WO2022229817A1

WO2022229817A1 - Vaccines comprising virus-like particles displaying sars-cov-2 antigens and methods of use

Info

Publication number: WO2022229817A1
Application number: PCT/IB2022/053821
Authority: WO
Inventors: Ariane Volkmann; Adam Frederik Sander Bertelsen
Original assignee: Bavarian Nordic A/S; Adaptvac Aps
Priority date: 2021-04-28
Filing date: 2022-04-25
Publication date: 2022-11-03

Abstract

The invention provides vaccines comprising virus-like particles displaying at least one SARS-CoV-2 antigen. Virus-like particles (VLPs) comprising AP205 protein display antigens such as the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using a connection comprising a peptide tag and binding partner. The invention also relates to methods of treatment using the VLPs of the invention and use of the VLPs to treat and/or prevent infection with the SARS-CoV-2 virus and variants thereof. The invention further relates to medical uses of the VLPs in the prevention and/or amelioration of COVID-19 symptoms.

Description

VACCINES COMPRISING VIRUS-LIKE PARTICLES DISPLAYING SARS-COV-2

ANTIGENS AND METHODS OF USE

FIELD OF THE INVENTION

The present invention relates to vaccines comprising virus-like particles displaying at least one SARS-CoV-2 antigen, such as the receptor-binding domain (RBD) of the SARS- CoV-2 spike protein. Antigens are displayed on virus-like particles (VLPs) comprising AP205 using a peptide tag and binding partner. The invention also relates to methods of treatment using the VLPs to treat and/or prevent infection with the SARS-CoV-2 virus and variants thereof. The invention further relates to medical uses of the recombinant VLPs in the prevention of COVID-19.

BACKGROUND OF THE INVENTION

SARS-CoV-2 was described soon after a series of unidentified pneumonia diseases had occurred in Wuhan, China, at the end of 2019 (Zhou et al. (2020) Nature 579: 270-3). Typical clinical symptoms were reported to be fever, dry cough, dyspnea, headache, and pneumonia, and the infection occasionally resulted in progressive respiratory failure due to alveolar damage and even death (Zhou et al. (2020) Nature 579: 270-3). Moreover, olfactory and gustatory disorders are regarded as strong specific symptoms (Lechien et al. (2020) Eur. Arch. Otorhinolaryngol. 6: 1-11). In March 2020, WHO characterized the disease caused by SARS-CoV-2 - meanwhile referred to as coronavirus disease 2019 (COVID-19) - as a pandemic. SARS-CoV-2 showed efficient transmission in the human population with a reproductive index R0 of more than 3 in the initial phase of the pandemic.

COVID-19, similar to the diseases caused by SARS-CoV-1 and MERS-CoV, is considered to have its origin in a zoonotic transfer of the causative virus from its natural reservoir host, most likely bats, to humans, possibly via an intermediate mammalian host.

Due to the fact that COVID-19 appeared only recently, the knowledge and understanding of the disease and its causative virus, SARS-CoV-2, is limited.

SARS-CoV-2 belongs to the Coronaviridae family, a family of positive-sense, single- stranded RNA viruses. Like other coronaviruses, SARS-CoV-2 is characterized by a crown like (“corona”) appearance when viewed by electron microscopy which is produced by the spikes extruding from the virus surface. Such spike (S) proteins are essential for attachment and entry of the virus into host cells. The SARS-CoV-2 S protein is a large type I transmembrane protein composed of two subunits, SI and S2. The SI subunit contains a receptor-binding domain (RBD) that mediates virus attachment to the host cell receptor. The S2 subunit (ectodomain) mediates fusion between the viral and host cell membranes.

It is assumed that the S protein plays a key role in the induction of neutralizing antibodies, T cell responses and protective immunity. The entry of SARS-CoV-2 into host cells involves a series of conformational changes upon binding to the cellular receptor angiotensin-converting enzyme 2 (ACE), and eventually the S protein undergoes a substantial structural rearrangement from the prefusion to the postfusion conformation (Wrapp et al. (2020) Science 367: 1260-3). To prevent entry of SARS-CoV-2 into host cells, antibodies against the prefusion form of S are considered to be much more effective than those against the postfusion form, which renders the prefusion form of SARS-CoV-2 S the preferred antigenic conformation of S for a vaccine. It has been reported that the RBD within the S protein forms the main target for the induction of neutralizing antibody responses, which correlate with disease outcome in macaques (Mercado et al. (2020) Nature 586: 583- 88).

The use of peptide tags and binding partners for linking or attaching proteins to each other and other entities is a useful tool of molecular biology and can be used, inter alia , for generating capsid-like particles or virus-like particles (VLPs) covered with proteins, for example, as described in WO 2016/112921. Peptide tags and binding partners can be used to display molecules such as antigens on the surface of VLPs, including for use in vaccines. Some peptide tag and binding partner pairs interact via an isopeptide bond that can form spontaneously and provide a stable or irreversible bond between the peptide tag and its binding partner. Isopeptide bonds are amide bonds formed between carboxyl/carboxamide and amino groups, where at least one of the carboxyl or amino groups is outside of the main chain of the protein that forms the “backbone” of the protein. These bonds are resistant to most proteases and chemically irreversible under normal biological conditions.

Using peptide tags and binding partners that form isopeptide bonds, other peptides or molecules that are attached to the peptide tag and/or the binding partner are also linked to each other via the interaction between the peptide tag and binding partner. In this manner, peptide tags and binding partners can be used to attach molecules such as antigens to VLPs, for example, for use in vaccines. VLPs decorated with the SARS-CoV-2 RBD have been described by Fougeroux et al. (2021) Nat. Commun. 12: 324.

However, despite global efforts and increasing knowledge about SARS-CoV-2, the ongoing pandemic and repeated emergence of variants with the ability to escape control by some current vaccines has created a need for effective prophylactic vaccines against SARS- CoV-2 variants.

By April 2021, several variants of the wild-type virus originally detected in Wuhan had been identified and their sequences determined, e.g .: B.l.1.7 (so-called “British variant,” later designated “Alpha”), B.1.351 (“South- African variant,” later designated “Beta”), P.l (“Brazilian variant,” later designated “Gamma”), B.1.525 (a combination of the British and South-African variants), and B.1.617 (“Indian variant,” of which B.1.617.2 was later designated “Delta”; also sometimes denoted Bl.617.2). By September 2021, additional identified variants included, e.g., P.l (“Gamma”), B.1.525 (“Eta”), B.1.526 (“Iota”),

B.1.617.1 (“Kappa”), C.37 (“Lambda”), and B.1.621 (“Mu).

For these reasons, there is an urgent need for the further development of vaccines against SARS-CoV-2 virus infections.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide a vaccine against SARS-CoV-2 infection and related diseases.

The invention provides vaccines comprising virus-like particles displaying at least one SARS-CoV-2 antigen. A SARS-CoV-2 antigen is any antigen that produces an immune response to SARS-CoV-2, such as, for example, the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein or a portion thereof. Antigens are displayed on virus-like particles (VLPs) comprising the RNA bacteriophage AP205 coat protein (“AP205”) using a peptide tag and binding partner.

In some embodiments of the invention, the peptide tag and binding partner comprise the amino acid sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 6, respectively.

The invention also provides methods of treatment using the VLPs of the invention and use of the VLPs to treat and/or prevent infection with the SARS-CoV-2 virus and variants thereof. The invention further provides medical uses of the VLPs in the prevention and/or amelioration of COVID-19 symptoms. Particularly, in one aspect the invention provides a virus-like particle (VLP) comprising an AP205 protein fused to a peptide tag and comprising a SARS-CoV-2 antigen fused to a peptide binding partner, whereby the SARS-CoV-2 antigen is displayed on the surface of said VLP.

In another aspect, the invention provides a virus-like particle (VLP) comprising: (a) an AP205 protein fused to a peptide tag having the amino acid sequence set forth in SEQ ID NO:4, or a derivative thereof; and (b) a SARS-CoV-2 antigen fused to a peptide binding partner having the amino acid sequence set forth in SEQ ID NO: 6, or a derivative thereof, whereby the SARS-CoV-2 antigen is displayed on the surface of said VLP, that when administered to a subject as a vaccine stimulates a response that prevents or alleviates symptoms of coronavirus infection caused by SARS-CoV-2 variants B.1.1.7 and/or B.1.351.

