Cyanobacterial vaccines

Abstract

There is provided an immunogenic composition comprising or consisting of a recombinant Cyanobacteria or lysate thereof wherein the recombinant Cyanobacteria comprises a nucleic acid encoding at least one VLP and at least one heterologous antigen, wherein the expressed VLP comprises the at least one heterologous antigen.

Description

CYANOBACTERIAL VACCINESTechnical Field

[0001] The technology relates to immunogenic compositions (such as vaccines) of Cyanobacteria suitable for oral administration, nasal administration or both. The compositions comprise Cyanobacteria expressing a virus-like particle having at least one antigen for a disease-causing agent.Cross-reference to related application

[0002] This application claims priority to Australian provisional patent application number 2024903937 filed 28 November 2024, the entire contents of which are incorporated by reference.Background

[0003] Vaccines based on inactivated (killed), live attenuated pathogens or recombinant organisms have been used effectively against various infectious diseases but have several limitations including a limited potential to induce a strong immune response and poor efficacy.

[0004] Recent outbreaks of infectious diseases such as Covid-19 have manifested the need for the development of robust vaccines to overcome these limitations. One challenge is to develop new approaches that enhance immunity without jeopardizing safety, efficacy, and tolerability.

[0005] Virus-like particle (VLP) technology presents an alternative platform for developing effective vaccines to combat infectious diseases of serious concern, and it is moving in parallel with mRNA and viral-vector based vaccines. VLPs are also far more immunogenic compared to other subunit vaccines as they present repetitive antigenic epitopes on their surface in a more authentic confirmation that the immune system can readily detect.

[0006] VLPs can overcome various problems that are usually associated with traditional vaccines; specifically, the infectious nature related to live attenuated vaccines, reversion to a virulent form, risk of mutation, reduced immunogenicity of inactivated vaccines, unstable toxicity, low yield, and lengthy formulation time

[0007] However, some of the key challenges associated with VLPs are lower stability, difficult downstream processing, high production costs, and sensitivity to environmental conditions.

[0008] Accordingly, there is a need for improved VLP based immunogenic compositions that are shelf stable, inexpensive to produce, quick to develop and can be administered without needles.Summary

[0009] In a first aspect, the invention relates to a composition comprising a recombinant Cyanobacteria or lysate thereof wherein the recombinant Cyanobacteria comprises a nucleic acid encoding at least one VLP protein and at least one heterologous antigen, wherein the expressed VLP protein comprises the at least one heterologous antigen.

[0010] The VLP comprises a structural protein of a virus, for example one or more of a capsid or envelope protein

[0011] The sequence encoding the heterologous antigen may be within the sequence encoding the capsid or envelope protein. Alternatively, the sequence encoding the heterologous antigen is 5' or 3' and in frame with the sequence encoding the capsid or envelope protein.

[0012] In some embodiments the nucleic acid encoding the VLP protein further comprises a linker sequence between the sequence encoding the heterologous antigen and the capsid or envelope protein

[0013] The nucleic acid may further comprise a sequence encoding a targeting peptide such as a Sec or Tat signal peptide for targeting the VLP protein to a thylakoid membrane

[0014] The heterologous antigen may be an antigen from one or more of malaria, SARS- CoV-2, influenza, Pneumococcal disease, diphtheria, dengue, hepatitis, Hib (Haemophilus influenzae type b) HPV (human papillomavirus), measles, meningococcal disease, Mpox, mumps, polio (poliomyelitis), rotavirus, RSV (respiratory syncytial virus), rubella (German measles), shingles (herpes zoster), tetanus, whooping cough (pertussis), adenovirus, anthrax, cholera, Japanese encephalitis (JE), rabies, smallpox, tuberculosis, typhoid fever, yellow fever Zika, Chikungunya, Lyme disease, MERS, herpes simplex, HIV, Ross River Virus, West Nile Fever, Ebola, Lassa Fever, Nipah, or Rift Valley Fever.

[0015] The recombinant Cyanobacteria may further comprise one or more nucleic acid sequences encoding an adjuvant, or an enzyme for the biosynthesis of an adjuvant. In one embodiment the adjuvant is a terpene adjuvant, such as squalene, dehydroisosqualene, farnesene thermal dimer, nonaprenol (solanesol), difarnesyl ether, geranyl diphosphate, farnesyl diphosphate, geranyl geranyl diphosphate, isopentenyl diphosphate, or derivates thereof. In one embodiment the enzyme is squalene synthase and the adjuvant is squalene.

[0016] Alternatively or in addition, the recombinant Cyanobacteria may comprise an inactivating mutation in the endogenous squalene hopene cyclase gene.

[0017] The immunogenic composition may comprise or consist of the recombinant Cyanobacteria or a lysate of the recombinant Cyanobacteria.

[0018] The immunogenic composition may be in an aqueous form or a dried form

[0019] In one embodiment the Cyanobacteria is not Spirulina or Arthospira and is from the order Chroococcales (for example Synechococcus sp. PCC 7002 or Synechocystis sp. PCC 6803) or Synechococcales ( e.g. Synechococcus elongatus PCC 7942)

[0020] The Cyanobacteria may be selected from the group consisting of Synechococcus sp.PCC 11901 , Synechococcus sp. UTEX 2434, Synechococcus sp. UTEX 2973, Synechococcus sp. LITEX 3153, and Synechococcus sp. LITEX 3154.

[0021] In a second aspect the invention relates to a dietary supplement containing the immunogenic composition of the first aspect.

[0022] In a third aspect the invention relates to a method of eliciting an immune response in subject comprising administering to the subject an effective amount of a composition of the first aspect, wherein the immune response is specific for the heterologous antigen.

[0023] The method may comprise administering a first dose of the composition and a second dose of the composition. One or more additional doses may also be administered.

[0024] The first dose may be administered orally, nasally or by intramuscular injection. The second dose may be administered orally, nasally or by intramuscular injection. The at least one additional dose may be administered orally, nasally or by intramuscular injection.

[0025] In one embodiment the second dose may be administered from 1 week to 2 months after the first dose.

[0026] The at least one additional dose may be administered 6 to 18 months after the second dose.

[0027] In one embodiment the oral administration comprises the subject ingesting the dietary supplement of the second aspect.

[0028] Preferably, the immune response protects the subject from infection, or lessens the severity of a disease caused by the organism from which the heterologous antigen is derived.

[0029] In a fourth aspect the invention relates to use of a composition of any one of claims 1 to 13 for the manufacture of a medicament for eliciting an immune response in subject, wherein the immune response is specific for the heterologous antigen.Definitions

[0030] Throughout this specification, unless the context clearly requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0031] Throughout this specification, the term 'consisting of' means consisting only of.

[0032] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present technology. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present technology as it existed before the priority date of each claim of this specification.

[0033] Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the technology recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

[0034] In the context of the present specification the terms 'a' and 'an' are used to refer to one or more than one (i.e. , at least one) of the grammatical object of the article. By way of example, reference to 'an element' means one element, or more than one element.

[0035] In the context of the present specification the term 'about' means that reference to a figure or value is not to be taken as an absolute figure or value, but includes margins of variation above or below the figure or value in line with what a skilled person would understand according to the art, including within typical margins of error or instrument limitation. In other words, use of the term 'about' is understood to refer to a range or approximation that a person or skilled in the art would consider to be equivalent to a recited value in the context of achieving the same function or result.

[0036] Those skilled in the art will appreciate that the technology described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the technology includes all such variations and modifications. For the avoidance of doubt, the technology also includes all of the steps, features, and compoundsreferred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps, features and compounds.

