WO2021210984A1

WO2021210984A1 - Coronavirus vaccine

Info

Publication number: WO2021210984A1
Application number: PCT/NL2021/050249
Authority: WO
Inventors: Bartholomeus Leonardus Haagmans; Berend Jan Bosch
Original assignee: Erasmus University Medical Center Rotterdam; Universiteit Utrecht Holding B.V.
Priority date: 2020-04-17
Filing date: 2021-04-19
Publication date: 2021-10-21

Abstract

The present invention provides inter alia a self-assembling nanoparticle that displays on the outer surface a receptor binding domain (RBD) of a coronavirus spike (S) protein. The present prevention also relates to vaccine formulations and methods for therapeutic and prophylactic interventions for coronaviral infection, more in particular SARS-CoV-2, the causal agent of COVID-19.

Description

Title: Coronavirus vaccine

FIELD OF THE INVENTION The invention is in the field of medicine, and relates to methods and products for providing immunity against viral disease. In particular, the present prevention relates to vaccine formulations and methods for therapeutic and prophylactic interventions for coronaviral infection, more in particular SARS-CoV-2, the causal agent of COVID-19.

BACKGROUND OF THE INVENTION

Given the current global emergency created by the Coronavirus disease 2019 (COVID-19) pandemic due to SARS-CoV-2, there is an unprecedented need for methods for treatment and prevention of coronalviral infection. Viral inhibitors including nucleotide analogs and protease inhibitors are currently tested in clinical trials, as well as recombinant human ACE2 to neutralize the virus and prevent lung damage. Another curative option is the use of convalescent serum. Despite the lack of clinical results for any of these therapeutic interventions, compassionate use of some drugs has already been reported for SARS-CoV-2 infections.

In addition to therapeutic methods, there is a critical need for prophylactic methods of controlling the disease, in particular in the form of vaccines. The need for coronavirus vaccines has in fact never been higher since the SARS-CoV-1 and MERS-CoV outbreaks in in 2003—2004 and 2012- 2015, respectively. As of 2020 there is no specific vaccine or treatment for either of these diseases, and it will take critical time to develop such vaccines. The difference in the management of the periodic influenza virus pandemics is the complete lack of vaccines or production processes for coronavirus vaccines. While each influenza vaccine is essentially a change of strain within an established and approved processes, with established release criteria, and existing correlates of protection, vaccine producers have no pipeline. Moreover, we are facing a new challenge in the form of a virus that has just emerged in humans, and the response will be more complex.

Vaccine technology has significantly evolved in the last decade, including the development of several RNA and DNA vaccine candidates, licensed vectored vaccines (e.g., Ervebo, a vesicular stomatitis virus [VSV]- vectored ebolavirus vaccine), recombinant protein vaccines (e.g., Flublok, an influenza virus vaccine made in insect cells), and cell-culture-based vaccines (e.g., Flucelvax, an influenza virus vaccine made in mammalian cells). It is known from studies on SARS-CoV-1 and the related MERS-CoV vaccines that the S protein on the surface of the virus is an ideal target for a vaccine. In SARS-CoV-1 and SARS-CoV-2, this protein interacts with the receptor ACE2, and antibodies targeting the spike can interfere with this binding, thereby neutralizing the virus. The structure of the S protein of SARS-CoV- 2 was solved in record time at high resolution, contributing to our understanding of this vaccine target. Hence, a target antigen exists that can be incorporated into advanced vaccine platforms.

Several vaccines for SARS-CoV-1 and MERS-CoV have been developed and tested in animal models, including inactivated, live- attenuated, and vectored vaccines. Many of these include recombinant S- protein-based vaccines. Most of these vaccines protect animals from challenge the virus, leading to greater survival, reduced virus titers, and/or less morbidity compared with that in unvaccinated animals. However, vaccination with the live virus may result in serious complications, or even enhancement of disease. Certain epitopes on the S protein may be protective, whereas immunity to others seemed to be enhancing disease. It is clear that global use of vaccines for SARS-CoV-2, need to be sufficiently safe. Many SARS-CoV-1 and MERS-CoV vaccines have not made it to clinical trials and are as such untested, because extinction of the virus from the human population also removed funding possibilities. Based on current research, MERS-CoV neutralizing antibodies (Abs) mainly recognize epitopes in the MERS-CoV spike (S) protein. The S protein consists of Si subunit (the N-terminal head) and an S2 subunit (the C-terminal stalk). The Si subunit mediates virus attachment and entry through its N-terminal Si A domain (comprising sialic acids, a viral attachment factor), and its C-terminal receptor binding domain (RBD), which binds to the viral receptor dipeptidyl peptidase 4 (DPP4) in the case of MERS and ACE2 receptor in the case of SARS. The S2 subunit is more conserved and mediates viral fusion to the host cell through the fusion peptide (FP) and the two heptad repeats HR1 and HR2. MERS-CoV neutralizing Abs mainly recognize epitopes in the RBD of the spike head Si subunit; and to a lower extent, epitopes in the sialic acid binding domain and the fusion-mediating S stalk S2 subunit. This makes RBD a preferred aspect for subunit vaccine production against MERS.

SUMMARY OF THE INVENTION

The present inventors have now obtained experimental evidence showing immunogenicity and protective capacity in animal studies against MERS-CoV infection for a recombinant subunit protein vaccine developed for MERS. The inventors presented the RBD subunit of the S protein on a protein scaffold. An advantage of such a recombinant subunit protein vaccine is its higher safety profile, its speed and ease of production, and its scale-up possibilities, relative to other vaccine platforms. Although in general, a lower immunogenicity may be associated with these subunit protein vaccine types, the present inventors have found that the use of self assembling multimeric protein scaffold particles (MPSP) as the presenting scaffold presents the subunit antigens in a multivalent virus -mimicking manner.

The multimeric self-assembling display of CoV RBD improves stability and immunogenicity of the RBD antigen, resulting in a focused and strong immune response with high induced titers of CoV neutralizing antibodies. Two approaches have been validated in ZAPI, with the non- enzymatic coupling and in-vitro assembly of the antigens using bacterial superglue SpyTag (ST) / SpyCatcher (SC) and with the genetic fusion and in-cell assembly of the antigens using multimeric protein scaffold particle Lumazine- Synthase (MPSP LS). The latter approach has achieved proof-of- concept with the preclinical evaluation of the MERS-RBD multimeric vaccine in rabbit immunization and challenge experiments. The RBD-LS vaccine candidate was shown to induce higher MERS-CoV neutralizing antibody titers than RBD non fused to LS and abrogated MERS-CoV replication.

By fusing either SpyTag or SpyCatcher to self-assembling molecules such as virus-like particles, antigens fused to the other pair can be decorated onto the molecule via the isopeptide bond formed. This enables fast production of vaccines as the central self-assembling molecule can be stocked up beforehand, whilst the antigen can be easily produced under optimal conditions to achieve proper protein folding.

In a first aspect, the present invention provides a self-assembling protein nanoparticle that displays on the outer surface a receptor binding domain (RBD) of a coronavirus spike (S) protein.

In a preferred embodiment of this aspect, said nanoparticle is a multimeric protein particle that self-assembles by multimerization of a protein monomer.

In another preferred embodiment of this aspect, said nanoparticle is a multimeric protein particle that self-assembles by homomultimerization of a protein monomer.

In another preferred embodiment of this aspect, said nanoparticle is a synthetic or artificial nanoparticle.

In another preferred embodiment of this aspect, said nanoparticle is non-viral and/or not a virus. In another preferred embodiment of this aspect, said nanoparticle is a 60-meric particle.

In another preferred embodiment of this aspect, said nanoparticle is a 60-meric particle with an icosahedral symmetry.

In another preferred embodiment of this aspect, said nanoparticle is a multimeric protein scaffold particle (MPSP).

In another preferred embodiment of this aspect, said nanoparticle comprises a multimer of a protein monomer that is selected from the group formed by an 13-01 protein monomer and a lumazine synthase protein monomer.

In another preferred embodiment of this aspect, said said 13-01 protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:2 or 17 and/or wherein said lumazine synthase protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:3, or sequences having at least 90%, preferably at least 95% sequence identity therewith and self-assemble by multimerization into a protein nanoparticle.

In another preferred embodiment of this aspect, said said RBD comprises or consists of:

- an amino acid sequence selected from SEQ ID NOs:l, 7, 13 and 28;

- an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:l, 7, 13 or 28, and which preferably binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle of the present invention as described above wherein said RBD comprises or consists of an amino acid sequence of SEQ ID NO:l, 7, 13 or 28, respectively; and/or

- an antigenic part of the amino acid sequence of SEQ ID NO:l, 7, 13 or 28 which preferably binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle of the present invention as described above wherein said RBD comprises or consists of an amino acid sequence of SEQ ID NO:l, 7, 13 or 28, respectively.

In another preferred embodiment of this aspect, said nanoparticle displays said RBD multivalently.

In another preferred embodiment of this aspect, each protein monomer in said self-assembling nanoparticle is coupled to an RBD thereby multivalently displaying said RBD on the outer surface of said nanoparticle.

In another preferred embodiment of this aspect, each protein monomer in said self-assembling nanoparticle is coupled to an RBD through

(i) a direct or indirect protein fusion between said protein monomer and said RBD; or

(ii) formation of an isopeptide bond between a SpyTag protein moiety that is directly or indirectly fused to said RBD and a SpyCatcher protein moiety that is directly or indirectly fused to said protein monomer.

In another preferred embodiment of this aspect, said RBD is indirectly fused to said protein monomer, preferably said RBD is indirectly fused to said protein monomer and separated by a linker protein such as a glycine/serine (GS) linker protein.

In another preferred embodiment of this aspect, said SpyTag protein moiety comprises or consists of the amino acid sequence as indicated in SEQ ID NO: 4 and/or wherein said SpyCatcher protein moiety comprises or consists of the amino acid sequence as indicated in SEQ ID NO:5 or SEQ ID NO:29.

In another preferred embodiment of this aspect, said SpyTag protein moiety is linked to said RBD through a glycine/serine (GS) linker and/or wherein said SpyCatcher protein moiety is linked to said protein monomer through a GS linker.

In another preferred embodiment of this aspect, said nanoparticle is obtainable by allowing (i) a protein that comprises or consists of the amino acid sequence of SEQ ID NO:30 and (ii) a protein that comprises or consists of the amino acid sequence of SEQ ID NO:31 to self-assemble into a nanoparticle that displays on the outer surface a receptor binding domain (RBD) of a coronavirus spike (S) protein, preferably wherein one or more, preferably each, protein monomer in said self-assembling nanoparticle is coupled to an RBD by allowing the formation of an isopeptide bond between said (i) protein that comprises or consists of the amino acid sequence of SEQ ID NO:30 and said (ii) protein that comprises or consists of the amino acid sequence of SEQ ID NO:31.

In another aspect, the present invention provides a fusion protein comprising a first part that is a receptor binding domain (RBD) of a coronavirus spike (S) protein and a second part that is a SpyTag protein moiety.

In a preferred embodiment of this aspect, said RBD comprises or consists of:

- an amino acid sequence of SEQ ID NO:l, 7, 13 or 28;

- an antigenic part of the amino acid sequence of SEQ ID NO: 1, 7 or 13 which preferably binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle of the present invention as described above wherein said RBD comprises or consists of an amino acid sequence of SEQ ID NO:l, 7, 13 or 28, respectively.

In another preferred embodiment of this aspect, said SpyTag protein moiety comprises or consists of the amino acid sequence as indicated in SEQ ID NO:4. In another preferred embodiment of this aspect, said first part and said second part are linked through a glycine/serine (GS) linker.

In another preferred embodiment of this aspect, said fusion protein further comprises a tag such as a Streptag or CTag.

In another preferred embodiment of this aspect, said RBD is located N-terminally relative to said SpyTag protein moiety.

In another preferred embodiment of this aspect, said fusion protein comprises or consists of the amino acid sequence of SEQ ID NO:30.

In another aspect, the present invention provides a fusion protein comprising a first part that is a receptor binding domain (RBD) of a coronavirus spike (S) protein and second part that is a protein monomer that is selected from the group formed by an 13-01 protein monomer and a lumazine synthase protein monomer; preferably wherein said RBD comprises or consists of

- an amino acid sequence of SEQ ID NO:l, 7, 13 or 28;

- an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:l, 7, 13 or 28, and which preferably binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle of the present invention as described above wherein said RBD comprises or consists of an amino acid sequence of SEQ SEQ ID NO:l, 7, 13 or 28, respectively; and/or

- an antigenic part of the amino acid sequence of SEQ ID NO:l, 7, 13 or 28 which preferably binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle as described above wherein said RBD comprises or consists of an amino acid sequence of SEQ ID NO:l, 7, 13 or 28, respectively.

In a preferred embodiment of this aspect, said 13-01 protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:2 or 17 and/or wherein said lumazine synthase protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:3.

In another preferred embodiment of this aspect, said first part and said second part are linked through a glycine/serine (GS) linker.

In another preferred embodiment of this aspect, said fusion protein further comprises a tag such as a Streptag.

In another aspect, the present invention provides a nucleic acid molecule encoding the fusion protein according to any one of the present invention as described above.

In another aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the present invention as described above.

In another aspect, the present invention provides a host cell comprising the nucleic acid molecule or expression vector according to the invention. The host cell may be a bacterial cell or an eukaryotic cell. For production of the RBD, the host cell is preferably a eukaryotic host cell, preferably selected from a fungal cell, a yeast cell or a mammalian cell such as a HEK293(T) cell, still more preferably a fungus cell. For production of the 13-01 protein monomer or the lumazine synthase protein monomer, the host cell is preferably a bacterial cell. When using such host cell, corresponding expression systems are used, such that a bacterial expression system is used in a bacterial host cell and a fungus expression system is used in a fungus. Such expression systems are well known in the art.

In another aspect, the present invention provides a method of producing a self-assembling nanoparticle according to the present invention, comprising the steps of:

- providing a first fusion protein that is a fusion protein according to present invention as described above;

- providing a second fusion protein that is fusion protein comprising - a first part that is a protein monomer selected from the group formed by an 13-01 protein monomer and a lumazine synthase protein monomer, preferably wherein said 13-01 protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:2 or 17 and/or wherein said lumazine synthase protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:3; and

- a second part that is a SpyCatcher protein moiety, preferably wherein said SpyCatcher protein moiety comprises or consists of the amino acid sequence as indicated in SEQ ID NO:5 or SEQ ID NO:29;

- optionally wherein said first part and second part are linked through a glycine/serine (GS) linker;

- optionally further comprising a tag such as a Streptag;

- allowing said first and second fusion protein to self-assemble into a nanoparticle that displays on the outer surface a receptor binding domain (RBD) of a coronavirus spike (S) protein.