In yet another aspect, the invention provides a virus-like particle (VLP) comprising:

(i) an AP205 protein fused to a peptide tag; and (ii) a SARS-CoV-2 antigen comprising the wild-type (Wuhan) spike protein Receptor Binding Domain (RBD), whereby the SARS-CoV- 2 antigen is displayed on the surface of said VLP; and that when administered to a subject as a vaccine stimulates an immune response that prevents or alleviates symptoms of coronavirus infection caused by SARS-CoV-2 variants such as B.1.1.7, B.1.351, and/or B 1.617.2.

In yet another aspect, the invention provides a vaccine comprising the VLP according to the invention in an aqueous solution that contains no squalene (i.e., that does not contain any squalene).

In yet another aspect, the invention provides a method of treating a subject to prevent or ameliorate symptoms of a coronavirus infection, preferably coronavirus disease 19 (COVID-19), comprising the step of administering the vaccine according to the invention to a subject.

In yet another aspect, the invention provides a VLP according to the invention, or a vaccine according to the invention, for use in the prevention or treatment of a coronavirus infection, preferably coronavirus disease 19 (COVID-19).

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description serve to explain the principles of the invention. Figure 1 diagrams the construction of VLPs; in this example, the VLP comprises AP205 coat protein fused to a peptide tag; the VLP is coated with SARS-CoV-2 RBD antigen genetically fused to a binding partner (“catcher”). The components were produced by recombinant expression; the Spike Receptor Binding Domain (“RBD”) antigen was produced in Drosophila S2 cells, and the virus-like particle was produced in E. coli. The components were then mixed, and the peptide tag and binding partner fused together in a spontaneous reaction that resulted in an isopeptide bond. This spontaneous covalent irreversible binding between the peptide tag and binding partner produces the VLP display, which provides high density, ordered, directional display of antigens and is highly immunogenic.

Figure 2 shows results from experiments described in Example 2, in which non human primates were vaccinated with VLPs according to the invention. Subjects were then monitored for production of RBD-binding antibodies and CoV-2 neutralizing antibodies pre vaccination (“pre”) and at intervals thereafter (shown on the x-axis as number of weeks). Treatment groups are as described in Example 2; groups A (high dose/MF59® adjuvant), B (high dose), C (low dose/MF59® adjuvant), and D (low dose) are shown from left to right on the x-axis in each panel.

Figure 3 shows upon challenge with SARS-CoV-2 a reduction of viral load in lung lavage (bronchoalveolar lavage, or “BAL”) in subjects treated with VLP vaccines. In the left hand panel, virus copies/mL are shown for treatment groups A, B, C, and D from Example 2; groups are shown from left to right on days 2, 4, and 6, followed by the control group. The right hand panel shows measurements of virus RNA for each treatment group and the control. These data show that the SARS-CoV-2 load (AUC, subgenomic RNA) was significantly reduced in all vaccinated groups compared to non-vaccinated controls. No virus could be detected at any timepoint in 4 out of 6 subjects that were vaccinated twice with 100 pg of vaccine; in the remaining two animals, copies were detected at a level reduced more than 1,500 fold at 2 days post challenge only.

Figure 4 shows the amount of SARS-CoV-2 neutralizing antibodies measured in serum from rhesus macaques that had been vaccinated with the VLP vaccine described in Example 1. The SARS-CoV-2 variants or strains against which the antibodies were tested were the wild-type strain from China (“Wuhan”) and two variants (“B.1.1.7”, “B.1.351”) thereof.

Figure 5 further shows that neutralizing antibodies in serum from these non-human primates were induced at comparable levels following vaccination with the VLP vaccine described in Example 1 (ABNCoV2) in different doses. The SARS-CoV-2 variants or strains against which the antibodies were tested included the Wuhan wild-type strain or variant as well as the variants designated B.l.1.7 (“Alpha”), B.1.351 (“Beta”), and Bl.617.2 (“Delta”). Cross-neutralization of variants by NHP serum was observed following immunization with ABNCoV2 at both the high dose (100 pg, data points and bars on left side of graph) and low dose (15pg, data points and bars on right side of graph).

Figure 6 shows that high-level neutralizing antibodies were induced in human patients in the Phase 1 clinical trial, at 14 days after the second dose of ABNCoV2 (non-adjuvanted dose groups). An increase in neutralizing antibody titer was seen with increasing doses of ABNCoV2 up to 25pg, when a plateau was reached. These antibody levels were up to 12- fold higher than titers in human convalescent sera samples (HCS).

Figure 7 shows the induction of high levels of neutralizing antibodies against SARS- CoV-2 in human subjects in the Phase I trial. Geometric mean titers with geometric standard deviations are shown 14 days after the second ABNCoV2 dose was administered for patients in the clinical trial (data points on left side of graph) in comparison to human convalescent samples (HCS, data points on right side of graph).

Figure 8 shows neutralizing antibodies in human patients in the Phase 2 trial. These patients were in the 100 pg dose group and were initially seropositive subjects. Figure 8 shows neutralizing antibody titers at baseline (“week 0”), week 1, and week 2 for the overall population (left) and for patients stratified by baseline antibody level (left to right) for the SARS-CoV-2 Wuhan strain/variant. (NT = neutralization titer; LLOQ = lower limit of quantitation)

Figure 9 shows the neutralizing antibody response from human patients in the Phase 2 trial. Neutralizing antibody response is shown for seropositive subjects at week 2 for SARS-CoV-2 variants Alpha, Beta, and Wuhan. The three bars shown for each variant indicate the percentage of subjects with at least 2-fold (left-most bar), at least 4-fold (middle bar), and at least 6-fold increase (right-most bar) for each variant tested. Upper left quadrant shows overall results, while the other three quadrants show results grouped by relationship of baseline NT (neutralization titer) to LLOQ (lower limit of quantitation). DETAILED DESCRIPTION OF THE INVENTION

The invention provides vaccines comprising virus-like particles displaying at least one SARS-CoV-2 antigen. A SARS-CoV-2 antigen is any antigen that produces an immune response to SARS-CoV-2, such as, for example, the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein or a portion thereof. Antigens are displayed on virus-like particles (VLPs). In some embodiments, the VLPs comprise the RNA bacteriophage AP205 coat protein (also referred to herein as “AP205”) linked to a SARS-CoV-2 antigen via a peptide tag and binding partner.

In some embodiments of the invention, the peptide tag and binding partner comprise the sequences set forth in SEQ ID NO:4 and SEQ ID NO:6, respectively.

The invention also provides methods of treatment using the VLPs of the invention and use of the VLPs to treat and/or prevent infection with the SARS-CoV-2 virus and variants thereof. The invention further provides medical uses of the VLPs in the prevention and/or amelioration of COVID-19 symptoms.

Notably, it has been shown that VLPs of the invention were immunogenic and provided protection against SARS-CoV-2 infection without using a squalene adjuvant. The effect was particularly prominent in a non-adjuvanted prime-boost regime. This finding involves several advantages. Customarily, squalene adjuvants are prepared from shark liver oil. The omittance of those adjuvants helps avoid killing of and exploiting live sharks and thus has a desirable environmental implication. Moreover, due to the natural source of shark liver oil, the source material of the squalene adjuvants is limited. This aspect may become relevant for example in case of a high demand for those adjuvants as during a pandemic such as the COVID-19 pandemic.

Furthermore, it has been shown that VLPs of the invention induced neutralizing antibodies not only against the wild-type strain of SARS-CoV-2 (“Wuhan”) but also against variants (“mutants”) that developed and emerged during the COVID-19 pandemic. Note that some publications in the art refer to the originally-identified (“Wuhan”) strain of SARS-CoV- 2 as “wild-type,” while others refer to it as another “variant,”; under the circumstances, both terms can be considered correct and are used interchangeably herein, along with the term “strain.” Because so many SARS-CoV-2 variants have arisen during the pandemic, the induction of broadly neutralizing antibodies by the VLPs of the invention is a very beneficial property because it helps avoid re-adapting the vaccine to each new SARS-CoV-2 variant and minimizes the numbers of vaccination every individual needs to receive for continued protection, particularly in circumstances where multiple variants and/or new variants may be present in a population.

