capsid protein were no longer found to be present.

[0083] This result suggests that the capsid protein became insoluble through denaturation and aggregation during the treatment. This is expected from previous studies on heat inactivation of gram negative and positive bacteria. For subsequent immunogenicity testing of heat-inactivated L. lactis, the 37°C condition was chosen to balance cell inactivation with protein solubility.Sodium hypochlorite inactivation of Lactococcus lactis cells

[0084] Lactococcus lactis L. lactis) cells were also inactivated using sodium hypochlorite at concentrations ranging from 1000 to 5000 mg / L for 2 hours. Colony counting revealed a concentration-dependent effect on cell viability (Table 2), with a 7.7-log reduction at 5000 mg / L. The total protein content in the lysate of untreated cells (0 mg / L) did not differ significantly from that of the cells exposed to 1000 mg / L to 3000 mg / L hypochlorite but was lower in the cells treated with 4000 mg / L to 5000 mg / L hypochlorite (FIG. 12). This result indicates that hypochlorite inactivation at concentrations up to 3000 mg / L does not cause detectable protein leakage from the cells. It had been previously shown that no significant protein leakage was found when E. coli viability was reduced by less than 7-log with hypochlorite. At higher concentrations, hypochlorite treatment caused substantial leakage, likely due to cell wall disruption. In addition, increasing hypochlorite concentrations reduced the amount of soluble NNV capsid protein, as determined by SDS-PAGE and western blot (FIG. 12). To balance antigen preservation with effective inactivation, a concentration of 2000 mg / L sodium hypochlorite was selected for inactivation of L. lactis cells used in feed preparation.Table 2. Viability reduction of L. lactis cells ((-Logio(N / No), where N and N° are the numbers of colonies (CFU / mL) present on the agar plates after and before sodium hypochlorite treatment, respectively) after a 2-hour incubation with sodium hypochlorite at a series of concentrations as indicated in the table below.Effect of hypochlorite inactivation on virus-like particle structure

[0085] To determine whether hypochlorite inactivation affected virus-like particle (VLP) integrity, capsid protein was extracted from L. lactis cells treated with 2000 mg / L sodium hypochlorite using a Capto Core 400 column. Transmission electron microscopy confirmed that the capsid protein retained its VLP structural integrity (FIG. 10B). Thus, sodium hypochlorite inactivation preserves both the solubility and the structural integrity of NNV VLPs.Immunogenicity of purified virus-like particles, live, and inactivated cells by heat or hypochlorite

[0086] To evaluate the immune response of nervous necrosis virus (NNV) virus-like particles (VLPs) in Asian seabass (ASB) fingerlings, sera were collected from vaccinated and control groups at 4 weeks post-vaccination. Nervous necrosis virus (NNV)-specific IgM levels were determined by indirect ELISA. Injection of the purified VLPs elicited the strongest antibody response, with IgM level approximately 4 times higher than that induced by oral administration (FIG. 13 A), even though the oral dose (100 pg / g FBW) was 10 times more than the injection dose (10 ug / g FBW). In contrast, oral delivery of live L. lactis cells expressing NNV VLPs induced marginally higher NNV-specific antibody levels compared to the negative control. These results indicate that although purified VLPs are immunogenic via both injection and oral immunization, encapsulation of VLPs within the live L. lactis cells appears to suppress the immune response in ASB.

[0087] To assess the functionality of the antibodies, a virus microneutralization assay (VMN assay) was performed. In groups vaccinated with the purified VLPs, neutralizing antibody titres (FIG. 13B) correlated strongly with NNV-specific IgM levels (FIG. 13A). By contrast, fish fed with live L. laclis cells expressing VLPs produced negligible neutralizing antibodies, comparable to non-vaccinated control, confirming that live L. lactis cells expressing VLPs is unsuitable as an oral vaccine.

[0088] To further investigate suitability of inactivated L. lactis cells as an oral vaccine, recombinant cells were heat-inactivated at 37°C and incorporated into fish feed. Fish fed with these inactivated cells produced no detectable NNV-specific IgM antibodies or neutralizing antibodies (FIG. 13C and 13D). Given that about 30% of the NNV capsid protein remained soluble after heat treatment at 37°C (FIG. 11), it was expected that antibody titres would reach about 30% of those in the oral group fed with the purified VLPs if antigen (NNV VLPs) release from the inactivated cells occurred. However, the absence of antibody production suggests that heat inactivation does not promote antigen release from bacterial cells in the fish gastrointestinal tract.