[0037] In order that the present technology may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.Brief description of the FiguresFigure 1 : Production and isolation of Synechococcus-expressed NANP-VLP

[0038] (A) Overview of components of Synec / iococcus-produced VLP constructs (1) NANP- VLP presenting ‘Control Antigen’ (x15-NANP; CSP ‘R’ epitope, Malaria), (2) ‘Naked’ VLP control. Both VLPs contain a C-terminally located short linker (“L”, grey box, x5 GGS) between the CoreN and CoreP VLP assembly domains (white). Each VLP includes a C- terminally located hexahistidine-peptide (“H”, black box) and a Nucleic Binding Domain (“NAB”, grey box). NANP-VLP (2) includes a ‘Control Antigen’ (“ANT”, CSP epitope, Malaria; grey box). (B) Transmission Electron Microscope negatively stained image of assembled NANP-VLPs from lysed Synechococcus. (C) Western Blots of lysed Synechococcus soluble fraction against (left) Anti-HIS IR800 Ab and (right) Anti-CSP Ab, following VLP purification via ultracentrifugation through a 30% sucrose cushion. (D) SDS PAGE (Left) & Western Blot against Anti-HIS IR800 Ab (right) of lysed Synechococcus fractions, following VLP purification via ultracentrifugation through a 30% sucrose cushion, followed subsequently by 30-70% sucrose gradient (Fractions: G1 :~30%, G2: -38% G3:~46%, G4:~54%, G5:~62%, G6:~70%). (E) Lysed Synechococcus soluble fraction following VLP purification via ultracentrifugation through a 30% sucrose cushion (left) and subsequently by 30-70% sucrose gradient (right).Figure 2 Weight monitoring after administration of Synechococcus lysed biomass.

[0039] Accompanies safety data (Table 1) for oral and nasal administration of cyanobacterial biomass, demonstrating that it is safe and non-toxic to rodents. Mice were orally administered 20mg Synechococcus lysed biomass (OR) or nasally administered 2.5mg of Synechococcus lysed biomass (IN). A second oral administration of 20mg lysed biomass was given after 14 days to these two groups of mice. Negative control mice were left untreated for the duration. The lack of reactogenicity with of our cyanobacterial platform avoids problematic bacterial endotoxins and removes both the costs, complexities and risks associated with bacterial or fungal produced vaccines.Figure 3 | Immunogenicity of Synechococcus-expressed VLP lysate

[0040] (A) Vaccination regimen & serum draw. x3 groups of mice (n=3 per group) were primed with 15pg of purified NANP-VLP administered intramuscularly (IM) and then boostedat Day 14 with either 15pg purified NANP-VLP administered IM, crude lysate containing 15pg NANP-VLP, administered intranasally (IN), or crude lysate containing 90pg NANP- VLP, administered by oral gavage (OD). 3 further groups of mice (n=3 per group) were nasally administered an initial prime of crude lysate containing 15pg NANP-VLP administered intranasally (IN) and boosted at Day 14 with either an identical IN dose of crude lysate containing 15 pg NANP-VLP, crude lysate containing 90pg NANP-VLP, administered by oral gavage (OD), or undisrupted whole cell material containing 30 pg NANP-VLP, administered by oral gavage (OW). All IM administered doses of VLPs were purified via sucrose gradient and mixed 1:1 with AddaVax (squalene-based) adjuvant. All IN doses were from soluble crude extract of lysed Synechococcus biomass containing the expressed VLP, dried & resuspended in water & mixed 1:1 with AddaVax. All OD doses were from soluble crude extract of lysed Synechococcus biomass containing the expressed VLP, dried & resuspended in water, & mixed 3:7 with Montenide GR gel to encapsulate the VLP for oral delivery. All OW doses comprised of whole cell Synechococcus biomass (not lysed) containing the expressed VLP. Biomass was dried and resuspended in HBSS buffer with no external adjuvant included.

[0041] (B) Exemplar ELISA data for IN Prime, IN Boost (IN / IN) underpinning IgG Endpoint Titre (shown in Figure 3C). Immunogenicity at 28 days was measured comparing immunized and pre-bleed mouse serum serially diluted from 1 / 25 to 1 / 128000 by ELISA with a 6XNANP peptide antigen. The pre-bleed negative controls were used to define a statistically defined (95% confidence) cut-off for every dilution as outlined by Frey et. al (J Immunol Methods. (1998); 221 (1-2):35-41)). The endpoint IgG titre shown in Fig. 1C is the average reciprocal of x3 individual mouse sera titres, of the highest dilution that has a median reading of x3 technical replicates above the cut-off.

[0042] (C) Immunogenicity to CSP control antigen by Western Blot against immunized mice serum. Western blot analysis of recombinant full-length CSP probed with anti-mouse serum collected from different immunization routes. Each lane contains recombinant full-length CSP, and the membrane was probed with serum from mice immunized with VLPs containing x15-NANP-repeats (control epitope for CSP). Lane 1: Mice primed with 15pg of purified NANP-VLP administered intramuscularly (IM) and then boosted with 15 pg purified NANP-VLP administered IM. Lane 2: Mice primed with 15pg of purified NANP-VLP administered IM, and then boosted with crude lysate containing 15pg NANP-VLP, administered intranasally (IN). Lane 3: Mice primed with 15pg of purified NANP-VLP administered IM, and then boosted with crude lysate containing 90pg NANP-VLP, administered by oral gavage (OD). Lane 4: Mice primed with crude lysate containing 15pgNANP-VLP, administered IN and boosted with an identical IN dose. Lane 5: Negative control (pre-bleeds from unimmunized mice); Lane 6: Positive control, anti-CSP monoclonal antibody 2A10. The blot shows the immune response elicited by different vaccination routes, with serum from vaccinated mice detecting the CSP antigen, indicating successful antigen recognition.

[0043] (D) Endpoint IgG titre at 28 days measured by ELISA with control peptide antigen. x3 groups of mice (n=3 per group) were primed with 15pg of purified NANP-VLP administered intramuscularly (IM) and then boosted at Day 14 with either 15pg purified NANP-VLP administered IM, crude lysate containing 15pg NANP-VLP, administered intranasally (IN), or crude lysate containing 90pg NANP-VLP, administered by oral gavage (OR). 3 further groups of mice were nasally administered an initial prime of crude lysate containing 15pg NANP-VLP administered intranasally (IN) and boosted at Day 14 with either an identical IN dose of crude lysate containing 15 pg NANP-VLP, crude lysate containing 90pg NANP-VLP, administered by oral gavage (OD), or undisrupted whole cell material containing 30 pg NANP-VLP, administered by oral gavage (OW). All IM administered doses of VLPs were purified via sucrose gradient and mixed 1:1 with AddaVax (squalene-based) adjuvant. All IN doses were from soluble crude extract of lysed Synechococcus biomass containing the expressed VLP, dried & resuspended in water & mixed 1:1 with AddaVax. All OD doses were from soluble crude extract of lysed Synechococcus biomass containing the expressed VLP, dried & resuspended in water, & mixed 3:7 with Montenide GR gel to encapsulate the VLP for oral delivery. All OW doses comprised of whole cell Synechococcus biomass (not lysed) containing the expressed VLP. Biomass was dried & resuspended in HBSS buffer with no external adjuvant included. Immunogenicity at 28 days was measured comparing immunized and pre-bleed mouse serum serially diluted from 1 / 25 to 1 / 128000 by ELISA with a 6XNANP peptide antigen (see Figure 3B for exemplar ELISA data). The pre-bleed negative controls were used to define a statistically defined (95% confidence) cut-off for every dilution as outlined by Frey et. al. (J Immunol Methods. (1998); 221 (1-2):35-41). The endpoint IgG titre shown is the average reciprocal of x3 individual mouse sera titres, of the highest dilution that has a median reading of x3 technical replicates above the cut-off.Fig 4. High-density lab- & large-scale photosynthetic cultivations of Synechococcus

[0044] (A) Lab-scale cultivation showing rapid, high-density cultivation of Synechococcus PCC7200 in tissue culture flasks with 12mL (black), 16mL (dashed) and 24mL (grey) volumes, grown in a CO2 photoincubator, showing impact of volume, culture depth & selfshading. In low volumes, where light access is non-limiting, cultures can reach 27g / L DCWbiomass density in a week in phototrophic growth. (B) High-density growth translated to industrial scale photobioreactors. 6L cultures grown in the Subitec LS6 FPA unit have reached ~16g / L (OD750 —70) in x1 week.Figure 5 | Immunogenicity of Synechococcus VLP-containing lysate & whole cell material