VLPs

VLPs of the invention display or are linked to a peptide of interest that is a SARS- CoV-2 antigen, as further discussed elsewhere herein. By “VLP” (z.e., Virus-Like Particle) is generally intended a self-assembling particle of viral capsid proteins that can also be referred to as Capsid-Like Particles (“CLPs”), or in some instances, just “particles.” VLPs are structures that resemble virions but do not contain viral genetic material necessary for infection of and replication in host cells. VLPs can be naturally occurring or can be synthesized via the expression or production of viral structural proteins, which can then self- assemble into the virus-like structure (also referred to in the art as capsid proteins and capsids or CLPs, respectively).

In some embodiments, a fusion protein comprising AP205 and the peptide tag of SEQ ID NO:4 is provided by expressing the fusion protein from an expression vector comprising a nucleotide sequence encoding the fusion protein. This fusion protein can then be mixed together with a binding partner linked to a SARS-CoV-2 antigen under conditions allowing self-assembly of the VLP to produce VLPs of the invention.

The structure of the VLP is provided by self-assembly of particle-forming proteins, such as, for example, the AP205 protein, as described in U.S. Pat. No. 7,138,252, herein incorporated by reference in its entirety. In some embodiments, a particle-forming protein is fused to a peptide tag and a peptide of interest is fused to a binding partner to produce two components that are capable of spontaneously binding to each other by forming an isopeptide bond (see diagram in Figure 1), while not interfering with the ability of the particle-forming protein to form particles. When these two components are mixed together in favorable conditions, particles are formed and an isopeptide bond forms spontaneously between the peptide tag and binding partner, resulting in the peptide of interest being displayed on the surface of the particle.

Figure 1 shows a diagram of the general idea of such an embodiment in which a particle-forming protein is fused to a peptide tag and the peptide to be displayed (here, a SARS-CoV-2 antigen) is fused to the peptide binding partner. The spontaneous formation of an isopeptide bond between the peptide tag and binding partner results in the SARS-CoV-2 antigen being displayed on the surface of the particle. This strategy has been used to generate VLPs displaying antigenic peptides, as described in detail in WO 2016/112921, hereby incorporated by reference in its entirety (see, e.g., section entitled “The AP205 VLP” and the working Examples). Bacteriophage capsid proteins are examples of suitable particle-forming proteins that can be used to generate these VLPs and include, for example, AP205, QB, MS2, and HBc; other suitable proteins are known in the art. Derivatives and/or fragments of known particle-forming proteins may also be used in the compositions and/or methods of the invention, so long as they retain the property of being capable of self-assembling into particles.

Thus, particle-forming proteins that are derivatives of bacteriophage capsid proteins can be used in the compositions and methods of the invention, and can have sequences that differ from those of a known sequence such as, for example, SEQ ID NO: 1 and/or SEQ ID NO:2 by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more amino acids so long as the protein is capable of forming a capsid on which antigens can be displayed. In some embodiments, particle-forming proteins can have sequences that share sequence identity with SEQ ID NO:l and/or SEQ ID NO:2 of 80% or more, or at least 85%, 90%, 95%, or 99% or more sequence identity with SEQ ID NO: 1 and/or SEQ ID NO:2. In some embodiments, a particle-forming protein can have a sequence that is a fragment or portion of SEQ ID NO: 1 or SEQ ID NO:2, so long as the protein is still capable of forming a capsid on which antigens can be displayed.

Thus, a protein capable of self-assembling into particles such as VLPs can be genetically modified by fusion with a peptide tag. The assembled particles will display the peptide tag on their surface and can then be coupled to a peptide binding partner that will react with the peptide tag to form an isopeptide bond. An antigen coupled to the peptide binding partner will then be displayed on the particles.

In some embodiments, the components are rearranged so that the protein capable of self-assembling into particles such as VLPs is coupled to a peptide binding partner, and the antigen to be displayed on the surface of the VLP is coupled to a peptide tag. Generally herein the terms “peptide binding partner” and “peptide tag” are interchangeable so long as the objects of the invention are met.

The coupling or fusion of the peptide tag to the protein capable of self-assembling into particles and of the peptide binding partner to the antigen, or vice versa, is readily performed using any suitable technique known in the art. For example, a fusion protein can be obtained by constructing a polynucleotide encoding the protein capable of self-assembling into particles fused to the peptide tag, and/or by constructing a polynucleotide encoding the peptide binding partner fused to the antigen that is to be displayed on the VLP, and expressing these in an expression vector in a suitable host cell. A spacer or linker may be included between the different portions of each fusion protein in this construct, for example, to enhance binding properties of the fusion proteins or assembly of the final VLP product.

The compounds of interest may be fused to the peptide tag or binding partner via an N- terminal fusion or a C-terminal fusion or via an internal fusion, for example, in a loop.

SARS-CoV-2 antigens

VLPs of the invention display or are linked to a peptide of interest that is a SARS- CoV-2 antigen. By “SARS-CoV-2 antigen” is intended that the peptide is capable of stimulating an immune response to SARS-CoV-2 in a subject. In some embodiments, a peptide that is a SARS-CoV-2 antigen is a portion of a spike protein of SARS-CoV-2. In some embodiments, a peptide that is a SARS-CoV-2 antigen comprises all or a portion of the receptor-binding domain (“RBD”) of the SARS-CoV-2 spike protein.

In some embodiments, the SARS-CoV-2 antigen has an amino acid sequence of a SARS-CoV-2 spike (S) protein or a part thereof, wherein the amino acid sequence is the sequence of a SARS-CoV-2 S full-length protein; or the amino acid sequence is the sequence of a part of a SARS-CoV-2 S protein SI domain that comprises or consists of a SARS-CoV-2 S receptor binding domain (RBD). In some embodiments, the entire RBD is included ( e.g ., corresponding to amino acids 319-591 or 330-583 of the protein reference sequence GenBank QIA20044.1, or other fragments of the full-length S receptor protein that comprise all or part of the RBD, for example, as set forth in SEQ ID NO: 13). An exemplary SARS-CoV-2 S full- length protein from the Wuhan strain (YP 009724390.1, SARS-CoV-2 isolate Wuhan-Hu-1, NC_045512.2) has the amino acid sequence set forth in SEQ ID NO:7. A SARS-CoV-2 antigen that is a derivative or fragment of a known SARS-CoV-2 antigen from any strain or variant may also be useful in the compositions and/or methods of the invention, for example, so long as it is capable of producing an immune response when used as a component of a VLP, or so long as it shares immunogenic properties with another known SARS-CoV-2 antigen so that an antibody that binds to one also binds to the other.

In some embodiments, the SARS-CoV-2 antigen has an amino acid sequence that is all or a portion of a SARS-CoV-2 S protein SI domain that comprises or consists of a SARS- CoV-2 S RBD (Receptor Binding Domain). In some embodiments, the SARS-CoV-2 antigen is a fusion protein comprising two or more portions of one or more SARS-CoV-2 proteins. In such fusion proteins, at least one portion can be from a part of the native full-length SARS- CoV-2 protein that is not normally exposed on the surface of a SARS-CoV-2 virion. In some embodiments, the SARS-CoV-2 protein comprises two consecutive non-native proline residues and/or has been otherwise modified to prevent proteolytic cleavage by furin-like proteases.

In some embodiments, the SARS-CoV-2 antigen is from a “variant” strain of SARS- CoV-2 that is known to differ from the first-discovered strain, sometimes referred to as the “Wuhan” strain. Several variant strains have been identified to date and, based on the rate of their appearance, additional variants are expected to arise in the future (see, e.g ., Guruprasad (2021) Proteins doi: 10.1002/prot.26042). The spike proteins, RBD domains, and other domains of proteins from these variant strains and nucleotide sequences encoding them are readily obtained by one of skill in the art and adapted for use in the VLPs of the invention.