In another aspect, the present invention provides a method of producing a nanoparticle according to the present invention, comprising the steps of:

- providing a fusion protein according to the present invention as described above;

- allowing said fusion protein to self-assemble into a nanoparticle that displays on the outer surface a receptor binding domain (RBD) of a coronavirus spike (S) protein.

In another aspect, the present invention provides a self assembling nanoparticle obtainable by a method according to the present invention as described above.

In another aspect, the present invention provides an immunogenic composition comprising a self-assembling nanoparticle according to the present invention as described above and a pharmaceutically acceptable carrier, preferably an aqueous liquid.

In a preferred embodiment of this aspect, said immunogenic composition further comprising a pharmaceutically acceptable adjuvant such as an alum, for instance aluminium hydroxide.

In another aspect, the present invention provides a method of preventing or treating an infectious disease caused by coronavirus, preferably a MERS or COVID such as COVID-19, in a subject, comprising the step of:

- administering a therapeutically effective amount of a self-assembling nanoparticle according to the present invention as described above or an immunogenic composition according to the present invention as described above.

In another aspect, the present invention provides a method of immunizing a subject, comprising the step of:

- administering (an effective amount) a self-assembling nanoparticle according to the present invention as described above or an immunogenic composition according to the present invention as described above.

In a preferred embodiment of this aspect, said self-assembling nanoparticle or said immunogenic composition is for administration according to a prime boost immunization schedule.

In another aspect, the present invention provides a nanoparticle according to the present invention as described above, the fusion protein according to the present invention as described above, or the immunogenic composition according to the present invention as described above, for use as a medicament, preferably for use in (i) treating or preventing an infectious disease caused by coronavirus, preferably a MERS or COVID such as COVID-19, in a subject or (ii) immunizing a subject.

In another aspect, the present invention provides the use of the nanoparticle according to the present invention as described above, the fusion protein according to the present invention as described above, or the immunogenic composition according to the present invention as described above, for use in the manufacture of a medicament for treating or preventing an infectious disease caused by coronavirus, preferably a MERS or COVID such as COVID-19, in a subject.

The invention also provides a protein comprising an amino acid sequence selected from the group formed by SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 25 and SEQ ID NO: 26. The invention also provides a composition (or a combination or a set) comprising a first protein that comprises an amino acid sequence of SEQ ID NO: 19, and a second protein that comprises an amino acid sequence of SEQ ID NO:25 or SEQ ID NO:26.

In aspects of this invention the term “a receptor binding domain (RBD) of a coronavirus spike (S) protein” refers only to the RBD portion of the full coronavirus spike (S) protein. Preferably, a nanoparticle of the invention displays on the outer surface only a receptor binding domain (RBD), or an antigenic part thereof, of said coronavirus spike (S) protein. In other words, in preferred embodiments, only a receptor binding domain (RBD), or an antigenic part thereof, is displayed by said nanoparticle and thus not (full length) coronavirus spike (S) protein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the generation of multimeric protein scaffold particles (MPSP)- based vaccines of this invention. (A) Schematic diagram of the MERS-CoV spike (S) protein mapping regions selected for vaccine generation; the receptor binding domain (RBD), the fusion peptide (FP) and heptad repeat 2 (HR2). (B, C) Schematic diagram illustrating the construct design and production of the lumazine synthase (LS) and 13-01 (I3)-based self-assembling MPSP vaccines. (D) Reducing SDS-PAGE showing generation of RBD-LS by covalent coupling of RBD-SpyTag (RBD-ST) and LS-SpyCatcher (LS-SC) at different molar ratios of LS-SC: RBD-ST with the last two lanes showing each in its free (uncoupled) form. (E) Reducing SDS- PAGE analysis of immunogens used in this study. The size of each protein (KDa) is given in Table 1. * Fuzzy bands due to heterogeneous glycosylation of HR2 or RBD.

Figure 2 shows the immunogenicity of MERS-CoV spike MPSP vaccines. (A) Vaccination scheme for rabbit immunizations. Six groups of rabbits (5/group) were vaccinated in a prime/boost regimen with 15 pg of adjuvanted vaccine at 4-week interval and challenged with MERS-CoV (EMC strain; accession no. NC_019843) 3 weeks post-boost. Anti-MERS-CoV spike S2 (B) and Si (D) IgG titers measured by ELISA in rabbits at different time points. Shown is the mean ± s.e.m. antibody titers from five rabbits per group. (C,E) MERS-CoV neutralizing antibody titers measured by a 90% reduction in a plaque reduction neutralization assay (PRNT90). (B-E)

Shown is the mean ± s.e.m. of five rabbits per group. (F,G) Vaccine-induced antibodies in nasal swabs of vaccinated rabbits. Anti MERS-CoV S2 (F) and Si (G) antibody responses in the nasal swabs (tested at a 1:50 dilution) of vaccinated rabbits pre-challenge (three weeks post-boost). The difference in antibody responses between monomeric (RBD+LS) and multimeric (RBD- LS) RBD was tested for statistical significance using a student’s t-test, with asterisks indicating the level of significance. *P < 0.05. Error bars indicate mean ± s.e.m. The dotted lines represent the limits of detection. HR2, heptad repeat 2; FP, fusion peptide; LS, lumazine synthase 60-meric particles; 13, 13-01 60-meric particles; RBD, receptor binding domain; RBD + LS, monomeric uncoupled RBD; RBD-LS, multimeric RBD coupled to LS through covalent SpyTag/SpyCatcher.

Figure 3 shows the avidity of vaccine-induced serum antibody responses. The avidity of serum IgG antibody responses after one (A, Day 28) and two immunizations (B, Day 46) with either monomeric RBD (RBD+LS, blue, n=5) or multimeric RBD (RBD-LS, green, n=5) was assessed using ammonium thiocyanate (SCN) avidity ELISA. (A, B) The percentage of serum antibodies bound following the addition of different concentration of SCN was used to determine (C) the avidity index (IC50). The difference in serum avidity between both groups was tested for statistical significance using a student’s t-test, with asterisks indicating the level of significance. ***p < 0.001, ****p < 0.0001. Error bars indicate mean ± s.e.m.

Figure 4 shows the anti- scaffold antibody responses in sera of vaccinated rabbits. Anti-lumazine synthase (LS) scaffold antibody titers following (A) homologous prime boost in monomeric RBD+LS and multimeric RBD-LS vs heterologous LS/I3 prime boost in control LS/I3 as well as (B) HR2-LS/I3 and FP-LS/I3. Shown are (average ±s.e.m. of n=5 rabbit/group) antibody titers 4 weeks after prime (day 28, D28) and 3 weeks after boost (day 46, D46) as measured by ELISA. (C) Fold increase (from prime, day 28) in anti-LS antibody titers following boost vaccination (day 46). A paired t-test was performed to determine significant increases in antibody titers post-prime and post-boost within groups (A, B), and an unpaired t-test was performed to determine significant changes in titers between groups (C), with asterisks indicating the level of significance.*? < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The dotted lines represent the limits of detection. HR2, heptad repeat 2; FP, fusion peptide; LS, lumazine synthase 60-meric particles; 13, 13-01 60-meric particles; RBD, receptor binding domain; RBD + LS, monomeric uncoupled RBD; RBD-LS, multimeric RBD coupled to LS through covalent SpyTag/SpyCatcher.

Figure 5 shows the protective capacity of MERS-CoV MPSP vaccines against upper respiratory tract infection in rabbits. Six groups of vaccinated and control rabbits (n = 5/group) were tested for the presence of viral RNA (A, C) and infectious virus particles (B, D) in the upper respiratory tract (nasal swabs) at days -3 and 1-4 post intranasal viral challenge (days 46 and 50-53 post first vaccination) with 106 TCID50 MERS-CoV EMC strain. Shown is the average ± s.e.m. of five animals per group. The dotted lines represent the limits of detection. HR2, hepad repeat 2; FP, fusion peptide; LS, lumazine synthase 60-meric particles; 13, 13-01 60- meric particles; RBD, receptor binding domain; RBD + LS, monomeric uncoupled RBD; RBD-LS, multimeric RBD coupled to LS through covalent SpyTag/SpyCatcher.

Figure 6. Protective capacity of MERS-CoV MPSP vaccines against lower respiratory tract infection in rabbits. Six groups of vaccinated and control rabbits (n = 5/group) were tested for the presence of viral RNA (A, B) in the lung tissue homogenates and viral nucleocapsid antigen in lung tissues (C) collected 4 days post intranasal viral challenge with 106 TCID50 of MERS-CoV (EMC isolate). (A, B) Shown are the average and SEM equivalent virus titers/gram of tissue. C) Representative pictures of immunohistochemical detection of MERS-CoV nucleoprotein (shown in red) in the lungs of PBS (left) vs RBD-LS (right) immunized rabbits four days post- viral challenge; the upper and lower panels show a 200X and 1000X magnification, respectively. HR2, hepad repeat 2; FP, fusion peptide; LS, lumazine synthase 60-meric particles; 13, 13-01 60-meric particles; RBD, receptor binding domain; RBD + LS, monomeric uncoupled RBD; RBD-LS, multimeric RBD coupled to LS through covalent SpyTag/SpyCatcher.

Fig. 7 shows the domains of the MERS-CoV spike protein that are presented on multimeric protein scaffold particles. Domains are color-coded: receptor binding domain (RBD, green), fusion peptide (FP, orange), Heptad repeat 2 (HR2, lilac).

Fig. 8 shows the amino acid sequences of protein constructs used in this study for immunization of rabbits. Different domains are color-coded: signal sequence (grey), lumazine synthase (LS, orange), 13-01 (13, pea green), Streptag (green), SpyTag (ST, light blue), SpyCatcher (SC, dark blue), MERS-FP (FP, lilac), MERS-HR2 (HR2, red), MERS-CoV RBD (RBD, purple). Fig. 9 shows the vaccine-induced MERS-CoV neutralizing antibodies in sera of vaccinated rabbits against the clade B Qatari 5 strain (GenBank accession no. MK280984.2) using plaque reduction neutralization assay (PRNT). The dotted line represents the lower limit of detection. Figure 10 shows non-enzymatic coupling between Mi3 and SARS-

CoV-2 RBD using SpyTag/SpyCather system. Assembly, and SDS-Page and Western blotting experiments of the constructs of the invention wherein the Mi3-SC was expressed in bacterial cells, and the SARS-CoV-2 RBD-ST was expressed in mammalian cells (HEK-293T), followed by in vitro coupling. SARS-CoV-2 RBD-ST was incubated with SC-Mi3 in different molar ratios to assess the optimal coupling of both components.

Figure 11 shows genetic fusion between Mi3 and SARS-CoV-2 RBD. In-cell assembly, and Western blotting experiments of expression of the SARS-CoV-2 RBS-Mi3 constructs of the invention in HEK-293T cells Figure 12 shows electron micrographs of the nanoparticles of the invention by non enzymatic coupling between Mi3 and SARS-CoV-2 RBD using the SpyTag/SpyCatcher system. Left: Mi3; Right RBD-ST coupled to SC-Mi3 in a 1:15 molar ratio.

Figure 13 shows the sequences of the expression constructs RBD- SpyTag (RBD-ST), SpyCatcher-Mi3 (SC-Mi3), and RBD-Mi3 genetic fusion for the SARS-CoV-2 nanoparticle according to the invention. Indicated are the signal sequence (SEQ ID NO: 14), the SARS-CoV-2 RBD sequence (SEQ ID NO:l), the linker regions (SGESG, GGGS, GGGSGGGSGGGS), two StrepTag sequences (SEQ ID NO: 12), SpyTag sequence (SEQ ID NO:4) for RBD-ST; the SpyCather sequence (SEQ ID NO:5 or SEQ ID NO:29), a linker region (GGSGGSGGSGGS), the mutant Ϊ3-01 (Mi3) sequence (SEQ ID NO:2), a linker region (GSG), a C-tag sequence (SEQ ID NO: 15) for SC-Mi3; and the signal sequence (SEQ ID NO: 14), the SARS-CoV-2 RBD sequence (SEQ ID NO:l), a linker region (GGSGGSGGSGGS), the mutant i3-01 (Mi3) sequence (SEQ ID NO:2), a linker region (GSG), and a StrepTag sequence (SEQ ID NO:12).

Figure 14 shows the receptor Binding Domain within the SARS- CoV-2 S protein and antigen minimization aimed at focused immune response.

Figure 15 shows the multimeric self-assembling vaccine of the invention wherein the SARS-CoV-2 RBD is displayed on the scaffold.

Figure 16 shows the multivalent antigen display: non-enzymatic coupling using SpyTag/SpyCatcher in the embodiment for the MERS-CoV antigen

Figure 17 shows the Multivalent antigen display method of the invention in the embodiment for MERS-CoV RBD wherein the coupling is arranged through a SpyTag/SpyCatcher system (left) or a Genetic fusion using LS as the self-assembling protein for display. The advantages of the multimeric RBD vaccines are indicated as a focused immune response, an increased stability and immunogenicity, a higher titer of neutralizing antibodies, a lower vaccine antigen dose, and easier production of the vaccine.

Figure 18 shows the protein sequences of antigens used in the hamster immunization/challenge study of Example 4.

Figure 19 shows an SDS-PAGE analysis of the antigens used in the hamster immunization/challenge study of Example 4.

Figure 20 shows the vaccination schedule that was applied in the hamster immunization/challenge study of Example 4. For the coupled RBD (RBD-SpyTag=SpyCat-NP), the RBD-SpyTag was mixed with the SpyCat- NP in a 1:3 molar ratio. For the non-coupled RBD, the RBD-SpyTag and SpyTag-NP were mixed in a 1:3 molar ratio. Each animal received each time 10 pg of RBD antigen. The volume injected was 150 mΐ (100 mΐ of antigen and 50 mΐ of adjuvant) for the RBD groups also receiving an adjuvant. Figure 21 shows that nanoparticles produced by coupling SARS- CoV-2 RBD to 13-01 through the use of SpyTag- SpyCatcher proteins induce strong virus neutralizing antibody responses in hamsters. Hamsters were assigned to eight groups of five animals each. Immunizations were performed intramuscularly with either PBS, RBD only, RBD and nanoparticle (NP) mixed (RBD+NP) or RBD coupled to the nanoparticle (RBD-NP) in the absence or presence of aluminium hydroxide (Alum) at day 0 and day 28. Shown are the RBD reactive antibodies detected in an ELISA (A-C) or virus neutralization assay (D) when testing sera obtained at day 0 (A), day 28 (B) and day 42 (C and D). Dashed lines indicate the detection limit of the assays.