[0045] (A) Vaccination regimen and serum draw. x2 groups of mice (n=3 per group) were primed with 15pg of purified NANP-VLP administered intramuscularly (IM) and then either boosted at Day 21 with 15pg purified NANP-VLP administered IM or provided no boost. 3 further groups of mice (n=3 per group) were nasally administered an initial prime of crude lysate containing ~84pg NANP-VLP administered intranasally (IN) and boosted twice at Day 21 and Day 42 with either an identical IN dose of crude lysate containing ~84 pg NANP- VLP, undisrupted whole cell material containing 330 pg NANP-VLP, administered by oral gavage (OW), or crude lysate containing -330 pg NANP-VLP, administered by oral gavage (OD). All IM administered doses of VLPs were purified via sucrose gradient and mixed 1 :1 with AddaVax (squalene-based) adjuvant. All IN doses were from soluble crude extract of lysed Synechococcus biomass containing the expressed VLP, dried & resuspended in water & mixed 1:1 with AddaVax. All OD doses were from soluble crude extract of lysed Synechococcus biomass containing the expressed VLP, dried & resuspended in water, & mixed 3:7 with Montenide GR gel to encapsulate the VLP for oral delivery. All OW doses comprised of whole cell Synechococcus biomass (not lysed) containing the expressed VLP. Biomass was dried and resuspended in HBSS buffer with no external adjuvant included.

[0046] (B) Exemplar ELISA data for IN Prime, IN Boost (IN / IN) underpinning IgG Endpoint Concentration Data (shown in Figure 5C). Immunogenicity at 63 days was measured comparing immunized and pre-bleed mouse serum (diluted to 1 / 800) by ELISA with a 6XNANP peptide antigen and quantified against an in-plate IgG Standard (Mouse anti- NANP mAb 2A10). Anti-NANP IgG antibody standards were prepared in triplicate between 15 ng / mL and 750 ng / mL and used to generate a standard curve (black circles). The prebleed negative controls were used to blank correct each serum read, before an IgG concentration for each sera sample was calculated by comparison to the Anti-NANP IgG antibody standard curve (Mouse #0, black squares; Mouse #1, hatched squares; Mouse #2, open squares). The endpoint IgG concentrations for each mouse (average cone., x3 technical replicates) are shown in Fig. 5C, alongside the average concentration of x3 individual mouse sera concentrations.

[0047] (C) Endpoint IgG concentration at 63 days measured by ELISA via comparison to Anti-NANP IgG antibody standards. x2 groups of mice (n=3 per group) were primed with15|jg of purified NANP-VLP administered intramuscularly (IM) and then either boosted at Day 21 with 15pg purified NANP-VLP administered IM or provided no boost. 3 further groups of mice (n=3 per group) were nasally administered an initial prime of crude lysate containing ~84 pg NANP-VLP administered intranasally (IN) and boosted twice at Day 21 and Day 42 with either an identical IN dose of crude lysate containing ~84 pg NANP-VLP, undisrupted whole cell material containing 330 pg NANP-VLP, administered by oral gavage (OW), or crude lysate containing -330 pg NANP-VLP, administered by oral gavage (OD). All IM administered doses of VLPs were purified via sucrose gradient and mixed 1:1 with AddaVax (squalene-based) adjuvant. All IN doses were from soluble crude extract of lysed Synechococcus biomass containing the expressed VLP, dried & resuspended in water & mixed 1:1 with AddaVax. All OD doses were from soluble crude extract of lysed Synechococcus biomass containing the expressed VLP, dried & resuspended in water, & mixed 3:7 with Montenide GR gel to encapsulate the VLP for oral delivery. All OW doses comprised of whole cell Synechococcus biomass (not lysed) containing the expressed VLP. Biomass was dried & resuspended in HBSS buffer with no external adjuvant included. Immunogenicity at 63 days was measured comparing immunized and pre-bleed mouse serum serially diluted to 1 / 800 by ELISA with a 6XNANP peptide antigen against an in-plate Anti-NANP IgG antibody standard (see Figure 5B for exemplar ELISA data). The endpoint IgG concentrations for each mouse (black squares, average concentration, x3 technical replicates) are shown, alongside the average concentration of x3 individual mouse sera concentrations (bar graph).Description of Embodiments

[0048] The present inventors have developed recombinant Cyanobacteria expressing Virus Like Particles (VLPs) engineered to present antigens from disease-causing organisms. These recombinant Cyanobacteria can be administered to a subject to elicit an immune response specific for the antigens. In addition, the specific immune response can be elicited by oral or nasal administration of the recombinant Cyanobacteria or compositions comprising the to a subject.Engineered VLPs

[0049] The recombinant Cyanobacteria of the present invention comprise a nucleic acid encoding one or more proteins that self-assemble into a VLP having at least one heterologous antigen.

[0050] VLPs are multi-subunit self-assembly-competent protein structures with identical or highly related overall structure to their corresponding native viruses. It is envisaged that any VLP known in the art can be used as the standard genetic engineering techniques can beused to incorporate a sequence encoding the heterologous antigen into the VLP sequence. That is, any virus can be utilized to develop engineered VLPs; for example VLPs can be developed by cloning the structural genes that code for the proteins of a virus of interest into a suitable expression vector noting that the structural proteins, envelope proteins, or capsid proteins, can, either independently or collectively, spontaneously self-assemble to form virus-like particles (VLPs) without the viral genome.

[0051] The coat protein of a virus can be recombinantly engineered using conventional methods to include essentially any heterologous antigen of interest. To maximize the immunogenic potential of platform technologies such as the recombinant Cyanobacteria described herein, target antigens are ideally displayed at high density on the surface of VLPs. A number of different approaches may be used to engineer VLPs to display heterologous antigens of various compositions, sizes, and structures. One approach is to generate recombinant fusions in which foreign antigens are inserted into sites within the viral structural protein so that the foreign antigen is displayed on the resulting VLP’s surface.

[0052] As with many VLPs, sites for the insertion of heterologous antigens in VLPs can be identified using rational design based on structural characteristics (i.e. surface exposed loops or disordered regions).