Thus, known SARS-CoV-2 antigens and proteins that are derivatives of known SARS-CoV-2 antigens can be used in the compositions and methods of the invention, and can have sequences that differ from those of a known sequence such as, for example, SEQ ID NO:7 and/or SEQ ID NO: 13 by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more amino acids so long as the protein is capable of stimulating an immune response in a subject, for example, when administered as a vaccine either by itself or displayed on a VLP. In some embodiments, antigens can have sequences that share sequence identity with SEQ ID NO: 7 or SEQ ID NO: 13 of 80% or more, or at least 85%, 90%, 95%, or 99% or more sequence identity with SEQ ID NO: 7 or SEQ ID NO: 13. In some embodiments, a protein can have a sequence that is a fragment or portion of SEQ ID NO:7 or SEQ ID NO: 13, so long as the protein is still capable of acting as an antigen (that is, of stimulating an immune response in a subject).

In some embodiments, the antigenic peptide is capable of eliciting an immune response in an animal such as a mammal; for example, a subject that can be vaccinated and/or immunized can be a cow, pig, horse, sheep, goat, llama, mouse, rat, monkey, dog, cat, bird, fish, or human patient. An immune response in a subject that has been vaccinated using a vaccine comprising VLPs may comprise the production of or an increase in neutralizing antibodies and/or T cell responses. Peptide tag and binding partner

In some embodiments, the peptide tag and binding partner comprise the sequences set forth in SEQ ID NO:4 and SEQ ID NO:6. Other peptide tags and binding partners may also be used in the compositions and methods of the invention so long as they are capable of spontaneous isopeptide bond formation so as to link the SARS-CoV-2 antigen to the VLP; for example, the binding partner may comprise the sequence set forth in SEQ ID NO: 10. Thus, peptide tag and binding partners may have sequences that differ from those set forth in SEQ ID NO:4 and SEQ ID NO:6 (that is, may be derivatives of SEQ ID NO:4 and SEQ ID NO:6) so long as they are capable of forming an isopeptide bond between the tag and partner. A “derivative” of a particular protein or sequence can share at least a particular percentage of sequence identity, or can differ at one or more amino acid or nucleotide residues from another protein or sequence. Thus, peptide tags and binding partners that are derivatives of SEQ ID NO:4 and/or SEQ ID NO:6 can be used in the compositions and methods of the invention, and can have sequences that differ from those of SEQ ID NO:4 and/or SEQ ID NO:6 by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more amino acids so long as the peptide tag and binding partner can be linked to each other by an isopeptide bond. In some embodiments, peptide tags and binding partners can have sequences that share sequence identity with SEQ ID NO: 4 and/or SEQ ID NO:6 of 80% or more, or at least 85%, 90%, 95%, or 99% or more sequence identity with SEQ ID NO:4 or SEQ ID NO:6. In some embodiments, a peptide tags and/or binding partners can have a sequence that is a fragment or portion of SEQ ID NO:4 or SEQ ID NO:6, respectively; for example, the binding partner may comprise the sequence set forth in SEQ ID NO: 10.

Thus, in some embodiments, the binding partner is attached at its N-terminus or C- terminus to the peptide of interest at the N-terminus or at the C-terminus, or alternatively is attached to the particle-forming protein at its N-terminus or C-terminus. In some embodiments, the peptide tag is attached at its N-terminus or C-terminus to the peptide of interest at the N-terminus or at C-terminus, or alternatively is attached to the particle-forming protein at its N-terminus or the C-terminus. Any configuration or combination of the components can be used in the compositions and methods of the invention so long as the VLPs of the invention can be formed so that the peptide of interest is displayed on the surface of VLPs, and is linked to the VLP particle-forming protein via a peptide tag and binding partner pair that are connected via an isopeptide bond. The assembly of the components can be assessed by in vivo assays of immunogenicity or by in vitro assays showing that the components have bound to each other.

Combinations

Thus, provided by the invention are compositions comprising a particle-forming protein linked or fused to a peptide tag and an antigen linked or fused to a binding partner, wherein the peptide tag and binding partner are capable of interacting by the spontaneous formation of an isopeptide bond and wherein the particle-forming protein and the antigen are linked via an isopeptide bond between the peptide tag and binding partner. Also provided are compositions comprising a particle-forming protein fused to a binding partner and an antigen fused to a peptide tag, wherein the binding partner and peptide are capable of interacting by the spontaneous formation of an isopeptide bond, wherein the particle-forming protein and antigen are linked via an isopeptide bond between the binding partner and the peptide tag, and wherein the particle-forming protein and antigen form a particle displaying said antigen; in some embodiments, the particle is a virus-like particle (VLP).

In some embodiments, the particle-forming protein is a VLP-subunit monomer that is an AP205 subunit monomer, for example, an AP205 subunit monomer comprising the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, the VLP- subunit monomer may have a sequence that is a subsequence of SEQ ID NO: 1 or SEQ ID NO:2, such as, for example, a sequence that is missing the initial methionine of SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, the binding partner can have the sequence set forth in SEQ ID NO:6, or it can have a truncated sequence such as the sequence set forth in SEQ ID NO: 10.

In some embodiments, protein linkers or tags are used to connect parts of constructs together; for example, a linker can be used to genetically connect an AP205 protein to a peptide tag, so that a VLP-subunit monomer can comprise a peptide tag having the sequence set forth in SEQ ID NO:4, a linker having the sequence set forth in SEQ ID NO:8, and an AP205 protein having the sequence set forth in SEQ ID NO:l. Thus, a VLP subunit monomer can have the sequence set forth in SEQ ID NO: 9. Similarly, in some embodiments, an RBD-catcher component (also sometimes referred to herein as an RBD- antigen component or “RBD-binding tag”) comprises a catcher or binding partner having the sequence of SEQ ID NO: 10, a linker having the sequence of SEQ ID NO:l 1, an RBD antigen having the sequence set forth in SEQ ID NO: 13, a linker having the sequence set forth in SEQ ID NO: 11, and a C-tag having the sequence set forth in SEQ ID NO: 12 ( e.g ., ABNCoV2). Thus, in some embodiments, an RBD-catcher component has the sequence set forth in SEQ ID NO: 14. A C-tag can be used to aid in purification of the protein to which it is attached, and a secretion signal sequence may also be genetically linked to this protein to facilitate production and later cleaved off prior to assembly of the VLP vaccine.

A “derivative” of a particular protein or nucleotide sequence can share at least a particular percentage of sequence identity, or can differ at one or more amino acid or nucleotide residues from a reference protein or sequence. Thus, for example, derivatives of proteins that comprise a subunit monomer of a VLP linked to an RBD-antigen component can be used in the compositions and methods of the invention, and can have sequences that differ from those of known sequences by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more amino acids. In some embodiments, derivatives can have sequences that share sequence identity with a reference sequence of 80% or more, or at least 85%, 90%, 95%, or 99% or more sequence identity with a particular reference sequence or protein. In some embodiments, a protein can be a fragment or portion of another known protein that includes less than the full length of the known protein. That is, for example, a protein that is a fragment or portion of another known protein may be missing amino acids from the N- or C- terminus of the known protein, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more amino acids, or 20, 50, or 100 amino acids, so long as a characteristic of the protein is retained, such as, for example, immunogenicity or binding to a particular antibody.

In some embodiments, a particle-forming protein that is a VLP subunit is produced by expression in E. coli and can be recovered following cell lysis, while the RBD or other SARS-CoV-2 antigen is expressed in Drosophila S2 cells and is secreted into the medium.

To obtain purified components, each of the RBD or other antigen and the particle-forming protein is separated from the medium and/or cell debris and purified by suitable techniques, for example, chromatography, ultrafiltration, and/or diafiltration. The particle-forming protein and the RBD or other antigen can then be coupled by mixing the components together and incubating for a suitable period of time (e.g., overnight) to produce VLPs displaying the antigen, followed by filtration such as ultrafiltration using tangential flow filtration or other suitable filtration to separate assembled VLPs displaying antigens from non-coupled particle forming protein and antigen components. The assembled VLPs can be frozen in solution and stored, then later diluted with formulation buffer to the required concentration for administration as a vaccine. Suitable formulation buffers are known in the art, for example, a formulation buffer can comprise or consist of PBS, Tris buffer, and sucrose.