Figure 22 shows in Table format the vaccine-induced antibody titers and fold changes following prime (4 weeks 705 post-prime) and booster (3 weeks following booster) vaccinations, as described in Example 1. In this Figure, antibody titers and fold increase relative to baseline (Day 0) are expressed in GMT (95% Cl) for n=5 rabbits/group. Cl, confidence interval; GMT, geometric mean titer FP, MERS-CoV fusion peptide; HR2, MERS-CoV heptad repeat 2; 13, 13-01; LS, Lumazine synthase; MERS-CoV, Middle East respiratory syndrome coronavirus, N/A, not applicable; neg, negative; PRNT90, 90% reduction in plaque reduction neutralization test using MERS-CoV EMC strain; RBD, MERS-CoV receptor binding domain; Si, MERS-CoV Spike protein Si subunit; S2, MERS-CoV spike protein S2 subunit; SC, SpyCatcher, ST, SpyTag.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “nanoparticle”, as used herein, includes reference to a synthetic or artificial particle of matter that has a particle size that is on the nanometer scale. Preferably a nanoparticle as described herein has a particle size, diameter or (hydrodynamic) radius of 1-100 nanometer as determinable by electron microscopy (such as TEM) analysis or dynamic light scattering. For instance, a nanoparticle or a multimeric protein scaffold particle as described herein such as 13-01 may have a hydrodynamic radius of 1-100 nm such as about 14 nm. Preferably, the nanoparticle is a multimeric protein scaffold particle (MPSP). The nanoparticle can be a virus-like particle (VLP), preferably a protein-based virus-like particle that mimics a virus but does not contain virus capsid or envelope proteins.

The phrase “displays on the outer surface”, as used herein, includes reference to displaying a receptor binding domain (RBD) of a coronavirus spike (S) protein on a nanoparticle such that it is presented to, and recognized by, the immune system of a subject when said nanoparticle is administered to a subject. In other words, the display of RBD is such that the immune system of a subject recognizes said RBD and/or mounts an immune response against said RBD.

The term “receptor binding domain (RBD) of a coronavirus spike (S) protein”, as used herein, includes reference to a part of the coronavirus spike (S) protein that is involved in viral attachment to a receptor on a cell of subject, and subsequent entry into the cell. The cell receptor may be an angiotensin-converting enzyme 2 (ACE2) receptor. Preferably, the RBD comprises or consists of the amino acid sequence of SEQ ID NOs:l, 7, 13 or 28. Within the definition of RBD is also foreseen an RBD that is an antigenic part of the amino acid sequence of SEQ ID NOs:l, 7, 13 or 28. An RBD as described herein can be modified in that 1-100, preferably 1-50, more preferably 1-10 amino acid residues are added, substituted or deleted from an amino acid sequence of SEQ ID NOs:l, 7, 13 or 28 or an antigenic part thereof, preferably wherein said modified RBD binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle as described herein, wherein the RBD of said nanoparticle comprises or consists of an amino acid sequence of SEQ ID NOs:l, 7, 13 or 28. The term RBD thus includes reference to a sequence having 80%, preferably 90%, more preferably 95% sequence identity with SEQ ID NOs:l, 7, 13 or 28 and binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle as described herein, wherein the RBD of said nanoparticle comprises or consists of an amino acid sequence of SEQ ID NOs:l, 7, 13 or 28.

Each individual amino acid sequence disclosed herein (e.g. in the Figures) forms part of the present invention and is explicitly foreseen as an aspect of the invention. This is especially the case for amino acid sequences that include both an RBD and a Spytag (ST) or an RBD and a protein monomer as described herein, which are also referred to herein as fusion proteins or protein conjugates. Also envisaged are nucleic acid molecules encodings said amino acid sequences.

The term “spike (S) protein”, or the equivalent term “spike (S) glycoprotein”, refers to the coronavirus S protein consisting of a Si subunit (the N-terminal head) and an S2 subunit (the C-terminal stalk). The Si subunit mediates virus attachment and entry through its N-terminal SlA domain (comprising sialic acids, a viral attachment factor), and its C- terminal receptor binding domain (RBD), which binds to the viral receptor dipeptidyl peptidase 4 (DPP4) in the case of MERS and ACE2 receptor in the case of SARS. The S2 subunit is more conserved and mediates viral fusion to the host cell through the fusion peptide (FP) and the two heptad repeats HR1 and HR2. Binding od the SARS-CoV S protein RBD to human ACE2 and CLEC4M/DC-SIGNR receptors results in and internalization of the virus into the endosomes of the host cell.

The term “multimeric protein particle” or “multimeric protein scaffold particle”, as used herein, includes reference to a nanoparticle that is composed of a multimer of protein monomers or protein subunits, preferably wherein the multimer of protein monomers or protein subunits is a homomultimer of (the same) protein monomer or protein subunit, more preferably is a homomultimer of protein monomers or protein subunits that consists of 60 protein monomers or subunits assembled into a 60(multi)mer. The reference to a scaffold in the definition of MPSP includes reference to the fact that the scaffold particle is the vehicle or substrate that is to present or display or present the RBD of a coronavirus spike (S) protein to the immune system of subject. It is well within the ambit of the skilled person to developed multimeric protein scaffold particles, for instance by using lumazine synthase (LS) or 13-01 (13) which self-assemble into 60- meric particles, which can be expressed in E. coli and have been used as scaffolds for development of multimeric vaccines with improved immune responses compared to monomeric forms.

The term “multimeric protein”, as used herein, includes reference to a multimer of protein monomers or protein subunits.

The term “protein monomer” or “protein subunit”, as used herein, includes reference to a protein monomer or protein subunit that has the capacity to multimerize into a protein multimer, also referred to as a multimeric protein, so as to form a nanoparticle of the invention, preferably so as to form a multimeric protein scaffold particle of the invention. Suitable examples of protein monomers or protein subunits that assemble into multimeric proteins useful for use in the invention are 13-01 (13) protein monomers or subunits and lumazine synthase (LS) protein monomers subunits, of which exemplary amino acid sequences are provided in SEQ ID NO:2 and SEQ ID NO:3, respectively. The skilled person is well aware of other protein monomers or protein subunits that can be used in the present invention and which self-assemble into a multimeric protein as described herein.

The term “self-assembling”, as used herein, includes reference to the property of certain protein monomers to self-assemble through multimerization when subjected to a condition of multimerization to thereby form a nanoparticle as described herein. As already indicated, the skilled person is well aware of suitable protein monomers that self-assemble through multimerization when allowed to, and is well aware of suitable multimerization conditions that allows for self-assembly of protein monomers.

The term “13-01”, as used herein, includes reference to a multimeric protein particle or the protein monomer constituent thereof. Particle 13-01 is a well-known icosahedral protein structure widely utilized in biological systems for packaging and transport. A suitable sequence of 13- 01 is provided in SEQ ID NO:17. The term “13-01” includes reference to mutant forms of the Ϊ3-01 protein, such as Mi3 (e.g., SEQ ID NO: 2), having improved functionality. The term “13-01”, as used herein, includes reference to monomeric proteins of 13-01 or Mi3 that are modified in that 1-100, preferably 1-50, more preferably 1-10 amino acid residues are added, substituted or deleted from an amino acid sequence of SEQ ID NO:2 or 17, and includes reference to a sequence having 80%, preferably 90%, more preferably 95% sequence identity with SEQ ID NO:2 or 17 and having the property of self-assembling through multimerization when subjected to a condition of multimerization to thereby form a nanoparticle as described herein.

The term “lumazine synthase”, as used herein, includes reference to a multimeric protein particle or the protein monomer constituent thereof. The particle lumazine synthase is a well-known icosahedral protein structure widely utilized in biological systems for packaging and transport. Preferred embodiments are the lumazine synthase the amino acid sequence of which is provided in SEQ ID NO:3. A lumazine synthase as described herein can be modified in that 1-100, preferably 1-50, more preferably 1-10 amino acid residues are added, substituted or deleted from an amino acid sequence of SEQ ID NO: 3, and includes reference to a sequence having 80%, preferably 90%, more preferably 95% sequence identity with SEQ ID NO:3 and having the property of self-assembling through multimerization when subjected to a condition of multimerization to thereby form a nanoparticle as described herein.

The term “multivalent display”, as used herein, includes references to displaying on a nanoparticle as described herein multiple copies of a RBD as described herein. Preferably, each protein monomer in a nanoparticle as described herein displays one RBD as described herein.

The term “SpyTag protein moiety”, as used herein, includes reference to a generally known and widely available protein moiety of the plug-and-display SpyTag/SpyCatcher system. An exemplary amino acid sequence of a SpyTag protein moiety is provided in SEQ ID NO:4.

The term “SpyCatcher protein moiety”, as used herein, includes reference to a generally known and widely available protein moiety of the plug-and-display SpyTag/SpyCatcher system. A Spycather protein is inter alia provided in GenBank: AFD50637.1 (amino acids 48-139). An exemplary amino acid sequence of a SpyCatcher protein moiety is provided in SEQ ID NO:5 or SEQ ID NO:29.

The term “glycine/serine (GS) linker protein”, as used herein, includes reference to a well-known linker peptide that is composed of glycine and/or serine amino acid residues, preferably a repetitive GS linker peptide.

The term “protein”, as used herein, includes reference to both polypeptides and peptides.

The term “fusion protein”, as used herein, includes reference to any kind of protein that contains at least two different regions of amino acid residues that constitute different domains that naturally do not occur together in the same protein, such as a combination of RBD with a (i)

Spytag protein moiety, (ii) Spycatcher protein moiety, (iii) protein monomer selected from i3, Mi3 and LS, (iv) tag such as a Streptag or a C-tag or (v) a peptide linker such as a GS-linker. The term “fusion protein”, as used herein, can be used interchangeably with the term “protein conjugate”. Preferably an RBD as described herein in relation to a nanoparticle or fusion protein of the invention is conjugated or fused to a Spytag protein moiety or a protein monomer as described herein such as selected from i3, Mi3 and LS, optionally wherein said RBD is linked to said Spytag protein moiety or a said protein monomer through a peptide linker such as a GS- linker peptide.

The term “virus”, as used herein, includes reference to a small infectious agent that replicates only inside the living cells of an organism. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea.

The term “infectious disease”, as used herein, includes reference to diseases that are caused by micro-organisms such as bacteria or viruses.

The term “corona virus”, as used herein, includes reference to a family of viruses also referred to as Coronaviridae or Coronavirinae, which belong to the order of Nidovirales. Preferably, the coronavirus is a virus of the subfamily of Coronaviridae, which inter alia comprises the genera of Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Preferably, the coronavirus is a severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), preferably a SARS coronavirus 2 (SARS-CoV-2). Such a virus is the causative virus for coronavirus disease (COVID), preferably coronavirus disease 2019 (COVID-19), respectively. Testing for positive cases of SARS-CoV-2 can be based on detection of virus RNA sequences by NAAT such as real-time reverse-transcription polymerase chain reaction (rRT-PCR) with confirmation by nucleic acid sequencing when necessary. The viral genes targeted so far include the N,

E, S and RdRP genes. The coronavirus may also be a Middle East respiratory syndrome coronavirus (MERS-CoV), which is the causative virus for Middle East respiratory syndrome. SARS-CoV- 1 is a known coronavirus closely related to SARS-CoV-2.

The term “COVID”, as used herein, includes reference to an infectious disease caused by severe acute respiratory syndrome coronavirus (SARS-CoV). Preferably, the SARS-CoV is SARS-CoV-2, and the COVID is COVID-2019. Common COVID symptoms include fever, cough, and shortness of breath. Muscle pain, sputum production and sore throat are less common. While the majority of cases result in mild symptoms, some progress to severe pneumonia and multi-organ failure. The infection is typically spread from one person to another via respiratory droplets produced during coughing. It may also be spread from touching contaminated surfaces and then touching ones face.

The term “subject”, as used herein, can be used interchangeably with the term “patient”, and includes reference to a mammal, preferably a human. Preferably, a subject is at least 30 or at least 40 years old. More preferably, the subject is at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or at least 65 years old. Even more preferably, a subject is at least 66, 67, 68, 69 or at least 70 or at least 80 years old. In addition to the aforementioned embodiments, or alternatively, the subject is an elderly human, preferably a frail elderly human.

The term “nucleic acid”, as used herein, refers to DNA and RNA including mRNA or cDNA, as well as synthetic congeners thereof. The nucleic acid can be a recombinant or synthetic nucleic acid. The term “antigen” or “antigenic”, as used herein, includes reference to a protein (including polypeptides and peptides) which contains at least one epitope specifically recognized by a product or effector of the immune system of a subject such as a T-cell receptor, an antibody or another element of humoral and/or cellular immunity. The whole protein may be recognized, or one or more portions of the protein, for instance following intracellular processing of a protein into an MHC peptide antigen complex and subsequent antigen presentation. The term “antigen” or “antigenic” includes reference to at least one, or more, antigenic epitopes of a RBD as described herein. The term “immunogenic composition”, as used herein, includes reference to a composition which elicits an immune response against a nanoparticle as described herein when administered to a subject who is in need of an immune response against said nanoparticle. The composition may include an adjuvant, for instance an alum such as aluminium hydroxide, and optionally one or more pharmaceutically-acceptable carriers, excipients and/or diluents. The immunogenic composition can be employed in prime-boost vaccination, such as at least 2, 3, 4 or at least 5 immunizations separated in time.

The term “adjuvant”, as used herein, includes reference to a compound or compounds that, when used in combination with specific vaccine antigens in formulations, augment or otherwise alter or modify the resultant immune responses. Preferably, the adjuvant is an alum (an aluminium salt) such as aluminium hydroxide.

The term “vaccine”, as used herein, includes reference to a composition of antigenic moieties, usually consisting of modified-live (attenuated) or inactivated infectious agents, or some part of the infectious agents, that is administered, most often with an adjuvant, into the body to produce active immunity. The present invention provides immunogenic compositions comprising a self-assembling nanoparticle as described above in a pharmaceutically acceptable carrier. Such a carrier may be an aqueous liquid, or an aerosol composition.

The term “therapeutically effective amount”, as used herein, refers to an amount of a therapeutic agent to treat, ameliorate, counteract, inhibit or prevent a desired disorder or condition, or to exhibit a detectable therapeutic or prophylactic effect. The precise effective amount needed for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. The therapeutically effective amount for a given situation can be determined by routine experimentation. The term “% sequence identity” is defined herein as the percentage of nucleotides in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the nucleotides, resp. amino acids, in a nucleic acid or amino acid sequence of interest, after aligning the sequences and optionally introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignments are well known in the art. Sequence identity is calculated over substantially the whole length, preferably the whole (full) length, of an amino acid sequence of interest. The skilled person understands that consecutive amino acid residues in one amino acid sequence are compared to consecutive amino acid residues in another amino acid sequence.