[0053] It is envisaged that any VLP can be used in the invention. For example a variety of VLPs can be engineered to display heterologous antigens as fusions with the viral structural protein. Suitable VLPs include Porcine circovirus 2 (PCV2), Human parvovirus B19, Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Goose parvovirus (GPV), Porcine parvovirus (PPV), canine parvovirus (CPV), Hepatitis E virus (HEV), Alfalfa mosaic virus (AIMV), Brome mosaic virus (BrMV), Cowpea chlorotic mottle virus (CCMV), cucumber-mosaic virus-like particles (CuMV), Enterobacteria phage MS2, Enterobacteria phage PP7, Enterobacteria phage fr, Enterobacteria phage T7, Enterobacteria phage P22, Enterobacteria phage lambda, Enterobacteria phage GA, Enterobacteria phage Q , Acinetobacter page AP205, Caulobacter phage phiCb5, Cowpea mosaic virus (CPMV), Porcine encephalomyocarditis virus (EMCV), Coxsackievirus B (CVB3), foot-and-mouth disease virus (FDMV), Flock house virus (FHV), Tomato bushy stunt virus, Malabaricus grouper nervous necrosis virus (MGNNV), Norwalk virus (NV), Rabbit haemorrhagic disease virus (RHDV), Murine polyomavirus (MuPV), Hamster polyomavirus (HaPV), Simian virus 40 (Sv40), JC polyomavirus, Human papillomavirus (HPV), Bovine papillomavirus (BPV), Cottontail rabbit papillomavirus (CRPV), Bluetongue virus (BTV), African horse sickness virus (AHSV), rotavirus (RT), Human adenovirus B (type 3) (Ad3),Papaya mosaic virus (PapMV), Potato virus X (PVX), bamboo mosaic virus (BaMV), Johnsongrass mosaic virus (JGMV), Plum pox virus (PPV), Potato virus Y (PVY), Tobacco mosaic virus (TMV), Hepatitis B virus (HBV), HBV core (HBc) particles, HBV surface (HBs) particles, Woodchuck hepatitis virus (WHV), Woodchuck hepatitis virus (WHV) core (WHc) particles, West Nile virus (WNV), Dengue virus, Hepatitis C virus (HCV), Japanese encephalitis virus (JEV), Tick-borne encephalitis virus (TEV), Sindbis virus (SIN), Ross river virus (RRV), Chikungunya virus (CHIKV), Salmonid alphavirus (SAV), SARS-CoV, Crimean- Congo hemorrhagic fever (CCHF) virus, Rift Valley fever virus (RVFV), Hantaan virus (HTNV), Human immunodeficiency virus 1 (HIV-1), Human immunodeficiency virus 2 (HIV- 2), Simian immunodeficiency virus (SIV), Rous-sarcoma virus (RSV), Murine leukemia virus (MLV), Human herpesvirus 4 (Epstein-Barr virus, EBV), Orthomyxoviridae Influenz avirus A, Lassa virus, Tacaribe and Junin viruses, Newcastle disease virus, Respiratory syncytial virus (RSV), Nipah virus (NiV), Sendai virus (SeV), Human parainfluenza virus 1, Human parainfluenza virus 3, mumps virus (MuV), Menangle virus (MeV), Tioman virus (TioV), Human metapneumovirus (hMPV), Measles virus (MV), Marburg marburgvirus (MARV), Zaire ebolavirus (EBOV)Nucleic acid sequences encoding the engineered VLP can be made by any method known in the art and inserted into a suitable vector or other delivery system to facilitate expression in a Cyanobacteria.

[0054] Woodchuck hepatitis virus (WHV) possesses a compact genome that encodes only four overlapping open reading frames: C (core-related structural and non-structural proteins), P (polymerase), S (surface-related structural proteins), and X (protein X).

[0055] Open reading frame C encodes four functionally relevant core-related polypeptides useful in the preparation of VLPs. Two of these proteins are major, the first is p21 , also called WHcAg , Cp, or structural WHc protein monomeric unit which is a constituent in WHc nucleocapsid formation. The second, p17, or WHcAg, is a non-structural protein.

[0056] The WHc protein has the unique capability to self-assemble and the correct folding of the WHc monomer and the formation of authentic WHc VLPs have been documented. The basic structural unit of the WHc protein is a dimer, and a disulfide bridge connecting two monomers can form, although this connection is not obligatory for self-assembly. Typically, the WHc protein assembles into VLPs containing 120 dimers.

[0057] The WHc protein contains four transmembrane helices. The sequence encoding the heterologous antigen can be positioned such that when expressed, it is between the helices the heterologous antigen is external to the VLP. For example, the heterologous antigen can be encoded by a sequence that is use to replace or be inserted into the sequence encoding WHc or WHcAg

[0058] Hepatitis B virus (HBV), the prototype of the Hepadnaviridae family also possesses a genome that encodes only four overlapping open reading frames: C (core-related structural and non-structural proteins), P (polymerase), S (surface-related structural proteins), and X (protein X).

[0059] Open reading frame C encodes four functionally relevant core-related polypeptides useful in the preparation of VLPs. Two of these proteins are major, the first is p21 , also called HBcAg, Cp, or structural HBc protein monomeric unit which is a constituent in HBV nucleocapsid formation. The second, p17, or HBeAg, is a non-structural but immunologically important HBe protein.

[0060] The HBc protein has the unique capability to self-assemble and the correct folding of the HBc monomer and the formation of authentic HBc VLPs have been documented in numerous eukaryotic cell lines and bacteria. The basic structural unit of the HBc protein is a dimer, and a disulfide bridge connecting two monomers can form, although this connection is not obligatory for self-assembly. Typically, the HBc protein assembles into VLPs containing 120 dimers.

[0061] The HBc protein contains four transmembrane helices. The sequence encoding the heterologous antigen can be positioned such that when expressed, it is between the helices the heterologous antigen is external to the VLP. For example, the heterologous antigen can be encoded by a sequence that is used to replace or be inserted into the sequence encoding the HBc protein.

[0062] When the VLPs are constructed the sequence encoding the heterologous antigen are inserted or added to the sequence encoding the VLP proteins without influencing the self-assembly.

[0063] In some embodiments, the VLP contains Hepatitis B virus (HBV) proteins such as HBV core (HBc) protein, HBV surface (HBs) protein.

[0064] Preferably the VLP contains Woodchuck hepatitis virus (WHV) proteins such as Woodchuck hepatitis virus core (WHc) protein.

[0065] A VLP of the present invention can be generated by expression of a viral structural protein engineered to include at least one heterologous antigen.

[0066] The sequence encoding the heterologous antigen may be located in frame (either 5' or 3') with the coding sequence for the structural protein such that when expressed the VLP contains an N-terminal or C-terminal fusion of the heterologous antigen and the structural protein. Structural proteins include multiple transmembrane helices joined by loop regions and in other embodiments the sequence encoding the heterologous antigen may be inserted into the sequence encoding the loop regions of structural protein such that whenexpressed the heterologous antigen is present on the outer or inner surface of the VLP, preferably on the outer surface.

[0067] The sequence for the heterologous antigen may be located in frame (either 5' or 3') with the coding sequence for the structural poteen such that when expressed the VLP contains an N-terminal or C-terminal fusion of the heterologous antigen and the structural protein.

[0068] The sequence comprising a nucleic acid encoding one or more proteins that selfassemble into a VLP having at least one heterologous antigen can be introduced into a Cyanobacteria using any method known in the art, for example using and extra- chromosomal plasmid or by homologous recombination into the Cyanobacterial genome.

[0069] The sequence encoding the VLP and heterologous can further comprise a targeting sequence to ensure the expressed protein is targeted to a specific subcellular location o the Cyanobacteria. For example the nucleic acid may further comprise a sequence encoding a signal peptide specific for the thylakoid membrane, for example a Sec or Tat signal peptide. In this way an enveloped VLP (eVLP) may be produced. This may also aid glycosylation of the heterologous antigen. In some embodiments, the methods described in the Applicant's co-pending PCT application, PCT / AU2021 / 051121 , titled 'Standardised cyanobacterial strain-engineering' can be used to prepare nucleic acid sequences encoding the engineered VLP of the present invention in a form suitable for expressing the engineered VLP in a Cyanobacteria,

[0070] Briefly, PCT / AU2021 / 051121 describes the preparation of a nucleic acid cassette comprising two portions of a neutral site sequence and a heterologous nucleic acid comprising at least two landing zones and a first selectable marker gene located between the landing zones; and wherein the heterologous nucleic acid is between the two portions of the neutral site, in th present case the heterologous nucleic in the caseate will be the sequence encoding the VLP and the heterologous antigen.

[0071] The neutral site is substantially homologous to at least a part of a non-essential region of the Cyanobacterial genome. For example the non-essential region may be selected from the NSC1 region of Synechocystis sp. strain PCC 6803, the slr0168 region of Synechocystis sp. strain PCC 6803, the A0159 region of Synechococcus sp. strain PCC 7002, the A2842 region of Synechococcus sp. strain PCC 7002, or a non-essential region of Synechococcus sp. strain PCC 7942.Recombinant Cyanobacteria

[0072] Once a nucleic acid sequence encoding the engineered VLP has been prepared it may be transformed into a Cyanobacteria using any known method, for example the methods described in the Applicants' co-pending PCT application PCT / AU2021 / 050300 titled 'Method for transforming Cyanobacteria'

[0073] Briefly, PCT / AU2021 / 050300 describes a method for transforming a Cyanobacteria comprising incubating the Cyanobacteria and a nucleic acid comprising a selectable marker (such as the nucleic acid encoding the engineered VLP above) under conditions suitable for transformation of the micro-organism with the nucleic acid; further incubating the microorganism in growth media under conditions suitable for recovery of the micro-organism; and selecting the transformed micro-organism using a selection agent. This method provides a population of fully segregated transformed Cyanobacteria and can be performed solely in aqueous media.