[0089] In contrast, inactivation with sodium hypochlorite (2000 mg / L), which preserves the structural integrity and solubility of VLPs inside the cells, resulted in an enhanced immune response Fish orally vaccinated with hypochlorite-inactivated L. lactis cells produced about two times as many VLP-specific antibodies and neutralizing antibody titres compared to the group orally immunized with the purified VLPs (FIG. 14A and 14B), despite identical antigen doses. This indicates that hypochlorite treatment enables efficient release of the NNV VLPs in the gastrointestinal tract and that encapsulation of VLPs in the cells enhances antigen availability for immune stimulation.

[0090] In another example, there is described a fish food comprising the recombinant cell as disclosed herein In another example, the fish food is provided in the form of feed pellets

[0091] In one example, the vaccine formulation disclosed herein was obtained by pelleting a mix of fish feed powder and the recombinant cells described into granules / pellets at room temperature.

[0092] The recombinant cells disclosed herein are incorporated into feed, for example, fish feed. Such fish feed can therefore be used as an oral vaccine against the virus from which the viral capsid proteins had been obtained.

[0093] The present disclosure also describes inactivated recombinant cells expressing and encapsulating viral capsid protein for use, for example, as an oral vaccine or a food supplement. In one example, such recombinant cells can be E. coli or L. lactis.

[0094] Thus, disclosed herein is a method of inoculating a subject against nervous necrosis virus, the method comprising administering the recombinant cell as disclosed herein to the subject. In another example, there is disclosed a method of treating or preventing viral nervous necrosis in a subject, the method comprising administering the recombinant cell of described herein to the subject.

[0095] Also described in one example is the use of the recombinant cell of as disclosed herein in the manufacture of a medicament for eliciting an immune response against nervous necrosis virus (NNV) in a subject. In another example, there is disclosed the use of the recombinant cell disclosed herein in the manufacture of a medicament for preventing or treating a nervous necrosis virus (NNV) infection in a subject.

[0096] In another example, there is described a fish feed additive comprising the recombinant cell as disclosed herein.

[0097] The protective efficacy of the hypochlorite-inactivated cell vaccine was further evaluated through viral challenge 30 days post-vaccination against greasy grouper nervous necrotic virus (GGNNV). In the non-vaccinated control group, NNV loads in the brain exceeded 105.5 TCIDso / mL on day 7 post-challenge (FIG. 14C). In contrast, fish immunized intraperitoneally with purified NNV VLPs exhibited a >3 ,5-log reduction in brain viral titres, while those orally vaccinated with hypochlorite-inactivated L. lactis cells expressing VLP showed a roughly 2.5-log reduction. Notably, the hypochlorite-inactivated cell vaccine provided greater viral reduction than the orally delivered purified VLPs (FIG. 14C). Over a 15-day observation period post-challenge, no mortality was recorded in any group. Nevertheless, the control group exhibited mild clinical symptoms while vaccinated groups were healthy. These results demonstrate that vaccination markedly reduced viral replication against NNV challenge and establishes the use of hypochlorite-inactivated . lactis cells expressing NNV VLPs as an oral vaccine candidate.

[0098] In summary, sodium hypochlorite-inactivated L. lactis cells were shown to provide an improvement in vaccine performance over conventional methods. Oral administration of these inactivated cells induced approximately twice the antibody and neutralizing titres compared to the purified VLPs at the same antigen dose.

[0099] In addition, challenge experiments showed that fish immunized with hypochlorite-inactivated cells achieved a roughly 2.5-log reduction in viral load, exceeding viral reduction conferred by orally delivered purified virus-like particles (VLPs), though still slightly below that achieved by injection. A reduction in the viral load in the brain following NNV challenge indicates effective viral suppression and correlates with enhanced protection. A lower brain viral load suggests that the vaccine disclosed herein reduces viral replication and spread, which is consistentwith previous findings in other fish species. Importantly, this viral reduction was achieved without mortality or visible disease symptoms, highlighting the safety of this approach.

[0100] Thus, disclosed herein is an oral vaccine comprising the recombinant cell as described herein.

[0101] In one example, the oral vaccine comprises fish feed powder, fish oil, and starch.