Emerging zoonotic viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have been able to cross the species barrier posing a threat to the human population. MERS-CoV causes severe respiratory disease and fatalities in humans [1,2], and the virus is continuously introduced into the human population through infected dromedary camels, the viral reservoir with resulting outbreaks [3] . The wide geographical distribution of this viral reservoir, the high case-fatality rate in humans (35%), and the lack of treatment and licensed vaccines, make the virus a threat to the human population. This has put MERS-CoV on the recent WHO list of diseases having an epidemic or even pandemic potential for which countermeasures are lacking and are urgently needed [4]

Vaccination is potentially one of the most effective ways to prevent the ongoing MERS-CoV outbreaks. Several MERS-CoV vaccine candidates have been developed using different platforms including inactivated, live-attenuated, and subunit vaccines [5] . Compared to other vaccine production platforms, recombinant subunit proteins have a higher safety profile, are relatively faster and easier to produce, and can be scaled- up in a more cost-effective manner; nonetheless, they tend to induce lower levels of protective immunity [6] . The use of self-assembling multimeric protein scaffold particles (MPSP) to present antigens in a multivalent virus- mimicking manner (size, repetitiveness, and geometry), has been shown to enhance vaccine-induced immune responses [7-11], and to offer advantages over other multimeric antigen presentation platforms (reviewed in [12]). Both lumazine synthase (LS) and 13-01 (13) can self-assemble into 60-meric particles, which can be expressed in E. coli and have been used as scaffolds for development of multimeric vaccines with improved immune responses compared to monomeric forms [13-15]. An LS-based HIV vaccine, (eOD- GT8), has recently advanced to a phase I human clinical trial (NCT03547245). Linking of antigens to these MPSP can be achieved through several mechanisms; as e.g. genetic fusion or the SypTag- SpyCatcher (ST/SC) system [16]. While the former requires the antigen and scaffold to be produced in the same expression system, the latter allows each to be expressed in its suitable system harnessing a rapid post-translational ‘plug-and-play’ assembly. This is advantageous, allowing scaffold-SC to be produced at scalable levels in E. coli and SpyTagged glycosylated antigens such as viral surface proteins to be produced in its optimal system, such as mammalian or insect cells. The antigen-ST can then be multivalently displayed on the surface of the SC-scaffolds through the spontaneous formation of a stable isopeptide bond. This can be a platform for rapid vaccine manufacturing in case of epidemics or pandemics, to create optimized vaccines at reduced costs and also with reduced development times.

The MERS-CoV spike (S) protein is the main target for subunit vaccine development [5] It assembles as a homotrimer and consists of an N- terminal head (Si subunit) and a C-terminal stalk (S2 subunit). The Si subunit mediates virus attachment and entry through its N-terminal SlA domain and its C-terminal receptor binding domain (RBD), respectively [17,18]. The SlA domain binds sialic acids, a viral attachment factor, while the RBD binds to the viral receptor, dipeptidyl peptidase 4 (DPP4).

Following attachment and entry, the S2 subunit mediates viral fusion to the host cell through its fusion machinery; comprised of the fusion peptide (FP) and the two heptad repeats - HR1 and HR2 [19]. MERS-CoV neutralizing antibodies (Abs) mainly recognize epitopes in the RBD of the spike head Si subunit; and to a lower extent, epitopes in the sialic acid binding domain and the fusion-mediating more conserved S stalk (S2).

Nonetheless, antibodies directed against the sialic acid binding SlA domain or the more conserved S2 subunit, although subdominant, may protect against MERS-CoV [20,21]

Immune focusing can enhance immune responses to subdominant regions [22] In the current study, using LS and 13 self-assembling particles, we evaluated whether immune focusing and multivalent presentation can induce immune responses to the more sequence-conserved S2 regions: FP and HR2. Furthermore, using a SypTag/SpyCatcher system and LS particles, we tested whether immune focusing with/without multivalent presentation of the viral RBD can lead to enhanced protection against a MERS-CoV challenge in rabbits.

Following this approach, the same was done for SARS-CoV-2. We herein provide nanoparticles that display coronavirus Spike protein receptor binding domains, and which can be used as vaccines against coronavirus disease.

Production of a nanoparticle of the invention

Self-assembling proteins for the production of nanoparticles of this invention, and their constituents, can be produced by methods generally known in the art, such as for instance described in reference [15].

As scaffolds for presenting the viral antigens in recombinant subunit protein vaccines of the present invention use is made of self- assembling molecules, which self-assemble into multimeric protein scaffold particles (MPSP). Suitable self-assembling molecules include lumazine synthase (LS) and 13-01 (13) of mutant versions thereof, such as mi3, which self-assemble into 60-meric particles, are genetically fused to moieties of a protein conjugation system such as SpyCathcher/SpyTag, which are, e.g., expressible in bacterial cells such as E. coli.

For instance, a suitable protein monomer such as an LS or 13-01 (13) can be synthesized using for instance human-preferred codons. One suitable example of an LS amino acid sequence is providing in SEQ ID NO:3. Alternatively, another suitable protein monomer for use in the invention, 13-01 (13; PDB 5KP9, amino acid residues 19-222) can also be synthesized using for instance human-preferred codons. An exemplary amino acid sequence of 13-01 is provided in SEQ ID NO:17. Further, a SpyCatcher protein moiety (SC; e.g. UniProt accession no. AFD50637.1; amino acid residues 48-139) and a SpyTag protein moiety (ST; e.g. UniProt accession no. WP_129284416.1; amino acid residues 981-994) can be synthesized using for instance human-preferred codons. One example of a suitable ST is provided in SEQ ID NO:4. An example of a suitable SC is provided in SEQ ID NO:5 or SEQ ID NO:29. The protein monomer constructs, such as the LS and 13-01 gene constructs can be cloned in a pGEX-2T bacterial expression vector. To generate an I3-HR2 expression vector, the heptad repeat 2 encoding region (HR2, amino acid residues 1215- 1287) of the MERS-CoV S gene can be ligated in-frame with an N-terminal sequence encoding the 13-01 and optionally a C-terminal streptag purification tag interspaced with a linker, and subsequent cloned into a suitable expression vector such as a pGEX-2T bacterial expression vector.

To generate a fusion peptide (FP)-I3 or a FP-LS expression vector, a fusion peptide (FP; e.g. amino acid residues 884-898) can be ligated in-frame with an N-terminal sequence encoding 13-01 or LS, and optionally a C-terminal Streptag purification tag. The construct can subsequently be cloned into a suitable bacterial expression vector such as a pGEX-2T bacterial expression vector. To generate a RBD-ST expression vector, the MERS-RBD (e.g. amino acid residues 358-588 or SEQ ID NO:7) or SARS-RBD as described herein (e.g. SEQ ID NO:l or SEQ ID NO: 13 or variants thereof) can be ligated in frame with a C-terminal sequence encoding an ST followed by for instance a double Streptag, and can optionally be ligated in frame with an N-terminal sequence encoding a signal sequence such as a CD5 signal sequence. Subsequently, the construct can be cloned into a suitable mammalian expression vector such as a pCAGGS mammalian expression vector. To generate a LS-SC expression vector, a (codon optimized) SC sequence (an exemplary sequence is provided in SEQ ID NO: 5), optionally equipped with an N-terminal FLAG-tag (DYKDDDDK), can be cloned to the N-terminus of an LS sequence in a suitable expression vector such as the pET15b bacterial expression vector.

Preferably, in relation to the Spytag/SpyCatcher embodiments described herein, the RBD of a coronavirus spike (S) protein — that is directly or indirectly fused to said SpyTag protein moiety — is produced in a eukaryotic, preferably mammalian, cell using a mammalian expression construct. Mammalian expression of a RBD-ST construct can be done using techniques widely available in the art, for instance as described previously [17]. As an example, a mammalian expression vector can be polyethylenimine (PEI)-transfected into HEK-293T cells, after which medium can be replaced with a suitable expression medium and for instance incubated at 37 °C in 5% C02. Tissue culture supernatants can be harvested 5—6 days post transfection, and expressed proteins can be purified using StrepTactin Sepharose beads (IBA).

Preferably, in relation to the Spytag/SpyCatcher embodiments described herein, the protein monomer — that is for instance an LS or 13-01 that is directly or indirectly fused to a SpyCatcher (SC) protein moiety — is produced in a prokaryotic, preferably bacterial cell using a bacterial expression construct. Bacterial expression of a protein monomer-SC construct can be done using techniques widely availablein the art. As an example, bacterial cells such as BL21 cells can be transformed with a suitable bacterial expression vector as described above, such as a pGEX-2T expression vector, and grown in a suitable medium to log phase (OD600 ~1.0). Subsequently, said cells can be induced by adding a suitable compound such as IPTG (isopropyl- 6- d-thiogalactopyranoside). Thereafter, the cells can be pelleted, resuspended and sonicated. The cell homogenates can be centrifuged at for instance 20,000 x g for 60 min at 4°C. Proteins can be purified from the cell lysate supernatant using for instance a StrepTactin Sepharose beads (IBA). The purified proteins can be analyzed on for instance a 12% SDS/PAGE gel under reducing conditions and stained with GelCodeBlue stain reagent (Thermo Scientific). Purified proteins can be stored at 4°C until further use. Expression of an LS-SC fused to FLAG can be performed as described above with the following modifications: 1) Cells are treated with lmg/ml lysozyme in lysis buffer (for instance 50mM Tris- HC1, 150mM NaCl, 1% Triton X-100) for lh at room temperature prior to sonification on ice. 2) Purification can be performed using for instance an ANTI- FLAG® M2 Affinity Gel (Sigma Aldrich). Purified proteins can be dialyzed against lx TBS buffer (for instance 50mM Tris-HCl, 150mM NaCl, pH 7.4) and stored at -80°C until further use. The above embodiment is described in detail in Example 1

Another embodiment is described in detail in Example 2, wherein bacterial expression and purification of the mutant Ϊ3-01 (mi3) protein fused to a SpyCatcher (SC)protein-conjugation moiety is described. As a general method, transformed bacterial cells are used to express the mi3 protein genetically fused to the SC protein-conjugation moiety. Following purification of the protein monomer, and mixing with RBD-SpyTag (RBD- ST) to form the Mi3-RBD conjugate, self-assembly in the form of multimerization of the proteins is allowed to provide the nanoparticle according to the invention.

Immunogenic composition of the invention and administration thereof

An immunogenic composition of the invention may further comprise, or can be administered in combination with, a suitable immune- potentiator, or vaccine adjuvants, to increase the immunogenicity of nanoparticles and the compositions of the invention. Such immune- potentiators, or vaccine adjuvants, include, but are not limited to Freund’s (in)complete adjuvant, lipopolysaccharides and lipopolysaccharide derivatives; Alums, including aluminum phosphate and aluminum hydroxide; mineral oil; squalene; emulsions such as squalene/water emulsions (e.g. MF59); Montanide©; calcium or calcium phosphate; muramyl dipeptide and analogues; glycosides including saponins or triterpenoid glycosides, such as Quil A, or purified fractions of Quil A including QS7 and QS21 (also known as QA7 and QA21), Tomatine, ISCOM, ISCOMATRIX(TM); stearyl tyrosine; sterols, including cholesterol, lanosterol, lumisterol, stigmasterol and sitosterol; liposomes; bacterial lipopeptide, lipoprotein and lipoteichoic acid; monophosphoryl lipid A

(MPL); pluronic polyols; trehalose dimycolate; amine containing compounds; cytokines, including GM-CSF, IL-2, IFN-gamma, Flt-3; bacterial DNA; CpG DNA; hemozoin; viral single or double stranded RNA; imidazoquinolines; mycobacterial lipoglycan; yeast zymosan; porin; uropathogenic bacteria; meningococcal outer membrane vesicles (OMV); protozoan profilin; bacterial toxins, such as diphtheria toxin, pertussis toxin (PT), cholera toxin (CT), the E. coli heat-labile toxin (LT1 and LT2), Pseudomonas endotoxin A, C. botulinum C2 and C3 toxins as well as toxins from C. perfringens, C. spiriforma and C. difficile, and non-toxic mutants of such toxins; and combinations of the above, such as AS21 (squalene+MPL+QS21), AS04 (Alum+MPL).

Use may be made of adjuvants that increase the mucoadhesive and adsorptive properties of the compositions of the invention, including, for instance chitosan or lecithin/polymer mixtures, wherein the polymer is selected from polyacrylic acid, methacrylic acid, methacrylate, acrylamide, acrylate, acryinitrile, alkyl-esters of poly acrylic acid, poly (acrylamide-co butyl, methacrylate), acrylic-methacrylic acid, acrylic-acrylamide and poly (methacrylate), polyvinyl acetate phthalate, cellulose acetate phthalate, methylcellulose, polyethylene glycol, polyvinyl alcohol, and polyoxyethylene, in concentrations of lecithin and polymer of 0.001-1.0% each by weight/volume.

Surfactants (emulsifiers) may be added to aid in the mixing or emulsification of the components of the composition of the invention. Examples of appropriate surfactants include polyoxyethylene sorbitan monooleate, sorbitan monolaurate, sodium stearate, non-ionic ether-linked surfactants such as Laureth®4 and Laureth®23, alkyl sulfate surfactants, alkyl alkoxylated sulfate surfactants, alkylbenzenesulphonates, alkanesulphonates, olefinsulphonates, sulphonated polycarboxylic acids, alkyl glycerol sulfonates, fatty acyl glycerol sulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isothionates such as the acyl isothionates, N-acyl taurates, fatty acid amides of methyl tauride, alkyl succinamates and sulfosuccinates, mono- and diesters of sulfosuccinate, N-acyl sarcosinates, sulfates of alkylpolysaccharides, branched primary alkyl sulfates, alkyl polyethoxy carboxylates, and fatty acids esterified with isethionic acid and neutralized with sodium hydroxide; or other suitable nonionic detergent surfactants. If included, the emulsifier should be added in a concentration ranging from about 0.001-0.05% by volume of the mixture. The addition of adjuvants may aid in the stimulation of an immune response and in particular a mucosal immune response. If included, adjuvants may be present in a concentration of up to about 10% by weight of the composition, for example, less than about 1% by weight of the composition. In most cases, adjuvants are included in an amount to cause an induction of an immune response. In some instances, adjuvants herein provide additional stimulation of TH1 (T helper cell 1), an important mediator in mucosal immunity.

The concentration of an RBD, fusion protein or nanoparticle as described herein may range from nanogram to milligram quantities with about 1 microgram to 1 milligram preferred.

In addition to all of the above, excipients that can be employed in compositions of the invention include for example, dyes, alcohol, buffers, stabilizers, wetting agents, dissolving agents, colors, etc. With the exception of diluents such as alcohols which are used at higher levels, the levels of these excipients are generally not more than 0.001% to 1.0% by weight of the composition.

The immunogenic compositions of the invention may be administered by topical administration, by subcutaneous or intramuscular injection or may be delivered to a mucosal surface of the subject, such as the mucous membranes of the oral cavity, gut, nose, rectum, or vagina.