Methods of Treatment

VLPs of the invention can be used in prophylactic treatment of any animal or subject in which they produce an immune response. VLPs of the invention can be used to vaccinate any animal, including, for example, a human subject or patient. Vaccines may be administered to a subject in a single dose or in more than one dose. If more than one dose of the vaccine is administered, the doses can be given several days or several weeks apart, or may be given several months apart. If more than one dose of the vaccine is administered, each dose can contain the same vaccine (“homologous prime-boost regime” as used herein) or alternatively, the first dose can contain or comprise a first VLP with a first SARS-CoV-2 antigen and the second or subsequent dose can contain or comprise a second VLP with a second SARS-CoV-2 antigen that can be from a different SARS-CoV-2 strain or variant than the antigen in the first dose.

The compositions of the inventions thus may be vaccine compositions with prophylactic applications and useful for prophylaxis or treatment of a disease or disorder caused by SARS-CoV-2, including symptoms referred to as COVID-19. The compositions may also be useful for inducing an immune response in a subject by administering said compositions at least once to the subject. Vaccination with compositions of the invention can be effective to reduce or prevent symptoms of infection by SARS-CoV-2 and/or related viruses, including MERS. Those of skill in the art appreciate that a dose of a vaccine may be administered to a subject before or after doses of the same vaccine or a different vaccine, and a dose of vaccine that is administered to a subject who has previously been treated with the same vaccine or a different vaccine may be referred to as a “booster.”

VLPs of the invention, or vaccines comprising them, are said to elicit an immune response, for example, if they elicit neutralizing antibodies in a subject following administration. In this manner, the effectiveness of a vaccine of the invention can be assessed by measurement of neutralizing antibodies in a subject following administration, wherein the presence of neutralizing antibodies indicates that an immune response has been produced in the subject. In some embodiments, the virus neutralization titers exceed those produced following natural infection with SARS-CoV-2 or a variant thereof. An immune response in a subject that has been vaccinated using a vaccine comprising VLPs of the invention may also, or alternatively, comprise the production of or an increase in T cell responses. Methods of measuring neutralizing antibodies and T cell responses are known in the art. For example, antigen-specific IgG titers can be measured by ELISA, and the levels of antigen-specific T cells can be assessed using FACS analysis.

Compositions and methods of the invention prevent, alleviate, or ameliorate at least one symptom of infection with SARS-CoV-2. These symptoms include, for example, fever; high viral load in tissues such as lung, nose, and/or throat; chills; muscle or body aches; congestion; need for hospitalization; death; and other symptoms that have been reported. In some embodiments, by “alleviates symptoms of COVID-19” or “alleviates symptoms of infection with SARS-CoV-2” is intended that hospitalization and death are avoided when a composition or method of the invention is used to treat a subject. In some embodiments, by “ameliorating” a symptom of COVID-19 or infection with SARS-CoV-2 is intended that that symptom is less severe than in a patient that was not treated with the same composition or method, or is less severe than would be expected for a patient that was not treated, for example, by statistical analysis of treated and untreated patient populations, wherein a symptom is ameliorated if it is increased if favorable or decreased if unfavorable by at least 10%, 20%, 25%, 30%, or more in a treated versus an untreated subject populations using appropriate statistical analysis. In some embodiments, a patient that was previously infected with SARS-CoV-2 can be vaccinated with a VLP of the invention and lingering symptoms of the earlier infection are reduced or diminished; for example, fatigue may be decreased.

Doses of active agent ( i.e ., VLPs) to be administered are in the range of from 5 to 200 pg, preferably from 10 to 150 pg, more preferably from 15 to 100 pg. In some embodiments, doses of VLPs to be administered are in the range from 10 to 20 pg, preferably 15 pg or about 15 pg (“low dose”), or in the range from 80 to 120 pg, preferably 100 pg or about 100 pg (“high dose”). When administered to patients that have pre-existing measurable levels of antibodies or neutralizing antibodies in their serum {i.e., patients who are seropositive) due to previous exposure to SARS-CoV-2 and/or previous vaccination with one or more other vaccines or the same vaccine, lower doses of active agent (VLPs) may be used.

It will be understood that serum titers of neutralizing antibodies in a patient will increase following administration of VLPs, but in patients with measurable pre-existing levels of antibodies and/or neutralizing antibodies (that is, patients who have a high baseline antibody titer), the increase in antibodies and/or neutralizing antibodies following administration of VLPs may appear to be lower than the increase observed in patients who were previously seronegative or who had serum antibody titers that were below a level that can be accurately measured. However, an increase in serum antibody titers above the pre immunization baseline can be measured to confirm stimulation of the immune response by VLPs, even if the increase is as little as 2-fold (see, e.g., Example 4 and Figure 8). In this manner, the invention provides methods of increasing antibodies and/or neutralizing antibodies against SARS-CoV-2 antigens such as RBD by at least 2-fold, at least 4-fold, at least 6-fold, or at least 10-fold or more in a patient comprising administering VLPs to the patient, and VLPs can be administered to patients regardless of their previous serum antibody titer levels to induce a broad immune response against SARS-CoV-2 variants. In some embodiments, to measure the increase in serum antibody titers in a patient, the serum antibody titers are measured at least or about one week after administration of the VLPs or at least or about two weeks after administration of the VLPs.

Some patients do not exhibit symptoms of COVID-19 even when infected with SARS-CoV-2, other than an increase in anti-SARS-CoV-2 antibodies and/or neutralizing antibodies, yet such patients can also benefit from vaccination with VLPs because the resulting induction in antibodies and/or neutralizing antibodies from the vaccination can nevertheless prevent or alleviate symptoms of subsequent coronavirus infection. In this manner, vaccination with VLPs produces an increase in anti-SARS-CoV-2 antibodies and/or neutralizing antibodies in a patient and thus is considered to produce an immune response that prevents or alleviates symptoms of coronavirus infection. Thus, it can be said that the invention provides compositions and methods for boosting an immune response in a subject.

Formulations

Formulations without adjuvant: In some embodiments, the VLPs of the invention are formulated in an aqueous solution for administration as a vaccine. In some embodiments, the aqueous solution can be formulated for use as a vaccine and can further comprise a pharmaceutically acceptable carrier, adjuvant, or excipient. In some embodiments, the aqueous solution does not include an adjuvant, such as, for example, squalene and/or MF59® adjuvant, but only includes components such as buffers, salts, and the like that are not expected to additionally boost the immune response. In some embodiments, squalene is excluded from the aqueous solution comprising the VLPs of the invention and is not a component of the vaccine. In some embodiments, an adjuvant such as, for example, MF59® adjuvant and/or AddaVax™ adjuvant is excluded from the aqueous solution comprising the VLPs of the invention and is not a component of the vaccine.

Exemplary generation of a recombinant VLP

In some embodiments, the method of producing a VLP comprises the steps of: (i) obtaining a first polypeptide comprising or consisting of a peptide binding partner fused to a particle-forming protein; and obtaining a second polypeptide comprising or consisting of a peptide tag fused to an antigen of interest; or obtaining a first polypeptide comprising or consisting of a peptide tag fused to a particle-forming protein and obtaining a second polypeptide comprising or consisting of a binding partner fused to an antigen of interest; (ii) subjecting the first and second polypeptides to conditions which enable formation of an isopeptide bond between the peptide tag and binding partner portions of the polypeptides, whereby particles are produced in which the antigen of interest is displayed on the surface of the particles; and (iii) generating a pharmaceutical composition comprising said particles.

The particle-forming protein may be any of those listed herein, including, for example, a capsid protein that is AP205. Exemplary AP205 amino acid sequences are set forth in SEQ ID NO:l and SEQ ID NO:2.