[0102] In another example, the oral vaccine of the present disclosure is provided in the form of pellets. In a further example, the pellets are dried at a temperature no higher than 37°C. In other words, the pellets are dried at a temperature of up to, or not exceeding, 37°C. Such temperature can be 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or 36°C.

[0103] In another example, the pellets are dried overnight. In one example, the pellets are dried for at least 4 hours. In another example, the pellets are dried for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more hours.

[0104] In one example, the pellets according to the present disclosure retain their shape in water for at least 10 minutes. In another example, the pellets retained their shape in water for 10, 11, 12, 15, 20, 25, 30, or more minutes.

[0105] Without being bound by theory, it is thought that the efficacy of hypochlorite-inactivated cells is likely attributable to their dual functional properties. Hypochlorite-inactivated cells can withstand the acidic and proteolytic environment of the stomach and remain structurally intact, thereby protecting the encapsulated antigen from premature degradation. Once in the intestinal environment, however, these cells undergo rapid lysis to efficiently release antigenic VLPs at sites favourable for immune recognition. Although inactivation at 2000 mg / L hypochlorite did not completely disrupt the cell wall or membrane, it likely weakened it through oxidative damage, making the cells wall more prone to lysis in the intestine. In contrast, heat-inactivated and live cells may not lyse as efficiently to ensure timely antigen release in the intestine.

[0106] Importantly, hypochlorite treatment is also cost-effective, scalable, and compatible with feed-based vaccine production, making it a practical solution for large-scale vaccination in aquaculture.

[0107] The oral hypochlorite-inactivated L. lactis vaccine can be applied at all stages of fish development, especially during early developmental stages of fish, such as larvae and juveniles, which are highly susceptible to viral nervous necrosis (VNN) and difficult to vaccinate by injection. Oral delivery through feed allows mass immunization without handling stress or injury,improving vaccine coverage and fish survival in hatchery conditions. This approach also compatible with standard feeding practices and can be easily applied in grow-out operations.

[0108] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0109] As used in this application, the singular form “a," “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.

[0110] As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically, + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0111] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range

[0112] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0113] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0114] Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.EXPERIMENTAL SECTIONCell line and vir us

[0115] The SB cell line (CVCL VK76), derived from fibroblast of Lates calcarifer larvae, was cultured in Leibovitz L-15 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO, USA) and 1* antibiotic-antimycotic (Gibco, USA) at 28°C.

[0116] Greasy grouper NNV (GGNNV), a representative of the red spotted grouper NNV (RGNNV) genotype, with GeneBank (GB) accession number RNA1 (AF319555) and RNA2 (AF318942), previously obtained, was used for this study.DNA cloning and transformation

[0117] The DNA sequence encoding the NNV capsid protein, derived from RGNNV (accession number: AAZ23223.1) was codon-optimized for / ., lactis and A. coli, respectively. The codon-optimised DNA was obtained from Bio Basic Asia Pacific. For the construct used for L. lactis expression, the DNA was cloned into pNZ8149 vector (MobiTec GmbH) using Ncol and Kpnl restriction sites, which contained lacF gene as a food grade selection marker for growth on lactose. The construct was transformed into L. lactis NZ3910 electrocompetent cells (MobiTec GmbH), a lacF deleted strain. Transformants were selected on Elliker plates (20g / L tryptone, 5g / L yeast extract, 4g / L sodium chloride, 1.5g / L sodium acetate anhydrous, 0.5g / L L(+) ascorbic acid, 0.5% lactose and 15g / L agar) containing 0.004% bromocresol purple. Successful transformants were able to utilize lactose as a carbon source, giving yellow colonies. Positive clones were verified via plasmid extraction and sequencing.

[0118] For the construct used for obtaining purified VLPs from E. coli expression, the DNA was cloned into the pET28b vector with a N-terminal SUMO-tag plus 6 His residues using BamHI and Xhol restriction sites. For the construct used for E. coli expression of tag-free NNV capsid protein, the DNA was cloned into the pET32a(+)sf vector using Ndel and Xhol restriction sites.These constructs were transformed into E. coli BL21 (DE3) competent cells for protein expression, respectively.Protein expression