The invention therefore further provides a method for the prevention or treatment of a disease in a subject comprising administering to the subject an immunologically effective dose of a composition of the invention comprising the nanoparticle, preferably by contacting a mucosal surface of the subject with said composition. For mucosal delivery, the administration may for example be carried out by inhalation, by means of a rectal or vaginal suppository, or a pessary, by feeding or other buccal administration, by means of an aerosol, by intranasal delivery or direct application to mucosal surfaces. Especially preferred are oral and intranasal administration.

Preferably, an immunogenic compositions as described herein is a vaccine composition. As shown in Figure 21 herein, the immunogenic composition of the invention was able to produce neutralizing antibodies against SARS- CoV-2. It is an important advantage of self-assembling nanoparticle approach as used in the present invention that individual fusion protein parts of the nanoparticle assembly can be produced independently of one another, and at different time points. This allows for quick adaptation of the subunit vaccine to novel virus outbreaks, wherein only the RBD part of the fusion protein needs to be tailored to the novel outbreak. The nanoparticles of this invention have very advantageous stability properties. Therefore, the present invention has as an object to provide corona virus vaccines of advantageous stability, either with respect to chemical stability, physical stability, biological stability, temperature stability, salt stability, or pH stability.

EXAMPLES

Example 1. Potent protection against MERS-CoV using a plug-and- display particulate multivalent receptor binding domain vaccine

Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusingapproach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein - fusion peptide, heptad repeat 2, and receptor binding domain (RBD) - and tested their immunogenicity and protective capacity in rabbits. Using a ‘plug-and-display’ SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS- CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses.

Materials and Methods Protein Design and Expression.

Expression constructs were cloned using standard PCR methods. The gene encoding the 6,7-dimethyl-8-ribityllumazine synthase (LS;

GenBank accession no. WP_010880027.1) of A. aeolicus was synthesized using human-preferred codons obtained from GenScript USA, Inc, as described previously [17]. The cysteine at position 37 and asparagine at position 102 of LS were mutated to alanine and glutamine, respectively. The gene encoding 13-01 (13; PDB 5KP9, amino acid residues 19-222) derived from Thermotoga maritima was synthesized using human-preferred codons obtained from GenScript USA. The gene fragments encoding the ANlSpyCatcher (SC; UniProt accession no. AFD50637.1; amino acid residues 48-139; [23]) and SpyTag (ST; UniProt accession no. WP_129284416.1 ; amino acid residues 981-994) based on the Cna B-type domain-containing protein of Streptococcus pyogenes were synthesized using human-preferred codons obtained from GenScript USA, Inc. The LS and 13 gene constructs were cloned into the pGEX-2T bacterial expression vector (Sigma Aldrich).

To generate the HR2-LS expression vector, the HR2 region (amino acid residues 1215- 1287) encoding sequence of the MERS-CoV S gene (accession no. NC_019843) wasligated in-frame with an N-terminal sequence encoding a CD5 signal sequence and streptag tag purification tag, and with a C-terminal sequence encoding the LS via alinker, and subsequent cloned into the pCAGGS mammalian expression vector.

To generate the I3-HR2 expression vector, the heptad repeat 2 encoding region (HR2, amino acid residues 1215-1287) of the MERS-CoV S gene was ligated in-frame with an N-terminal sequence encoding the 13-01 and a C-terminal streptag purification tag interspaced with a linker, and subsequent cloned into the pGEX-2T bacterial expression vector (Sigma Aldrich).

To generate the FP-I3 and FP-LS expression vectors, the fusion peptide (FP; amino acid residues 884-898) encoding sequence of the MERS- CoV S gene was ligated in-frame with an N-terminal sequence encoding the 13-01 or LS, and a C-terminal Streptag purification tag and subsequently cloned into the pGEX-2T bacterial expression vector (Sigma Aldrich).

To generate the RBD-ST expression vector, the MERS-RBD (amino acid residues 358-588) encoding sequence of the MERS-CoV S gene was ligated in-frame with an N-terminal sequence encoding a CD5 signal sequence and with a C-terminal sequence encoding the ST followed by a double Streptag, and subsequently cloned into the pCAGGS mammalian expression vector.

To generate the LS-SC expression vector, the codon optimized SC sequence equipped with an N-terminal FLAG-tag (DYKDDDDK) was cloned to the N-terminus of the LS sequence in the pET15b bacterial expression vector (Novagen). All protein sequences are provided in Fig.7 and 8.

Mammalian expression.

Mammalian expression of the HR2-LS and RBD-ST constructs was done, as described previously [17]. In short, expression plasmids were polyethylenimine (PEI)-transfected into 60% confluent HEK-293T cells for 6 h, after which transfections were removed and medium was replaced with 293 SFM Il-based expression medium (Gibco Life Technologies) and incubated at 37 °C in 5% C02. Tissue culture supernatants were harvested 5—6 d post transfection, and expressed proteins were purified using

StrepTactin Sepharose beads (IBA) according to the manufacturer’s instruction.

Bacterial protein expression. BL21 cells (Novagen) were transformed with pGEX-2T expression vectors and grown in 2x yeast-tryptone medium to log phase (OD600 ~1.0) and subsequently induced by adding IPTG (isopropyl-6-d- thiogalactopyranoside) (GIBCO BRL) to a final concentration of 1 mM. Two hours later, the cells were pelleted, resuspended in 1/25 volume of 10 mM Tris (pH 8.0)-10 mM EDTA-1 mM phenylmethylsulfonyl fluoride, and sonicated on ice (five times, 2 min each). The cell homogenates were centrifuged at 20,000 x g for 60 min at 4°C. Proteins were purified from the cell lysate supernatant using StrepTactin Sepharose beads (IBA) according to the manufacturer’s instruction. All purified proteins were analyzed on a 12% SDS/PAGE gel under reducing conditions and stained with GelCodeBlue stain reagent (Thermo Scientific). Purified proteins were stored at 4°C until further use.

Expression of the FLAG-LS-SC was performed as described above with the following modifications: 1) Cells were treated with lmg/ml lysozyme in lysis buffer (50mM Tris-HCl, 150mM NaCl, 1% Triton X-100) for lh at room temperature prior to sonification on ice. 2) Purification was performed using ANTI-FLAG® M2 Affinity Gel (Sigma Aldrich) as recommended by the manufacturer. Purified proteins were dialyzed against lx TBS buffer (50mM Tris-HCl, 150mM NaCl, pH 7.4) and stored at -80°C until further use.

Rabbit immunizations

Rabbit immunizations and challenge were carried out at Viroclinics Bioscience B.V. Rotterdam, The Netherlands, under permit no. AVD277002015283-WP03, using BSL-3 containment facilities.

Female New Zealand White rabbits (Envigo, Venray, the Netherlands) of 11 weeks age were assigned to six groups (i-vi) of five animals each. Immunizations were performed intramuscularly with either i) HR2-LS, ii) FP-LS, iii) LS, at day 0 and boosted with either i) HR2-I3, ii) FP-I3, iii) 13 on day28 or iv) PBS, v) RBD+LS, vi) RBD-LS on days 0 and 28. Each animal received each time 15 pg of antigen adjuvanted with Adjuplex (5%; Sigma-Aldrich, Zwijndrecht, the Netherlands) in a total volume of 500 pL. Three weeks after the last vaccination (day 49 of the study), all animals were challenged intranasally under anesthesia with MERS-CoV (106 50% tissue culture infectious dose (TCID50) MERS-CoV EMC strain (accession no. NC_019843) in a volume of 1 mL divided over both nostrils). The animals were euthanized on day 4 post-challenge (day 53 of the study). Serum samples were collected on days 0, 28, and 46. Nasal swabs were collected on day 46 (pre-challenge) and on days 1 through 4 post-challenge. Following euthanasia, lungs were examined for gross pathology and lung tissue samples were collected for virus detection, and in 10% formalin histopathology and immunohistochemistry.

Enzyme-linked, immunosorbent assay (ELISA)

Antigen-binding and anti-LS (scaffold) antibodies produced after vaccination were tested in the sera collected at different time points as well as in pre-challenge nasal swabs using ELISA. Costar high-binding 96-well ELISA plates were coated overnight at 4°C with 1 pg/ml of either recombinant LS, MERS-CoV Si or S2 proteins in PBS.

The plates were washed with PBS and blocked for 1 hr using l%BSA/0.5%Tween-20/PBS. Following blocking, diluted samples (1:100 or serially diluted) were added and further incubated for 1 hr. The plates were then washed and and probed with an HRP-labeled goat anti-rabbit Ig (1:2000, Dako) secondary antibody. TMB was used for signal development and the absorbance of each sample was measured at 450 nm (OD450).

Antibody Avidity ELISA

Antibody avidity was assessed using an ammonium thiocyanate (NH4SCN)-displacement ELISA. This was carried out as described above using serum dilutions containing same level of Si absorbance units added in triplicates. Following serum incubation and washing, NH4SCN (0-5 M) was added to the wells for 15 minutes. The plates were then washed and further developed as described above. The concentration of NH4SCN resulting in a 50% reduction in signal was taken as the avidity index (IC50).

Focus reduction neutralization assay

The presence of MERS-CoV neutralizing antibodies in the sera and nasal swabs of vaccinated animals was tested using a plaque reduction neutralization assay (PRNT). Heat —inactivated two-fold serially diluted samples (starting 1:10) were mixed 1:1 with 400 PFU of MERS-CoV (EMC/2012) and incubated for one hour. The mix was then overlaid on HuH-7 cells in 96-well plates. Following one hour of incubation, the mix was removed and the cells were incubated for 8 hr. The cells were then fixed, permeabilized and stained using a mouse anti-MERS-CoV N protein monoclonal antibody (Sino Biological) followed by an HRP-labelled goat anti-mouse IgGl (SouthernBiotech). The signal was developed using a precipitate forming peroxidase substrate (True Blue, KPL). The ImmunoSpot® Image analyzer (CTL Europe GmbH) was used to count the number of infected cells per well. The neutralization titer of each serum sample was determined as the reciprocal of the highest dilution resulting in a >50% (PRNT50) or >90% (PRNT90) reduction in the number of infected cells. A titer of > 20 was considered to be positive.

Viral RNA detection

To evaluate the protective efficacy of vaccination against MERS- CoV challenge, nasal swabs, and homogenated lung tissues were tested for the presence of MERS-CoV RNA using RT-qPCR for and for the presence of infectious virus by virus titration.

The presence of viral RNA in nasal swabs and lung tissues was tested using UpE RT-qPCR as previously described [24] RNA was extracted from samples using Magnapure LC total nucleic acid isolation kit (Roche). RNA amplification and quantification were carried out using a 7500 Real- Time PCR System (Applied biosystems). Samples with a Ct value <40 were considered positive. RNA dilutions extracted from a MERS-CoV stock of known titer was used to generate a standard curve in order to calculate the TCID50 equivalent of RNA detected in samples. Concentrations of viral RNA in lung tissue are expressed in as TCID50 equivalents per gram tissue (TCID50 eq/g), and in the nasal swabs as TCID50 eq/mL. Virus titration

The presence of MERS-CoV infectious viral particles in respiratory tract samples (nasal swabs and lung tissue homogenates) was detected by titration on Vero cells as described previously [24] Briefly, 10- fold serially diluted samples (starting undiluted) were overlaid on Vero cells and the plates were incubated for five days at 37oC and the cytopathic effect was recorded. Infectious virus titers in lung tissue are expressed as TCID50 per gram tissue (TCID50/g), and infectious virus titer in nose swabs are expressed as TCID50/mL.

Histopathology and Immunohistochemistry

Lung tissue samples were collected in formalin and embedded in paraffin for pathological analysis. Hematoxylin-eosin staining was carried out for histopathological analysis. The presence of MERS-CoV nucleoprotein was detected by immunohistochemistry as previously published [24]

Statistical analysis

Statistical analyses were performed using Prism 7 (GraphPad Software Inc, USA). Data were compared using Mann- Whitney U test or Student’s t-test.P-values < 0.05 were considered significant.

Results

Generation of MERS-CoV spike particles

Particulate multivalent antigen display can enhance immunogenicity through different mechanisms, allowing for induction of immune responses against otherwise weakly immunogenic antigens [7,25]. We sought to design antigens capable of inducing strong immune responses against critical parts of the viral entry and fusion machinery within the MERS-CoV spike protein through immune focusing and multivalent presentation on self-assembling particles (Fig. 1). Within the Si subunit, the RBD is the main target for the induction of neutralizing antibodies and has been used to develop several vaccine candidates for MERS-CoV [5,26]. Indeed, the immunogenicity of RBD can be enhanced by its presentation on ferritin nanoparticles [27]. Likewise, the fusion peptide (FP) and the HR2, which show a high degree of sequence conservation among CoVs relative to the RBD, play crucial roles in the CoV spike-mediated fusion machinery, and can be targets for CoV protective antibodies [28-32]

Two 60-meric hyperstable self-assembling particles with icosahedral symmetry were used for multivalent display of MERS-CoV domains. The lumazine synthase (LS) particle, an icosahedron with a diameter of 15nm (PMID: 23539181) and the 13-01 (13) particle, a dodecahedron with a diameter of 25nm (PMID: 27309817). The N- and C- termini of both scaffolds are surface exposed, providing a platform to multivalently present (antigenic) domains. Two functional segments of the S2 subunit of the MERS-CoV spike protein were genetically fused to these nanoparticles; the fusion peptide containing region (amino acid residues 884-898) and the HR2 containing region (amino acid residues 1215-1287) (Fig. 1, Fig. 7). Chimeric nanoparticles were purified after expression in eukaryotic (mammalian) or prokaryotic systems (Fig. 1).

Generation of multimeric RBD-ST/ LS-SC (RBD-LS)

In addition, we used the SpyTag/SpyCatcher system to multivalently display the MERS-CoV RBD on LS nanoparticle via covalent bonding [8]. For this purpose, the SpyCatcher (SC) was genetically fused to LS and expressed and purified from E. coli.

The SpyTag (ST) was genetically fused to the MERS-RBD (amino acid residues 358-588) and expressed and purified from HEK-293T cells (Fig. IB). RBD-ST was incubated with LS-SC in different molar ratios to assess the optimal coupling of both components. A 1:2 molar ratio of RBD- ST and LS-SC allowed the optimal coupling of all of the provided RBD-ST antigens to the SC-LS particles (Fig. ID). The resulting conjugation products were used for immunization. In order to assess the effect of the particle-based multivalent antigen display on immunogenicity, a mixture of non-coupled RBD-ST and LS (without SC) was taken along for immunization in the same molar ratio. All particulate preparations displaying MERS-S antigenic domains (genetically fused or SC/ST coupled) were analyzed by SDS-PAGE (Fig. IE, Table 1), confirming their molecular integrity. Table 1. Immunogens used in this Example

13 226 24.10

FP-LS 210 22.27

FP-I3 261 27.71

HR2-LS 27. I

HR2-I3 319 34.07

RBD-ST 30.3 1

LS-SC 270 28.80

* These proteins appear larger in SDS-PAGE analysis due to N- glycosylation of HR2/RBD domain.