[0074] In some embodiments the nucleic acid encoding the engineered VLP is present as part of an extra-chromosomal plasmid, for example a native plasmid.

[0075] In other embodiments the nucleic acid encoding the engineered VLP is integrated into the genome of the Cyanobacteria, preferably multiple copies of the the nucleic acid integrated into the genome of the Cyanobacteria.

[0076] In one aspect the invention relates to a Cyanobacterial cell comprising the nucleic acid encoding the engineered VLP integrated into the genome of the cell.

[0077] Alternatively, or in addition, the nucleic acid encoding the engineered VLP is integrated into a native plasmid of the Cyanobacteria. The inventors have shown that this increases VLP expression and cellular VLP content. Any competent Cyanobacteria form the order Gloeobacterales, Synechococcales, pleurocapsales, Chroococcidiopsidales, Chroococcales or Oscillatoriales, may be used in the invention. That is, in some embodiments, the invention excludes the use of Spirulinales (such as Spirulina and Arthospira) which are filamentous and have high production costs and low production capacity.

[0078] Preferably, the Cyanobacteria is a Synechococcales or a Chroococcales.

[0079] For example suitable Synechococcales genera include Synechococcus, Prochlorococcus, Leptolyngbya, Oscillatoriales, Geitlerinema, Acaryochloris, Cyanothece, Thermosynechococcus, Pseudanabaena, and Gloeobacter. In some embodiments the Synechococcales is Synechococcus elongatus PCC 7942, Leptolyngbya sp. BL0902

[0080] For example suitable Chroococcales genera include Cyanobacterium, Geminocystis, Leptolyngbya, Synechococcus, Gloeocapsa, Microcystis, Cyanthece, Synechocystis,Candidatus, and Crocosphaera. In some embodiments the Chroococcales is Synechocystis sp. PCC 6803, or Synechococcus sp. PCC 7002.

[0081] Other suitable Cyanobacterial genera include those selected from the group comprising Collenia, Girvanella, Gunflintia, Morania, Sphaerocodium, Acaryochloris, Anabaena, Anabaenopsis, Aphanizomenon, Arthrospira, Aulosira, Borzia, Calothrix, Chlorogloeopsis, Chroococcidiopsis, Cyanonephron, Cylindrospermopsis, Cylindrospermum, Gloeocapsa, Gloeotrichia, Homoeothrix, Jakutophyton, Johannesbaptistia, Loefgrenia, Lyngbya, Merismopedia, , Nodularia, Nostoc, Oscillatoria, Ozarkcollenia, Palaeolyngbya, Petalonema, Planktothrix, Prochlorococcus, Prochloron, Radaisia, Rivularia, Rothpletzella, Scytonema, , Trichodesmium, and Wollea.

[0082] In some embodiments suitable strains include Synechococcus sp. PCC 11901 , Synechococcus sp. LITEX 2434, Synechococcus sp. LITEX 2973, Synechococcus sp. LITEX 3153, and Synechococcus sp. LITEX 3154.Adjuvants

[0083] In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one adjuvant.

[0084] The term 'adjuvant' refers to a compound or mixture that enhances the immune response to an antigen. Adjuvants may act primarily as a delivery system, primarily as an immune modulator or have features of both. Suitable adjuvants are known in the art and include those suitable for use in mammals, including humans.

[0085] Examples of known suitable delivery-system type adjuvants that can be used in humans include, but are not limited to, alum (e.g., aluminum phosphate, aluminum sulfate or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions such as MF59 (4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80), 0.5% w / v sorbitan trioleate (Span 85)), water-in-oil emulsions such as Montanide, and poly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

[0086] Examples of known suitable immune modulatory type adjuvants that can be used in humans include, but are not limited to, saponin extracts from the bark of the Aquilla tree (QS21, Quil A), TLR4 agonists such as MPL (Monophosphoryl Lipid A), 3DMPL (3-O- deacylated MPL) or GLA-AQ, LT / CT mutants, cytokines such as the various interleukins (e.g., IL-2, IL-12) or GM-CSF, and the like.

[0087] For veterinary applications including but not limited to animal experimentation, one can use Complete Freund’s Adjuvant (CFA), Freund’s Incomplete Adjuvant (IFA) , Emulsigen, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl- D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween 80 emulsion.

[0088] In an embodiment of the present invention, the immunogenic compositions disclosed herein comprise as an adjuvant one or more terpenes such as squalene, dehydroisosqualene, farnesene thermal dimers, nonaprenol (solanesol), difarnesyl ether, geranyl diphosphate, farnesyl diphosphate, geranyl geranyl diphosphate, isopentenyl diphosphate, those described in Fisher et al npj Vaccines (2023)8:14, or derivates thereof.

[0089] Suitable squalene based adjuvants are known in the art and include squalene emulsion adjuvants which are oil-in-water emulsions formulated with metabolizable squalene droplets and nontoxic surfactants in the presence or absence of the immune- costimulatory a-tocopherol (Table 1)Table 1 : Squalene based emulsion adjuvants

[0090] In one embodiment the squalene adjuvant is added to the immunogenic composition during formulation.

[0091] Alternatively, or in addition the squalene may be produced by the recombinant Cyanobacteria. In this regard the recombinant Cyanobacteria may be further modified to comprise one or more of the biosynthetic enzymes for squalene, such as squalene synthase or farnesyl-diphosphate synthase; which catalyses a two-step reaction in which two molecules of farnesyl pyrophosphate are converted into squalene, with the consumption of NADPH.

[0092] In some embodiments the recombinant Cyanobacteria may alternatively or additionally be modified to inactivate the endogenous squalene hopene cyclase which converts squalene into hopene. This modification may comprise any mutation (including a deletion) of the gene encoding squalene hopene cyclase.

[0093] The recombinant Cyanobacteria can further comprise modifications to express additional enzymes for the synthesis of an adjuvant such as squalene. For example the recombinant Cyanobacteria may express one or more enzymes in the 2C-methyl-d- erythritol-4-phosphate (MEP) pathway which provides farnesyl diphosphate, the substrate of squalene synthase. In a particular embodiment of the present invention, any of the immunogenic compositions disclosed herein comprise from 1 pg to 500 mg of squalene per mL of liquid composition or per gram of lyophilised composition when reconstituted. In some embodiments the liquid formulations comprise 1-50% (vol / vol) squalene, for example, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17,5, 10, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, or about 50% (vol / vol) squalene.Formulations

[0094] The immunogenic compositions may be in liquid form (i.e. , solutions or suspensions) or in a dried or lyophilized form and contain either whole recombinant Cyanobacteria or lysates of the recombinant Cyanobacteria.

[0095] In one embodiment the lysate is a soluble lysate or a total lysate. A total lysate may be prepare by any means known in the art (for example sonication, freeze / thaw cycles, osmotic stress, homogenisation, or high pressure methods such as a French press). In other embodiments a soluble lysate is used. A soluble lysate can be prepared by removal of insoluble and particulate material from a total lysate, for example by centrifugation or filtration. The lysates (soluble or total) may further be concentrated or dried.

[0096] The liquid or dried formulations may simply be prepared from whole recombinant Cyanobacteria, or a lysate of the recombinant Cyanobacteria.

[0097] It is advantageous for the formulations to contain a relatively high concentration of VLP compared to total biomass, in this regard the recombinant Cyanobacteria disclosed herein can express significant amounts of VLP per gram of biomass, for example -0.2-0.3% DCW (2-3pg VLP per mg whole-cell biomass), equivalent to -0.4-0.6% of soluble protein (4- 6pg VLP per mg soluble cell lysate) when the VLP is chromosomally-encoded.