[0119] For L. lactis expression, selected clones were cultured in Elliker medium at 30°C until ODeoo reached 0.6. The cell culture was then induced with 20 ng / ml nisin and incubated for 4 hours at 30°C for NNV expression. For E. coli expression, cells were cultured in Luria Bertani (LB) broth Miller (Merck) at 37°C until ODeoo reached around 0.6, and then induced with 0.5 mM isopropyl [3-d- 1 -thiogalactopyranoside (IPTG) The induced cell culture was incubated overnight at 18°C for protein expression. Cells were harvested by centrifugation and washed with PB S buffer before further use.Protein purification

[0120] The harvested L. lactis cells were resuspended in PBS buffer and disrupted by sonication in water-ice bath at 29% amplitude in 1 s on-off pulses for 60 minutes. Lysates were centrifugated at 39,000 g, 4°C for 30 minutes. The supernatants were loaded to a Capto Core 400 column (Cytiva) and incubated for 30 minutes. The flow-through was collected, then the column was washed with PBS. This column traps smaller protein molecules (<400 kDa) and allows larger molecules (<400 kDa) to pass through the column. The flow-through was further purified using a Superose 6 10 / 300 GL size-exclusion chromatography column (Cytiva). The harvested E. coli cells were disrupted similarly. For the tag-free protein cloned into the modified pET32a(+)sf vector and expressed in E. coli, the purification procedure was the same as that for the protein expressed in / .. lactis. For the SUMO-tag protein expressed in E. coli, the protein was purified from the soluble fraction using a Ni-NTA affinity column (Qiagen). SUMO-tag was cleaved with SUMO protease at 4°C and separated from NNV capsid protein using a Ni-NTA column. The resulting tag-free capsid protein was assembled into VLPs by dialysis against a VLP formation buffer (20 mM Tris, 200 mM NaCl, 1 mM CaC12, 1% glycerol, pH 7.4). These VLPs are hereafter referred to as purified VLPs.Transmission Electron Microscopy

[0121] Carbon film-coated 300-mesh copper TEM grids (Electron Microscopy Sciences) were glow-discharged (15 mA, 30 s) to render them hydrophilic. 3 uL of NNV capsid protein solution (about 0.5 mg / ml) was applied to the grids and allowed to adhere for 1 minute before excess sample was wicked away with filter paper. Grids were washed twice with distilled water and stained with 1% uranyl acetate for 1 minute before removal by filter paper for air-drying. Samples were visualized using a single tilt holder in FE1 Tecnai T12 at 120 kV at room temperature. Images were processed in ImageJ.Inactivation by heat

[0122] Cells were resuspended in PBS to a final concentration of 109CFU / mL and then incubated in a water bath at three temperatures: 37°C, 40°C, and 50°C. After the heat treatment, colony counting was used to estimate inactivation efficacy. To examine the effect of heating on NNV VLP solubility inside cells, treated cells were washed with PBS buffer and then lysed by sonication. Subsequently, soluble and insoluble fractions of the lysate were separated and then analysed using SDS PAGE.Inactivation by hypochlorite

[0123] Inactivation of both L. lactis and E. coli cells was performed as follows. Briefly, cell cultures with OD-„n:i of 1.3 to 1.5 were treated using sodium hypochlorite at a series of concentrations from 1000 to 5000 mg / L for 2 hours at room temperature. After the treatment, colony counting was used to estimate inactivation efficacy. Treated cells were washed with PBS buffer four times to remove residual hypochlorite. Subsequently, the cells were resuspended in PBS buffer and then lysed by sonication. The soluble and insoluble fractions of the lysate were separated and then analysed using SDS-PAGE and Western blot, respectively. Western blot was conducted by using 5% BSA as a blocking solution, mouse anti -NNV serum as primary antibody and goat anti-mouse IgG conjugated with HRP as secondary antibody. Bands were visualized using the PICO chemiluminescence substrate.Preparation of fish feed

[0124] Commercial seabass fish feed (Sheng Long Bio-Tech International Co., Ltd) was ground into a fine powder. This powder (44% w / w) was mixed with 15% cornstarch, 1% Menhaden fish oil, and 40% L. lactis cell suspension. For one gram of the feed powder, 1.4xlO10CFU of L. lactis or 5.0xl09E. coli cells containing 670 pg VLPs were added. To incorporate purified NNV VLPs into the feed, the cell suspension was replaced with the VLPs purified from E. coli expression, with a VLP content of 670 pg per gram of the feed powder. The powder mixture was pelleted into 1.5 mm feed pellets using a manual pellet maker. The pellets were dried at 30°C for 20 hours.Experimental fish, vaccination, and virus challenge