FP, MERS-CoV fusion peptide; HR2, MERS-CoV heptad repeat

2; 13, 13-01; LS, Lumazine synthase; MERS-CoV, Middle East respiratory syndrome coronavirus, RBD, MERS-CoV receptor binding domain; SC, SpyCatcher, ST, SpyTag. Immunogenicity of particulate MERS-CoV spike vaccines in rabbits.

We then evaluated the immunogenicity of the multimeric spike antigens using six groups of rabbits (n = 5 per group), which were intramuscularly immunized twice at a 4-week interval (Fig. 2A). To test the immunogenicity of uncoupled monomeric RBD versus the RBD-ST/LS-SC (RBD-LS) 60-meric particles, we vaccinated rabbits on days 0 and 28 with 15 pg of either monomeric RBD and LS (RBD+LS), RBD-LS conjugation products (RBD-LS) or PBS adjuvanted with 5% AdjuplexTM (Fig. 2A). In the same experiment, we also tested the immunogenicity of the multimeric FP and HR2 in a heterologous scaffold prime-boost scheme. Rabbits were vaccinated at day 0 with 15 pg of either HR2-LS, FP-LS or LS MPSPs; and boosted at day 28 with 15 pg of either HR2-I3, FP-I3 or 13 MPSPs, respectively, adjuvanted with 5% AdjuplexTM (Fig. 2A). The LS/I3 and PBS immunized groups served as controls. Serum samples were collected before vaccination (day 0), four weeks following the prime vaccination (day 28), and three weeks following the second (booster) vaccination (day 46) and were tested using ELISAs for Si and S2 antibodies, and PRNT90 for neutralizing antibodies. After the first immunization, we detected antibody responses against the corresponding S subunit (Si or S2) in the vaccinated rabbits while the control groups remained negative (Fig. 2B-E). Endpoint antibody titers for the vaccinated groups are shown as geometric mean titers (GMT) in Figure 22. The antibody responses were further boosted after the second immunization in all groups, while no responses were detected in the control groups, confirming the immunogenicity of the tested antigens in rabbits.

Anti-S2 antibody responses were detected in the HR2 and FP vaccinated groups with weak to no MERS-CoV neutralizing capacity (Fig. 2B,C). Only HR2 vaccination induced low levels of MERS-CoV neutralizing antibodies (PRNT90 titers: 20 - 40) in 4/5 rabbits; all 5 had MERS-CoV neutralizing antibodies at a 50 % cut-off (data not shown).

Likewise, both the monomeric RBD (RBD+LS) and the multimeric RBD-LS were immunogenic and elicited high Sl-specific antibody titers which were further boosted after the second immunization. The RBD-LS- induced Si antibody titers were significantly higher than those induced by the monomeric RBD following the prime- as well as booster-vaccination (P = 0.0397 and P = 0.0317, respectively by Mann-Whitney U test) (Fig. 2D). Multimeric RBD-LS vaccination elicited higher MERS-CoV neutralizing antibodies, a main correlate of protection, than the monomeric RBD+LS when tested for live virus neutralization using PRNT90 assay (P = 0.0109, and P =0.0079, post-prime and boost, respectively by Mann-Whitney U test) (Fig. 2E). The vaccine induced antibodies were able to neutralize clade A (EMC/2012 strain; Fig. 2E) as well as the more recently circulating clade B (Qatarl5/2015 strain; Fig. 9) viruses.

Following a single immunization, binding antibody titers were four-fold higher and neutralizing antibodies were eleven-fold higher in the coupled multimeric RBD-LS group than in the uncoupled monomeric RBD+LS (Figure 22). Three weeks after the boost, binding antibody responses were seven-fold higher (P = 0.0079, Mann-Whitney U test) and neutralizing antibodies were ten-fold higher (p= 0.0079, Mann-Whitney U test) in the coupled RBD-LS group than in the uncoupled RBD+LS (Fig. 2D,E; Figure 22). Additionally, we tested for vaccine induced mucosal immunity in the respiratory tract of vaccinated rabbits pre-challenge (Day 49) using ELISA. MERS-CoV specific antibodies were only detected in the nasal swabs of the groups vaccinated with conjugated or non-conjugated RBD (Fig. 2 F,G). Antibody responses detected in the RBD-LS vaccinated group were higher than those in the RBD + LS vaccinated group (P =

0.0357, Student’s t-test). This demonstrates that RBD-LS induces improved local mucosal immune responses compared to the monomeric RBD. Thus, vaccination with the newly produced RBD-LS MERS-CoV MPSP vaccines induce a robust immune response.

Avidity of RBD-LS induced antibodies

The avidity of MERS-CoV spike- specific antibodies in the monomeric versus the multimeric RBD vaccinated groups was analyzed at days 28 (4 weeks after prime) and 46 (3 weeks after boost) using an ammonium thiocyanate (NH4SCN)-displacement ELISA [33]. The avidity index IC50 was determined for each vaccinated rabbit and compared between the two groups. The avidity of the Sl-specific antibody responses was higher following RBD-LS vaccination compared to the monomeric RBD+LS vaccination (p<0.0001, Student’s t-test) (Fig. 3), indicating that a multimeric RBD-LS vaccine can induce antibody responses of both higher quantity and quality (Fig. 2D,E; Fig. 3)

Antibody responses to lumazine synthase scaffolds

In addition to evaluating anti-S (antigen) responses, we also tested for the induction on LS-specific (scaffold) antibodies. Antibody responses were elicited against the LS-particle in all groups except the PBS group, indicating that the particle was accessible and not sterically hidden by antigens displayed on its surface; even when RBD was displayed on its surface using SpyTag:SpyCatcher linkage (Fig. 4). Despite that, antigen- specific responses were not adversely affected by the presence of these anti scaffold antibodies, as demonstrated by the booster effect after the second immunization (Fig. 2D,E). Nonetheless, we tested whether a heterologous scaffold boost could help in minimizing such anti-scaffold responses using an LS/I3 prime-boost scheme. Using this approach, we found no significant increase in anti- scaffold antibody responses compared to the homologous prime-boost scheme (Fig. 4C). This indicates that a heterologous scaffold prime-boost approach could be advantageous for limiting unnecessary anti scaffold responses.

Efficacy of RBD-LS in preventing virus shedding and infection in rabbits To evaluate the protective efficacy of the immune responses induced by the different MERS-CoV spike MPSP vaccines, rabbits were challenged intranasally with 10⁶ TCID50 of MERS-CoV (strain HCoV- EMC/2012) and nasal swabs were collected up to 4 days post inoculation (pi) (Fig. 2A). On day 4 pi, the animals were euthanized, and lung tissue samples were collected. Consistent with earlier reports [34,35], none of the rabbits in the control group developed any clinical signs of infection upon MERS-CoV inoculation, and titration of infectious virus from lung tissues and nasal swabs was variable. Thus, to evaluate protection, we tested for MERS-CoV RNA by qRT-PCR, for MERS-CoV infectious virus by virus titration, and for MERS-CoV antigen (N protein) in lung tissues by immunohistochemistry (IHC). Except for the RBD-LS vaccinated group, viral RNA was detected in all vaccinated groups from day 1 through day 4 post-challenge at levels similar to control groups (Fig. 5, and 6). Viral RNA titers were significantly reduced in the nasal swabs of the RBD-LS vaccinated groups as early as day 1 post-challenge and were undetectable by day 4, in line with the absence of detectable infectious virus particles (Fig.

5). Viral RNA was also reduced in the lungs of RBD-LS-vaccinated rabbits (Fig. 6). Consistently, IHC revealed no viral antigen in the lungs of the RBD-LS vaccinated rabbits (Fig. 6C). Thus, in contrast to the monomeric form, the antigen-focused multimeric RBD-LS vaccine was able to block MERS-CoV replication significantly in the nose and lungs of the infected rabbits. Discussion

Recombinant subunit proteins provide advantages regarding safety, costs, and speed of vaccine production, making them very attractive platforms for the development of vaccines for emerging viruses. Multivalent antigen display allows for virus -mimicking presentation of antigens and has been shown to induce antibodies of high avidity and magnitude [7,10,11,27,36]; with non-viral self-assembling MPSP providing advantages over other multimeric antigen presentation platforms [8,12] Among the MERS-CoV vaccine candidates developed so far, the latter approach has been used to design two candidates, both are based on the receptor-binding domain [27,37], the main target for MERS-CoV protective antibodies [26]. One used self-assembling ferritin nanoparticles [27] and the second used canine parvovirus (CPV) VP2 structural protein forming virus like particles [37] as scaffolds. Both vaccine candidates were able to induce humoral and cellular immune responses in mice, nonetheless none has been tested for its protective capacity in a viral-challenge animal model. In our study, using an immune-focusing approach to target protective epitopes and domains along with multivalent presentation on self-assembling LS particles using a spontaneous covalent linker (SpyTag/Spy Catcher). We report for the first time the in-vivo protective capacity of a multimeric MERS-CoV RBD particle vaccine. We used self-assembling LS and 13 particles to generate chimeric multimeric protein scaffold particle displaying critical domains in the MERS-CoV spike protein and evaluated their immunogenicity and protective efficacy in rabbits. Multimeric FP and HR2 vaccinations induced high levels of anti-S2 antibodies, nonetheless, with low to undetectable virus neutralizing capacities and couldn’t protect rabbits against virus challenge. Meanwhile, multimeric RBD-LS vaccination was highly immunogenic and induced robust antibody responses of high magnitude, avidity and neutralizing capacity. Following a live virus challenge, it protected upper and lower respiratory tract of rabbits as detected by decrease in viral RNA titers, with an associated lack of MERS-CoV antigen (Fig. 5, 6). Despite producing strong antibody responses, the monomeric RBD failed to protect rabbits against MERS-CoV following an intranasal challenge. The presence of LS did not seem to influence the outcome, as it was included in the formulation of the monomeric form (RBD+LS), indicating that the coupling and the multimeric presentation are responsible for the enhanced response seen with the multimeric RBD-LS vaccine. The “plug-and- display” SpyTag/SpyCatcher system [8] used to generate these multimeric RBD-LS particles allows for rapid and robust production of vaccines in a cost- effective manner. This enables the development of vaccines in a timely manner, which is crucial to prevent global public health consequences of evolving, emerging and re-emerging viruses. When using scaffolds as antigen carriers, anti-scaffold antibody responses need to be considered to avoid their potential to compromise the targeted antigen-induced responses or to induce potential auto-antibodies against human antigens. Antibody responses were induced against the LS protein scaffold used in this study. However, antigen-specific responses were boosted following the second immunization and were not adversely affected by the presence of these anti scaffold antibodies (Fig. 4), similar to other reports [38]. Since the sequence of the LS protein does not show any similarity to any human sequences, it is unlikely that they will induce unwanted auto-(antihuman) antibodies. An LS-based vaccine for HIV, in a current phase 1 clinical trial (NCT03547245), can provide further evidence for the safety of this platform. Nonetheless, we developed a heterologous scaffold prime-boost using LS and 13 which can help in reducing anti-scaffold responses. Another challenge facing MERS-CoV vaccine development is the limited number of appropriate animal models for testing protection against clinical virus isolates. Rabbits provide some advantages as an animal model for MERS-CoV. By having the MERS-CoV receptor DPP4 expressed in both the upper and lower respiratory tract epithelium [24], the rabbits can be naturally infected. This allows the evaluation of both upper and lower respiratory tract MERS-CoV infection and in turn protection using natural field virus isolates rather than adapted strains. However, the animals are not able to develop severe infection such as that seen in severe human cases 34] Nonetheless, severe infection, thus far, has not been established consistently in any of the other animal models without genetic modification and/or virus adaptation, except for marmosets [39]. In addition to the aforementioned, rabbits are readily available and easier to handle compared to other species that can be naturally infected such as non-human primates. Following the addition of MERS-CoV as a priority pathogen in the WHO R&D Blueprint for action to prevent epidemics, a target product profile was developed which called for three types of MERS-CoV vaccines [40]. These include one for camels to prevent virus shedding and transmission, and two for humans: a two-dose vaccine for long-term protection of those at continuous high risk such as camel handlers and health-care workers, and a single-dose vaccine for rapid onset of immune responses to protect those at acute risk in outbreak settings. The RBD-LS can be used to develop the two-dose vaccine required to protect the high-risk populations, and can be further optimized using the heterologous scaffold prime/boost scheme developed in this study. Nonetheless, evaluating the longevity of the induced immune responses is warranted. Following the prime, RBD-LS vaccination induced antibody responses of high avidity and MERS-CoV neutralizing capacity. Owing to the robust immune responses induced after one dose, the RBD-LS can be a candidate for developing a rapid single-dose vaccine for MERS-CoV, which is required for reactive use in outbreak situations [40]. Additionally, this vaccine candidate was able to block MERS-CoV replication in the upper respiratory tract of infected rabbit, thus it could potentially be of use as a dromedary vaccine to block MERS-CoV transmission. Example 2: Expression and purification of SpyCatcher-mi3 recombinant protein, production of Mi3-SC/ST-RBD conjugate (SARS-CoV-2) and production of nanoparticle

BL21(DE3) competent E. coli cells were transformed with pET28a plasmids (Novagen) wherein nucleic acid sequences encoding the SpyCatcher-mi3 recombinant protein supplied with a C-tag sequence (Figure 13) were placed under control of an IPTG inducible promoter.

Cells were grown on LB medium agar plates supplemented with kanamycin (LB:Kanamycin, 1:1000) and single colonies were picked and grown in liquid LB/Kanamycin-medium. Subsequently, IPTG was used to induce expression of the recombinant proteins.

Following expression, cells were pelleted and lysed in a lysis buffer (lOmM Tris pH 8, 150mM NaCl, freshly supplemented with cOmplete™, Mini Protease Inhibitor Cocktail [Sigma- Aldrich], 100pg/mL lysozyme) by sonication. The cell lysate was clarified by ultracentrifugation, and the clarified lysate was mix with C-tag beads. After washing, the recombinant proteins were purified using the CaptureSelect™ C-tag Affinity Matrix (ThermoFisher Scientific). A SpyCatcher-mi3 recombinant protein of 33.6 kDa was thereby produced.

A nucleic acid construct encoding the fusion protein SARS-CoV-2 RBD-SpyTag (RBD-ST, Figure 13) was expressed in mammalian cells (HEK- 293T) largely as described in Example 1 and as described previously [17]. In short, expression plasmids comprising the RBD-ST sequence (Figure 13) were polyethylenimine (PEI)-transfected into 60% confluent HEK-293T cells for 6 h, after which transfections were removed and medium was replaced with 293 SFM Il-based expression medium (Gibco Life Technologies) and incubated at 37 °C in 5% CO2. Tissue culture supernatants were harvested 5—6 d post transfection, and expressed proteins were purified using StrepTactin Sepharose beads (IBA) according to the manufacturer’s instruction. An RBD-SpyTag recombinant protein for SARS-CoV-2 was provided.