[0098] In some embodiments the invention uses strains where the same VLP is in inserted onto a native plasmid. These strains produce more VLP, for example -1-3% DCW (10-30pg VLP per mg whole-cell biomass), equivalent to -2-6% of soluble protein (20-60pg VLP per mg soluble cell lysate).

[0099] Liquid formulations may advantageously be administered directly from their packaged form and are thus ideal for injection, or oral or nasal administration without the need for reconstitution in aqueous medium as otherwise required for lyophilized compositions of the invention.

[0100] Formulation of the immunogenic composition of the present invention can be accomplished using methods known in the art. For instance, the recombinant Cyanobacteria or lysates thereof can be formulated with a physiologically acceptable vehicle to prepare the composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.

[0101] In some embodiments the present disclosure provides an immunogenic composition comprising one or more recombinant Cyanobacteria or lysates thereof disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0102] In an embodiment, the immunogenic composition of the invention is in liquid form, preferably in aqueous liquid form.

[0103] Immunogenic compositions (liquid or dried) may comprise one or more of a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant such as a sugar, and an antioxidant such as a free radical scavenger or chelating agent, or any multiple combinations thereof.

[0104] In an embodiment, the immunogenic compositions of the invention comprise a buffer. In an embodiment, said buffer has a pKa of about 3.5 to about 7.5. In some embodiments, the buffer is phosphate, succinate, histidine or citrate. In certain embodiments, the buffer is succinate at a final concentration of 1 mM to 10 mM.

[0105] In an embodiment, the immunogenic compositions of the invention comprise a salt. In some embodiments, the salt is selected from the groups consisting of magnesiumchloride, potassium chloride, sodium chloride and a combination thereof. In one particular embodiment, the salt is sodium chloride.

[0106] In an embodiment, the immunogenic compositions of the invention comprise a surfactant. In an embodiment, the surfactant is selected from the group consisting of polysorbate 20 (TWEENTM20), polysorbate 40 (TWEENTM40), polysorbate 60 (TWEEN™60), polysorbate 65 (TWEEN™65), polysorbate 80 (TWEEN™80), polysorbate 85 (TWEEN™85), TRITON™ N-101, TRITON™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL), soy lecithin and a poloxamer. In one particular embodiment, the surfactant is a polysorbate.

[0107] In certain embodiments, the immunogenic composition of the invention has a pH of 5.5 to 7.5, more preferably a pH of 6.0 to 7.0.

[0108] In one embodiment, the present invention provides a container filled with any of the immunogenic compositions or recombinant Cyanobacteria disclosed herein. In one embodiment, the container is selected from the group consisting of a vial, a syringe, a flask, a fermentor, a bioreactor, a bag, a jar, an ampoule, a cartridge, and a disposable pen.

[0109] In an embodiment, the container of the present invention is made of glass, metals (e.g., steel, stainless steel, aluminium, etc.) and / or polymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers). In an embodiment, the container of the present invention is made of glass.

[0110] In one embodiment, the present invention provides a syringe filled with any of the immunogenic compositions, recombinant Cyanobacteria (or lysates thereof) disclosed herein.

[0111] A typical dose of the immunogenic composition of the invention for injection has a volume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1 mL.

[0112] Therefore the container or syringe as defined above is filed with a volume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1 mL, of any of the immunogenic compositions, recombinant Cyanobacteria (or lysates thereof) defined herein.

[0113] In one embodiment, for nasal or oral administration of a Cyanobacterial lysate each dose comprises from 1 - 1000 mg of lysate (which includes the VLP), for example, about 1 , 2.5, 5, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or at least 1000 mg per dose.

[0114] In another embodiment, for administration (e.g. intramuscular injection) of purified VLPs each dose comprises from 1 pg - 10mg of purified VLP, for example, about 1, 5, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 200, 250, 300, 2350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000pg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg or at least 10mg of purified VLP, or more per dose.Uses

[0115] In an embodiment, the recombinant Cyanobacteria and immunogenic compositions disclosed herein are for use as a medicament.

[0116] The immunogenic compositions described herein may be used in various therapeutic or prophylactic methods for preventing, treating or ameliorating an infection, disease or condition in a subject. In particular, immunogenic compositions described herein may be used to prevent, treat or ameliorate an infection, disease or condition in a subject. The infection, disease or condition is one that is associated with organism from which the antigen that forms part of the engineered VLP is derived.

[0117] Thus in one aspect, the invention provides a method of preventing, treating or ameliorating an infection, disease or condition associated with the malaria parasite in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention wherein the immunogenic composition comprises the malaria CSP (circumsporozoite protein) antigen.

[0118] In some embodiments, the infection, disease or condition is selected from the group consisting of malaria, SARS-CoV-2, influenza, Pneumococcal disease, diphtheria, dengue, hepatitis, Hib (Haemophilus influenzae type b) HPV (human papillomavirus), measles, meningococcal disease, Mpox, mumps, polio (poliomyelitis), rotavirus, RSV (respiratory syncytial virus), rubella (German measles), shingles (herpes zoster), tetanus, whooping cough (pertussis), adenovirus, anthrax, cholera, Japanese encephalitis (JEV), rabies, smallpox, tuberculosis, typhoid fever, yellow fever, Zika, Chikungunya, Lyme disease, MERS, herpes simplex, HIV, Ross River Virus, West Nile Fever, Ebola, Lassa Fever, Nipah, and Rift Valley Fever.Subjects

[0119] The immunogenic compositions described herein may be used in various therapeutic or prophylactic methods for preventing, treating or ameliorating an infection, disease or condition in a subject. In a preferred embodiment, said subject is a human.

[0120] In an embodiment, the immunogenic compositions disclosed herein are for use as a vaccine.

[0121] In some cases, as little as one dose of the immunogenic composition according to the invention is needed, but under some circumstances, a second, third, fourth or further doses may be given.Regimen

[0122] In some cases, as little as one dose of the immunogenic composition according to the invention is needed, although typically a prime / boost strategy is used. In some embodiments a second, third, fourth or further dose may be given. Following an initial administration (vaccination), subjects can receive one or several adequately spaced booster administrations or immunizations.

[0123] In an embodiment, the schedule of vaccination of the immunogenic composition according to the invention is a single dose.

[0124] In an embodiment, the schedule of vaccination of the immunogenic composition according to the invention is a multiple dose schedule. In a particular embodiment, the multiple dose schedule consists of a series of 2 doses separated by an interval of about 1 week to about 2 months. In a particular embodiment, said multiple dose schedule consists of a series of 2 doses separated by an interval of about 2 weeks, or a series of 2 doses separated by an interval of about 1 month.

[0125] In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 week to about 2 months. In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 2 weeks, or a series of 3 doses separated by an interval of about 1 month.

[0126] In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 week to about 2 months followed by a fourth dose about 10 months to about 14 months after the first dose. In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 or 2 weeks followed by a fourth dose about 10 months to about 14 months after the first dose, or a series of 3 doses separated by an interval of about 1 month followed by a fourth dose about 10 months to about 14 months after the first dose.

[0127] In an embodiment, a prime dose is given at day 0 and one or more boosts are given at intervals that range from about 1 to about 24 weeks, preferably with a dosing interval of 1-8 weeks.

[0128] The prime dose may be administered intramuscularly and the boost doses administered orally and / or nasally, for example an intramuscular prime with an oral boost, a nasal boost or a combination of oral and nasal boost.

[0129] In an alternate embodiment the prime dose may be administered orally and the boost doses administered orally and / or nasally, for example all oral dosing or a oral prime and at least one nasal boost.

[0130] In an alternate embodiment the prime dose may be administered nasally and the boost doses administered orally and / or nasally, for example all nasal dosing or a nasal prime and at least one oral boost.