[0125] Healthy Asian seabass (ASB) fingerlings were obtained from a commercial hatchery (Allegro Aqua, Singapore). The ASB, weighing 2.2 ± 0.2 g, were allocated to four 200 L recirculating tanks with 50 fingerlings per tank and acclimated for one week before the start of the experiment. The tanks were equipped with a standard biofiltration system at the experimental marine aquarium facilities of Temasek Life Sciences Laboratory, Singapore. Fish were maintained in UV-irradiated seawater at 30 ± 1°C, with a 12:12 light and dark photoperiod. To evaluatedifferent oral vaccination strategies, three independent immunization trials were conducted. In the first trial, livelactis cells expressing NNV VLPs were incorporated into feed, with additional groups receiving purified VLP vaccine either intraperitoneally or orally, and an unvaccinated control group. In the second and third trials followed the same design, except that heat-inactivated and sodium hypochlorite-inactivated L. lactis cells, respectively, were incorporated into feed, with the same control groups included. For intraperitoneal injection, the group of fish was immunized with 22 pg of purified NNV VLP in 40 pl of PBS per fish or 10 pg VLP / g fish body weight (FBW). For oral vaccination, two groups were used: the first oral group was fed with feed incorporated with purified VLPs, while the second oral group was fed with the feed made from L. lactis cells containing NNV VLPs. The feed was administered at a rate of 5% of fish’s body weight per day for three consecutive days, equivalent to 100 pg VLPs / g FBW for 3 days. Two weeks after the initial vaccination, all groups were vaccinated again in the same manner as the primary vaccination. The control group was fed with commercial feed. Blood samples were collected from 10 fish per experimental group on day 28. For viral challenge, in the final trial, thirty-five (35) fish from each group were challenged with 20 pl of 107TCID50 (50% tissue culture infectious dose) of GGNNV on day 30 and monitored for diseases progression until day 28 post-challenge.Measurement of anti -NNV specific antibodies by indirect ELISA

[0126] Serum NNV specific antibodies were determined by indirect ELISA using purified VLPs as the coating antigen. Briefly, 96-well microtiter ELISA plates were coated with 300 ng / well of NNV VLP antigen in coating buffer (0.1 M carbonate-bicarbonate, pH 9.6) and incubated overnight at 4°C, then washed thrice with PBS containing 0.05% Tween 20 (PBS-T). Non-specific binding sites were blocked with 2% (w / v) bovine serum albumin (BSA) in PBS and incubated at 37°C for 1 hour. After washing with PBS-T, samples of test sera were serially diluted to 1 :20 in PBS and added to triplicate wells and incubated for 2 h at 37 °C. After washing thrice with PBS-T, mouse anti -Asian seabass IgM monoclonal antibody (Aquatic Diagnostics Ltd., UK) diluted 1 :50 using PBS was added to each well and incubated for 1 h at 37 °C followed by washing thrice with PBS-T.

[0127] Then, 1:2000 dilutions of goat anti-mouse Ig (Dako Cytomation) conjugated with horseradish peroxidase was added to the respective wells and incubated for 1 h at 28 °C. The colour development was then visualized by adding 3,3’,5,5’-tetramethylbenzidine (Sigma), and the reaction was stopped by addition of diluted sulfuric acid solution. Absorbance was measured at 450 nm using a microwell plate reader.Determination of virus microneutralization (VMN) antibody titres against NNV virus

[0128] Virus neutralization activity of sera from groups of the experimental fish at day 28 (prechallenge) and day 35 (post-challenge) was assessed as follows.

[0129] Briefly, SB cells were seeded at 2 x 104cells / well in 96-well plates one day prior to infection. Triplicate two-fold serial-dilutions of fish sera (1:10 to 1:640) were prepared in serum-free L-15 medium (Gibco) and incubated with lOOx the 50% tissue culture infective dose (TCZD50) of GGNNV for 2 hours at 28°C in separate 96-well plates. The virus-antibody mixtures were then added into wells containing the monolayer of SB cells and incubated for 2 hours at 28°C. After incubation, the inoculum was removed and replaced with L-15 medium supplemented with 10% fetal bovine serum (FBS), and cells were further incubated at 28°C for 4 days. The cytopathic effect (CPE) was observed under an inverted microscope (Olympus 1X71), and the neutralizing titer was defined as the highest serum dilution that provided complete protection against CPE. For further confirmation, cells were fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature (RT). The fixed cells were analysed by indirect immunofluorescent assay (IF A) with mouse anti-NNV polyclonal serum for 1 hour at room temperature, followed by incubation with a 1 : 100 dilution of fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (Dako, Denmark) for 1 hour at room temperature. The fluorescence signal was evaluated by wide-field epi-fluorescence microscopy (Olympus 1X71).Quantification of brain NNV viral load post-challenge