The SpyCatcher-mi3 recombinant protein (Mi3-SC) was subsequently coupled to RBD-SpyTag (RBD-ST) through irreversible conjugation of their SpyTag/SpyCatch moieties. For this, different molar ratio’s of Mi3-SC and RBD-ST were used. Successful in vitro coupling was confirmed by SDS-Page and Western blotting as shown in Figure 10. The Mi3-SC/ST-RBD conjugate was subsequently allowed to self-assemble into a nanoparticle according to the invention. The resulting nanoparticles are displayed in Figure 12.

Example 3: Production of RBD-Mi3 genetic fusion (SARS-CoV-2)

In this example, a nucleic acid construct encoding the direct fusion between SARS-CoV-2 RBD and Mi3 (RBD-Mi3; Figure 13) was expressed in mammalian cells (HEK-293T) largely as described above. In short, expression plasmids comprising the RBD-Mi3 sequence (Figure 13) either with a StrepTag or with a C-Tag sequence were polyethylenimine (PEI)-transfected into 60% confluent HEK-293T cells for 6 h, after which transfections were removed and medium was replaced with 293 SFM II- based expression medium (Gibco Life Technologies) and incubated at 37 °C in 5% CO2. Tissue culture supernatants were harvested 5-6 d post transfection, and expressed proteins were purified using StrepTactin Sepharose beads (IBA) according to the manufacturer’s instruction (Figure 12, lanes 1, 2, and 3). or using the CaptureSelect™ C-tag Affinity Matrix (ThermoFisher Scientific) (Figure 12, lanes 4, 5, and 6). An RBD-Mi3 genetic fusion protein for SARS-CoV-2 was provided, which could form into a nanoparticle of the present invention as described above. Example 4. SARS-CoV-2 RBD-Mi3 nanoparticles elicit a neutralizing antibody response in hamsters.

SARS-CoV-2 RBD was coupled to nanoparticle (NP) 13-01 (Mi3) through the use of SpyTag- SpyCatcher proteins. SARS-CoV-2 RBD was produced in the Cl fungal expression system, the nanoparticle in E.coli. Figure 18 shows the protein sequences of the constructs used. RBD-SpyTag was coupled to SpyCat-NP which constitutes the coupled nanoparticle (RBD- SpyTag=SpyCat-NP).

SDS-PAGE analysis was performed to confirm correct expression of the protein constructs (Figure 19).

Hamsters were assigned to eight groups of five animals each.

Immunizations were performed intramuscularly with either PBS, RBD only (RBD-SpyTag), RBD (RBD-SpyTag) and nanoparticle (SpyTag-NP) mixed (i.e. uncoupled) or RBD (RBD-SpyTag) coupled to the nanoparticle (SpyCat- NP) in the absence or presence of aluminium hydroxide at day 0 and day 28. The coupled nanoparticle is referred to as RBD-SpyTag=SpyCat-NP. Each animal received each time 10 pg of RBD antigen. For the coupled RBD (RBD-SpyTag=SpyCat-NP), the RBD-SpyTag was mixed with the SpyCat- NP in a 1:3 molar ratio. For the non-coupled RBD, the RBD-SpyTag and SpyTag-NP were mixed in a 1:3 molar ratio. Each animal received each time 10 pg of RBD antigen. The vaccination schedule is shown in Figure 20.

Serum samples were collected on days 0, 28, and 42. We performed ELISAs by coating 96-well microtiter ELISA plates with SARS-CoV-2 RBD antigen in PBS overnight at 4°C. After blocking, we added diluted serum (diluted 1:100 or 2-fold serially diluted for titers) and incubated at 37°C for 1 h. Antigen- specific antibodies were detected by using peroxidase-labeled rabbit anti-hamster IgG (Dako) and 3,3',5,5'-tetramethylbenzidine as a substrate. The absorbance of each sample was measured at 450 nm, and we set the cutoff value at 6 SD above the mean value for the negative serum samples.

All animals immunized with the RBD- nanoparticle (RBD-SpyTag=SpyCat- NP / RBD-NP) in the presence of aluminiumhydroxide produced SARS-CoV- 2 RBD specific antibodies after one immunization (Fig. 2 IB) whereas no antibodies were detected at the start of the experiment (Fig. 21A). Virus specific antibodies were also detected in animals immunized with RBD- nanoparticles (RBD-SpyTag=SpyCat-NP / RBD-NP) in the absence of aluminiumhydroxide, after the boost immunization (Fig 21C).

Next we tested serum samples for their neutralization capacity against SARS-CoV-2 (German isolate; GISAID ID EPI_ISL 406862; European Virus Archive Global #026V-03883) by using PRNT. We 2-fold serially diluted heat- inactivated samples in Dulbecco modified Eagle medium supplemented with NaHC03, HEPES buffer, penicillin, streptomycin, and 1% fetal bovine serum, starting at a dilution of 1:10 in 50 pL. We then added 50 pL of virus suspension (400 plaque-forming units) to each well and incubated at 37°C for 1 h before placing the mixtures on Vero-E6 cells. After incubation for 1 h, we washed cells, supplemented them with medium, and incubated for 8 h. After incubation, we fixed the cells with 4% formaldehyde/phosphate- buffered saline (PBS) and stained the cells with polyclonal rabbit anti- SARS-CoV antibody and a secondary peroxidase-labeled goat anti-rabbit IgG. We developed signal by using a precipitate forming 3, 3', 5,5'- tetramethylbenzidine substrate and counted the number of infected cells per well by using an ImmunoSpot Image Analyzer. The serum neutralization titer is the reciprocal of the highest dilution resulting in an infection reduction of >50% (PRNT50). We considered a titer >20 to be positive. High levels of neutralizing antibodies were detected in animals immunized with the RBD-nanoparticle in the presence of aluminiumhydroxide (Fig. 2 ID). References

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SEQUENCE LISTING

SEQ ID NO:1

Definition: SARS-CoV-2 receptor binding domain (RBD) of spike (S) protein

TNLCPFGEVF NATRFASVYA WNRKRISNCV ADYSVLYNSA SFSTFKCYGV SPTKLNDLCF TNVYADSFVI RGDEVRQIAP GQTGKIADYN YKLPDDFTGC VIAWNSNNLD SKVGGNYNYL YRLFRKSNLK PFERDISTEI YQAGSTPCNG VEGFNCYFPL QSYGFQPTNG VGYQPYRVW LSFELLHAPA TVCGP

SEQ ID NO:2

Definition: mutant 13-01 (Mi3)

MKMEELFKKH KIVAVLRANS VEEAKKKALA VFLGGVHLIE ITFTVPDADT VIKELSFLKE MGAIIGAGTV TSVEQARKAV ESGAEFIVSP HLDEEISQFA KEKGVFYMPG VMTPTELVKA MKLGHTILKL FPGEW GPQF VKAMKGPFPN VKFVPTGGVN LDNVCEWFKA GVLAVGVGSA LVKGTPVEVA EKAKAFVEKI RGCTE

SEQ ID NO:3 Definition: Lumazine Synthase

MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEI PVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITA DTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR SEQ ID NO:4

Definition : SpyTag

AHIVMVDAYK PTK

SEQ ID NO:5 Definition: SpyCatcher

DSATHIKFSK RDEDGKELAG ATMELRDSSG KTISTWISDG QVKDFYLYPG KYTFVETAAP DGYEVATAIT FTVNEQGQVT VNGKATKGDA HI SEQ ID NO:6

Definition: MERS-CoV Spike (S) glycoprotein YP_009047204.1 [Middle East respiratory syndrome-related coronavirus]

MIHSVFLLMF LLTPTESYVD VGPDSVKSAC IEVDIQQTFF DKTWPRPIDV SKADGIIYPQ GRTYSNITIT YQGLFPYQGD HGDMYVYSAG HATGTTPQKL

FVANYSQDVK QFANGFW RI GAAANSTGTV IISPSTSATI RKIYPAFMLG

SSVGNFSDGK MGRFFNHTLV LLPDGCGTLL RAFYCILEPR SGNHCPAGNS

YTSFATYHTP ATDCSDGNYN RNASLNSFKE YFNLRNCTFM YTYNITEDEI

LEWFGITQTA QGVHLFSSRY VDLYGGNMFQ FATLPVYDTI KYYSIIPHSI RSIQSDRKAW AAFYVYKLQP LTFLLDFSVD GYIRRAIDCG FNDLSQLHCS

YESFDVESGV YSVSSFEAKP SGSW EQAEG VECDFSPLLS GTPPQVYNFK

RLVFTNCNYN LTKLLSLFSV NDFTCSQISP AAIASNCYSS LILDYFSYPL

SMKSDLSVSS AGPISQFNYK QSFSNPTCLI LATVPHNLTT ITKPLKYSYI

NKCSRLLSDD RTEVPQLVNA NQYSPCVSIV PSTVWEDGDY YRKQLSPLEG GGWLVASGST VAMTEQLQMG FGITVQYGTD TNSVCPKLEF ANDTKIASQL

GNCVEYSLYG VSGRGVFQNC TAVGVRQQRF VYDAYQNLVG YYSDDGNYYC

LRACVSVPVS VIYDKETKTH ATLFGSVACE HISSTMSQYS RSTRSMLKRR

DSTYGPLQTP VGCVLGLVNS SLFVEDCKLP LGQSLCALPD TPSTLTPRSV

RSVPGEMRLA SIAFNHPIQV DQLNSSYFKL SIPTNFSFGV TQEYIQTTIQ KVTVDCKQYV CNGFQKCEQL LREYGQFCSK INQALHGANL RQDDSVRNLF

ASVKSSQSSP IIPGFGGDFN LTLLEPVSIS TGSRSARSAI EDLLFDKVTI

ADPGYMQGYD DCMQQGPASA RDLICAQYVA GYKVLPPLMD VNMEAAYTSS

LLGSIAGVGW TAGLSSFAAI PFAQSIFYRL NGVGITQQVL SENQKLIANK

FNQALGAMQT GFTTTNEAFQ KVQDAVNNNA QALSKLASEL SNTFGAISAS IGDIIQRLDV LEQDAQIDRL INGRLTTLNA FVAQQLVRSE SAALSAQLAK

DKVNECVKAQ SKRSGFCGQG THIVSFW NA PNGLYFMHVG YYPSNHIEW

SAYGLCDAAN PTNCIAPVNG YFIKTNNTRI VDEWSYTGSS FYAPEPITSL

NTKYVAPQVT YQNISTNLPP PLLGNSTGID FQDELDEFFK NVSTSIPNFG

SLTQINTTLL DLTYEMLSLQ QW KALNESY IDLKELGNYT YYNKWPWYIW LGFIAGLVAL ALCVFFILCC TGCGTNCMGK LKCNRCCDRY EEYDLEPHKV

HVH SEQ ID NO:7

Definition: Receptor Binding Domain (RBD) of MERS-CoV spike (S) glycoprotein [Middle East respiratory syndrome-related coronavirus] SGVYSVSSFE AKPSGSW EQ AEGVECDFSP LLSGTPPQVY NFKRLVFTNC

NYNLTKLLSL FSVNDFTCSQ ISPAAIASNC YSSLILDYFS YPLSMKSDLS

VSSAGPISQF NYKQSFSNPT CLILATVPHN LTTITKPLKY SYINKCSRLL

SDDRTEVPQL VNANQYSPCV SIVPSTVWED GDYYRKQLSP LEGGGWLVAS

GSTVAMTEQL QMGFGITVQY GTDTNSVCPKL

SEQ ID NO:8

Definition: Fusion Peptide (FP) of MERS-CoV spike (S) glycoprotein [Middle East respiratory syndrome-related coronavirus] RSARSAIEDL LFDKV

SEQ ID NO:9

Definition: Heptad repeat 2 (HR2) of MERS-CoV spike (S) glycoprotein [Middle East respiratory syndrome-related coronavirus]

STNLPPPLLG NSTGIDFQDE LDEFFKNVST SIPNFGSLTQ INTTLLDLTY

EMLSLQQW K ALNESYIDLK ELG

SEQ ID NO:10 Definition: LS-Linker-StrepTag construct

MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEI PVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITA DTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGSGGSGGSGGGASLINDYK DDDDKAGPGWSHPQFEK

SEQ ID NO:11

Definition: Linker of LS-Linker-StrepTag construct GGSGGSGGSGGSGGGASLINDYKDDDDKAGPG

SEQ ID NO:12

Definition: StrepTag of LS-Linker-StrepTag construct

WSHPQFEK

SEQ ID NO:13

Definition: Receptor Binding Domain (RBD) of SARS-CoV-1 spike (S) glycoprotein

TNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLC

FSNVYADSFW KGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYN

YKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRV

W LSFELLNAPATV

SEQ ID NO:14

Definition: signal sequence

MFVFLVLLPLVSS

SEQ ID NO:15

Definition: C-tag sequence

EPEA

SEQ ID NO:16

Definition: Synthetic self-assembling protein of SARS-CoV-2 RBD fused N-terminally to Mi3

MFVFLVLLPLVSSTNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFK CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWN SNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYG FQPTNGVGYQPYRVW LSFELLHAPATVCGPGGSGGSGGSGGSMKMEELFKKHKIVAVL RANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAI IGAGTVTSVEQ ARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGE W GPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKA

KAFVEKIRGCTEGSGWSHPQFEK

SEQ ID NO:17 Definition: 13-01

MKMEELFKKH KIVAVLRANS VEEAKKKALA VFLGGVHLIE ITFTVPDADT VIKELSFLKE MGAIIGAGTV TSVEQCRKAV ESGAEFIVSP HLDEEISQFC KEKGVFYMPG VMTPTELVKA MKLGHTILKL FPGEW GPQF VKAMKGPFPN VKFVPTGGVN LDNVCEWFKA GVLAVGVGSA LVKGTPVEVA EKAKAFVEKI RGCTE

SEQ ID NO: 18

Definition: RBD-Mi3 SARS-CoV-2 fusion protein

TNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLC FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYR W VLSFELLHAPATVCGPGGSGGSGGSGGSMKMEELFKKHKIVAVLRANSVEEAKKKAL AVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAI IGAGTVTSVEQARKAVESGAEFIV SPHLDEEISQFAREKGVFYMPGVMTPTELVKAMKLGHT ILKLFPGEVVGPQFVKAMKGP FPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKI RGCTE

SEQ ID NO: 19

Definition: RBD-ST SARS-CoV-2 fusion protein

TNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLC FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYR W VLSFELLHAPATVCGPSGESGAHIVMVDAYKPTK