The SARS-CoV-2 antigens, particularly those comprising part or all of the RBD, can be produced in cell culture such as, for example, Schneider-2 insect cells (also referred to as S2 cells) (see, e.g., Moraes etal. (2012) Biotech. Adv. 30: 613-28). The AP205 component can be expressed in and prepared from E. coli cultures (see, e.g., Thrane el al. (2016) J. Nanobiotechnology 14: 30). These components can then be mixed together under suitable conditions, resulting in the formation of an isopeptide bond between the peptide tag and binding partner, which can be confirmed by SDS-PAGE analysis and other techniques such as affinity for binding, densitometry, and/or electron microscopy.

DEFINITIONS AND TERMINOLOGY

It is to be understood that both the foregoing summary and the detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. It is to be understood that this invention is not limited to a particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Terms are defined and explained so that the invention may be understood more readily. Additional definitions are set forth throughout the detailed description.

It must be noted that, as used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a nucleic acid” includes one or more nucleic acid sequences and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used in the context of an aspect or embodiment in the description of the present invention the term “comprising” can be amended and thus replaced with the term “containing” or “including” or when used herein with the term “having.” Similarly, any of the aforementioned terms (comprising, containing, including, having), whenever used in the context of an aspect or embodiment in the description of the present invention also include, the terms “consisting of’ or “consisting essentially of,” each of which denotes a specific legal meaning depending on jurisdiction.

When used herein, “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

The term “substantially free of’ an ingredient as used herein does not exclude trace amounts of the ingredient which does not materially affect the stability of the composition unless stated otherwise herein. The term “free of’ in front of for example mannitol means that the aqueous composition of the present invention does not contain mannitol.

"About" as used in the present application means ±10%, unless stated otherwise. It must also be noted that unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Through the specification the term “about” with respect to any quantity or concentration is contemplated to include that quantity. For example, “about 5mM” is contemplated herein to include 5mM as well as values understood to be approximately 5mM with respect to the entity described. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise. Likewise, the term “about” preceding any numerical value or range used herein in the context of the invention can be deleted and be replaced by the numerical value or range without the term “about” though less preferred.

The terms "nucleic acid," “nucleotide sequence,” “nucleic acid sequence,” and "polynucleotide" can be used interchangeably and refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The polynucleotides can be obtained by chemical synthesis or derived from a microorganism. The term “exogenous” nucleic acid sequences when used in connection with a recombinant virus means a foreign nucleic acid sequence, a nucleic acid sequence not contained in the non-recombinant virus used for generating the recombinant virus, or a nucleic acid sequence inserted into the virus genome while generating the recombinant virus.

A “derivative” of a particular protein or nucleotide sequence can share at least a particular percentage of sequence identity, or can differ at one or more amino acid or nucleotide residues from a reference protein or sequence. Thus, for example, proteins that are derivatives of AP205 can be used in the compositions and methods of the invention, and can have sequences that differ from those of known AP205 sequences by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more amino acids so long as the derivatives are capable of self-assembly into a particle. In some embodiments, derivatives can have sequences that share sequence identity with a reference sequence of 80% or more, or at least 85%, 90%, 95%, or 99% or more sequence identity with a particular reference sequence or protein. In some embodiments, a protein can be a fragment or portion of another known protein that includes less than the full length of the known protein. That is, for example, a protein that is a fragment or portion of another known protein may be missing amino acids from the N- or C- terminus of the known protein, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more amino acids, or 20, 50, or 100 amino acids, so long as a characteristic of the protein is retained, such as, for example, immunogenicity or binding to a particular antibody. For example, a protein that is a fragment of the binding partner of SEQ ID NO:6 can be missing the first 3 amino acids of that sequence and so has the sequence set forth in SEQ ID NO: 10.

“Pharmaceutically acceptable" means that the carrier, additive, antibiotic, preservative, adjuvant, diluent, stabilizer or excipient, at the dosages and concentrations employed, will substantially not cause an unwanted or harmful effect(s) in the subject(s) to which they are administered. While the instant vaccine was shown to be effective even in the absence of adjuvant, in some embodiments, the vaccine is formulated with an adjuvant such as, for example, MF59® adjuvant (Novartis, Cambridge, MA, USA) or AddaVax™ adjuvant (InvivoGen, San Diego, CA, USA). A “pharmaceutically acceptable” excipient is any inert substance that is combined with an active molecule such as a virus for preparing an agreeable or convenient dosage form. The "pharmaceutically acceptable” excipient is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation comprising the viral preparation. Examples of excipients are cryoprotectants, non-ionic detergents, buffers, salts and inhibitors of free radical oxidation. “Pharmaceutically acceptable carriers” are for example described in Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company (1990); Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, eds., Taylor & Francis (2000); and Handbook of Pharmaceutical Excipients,

3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000). Administration of the vaccine can be by any suitable route of administration, for example, by the intramuscular route. The terms "subject" and "patient" are used herein interchangeably. As used herein, a subject is typically a mammal, such as a non-primate ( e.g ., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human), and in some preferred embodiments is a human.

The term “prime-boost regime” as referred to herein means that the active agent, i.e. VLPs, are administered in a first vaccination and later after a certain period of time in a second vaccination or still further vaccinations. As is well understood in the art, a patient may be administered one or more different vaccines over time. In this context, the term “homologous” prime-boost regime as referred to herein means that the active agent (i.e., VLPs) used in the first vaccination are the same as those used in the second or further vaccinations, and “heterologous” prime-boost means that the vaccine that is first administered to a patient is different from the vaccine that is subsequently administered to a patient. Because the VLPs of the invention provide broad protection against SARS-CoV-2 variants (see, e.g., Figures 5 and 7), they are suitable for administration as either a priming vaccine or as a booster in homologous or heterologous prime-boost regimes, or for administration to any patient for whom an increase in neutralizing antibody titer is desirable regardless of vaccination or infection history. Further, this broad protection against SARS-CoV-2 variants necessarily means that an immune response in a subject is induced against multiple SARS- CoV-2 variants as a result of immunization with the VLPs of the invention.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The practice of the invention will employ, if not otherwise specified, conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant technology, which are all within the skill of the art. See e.g. Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, 1989; Current Protocols in Molecular Biology, Ausubel FM, et ak, eds, 1987; the series Methods in Enzymology (Academic Press, Inc.); PCR2: A Practical Approach, MacPherson MJ, Hams BD, Taylor GR, eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988. Further aspects and embodiments

The embodiments of the invention include the following items:

Item l is a virus-like particle (VLP) comprising an AP205 protein fused to a peptide tag and comprising a SARS-CoV-2 antigen fused to a peptide binding partner, whereby the SARS-CoV-2 antigen is displayed on the surface of said VLP.

Item 2 is the VLP of item 1, wherein said peptide tag has the sequence set forth in SEQ ID NO:4 and/or is encoded by the sequence set forth in SEQ ID NO:3.

Item 3 is the VLP of item 1 or 2, wherein said peptide binding partner has the sequence set forth in SEQ ID NO:6 or SEQ ID NO: 10 and/or is encoded by the sequence set forth in SEQ ID NO:5.

Item 4 is the VLP of any one of items 1 to 3, wherein said AP205 has the sequence set forth in SEQ ID NO:l or SEQ ID NO:2, or is missing the initial methionine from SEQ ID NO:l or SEQ ID NO:2.

Item 5 is the VLP of any one of items 1 to 4, wherein said antigen has the sequence set forth in SEQ ID NO:7 or SEQ ID NO: 13, or is a fragment thereof.

Item 6 is the VLP of item 5, wherein said antigen comprises amino acids 319-591 of SEQ ID NO:7 or is set forth in SEQ ID NO: 13.

Item 7 is the VLP of any one of items 1 to 6 that comprises an isopeptide bond between said binding partner and said peptide tag.

Item 8 is a virus-like particle (VLP) comprising: (i) an AP205 protein fused to a peptide tag; and (ii) a SARS-CoV-2 antigen comprising the wild-type (Wuhan) spike protein Receptor Binding Domain (RBD), whereby the SARS-CoV-2 antigen is displayed on the surface of said VLP; and that when administered to a subject as a vaccine stimulates an immune response that prevents or alleviates symptoms of coronavirus infection caused by any one of SARS-CoV-2 variants B.1.1.7, B.1.351, B 1.617.2, and/or another variant. In some embodiments, this immune response is a measurable increase in anti-SARS-CoV-2 antibodies and/or neutralizing antibodies in the patient following immunization, for example one or two weeks following immunization.