[0131] In embodiments involving oral administration, no or minimal formulation of the cyanobacteria is necessary. As the Cyanobacteria are non-toxic and can be safely ingested, it is sufficient to the recombinant Cyanobacteria or lysate thereof to be administered orally to the subject and do not necessarily need to be formulated with excipients before administration. Preferably the recombinant Cyanobacteria may be separated from their culture media, for example by centrifugation or filtration before being washed.

[0132] Similarly, a lysate of the recombinant Cyanobacteria does not need further formulation, although in preferred embodiments particulate material is removed from the lysate.

[0133] In some embodiments a lysate of the recombinant Cyanobacteria may be administered using a nasal spray device.Advantages

[0134] The invention provides a number of advantages which may include one or more of the following:• Oral / nasal delivery of whole-cells expressing chimeric VLPs lowers production and deployment costs• The compositions are thermostable thereby reducing dependence of cold-chain logistics and reducing costs.• In some embodiments the compositions comprise live or freeze-dried recombinant Cyanobacteria which can be used as a vaccine with minimal processing or purification, for example taken as whole-cell food or supplement• the compositions are safe as they include non-pathogenic / non-immunogenic Cyanobacteria, which do not contain problematic bacterial endotoxins• The engineered VLPs and recombinant Cyanobacteria can be produced quickly which provides an efficient and adaptable system for responding to emerging virus threats & mutations.• the compositions are cost effective to produce and distribute as they avoid coldchain logistics which are estimated to contribute approximately half of the total delivery costs for vaccinations• Rapid production of large amounts of VLP containing cyanobacteria. For example, a 180L photobioreactor has reached 12 g / L in 7 days and would produce -432,000 doses per 180L panel per week at 100pg VLP / dose or 10.8M doses per 180L panel per week at 4pg of purified VLP / dose.

[0135] The invention is illustrated in the accompanying examples. The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention.ExamplesExample 1 : Construction of engineered VLP and recombinant CyanobacteriaDNA sequences were designed encoding the Woodchuck Hepatis ‘core’ protein antigen (WHcAg) with (NANP-VLP) and without (‘Naked’ VLP) the ‘control antigen’ - a x15-NANP peptide repeat from the CSP protein of the malaria parasite Plasmodium. The ‘control antigen’ was inserted in the Major Immunodominant Region (MIR) between AA78 & AA79 of the ‘CoreC’ section of WHcAg (see Figure 1A). Sequences were codon-optimised for Synechococcus and split into sections for DNA synthesis (IDT). Integrating plasmids encoding synthetic landing zones (as described in PCT / AU2021 / 051121) were amplified by high-fidelity PCR and plasmids constructed via Gibson Assembly and transformed into competent E. coli via heatshock transformation. E.coli clones were screened via cPCR and length-validated against target sequences. Plasmids from overnight E.coli cultures were purified by filter column (Qiagen) and sequence-confirmed via NGS. Chromosomal VLP cassettes were integrated in cyanobacterial chromosomes (or native plasmid regions) via homologous recombination (as per PCT / AU2021 / 050300). Candidate VLP-expressing Synechococcus clones were screened by cPCR, length-validated against target sequences, and chromosomal insertions were sequence-confirmed via NGS of relevant PCR-amplified chromosomal regions.Example 2: Toxicity testing

[0136] Mice were orally administered 20mg Synechococcus lysed biomass or nasally administered 2.5mg of Synechococcus lysed biomass. A second oral administration of 20mg lysed biomass was given after 14 days to these two groups of mice. Negative control mice were left untreated for the duration.

[0137] The results are shown in Figure 2 and Tables 2 and 3Table 2: Oral prime, oral boost. Clinical signs after administration of Synechococcus lysed biomass1 Activity: Normal I Isolated I Inactive2 Posture: Normal / Hunched / Prostrate3 Coat: Normal / Mild pilo-erection / Severe pilo-erection4 Breathing: Normal / Rapid / LabouredOR Oral Boost (Day 14)Table 3: Nasal prime, oral boost. Clinical signs after administration of Synechococcus lysed biomass1 Activity: Normal I Isolated I Inactive2 Posture: Normal / Hunched / Prostrate3 Coat: Normal / Mild pilo-erection / Severe pilo-erection4 Breathing: Normal / Rapid / LabouredOR Oral Boost (Day 14)Example 3: VLP Delivery and preclinical Trials

[0138] Six groups of three mice each were selected to receive either purified VLPs, lysates of the recombinant Cyanobacteria, or whole cell undisputed cyanobacteria.

[0139] Prime doses were administered on day 0 followed by Boost doses at two weeks after the prime doses. Blood sera drawn at 2 weeks (pre-boost) and at 4 weeks (i.e. 2 weeks post-boost).

[0140] The formulations and administration routes are described in Table 4 and the dosing regime in Table 5Table 4: Formulations and administration routesTable 5: Dosing regime

[0141] Blood sera were taken at 28 days. Immunized and non-immunized mouse serum was serially diluted from 1 / 25 to 1 / 128000, tested by ELISA with a 6XNANP peptide antigen and endpoint IgG titres calculated.

[0142] These tests indicate that strong and comparable immune responses were generated for the following prime / boost regimens: IM / IM, IM / IN, IM / OD, IN / IN.

[0143] This establishes that intranasal (IN) and oral lysate (OD) delivery is effective to boost an IM prime and elicited comparable IgG responses (Titre (Logw): 3.8, 3.6) to IM / IM (3.9).

[0144] The IM / OD 28 day IgG response improved on the 14 day 'first bleed' following IM prime. A much greater IgG response was detected for IM / IM in this trial, compared to our first preclinical work.

[0145] These results demonstrate that 'needle-free' delivery, with a nasal / nasal (prime / boost) elicits an immune response having an IgG titre of 3.5 (Logw) which is equivalent to the IM / OD regimen (3.6).

[0146] The nasal / oral(disrupted) and nasal / oral(whole cell) regimens also produced immune responses (Titres 2.6 (Logw), 2.6 (Logw) respectively)

[0147] With reference to Figure 3D, Table 6 contains the data for endpoint IgG titre at 28 days as measured by ELISA with a control peptide antigen. Pre-bleed negative controls were used to define a statistically defined (95% confidence) cut-off for every dilution as outlined by Frey et. al. (J Immunol Methods. (1998); 221(1-2):35-41). Individual mouse sera titres (#1-3) show the median titre of x3 technical replicates of the highest dilution above the cut-off. The endpoint IgG titre shown is the average of the x3 individual mouse sera titres for each prime / boost regimen.Table 6: Endpoint IgG titre at 28 days measured by ELISA with control peptide antigenExample 4: Cultivation densities

[0148] Synechococcus PCC 7002 cultures were grown in MAD media at 33°C in 2-10% CO2 under 300-1 OOOuE of light for x7 days. Optical density (OD750) and biomass samples were taken every 24 hrs. Media salts were removed from biomass samples (500uL) by washing (x3 times) via centrifugation & resuspension in sterile water, in pre-weighed microtubes (1.5mL) before being lyophilised by freeze-drying and weighed to establish culture density (g / L).

[0149] Growth rates and cultivation densities are shown in Figure 4.Example 5: VLP Delivery and Preclinical Trial #2

[0150] Five groups of three mice each were selected to receive either purified VLPs, lysates of the recombinant Cyanobacteria, or whole cell undisputed cyanobacteria. For the IM Prime, doses were administered on day 0 followed by x1 Boost dose at three weeks after the prime dose. For the IN Prime followed by IN Boost, doses were administered on day 0 followed by x1 IN Boost dose at three weeks after the prime dose. For the IN Prime followed by oral boost, doses were administered on day 0 followed by x2 oral Boosts (OW or OD) dose at three weeks and six weeks after the prime dose. Blood sera was drawn at 2 and 3 weeks (pre-boost), at 5 weeks (i.e. 2 weeks post-boost), 6 weeks (i.e. 3 weeks postboost), 8 weeks and 9 weeks (Endpoint).