SEQ ID NO: 20

Definition: RBD-Mi3 SARS-CoV-1 fusion protein

NITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLND

LCFSNVYADSFW KGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGN YNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPY RVW LSFELLNAPATVGGSGGSGGSGGSMKMEELFKKHKIVAVLRANSVEEAKKKALAV FLGGVHLIEITFTVPDADTVIKELSFLKEMGAI IGAGTVTSVEQARKAVESGAEFIVSP HLDEEISQFAREKGVFYMPGVMTPTELVKAMKLGHT ILKLFPGEW GPQFVKAMKGPFP NVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTE

SEQ ID NO: 21

Definition: RBD-ST SARS-CoV-1 fusion protein

NITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLND

LCFSNVYADSFW KGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGN

YNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPY

RVW LSFELLNAPATVSGESGAHIVMVDAYKPTK

SEQ ID NO: 22

Definition: RBD-Mi3 MERS-CoV fusion protein

SGVYSVSSFEAKPSGSW EQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLS LFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSN PTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTV WEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLGGSGG SGGSGGSMKMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIE ITFTVPDADTVI KELSFLKEMGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGV MTPTELVKAMKLGHTILKLFPGEW GPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKA GVLAVGVGSALVKGTPVEVAEKAKAFVEK IRGCTE

SEQ ID NO: 23

Definition: RBD-ST MERS-CoV fusion protein

SGVYSVSSFEAKPSGSW EQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLL FSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPI SQFNYKQSFSNP TCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVW EDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLSGESGA

HIVMVDAYKPTK SEQ ID NO: 24

Definition: RBD-i3 MERS-CoV fusion protein

SGVYSVSSFEAKPSGSW EQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLS LFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSN PTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTV WEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLGGSGG SGGSGGSMKMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIE ITFTVPDADTVI KELSFLKEMGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGV MTPTELVKAMKLGHTILKLFPGEW GPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKA GVLAVGVGSALVKGTPVEVAEKAKAFVEK IRGCTE

SEQ ID NO: 25 Definition: SC-Mi3

DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAA PDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGSGGSGGSGGSMKMEELFKKHKIVA VLRANSVEEAKKKALAVFLGGVHL IEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSV EQARKAVESGAEFIVSPHLDEEISQFAREKGVFYMP GVMTPTELVKAMKLGHTILKLFP GEW GPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAE KAKAFVEKIRGCTEGSGEPEA

SEQ ID NO: 26

Definition: SC-Mi3 without CTag and linker

DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAA PDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGSGGSGGSGGSMKMEELFKKHKIVA VLRANSVEEAKKKALAVFLGGVHL IEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSV EQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMP GVMTPTELVKAMKLGHTILKLFP GEW GPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAE KAKAFVEKIRGCTE SEQ ID NO: 27

Definition: SC-LS without CTag and linker DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAA PDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGSGGSGGSGGSMQI YEGKLTAEGLR FGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDID AVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKH GNKGWEAALSAIEMANLFKSLR

SEQ ID NO:28

Definition: SARS-CoV-2 receptor binding domain (RBD) of spike (S) protein

TNLCPFGEVF NATRFASVYA WNRKRISNCV ADYSVLYNSA SFSTFKCYGV SPTKLNDLCF TNVYADSFVI RGDEVRQIAP GQTGKIADYN YKLPDDFTGC VIAWNSNNLD SKVGGNYNYL YRLFRKSNLK PFERDISTEI YQAGSTPCNG VEGFNCYFPL QSYGFQPTNG VGYQPYRVW LSFELLHAPA TV

SEQ ID NO:29

Definition: SpyCatcher protein moiety

MGSSDSATHI KFSKRDEDGK ELAGATMELR DSSGKTISTW ISDGQVKDFY LYPGKYTFVE TAAPDGYEVA TAITFTVNEQ GQVTVNGKAT KGDAHI

SEQ ID NO:30

Definition: SARS-CoV-2 RBD-SpyTag protein

MYAKFATLAA LVAGAAATNL CPFGEVFNAT RFASVYAWNR KRISNCVADY SVLYNSASFS TFKCYGVSPT KLNDLCFTNV YADSFVIRGD EVRQIAPGQT GKIADYNYKL PDDFTGCVIA WNSNNLDSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPCNGVEG FNCYFPLQSY GFQPTNGVGY QPYRVW LSF ELLHAPATVC GPGGGGSAHI VMVDAYKPTK GGGGSEPEA

SEQ ID NO:31

Definition: SpyCatch-Mi3 protein

MGSSDSATHI KFSKRDEDGK ELAGATMELR DSSGKTISTW ISDGQVKDFY LYPGKYTFVE TAAPDGYEVA TAITFTVNEQ GQVTVNGKAT KGDAHIGGSG GSGGSGGSMK MEELFKKHKI VAVLRANSVE EAKKKALAVF LGGVHLIEIT FTVPDADTVI KELSFLKEMG AIIGAGTVTS VEQARKAVES GAEFIVSPHL

DEEISQFAKE KGVFYMPGVM TPTELVKAMK LGHTILKLFP GEVVGPQFVK

AMKGPFPNVK FVPTGGVNLD NVCEWFKAGV LAVGVGSALV KGTPVEVAEK

AKAFVEKIRG CTEGSGEPEA

Claims

1. A self-assembling protein nanoparticle that displays on the outer surface a receptor binding domain (RBD) of a coronavirus spike (S) protein.

2. The nanoparticle according to claim 1, wherein said nanoparticle is a multimeric protein particle that self-assembles by multimerization of a protein monomer.

3. The nanoparticle according to any one of the preceding claims, wherein said nanoparticle is a multimeric protein particle that self- assembles by homomultimerization of a protein monomer.

4. The nanoparticle according to any one of the preceding claims, wherein said nanoparticle is a synthetic or artificial nanoparticle.

5. The nanoparticle according to any one of the preceding claims, wherein said nanoparticle is non-viral and/or not a virus.

6. The nanoparticle according to any one of the preceding claims, wherein said nanoparticle is a 60-meric particle.

7. The nanoparticle according to any one of the preceding claims, wherein said nanoparticle is a 60-meric particle with an icosahedral symmetry.

8. The nanoparticle according to any one of the preceding claims, wherein said nanoparticle is a multimeric protein scaffold particle (MPSP).

9. The nanoparticle according to any one of the preceding claims, wherein said nanoparticle comprises a multimer of a protein monomer that is selected from the group formed by an 13-01 protein monomer and a lumazine synthase protein monomer.

10. The nanoparticle according to any one of the preceding claims, wherein said 13-01 protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:2 or 17 and/or wherein said lumazine synthase protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:3, or sequences having at least 90%, preferably at least 95% sequence identity therewith and self-assemble by multimerization into a protein nanoparticle.

11. The nanoparticle according to any one of the preceding claims, wherein said RBD comprises or consists of:

- an amino acid sequence selected from SEQ ID NOs:l, 7, 13 and 28;

- an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:l, 7, 13 or 28, and which preferably binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle of any one of claims 1-10 wherein said RBD comprises or consists of an amino acid sequence of SEQ ID NO:l, 7, 13 or 28, respectively; and/or

- an antigenic part of the amino acid sequence of SEQ ID NO:l, 7, 13 or 28 which preferably binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle of any one of claims 1-10 wherein said RBD comprises or consists of an amino acid sequence of SEQ ID NO:l, 7, 13 or 28, respectively.

12. The nanoparticle according to any one of the preceding claims, wherein said nanoparticle displays said RBD multivalently.

13. The nanoparticle according to any one of the preceding claims, wherein each protein monomer in said self-assembling nanoparticle is coupled to an RBD thereby multivalently displaying said RBD on the outer surface of said nanoparticle.

14. The nanoparticle according to any one of the preceding claims, wherein each protein monomer in said self-assembling nanoparticle is coupled to an RBD through

15. The nanoparticle according to any one of the preceding claims, wherein said RBD is indirectly fused to said protein monomer, preferably said RBD is indirectly fused to said protein monomer and separated by a linker protein such as a glycine/serine (GS) linker protein.

16. The nanoparticle according to any one of the preceding claims, wherein said SpyTag protein moiety comprises or consists of the amino acid sequence as indicated in SEQ ID NO:4 and/or wherein said SpyCatcher protein moiety comprises or consists of the amino acid sequence as indicated in SEQ ID NO:5 or SEQ ID NO:29.

17. The nanoparticle according to any one of the preceding claims, wherein said SpyTag protein moiety is linked to said RBD through a glycine/serine (GS) linker and/or wherein said SpyCatcher protein moiety is linked to said protein monomer through a GS linker.

18. The nanoparticle according to any one of the preceding claims, wherein said nanoparticle is obtainable by allowing (i) a protein that comprises or consists of the amino acid sequence of SEQ ID NO:30 and (ii) a protein that comprises or consists of the amino acid sequence of SEQ ID NO:31 to self-assemble into a nanoparticle that displays on the outer surface a receptor binding domain (RBD) of a coronavirus spike (S) protein, preferably wherein one or more, preferably each, protein monomer in said self-assembling nanoparticle is coupled to an RBD by allowing the formation of an isopeptide bond between said (i) protein that comprises or consists of the amino acid sequence of SEQ ID NO:30 and said (ii) protein that comprises or consists of the amino acid sequence of SEQ ID NO:31.

19. A fusion protein comprising a first part that is a receptor binding domain (RBD) of a coronavirus spike (S) protein and a second part that is a SpyTag protein moiety.

20. The fusion protein according to claim 19, wherein said RBD comprises or consists of:

- an amino acid sequence of SEQ ID NO:l, 7, 13 or 28;

- an antigenic part of the amino acid sequence of SEQ ID NO: 1, 7 or 13 which preferably binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle of any one of claims 1-10 wherein said RBD comprises or consists of an amino acid sequence of SEQ ID NO:l, 7, 13 or 28, respectively.

21. The fusion protein according to any one of the preceding claims, wherein said SpyTag protein moiety comprises or consists of the amino acid sequence as indicated in SEQ ID NO:4.

22. The fusion protein according to any one of the preceding claims, wherein said first part and said second part are linked through a glycine/serine (GS) linker.

23. The fusion protein according to any one of the preceding claims, wherein said fusion protein further comprises a tag such as a Streptag.

24. The fusion protein according to any one of the preceding claims, wherein said RBD is located N-terminally relative to said SpyTag protein moiety.

25. The fusion protein according to any one of the preceding claims, wherein said fusion protein comprises or consists of the amino acid sequence of SEQ ID NO:30.

26. A fusion protein comprising a first part that is a receptor binding domain (RBD) of a coronavirus spike (S) protein and second part that is a protein monomer that is selected from the group formed by an 13-01 protein monomer and a lumazine synthase protein monomer; preferably wherein said RBD comprises or consists of - an amino acid sequence of SEQ ID NO:l, 7, 13 or 28;

- an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:l, 7, 13 or 28, and which preferably binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle of any one of claims 1-10 wherein said RBD comprises or consists of an amino acid sequence of SEQ SEQ ID NO:l, 7, 13 or 28, respectively; and/or - an antigenic part of the amino acid sequence of SEQ ID NO:l, 7, 13 or 28 which preferably binds to a product of an immune response, preferably an antibody, that is elicited when a subject is immunized with a nanoparticle of any one of claims 1-10 wherein said RBD comprises or consists of an amino acid sequence of SEQ ID NO:l, 7, 13 or 28, respectively.

27. The fusion protein according to claim 26, wherein said 13-01 protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:2 or 17 and/or wherein said lumazine synthase protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:3.

28. The fusion protein according to any one of claims 26 or 27, wherein said first part and said second part are linked through a glycine/serine (GS) linker.

29. The fusion protein according to any one of claims 27-28, wherein said fusion protein further comprises a tag such as a Streptag.

30. A nucleic acid molecule encoding the fusion protein according to any one of claims 19-29.

31. An expression vector comprising the nucleic acid molecule of claim 30.

32. A host cell comprising the nucleic acid molecule or expression vector according to claim 30 or claim 31, preferably wherein said host cell is a eukaryotic host cell, preferably selected from a fungal cell, a yeast cell or a mammalian cell, still more preferably a fungus cell.

33. A method of producing a self-assembling nanoparticle according to any one of claims 1-18, comprising the steps of:

- providing a first fusion protein that is a fusion protein according to any one of claims 19-25; - providing a second fusion protein that is fusion protein comprising

- a first part that is a protein monomer selected from the group formed by an 13-01 protein monomer and a lumazine synthase protein monomer, preferably wherein said 13-01 protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:2 or 17 and/or wherein said lumazine synthase protein monomer comprises or consists of the amino acid sequence of SEQ ID NO:3; and

- a second part that is a SpyCatcher protein moiety, preferably wherein said SpyCatcher protein moiety comprises or consists of the amino acid sequence as indicated in SEQ ID NO:5 or SEQ ID

NO:29;

- optionally further comprising a tag such as a Streptag; - allowing said first and second fusion protein to self-assemble into a nanoparticle that displays on the outer surface a receptor binding domain (RBD) of a coronavirus spike (S) protein.

34. A method of producing a nanoparticle according to any one of claims 1-18, comprising the steps of:

- providing a fusion protein according to any one of claims 26-29;

35. A self-assembling nanoparticle obtainable by a method according to claim 33 or 34.

36 . An immunogenic composition comprising a self-assembling nanoparticle according to any one of claims 1-18 or 35 and a pharmaceutically acceptable carrier, preferably an aqueous liquid.

37. The immunogenic composition according to claim 36, further comprising a pharmaceutically acceptable adjuvant.

38. A method of preventing or treating an infectious disease caused by coronavirus, preferably a MERS or SARS-CoV-2, such as COVID-19, in a subject, comprising the step of:

- administering a therapeutically effective amount of a self-assembling nanoparticle according to any one of claims 1-18 or 35 or an immunogenic composition according to any one of claims 36-37.

39. A method of immunizing a subject, comprising the step of:

- administering (an effective amount) a self-assembling nanoparticle according to any one of claims 1-18 or 35 or an immunogenic composition according to any one of claims 36-37.

40. The method according to any one of claims 38 or 39, wherein said self-assembling nanoparticle or said immunogenic composition is for administration according to a prime boost immunization schedule.

41. The nanoparticle according to any one of claims 1-18 or 35, the fusion protein according to any one of claims 19-29, or the immunogenic composition according to any one of claims 36 or 37, for use as a medicament, preferably for use in (i) treating or preventing an infectious disease caused by coronavirus, preferably a MERS or SARS-CoV-2, such as COVID-19, in a subject or (ii) immunizing a subject.

42. Use of the nanoparticle according to any one of claims 1-18 or 35, the fusion protein according to any one of claims 19-29, or the immunogenic composition according to any one of claims 36 or 37, for use in the manufacture of a medicament for treating or preventing an infectious disease caused by coronavirus, preferably a MERS or SARS-CoV-2, such as COVID-19, in a subject.