Item 9 is the VLP of item 8, wherein said peptide tag has the sequence set forth in SEQ ID NO:4 or is encoded by the sequence set forth in SEQ ID NO:3. Item 10 is the VLP of items 8 or 9, wherein said AP205 has the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, or has the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2 but without the initial methionine.

Item 11 is the VLP of any one of items 8 to 10, wherein said antigen has the sequence set forth in SEQ ID NO:7 or SEQ ID NO: 13, or is a fragment thereof.

Item 12 is the VLP of item 11, wherein said antigen comprises amino acids 319-591 of SEQ ID NO:7 or is as set forth in SEQ ID NO: 13.

Item 13 is a virus-like particle (VLP) comprising: (a) an AP205 protein fused to a peptide tag having the amino acid sequence set forth in SEQ ID NO:4, or a derivative thereof; and (b) a SARS-CoV-2 antigen fused to a peptide binding partner having the amino acid sequence set forth in SEQ ID NO:6, or a derivative thereof, whereby the SARS-CoV-2 antigen is displayed on the surface of said VLP; that when administered to a subject as a vaccine stimulates a response that prevents or alleviates symptoms of coronavirus infection caused by any one of SARS-CoV-2 variants B.l.1.7, B.1.351, B 1.617.2, and/or another variant. In some embodiments, this immune response is a measurable increase in anti-SARS- CoV-2 antibodies and/or neutralizing antibodies in the patient following immunization, for example one or two weeks following immunization.

Item 14 is the VLP of item 13, wherein said AP205 has the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, or has the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2 without the initial methionine.

Item 15 is the VLP of items 13 or 14, wherein said antigen has the sequence set forth in SEQ ID NO:7 or SEQ ID NO: 13, or is a fragment thereof.

Item 16 is the VLP of item 15, wherein said antigen comprises the sequence set forth in SEQ ID NO: 13, or amino acids 319-591 of SEQ ID NO:7.

Item 17 is the VLP of any one of items 13 to 16 that comprises an isopeptide bond between said binding partner and said peptide tag.

Item 18 is a pharmaceutical composition, or a vaccine, comprising the VLP of anyone of items 1 to 17, optionally further comprising a pharmaceutically acceptable carrier or excipient. Item 19 is the pharmaceutical composition of item 18, or the vaccine of item 18, that does not contain an immunologic adjuvant.

Item 20 is the pharmaceutical composition of item 18, or the vaccine of item 18, comprising the VLP in an aqueous solution that does not contain shark liver oil.

Item 21 is the pharmaceutical composition of item 18, or the vaccine of item 18, comprising the VLP in an aqueous solution that contains no squalene.

Item 22 is the pharmaceutical composition of item 18, or the vaccine of item 18, comprising the VLP in an aqueous solution that does not contain a squalene-based oil-in water nano-emulsion.

Item 23 is the pharmaceutical composition of item 18, or the vaccine of item 18, comprising the VLP in an aqueous solution that does not contain MF59® adjuvant or AddaVax™ adjuvant.

Item 24 is a method of preparation of the pharmaceutical composition or the vaccine of any of items 18 to 23, which method does not contain a step of including a squalene in the composition or vaccine.

Item 25 is a method of treating a subject to prevent or ameliorate symptoms of a coronavirus infection, preferably coronavirus disease 19 (COVID-19), comprising the step of administering the vaccine of any of items 18 to 23 or a vaccine comprising the VLP of any of items 1 to 17 to a subject.

Item 26 is the method of item 25, wherein the step of administering the vaccine produces an immune response in said subject that prevents or ameliorates symptoms of infection with SARS-CoV-2, preferably SARS-CoV-2 wild-type strain (Wuhan) or a variant thereof. In some embodiments, this immune response is a measurable increase in anti-SARS- CoV-2 antibodies and/or neutralizing antibodies in the patient following immunization, for example one or two weeks following immunization. Thus, it will be appreciated that the invention also provides a method of stimulating the immune response in a subject by administering said vaccine of Item 25 to said subject. It will also be appreciated that because administration of said vaccine of Item 25 stimulates a broad immune response in a subject to multiple SARS-CoV-2 variants that the invention provides methods of stimulating an immune response to multiple variants at once, including previously unidentified variants. Item 27 is the method of item 26, wherein the SARS-CoV-2 variant is B.1.1.7,

B.1.351, B 1.617.2, or another variant.

Item 28 is the method of item 25, wherein the step of administering the vaccine produces an immune response in said subject that prevents or ameliorates symptoms of infection with a strain of SARS-CoV-2 that is not the SARS-CoV-2 wild-type strain (Wuhan).

Item 29 is a VLP of any one of items 1 to 17, or a pharmaceutical composition or a vaccine of any one of items 18 to 23, for use in the prevention or treatment of a coronavirus infection, preferably coronavirus disease 19 (COVID-19).

Item 30 is the VLP or the pharmaceutical composition or the vaccine for use of item

29, wherein the coronavirus infection is caused by SARS-CoV-2, preferably SARS-CoV-2 wild-type strain (Wuhan) or a variant thereof.

Item 31 is the VLP or the pharmaceutical composition or the vaccine for use of item

30, wherein the SARS-CoV-2 variant is B.1.1.7, B.1.351, B 1.617.2, or another variant.

Item 32 is the VLP or the pharmaceutical composition or the vaccine for use of item 29, wherein the coronavirus infection is not caused by the SARS-CoV-2 wild-type strain (Wuhan).

Item 33 is the VLP or the pharmaceutical composition or the vaccine for use of anyone of items 29 to 32 that is used or administered in a prime-boost regime, for example a homologous prime-boost regime or a heterologous prime-boost regime.

Item 34 is the use of a VLP of anyone of items 1 to 17 for the preparation of a pharmaceutical composition or a vaccine for use in the prevention or treatment of a coronavirus infection, preferably coronavirus disease 19 (COVID-19).

Item 35 is the use of item 34, wherein the coronavirus infection is caused by SARS- CoV-2, preferably SARS-CoV-2 wild-type strain (Wuhan) or a variant thereof.

Item 36 is the use of item 35, wherein the SARS-CoV-2 variant is B.1.1.7, B.1.351, or Bl.617.2.

Item 37 is the use of item 34, wherein the coronavirus infection is not caused by the SARS-CoV-2 wild-type strain (Wuhan). Item 38 is the use of any one of items 34 to 37, wherein the pharmaceutical composition or vaccine is used or administered in a prime-boost regime, for example a homologous prime-boost regime or a heterologous prime-boost regime.

Item 39 is a VLP of any one of items 1 to 17, for use in the prevention or treatment of a coronavirus infection, preferably coronavirus disease 19 (COVID-19), wherein the VLP is used or administered without simultaneously using or administering an immunologic adjuvant selected from the group consisting of shark liver oil, squalene, a squalene-based oil- in-water nano-emulsion, MF59^® adjuvant and AddaVax™ adjuvant.

Item 40 is the VLP of item 39, wherein the VLP is used or administered in a prime- boost regime, for example a homologous prime-boost regime or a heterologous prime-boost regime.

Item 41 is a virus-like particle (VLP) comprising: (a) an AP205 protein comprising the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, or comprising the sequence set forth in SEQ ID NO:l or SEQ ID NO:2 without the initial methionine; (b) a linker having the sequence set forth in SEQ ID NO: 8 fused to a peptide tag having the amino acid sequence set forth in SEQ ID NO:4, or a derivative thereof; and (c) a SARS-CoV-2 antigen having the sequence set forth in SEQ ID NO: 13 or amino acids 319-591 of SEQ ID NO:7 fused to a peptide binding partner having the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 10, or a derivative thereof, whereby the SARS-CoV-2 antigen is displayed on the surface of said VLP; that when administered to a subject as a vaccine stimulates a response that prevents or alleviates symptoms of coronavirus infection caused by SARS-CoV-2 variants B.1.1.7, B.1.351, B 1.617.2, and/or another variant.