[0130] Viral load in the brains of post-challenge ASB were assessed using RT-qPCR. Brains from 10 fish were collected on day 7 post-challenge, and viral RNA was extracted using Trizol. Viral RNA was reverse transcribed into cDNA, and 100 ng of cDNA was used as the template for amplification of NNV capsid RNA2. NNV standards, serially diluted 10-fold from 107 to 100, were used to obtain a standard curve. Viral titres of vaccinated groups were compared to that of the control group.Statistical analysis

[0131] The two-tailed Student’ s t-test was performed to evaluate the significance of differences between the means of two groups using GraphPad Prism Software. A p-value of less than 0.05 was considered statistically significant.SEQUENCE LISTING

Claims

CLAIMS1. A recombinant cell comprising virus-like particles, wherein the recombinant cell is intact, inactivated, and does not exhibit antibiotic resistance2. The recombinant cell of claim 1, wherein the cell is Escherichia coh (E. coli) or Lactococcus lactis (L. lactis).

3. The recombinant cell of any of the preceding claims, wherein the virus-like particles comprise viral capsid proteins.

4. The recombinant cell of claim 3, wherein the viral capsid proteins are soluble viral capsid proteins.

5. The recombinant cell of any one of claims 3 to 4, wherein the virus-like particles self- assemble from viral capsid proteins6 The recombinant cell of any one of claims 1 to 5, wherein the virus-like particles comprise nervous necrosis virus (NNV) capsid proteins.7 The recombinant cell of claim 2, wherein the Escherichia coli cell is BL21DE38. The recombinant cell of claim 2, wherein the Lactococcus lactis cell is N73910.

9. An oral vaccine comprising the recombinant cell of any one of the preceding claims.

10. The oral vaccine of claim 9 comprising fish feed powder, fish oil, and starch.

11. The oral vaccine of any one of claims 9 to 10, wherein the oral vaccine is provided in the form of pellets.

12. A fish feed additive comprising the recombinant cell of any one of claims 1 to 8.

13. A method of inoculating a subject against nervous necrosis virus, the method comprising administering the recombinant cell of any one of claims 1 to 8 to the subject.

14. A method of treating or preventing viral nervous necrosis in a subject, the method comprising administering the recombinant cell of any one of claims 1 to 8 to the subject.

15. The method of any one of claims 13 to 14, wherein the subject is a fish.

16. The method of claim 15, wherein the fish is selected from the group consisting of Asian seabass ates calcarifer), European Seabass ( icentrarchus labrax), Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus). haddock (Melanogramrmis aeglefinus), greasy grouper (Epinephehis tauvina), yellow tang (Zebrasoma flavescens}, three spot damsel (Dascyllus trimaculatus'), and guppy (Poecilia reticulata).

17. A method of obtaining a recombinant cell comprising virus-like particles, wherein the recombinant cell is intact, inactivated, and does not exhibit antibiotic resistance; the method comprisinga. Removing antibiotic resistance genes from a plasmid to obtain an antibiotic resistance-free plasmid,b. Inserting the gene encoding a viral capsid protein into the antibiotic resistance free plasmid of step a. to obtain an overexpression plasmid;c. Transforming the overexpression plasmid of step b. into competent cells;d. Growing and selecting the transformed competent cells of step c. under conditions that allow overexpression of the gene encoding the viral capsid protein and selfassembly of the virus-like particles;e. Inactivating the cells of step d., wherein the inactivation leaves the recombinant cell intact; thereby obtaining intact recombinant cell comprising virus-like particles.

18. The method of claim 17, wherein step a. is optional.

19. The method of any one of claims 17 to 18, wherein the inactivation in step e. is performed using sodium hypochlorite.

20. The method of any one of claims 17 to 19, wherein the inactivation in step e. is performed for at least 0.25 hour.

21. A recombinant cell obtained using the method of any one of claims 17 to 20.

Images