[0151] The formulations and administration routes are described in Table 7 and the dosing regimen in Table 8Table 7: Formulations and administration routesTable 8: Dosing regimen

[0152] Blood sera were taken at 63 days. Immunized and non-immunized mouse serum was serially diluted to 1 / 800, tested by ELISA with a 6XNANP peptide antigen and endpoint antigen-specific IgG concentrations were calculated against an in-plate antibody standard (Mouse anti-NANP IgG 2A10), scaling for the 1 / 800 dilution of the undiluted sera.

[0153] These tests indicate that strong and comparable immune responses were generated for the following prime / boost regimens: IM / IM, IN / IN, IN / OW

[0154] This establishes that intranasal (IN) and oral Whole Cell (OW) delivery is effective to boost an IN prime and elicited comparable IgG responses (51.4 pg / mL, 14 pg / mL) to IM / IM (66.3 pg / mL).

[0155] The IN / IN (Group C), IN / OW (Group D) and IN / OD (Group E) Day 63 IgG response improved on the Day 63 single IM prime (Group B).

[0156] These results demonstrate that 'needle-free' delivery, with a IN / IN (prime / boost) and IN / OW (prime / boost) elicit strong immune responses, having an IgG concentration of 51.4 pg / mL and 14 pg / mL respectively, which is equivalent to the IM / IM regimen (66.3 pg / mL).

[0157] The nasal / oral(disrupted) regimen also produced an immune response (4.1 pg / mL)

[0158] With reference to Figure 5C, Table 9 contains the data for endpoint IgG concentration at 63 days as measured by ELISA with a control peptide antigen. Endpoint antigen-specific IgG concentrations were calculated against an in-plate antibody standard (Mouse anti-NANP IgG 2A10). Individual mouse sera IgG concentrations (#0-2) are shown (mean of x3 technical replicates), alongside the mean IgG concentration of x3 mice withinthe. The mean endpoint IgG concentration shown is the average of the x3 individual mouse sera concentrations for each prime / boost regimen. Mice where no immune response was detected (vs the pre-bleed control) are indicated as “NRD”, No Response Detected.Table 9: Endpoint IgG concentration at 63 days measured by ELISA with control peptide antigen*NRD -No Response Detected

Claims

Claims:

1. An immunogenic composition comprising a recombinant Cyanobacteria or lysate thereof wherein the recombinant Cyanobacteria comprises a nucleic acid encoding at least one VLP protein and at least one heterologous antigen, wherein the expressed VLP protein comprises the at least one heterologous antigen.

2. The immunogenic composition of claim 1, wherein the VLP protein comprises a structural protein of a virus3. The immunogenic composition of claim 2, wherein the structural protein is one or more of a capsid or envelope protein4. The immunogenic composition of claim 3, wherein the sequence encoding the heterologous antigen is within the sequence encoding the capsid or envelope protein.

5. The immunogenic composition of claim 3, wherein the sequence encoding the heterologous antigen is 5' or 3' and in frame with the sequence encoding the capsid or envelope protein.

6. The immunogenic composition of any one of claims 1 to 5, wherein the nucleic acid further comprises a linker sequence between the sequence encoding the heterologous antigen and the capsid or envelope protein.

7. The immunogenic composition of any one of claims 1 to 6, wherein the nucleic acid further comprises a sequence encoding a targeting peptide8. The immunogenic composition of claim 7, wherein the targeting peptide is a Sec or Tat signal peptide for targeting the VLP protein to a thylakoid membrane9. The immunogenic composition of any one of claims 1 to 9, wherein the heterologous antigen is an antigen from one or more of malaria, SARS-CoV-2, influenza, Pneumococcal disease, diphtheria, dengue, hepatitis, Hib (Haemophilus influenzae type b) HPV (human papillomavirus), measles, meningococcal disease, Mpox, mumps, polio (poliomyelitis), rotavirus, RSV (respiratory syncytial virus), rubella (German measles), shingles (herpes zoster), tetanus, whooping cough (pertussis), adenovirus, anthrax, cholera, Japanese encephalitis (JE), rabies, smallpox, tuberculosis, typhoid fever, yellow fever, Zika, Chikungunya, Lyme disease, MERS, herpes simplex, HIV, Ross River Virus, West Nile Fever, Ebola, Lassa Fever, Nipah, and Rift Valley Fever.

10. The immunogenic composition of any one of claims 1 to 9, wherein the recombinant Cyanobacteria further comprises one or more nucleic acid sequences encoding an adjuvant, or an enzyme for the biosynthesis of an adjuvant.

11. The immunogenic composition of claim 10, wherein the adjuvant is a terpene adjuvant.

12. The immunogenic composition of claim 10, wherein the terpene adjuvant is squalene, dehydroisosqualene, farnesene thermal dimer, nonaprenol (solanesol), difarnesyl ether, geranyl diphosphate, farnesyl diphosphate, geranyl geranyl diphosphate, isopentenyl diphosphate, or derivates thereof.

13. The immunogenic composition of claim 10, wherein the enzyme is squalene synthase and the adjuvant is squalene.

14. The immunogenic composition of any one of claims 1 to 9 wherein the recombinant cyanobacteria comprises an inactivating mutation in the endogenous squalene hopene cyclase gene.

15. The immunogenic composition of any one any one of claims 1 to 12, comprising or consisting of the recombinant Cyanobacteria, or a lysate of the recombinant Cyanobacteria.

16. The immunogenic composition of any one of claims 1 to 12, wherein the composition is in an aqueous form or a dried form17. The immunogenic composition of any one of claims 1 to 16 wherein the Cyanobacteria is not Spirulina or Arthospira and is from the order Chroococcales or Synechococcales.

18. The immunogenic composition of claim 17, wherein the Chroococcales is Synechococcus sp. PCC 7002 or Synechocystis sp. PCC 680319. The immunogenic composition of claim 17, wherein the Synechococcales is Synechococcus elongatus PCC 794220. The immunogenic composition of claim 17, wherein the Cyanobacteria is selected from the group consisting of , Synechococcus sp.PCC 11901 , Synechococcus sp. LITEX 2434, Synechococcus sp. UTEX 2973, Synechococcus sp. UTEX 3153, and Synechococcus sp. II TEX 3154.

21. The immunogenic composition of any one of claims 1 - 20, wherein the nucleic acid is integrated into the genome of the Cyanobacteria or is in an extra-chromosomal plasmid.

22. The immunogenic composition of claim 21, wherein the extra-chromosomal plasmid is a native plasmid.

23. A dietary supplement containing the immunogenic composition of any one of claims 1 to 22.

24. A method of eliciting an immune response in subject comprising administering to the subject an effective amount of a composition of any one of claims 1 to 22, wherein the immune response is specific for the heterologous antigen.

25. The method of claim 24, wherein the method comprises administering a first dose of the composition and a second dose of the composition.

26. The method of claim 25, wherein the method comprises administering at least one additional dose.

27. The method of claim 25 or 26, wherein the first dose is administered orally, nasally or by intramuscular injection.

28. The method of any one of claims 25 to 27, wherein the second dose is administered orally, nasally or by intramuscular injection.

29. The method of any one of claims 26 to 28, wherein the at least one additional dose is administered orally, nasally or by intramuscular injection.

30. The method of any one of claims 25 to 29 wherein the second dose is administered from 1 week to 2 months after the first dose.

31. The method of any one of claims 26 to 30 wherein the at least one additional dose is administered 6 to 18 months after the second dose.

32. The method of any one of claims 25 to 31 wherein the oral administration comprises the subject ingesting the dietary supplement of claim 14.

33. The method of any one of claims 24 to 32 wherein the immune response protects the subject from infection, or lessens the severity of a disease caused by, by the organism from which the heterologous antigen is derived.

34. Use of a composition of any one of claims 1 to 22 for the manufacture of a medicament for eliciting an immune response in subject, wherein the immune response is specific for the heterologous antigen.

Images