Item 42 is a subunit monomer of a virus-like particle (VLP) comprising: a peptide tag having the sequence set forth in SEQ ID NO:4, a linker having the sequence set forth in SEQ ID NO:8, and an AP205 protein comprising the sequence set forth in SEQ ID NO: 1 but missing the initial methionine.

Item 43 is the subunit monomer of a VLP of item 42 that has the sequence set forth in SEQ ID NO:9.

Item 44 is an RBD-antigen component comprising: a binding partner having the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 10, a linker having the sequence set forth in SEQ ID NO: 11, the SARS-CoV-2 antigen sequence set forth in SEQ ID NO: 13, a linker having the sequence set forth in SEQ ID NO: 11, and a C-tag having the sequence set forth in SEQ ID NO: 12.

Item 45 is the RBD-antigen component of item 44 that has the sequence set forth in SEQ ID NO: 14.

Item 46 is a VLP comprising a subunit monomer of item 42 or 43 and an RBD- antigen component of item 44 or 45. In some embodiments, the subunit monomer and RBD- antigen component are linked at their N-termini.

Item 47 is a pharmaceutical composition, or a vaccine, comprising the VLP of item 46, optionally further comprising a pharmaceutically acceptable carrier or excipient.

Item 48 is the use of a subunit monomer of a VLP of item 42 or item 43 or an RBD- antigen component of item 44 or 45 or a VLP of item 46 for the preparation of a pharmaceutical composition or a vaccine for use in the prevention or treatment of a coronavirus infection, preferably coronavirus disease 19 (COVID-19).

Item 49 is a method of treating a subject to prevent or ameliorate symptoms of a coronavirus infection, preferably coronavirus disease 19 (COVID-19), comprising the step of administering the pharmaceutical composition or vaccine of item 47 to a subject.

EXAMPLES

The following examples illustrate the invention but should not be construed as in any way limiting the scope of the claims. They merely serve to clarify the invention.

It will be apparent that the precise details of the methods or compositions described herein may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

EXAMPLE 1: Preparation of RBD-VLP vaccines

Design, expression, and purification of recombinant protein.

RBD antigens were designed with boundaries aa 319-591 of the SARS-CoV-2 sequence (Sequence ID: QIA20044.1). The RBD antigens were genetically fused with the split-protein Catcher (or “binding partner”) at the N-terminus. The antigen constructs had an N-terminal BiP secretion signal and a C-terminal C-tag (N-RBD-EPEA-C) used for purification. A GSGS linker was inserted between the RBD and the Catcher. The final gene sequences were codon optimized for expression in Drosophila melanogaster. The ExpreS2 platform was used to produce all proteins by transient transfection. Briefly, Schneider-2 (ExpreS2) cells were transiently transfected using transfection reagent (ExpreS2 Insect TRx5, ExpreS2ion Biotechnologies) according to manufacturer’s protocol. Cells were grown at 25 °C in shake flasks for 3 days before harvest of the supernatant containing the secreted protein of interest. Cells and debris were pelleted by centrifugation (5000 rpm for 10 min at 4 °C) in a Beckman Avanti JXN-26 centrifuge equipped with a JLA 8.1000 swing-out rotor. The supernatant was decanted and passed through a 0.22 pm vacuum filter (PES) before further processing. The supernatant was passed over a Centramate tangential flow filtration (TFF) membrane (0. Im2, 10 kDa MWCO, PALL) mounted in a SIUS-LS filter holder atop a SIUS- LS filter plate insert (Repligen/TangenX). The retentate was concentrated ten-fold by recirculation through a concentration vessel of 1 liter volume without stirring. Buffer exchange was performed by diafiltration until achieving a tum-over-volume of 10. The crude protein was loaded onto a Capture Select C tag resin (Thermo Fisher) affinity column and washed with capture buffer (25 mM Tris-HCl, 100 mM NaCl, pH7.5). The captured protein was step-eluted in 25 mM Tris-HCl (pH7.5) containing increasing concentrations of MgC12 (0.25 M, 0.5 M, 1 M and 2 M). Data were collected on Unicorn software (Cytivalifesciences, Marlborough, USA, version 5.11) and fractions containing the protein of interest were pooled and concentrated (Amicon 15 ml, 10 kDa or 30 kDa MWCO). Concentrated protein was loaded onto a preparative Superdex-200pg 26/600 (Cytiva) SEC column equilibrated in lx PBS (Gibco) and eluted in the same buffer. Fractions containing the monomeric RBD protein were pooled and concentrated as above.

Design, expression, and purification of Tag-VLP.

The peptide binding Tag and a linker (GSGTAGGGSGS) was added to the N- terminus of the Acinetobacter phage AP205 coat protein (Gene ID: 956335). The gene sequence was inserted into the pET28a(+) vector (Novagen) using Ncol (New England Biolabs) and Notl (New England Biolabs) restriction sites. The Tag-VLP was expressed in BL21 (DE3) competent A. coli cells (New England Biolabas) according to manufacturer’s protocols, and purified as described below for the VLP vaccines. Formulation and purification of the RBD-VLP vaccines.

Tag-VLP and RBD antigen were mixed in a 1:1 molar ratio in lxPBS, 5% glycerol and incubated overnight at room temperature. PBS buffer, pH 7.4, supplemented by 400 mM xylitol was chosen for quality assessment of the RBD vaccine. The mixture of RBD and VLP was subjected to a spin test to assess stability. Specifically, a fraction of the sample was spun at 16000 g for 2 min, and equal amounts of pre- and post-spin samples were subsequently loaded on a reduced SDS-PAGE to assess potential loss in the post-spin sample due to precipitation of aggregated RBD-VLP complexes. The RBD-Catcher (or “binding partner”) coupling efficiency was calculated as percentage conjugation ( i.e number of bound antigens divided by the total available binding sites (=180) per VLP) by densitometric analysis of on the SDS-PAGE gel, using ImagequantTL. The conjugated RBD-VLP was purified by dialysis (cutoff 1000 kDa) in a lxPBS with 5% (v/v) glycerol for immunization studies or 400 mM xylitol for quality assessment.

Quality assessment of the RBD-VLP vaccines.

Purified RBD-VLP were both quality checked by negative stain Transmission electron microscopy (TEM) (detailed description 10.1038/s41598-019-41522-5) as well as by Dynamic Light Scattering (DLS) analysis (DynaPro Nanostar, Wyatt technology). For DLS analysis, the RBD-VLP sample was first spun at 21,000 g for 2.5 min and then loaded into a disposable cuvette. The sample was then run with 20 acquisitions of 7 sec each. The estimated diameter of the RBD-VLP particle population and the percent polydispersity (%Pd) was calculated by Wyatt DYNAMICS software (v7.10.0.21, US).

EXAMPLE 2: Study of VLP vaccines in non-human primates

Study design

A vaccine comprising a VLP as described above was evaluated for its ability to induce an immune response in non-human primates (NHPs) (here, rhesus macaques) and for its ability to protect vaccinated NHPs from infection with and/or symptoms produced by SARS-CoV-2.

Specifically, the VLPs used in these experiments comprised: (1) the RBD of the SARS-CoV-2 spike protein (amino acids 319-591 of Sequence ID QIA220044.1) genetically fused at the N-terminus to a binding partner comprising the amino acid sequence set forth in SEQ ID NO: 6; and (2) a peptide tag having the amino acid sequence set forth in SEQ ID NO:4 genetically fused to the AP205 coat protein, wherein these components were linked by an isopeptide bond between the binding partner and the peptide tag (diagrammed in Figure 1; also referred to herein as “ABNCoV2”).

Four groups of six rhesus macaques were immunized intramuscularly with one of the following treatments: (A) 100 pg VLPs in MF59® adjuvant; (B) 100 pg VLPs with no adjuvant (in 0.4 mL PBS); (C) 15 pg VLPs in MF59® adjuvant; or (D) 15 pg VLPs without adjuvant (in 0.4 mL PBS). Vaccines formulated with adjuvant were at a higher concentration initially so that the final volume and concentration of VLP would be the same as in the groups without adjuvant (z.e., 0.4 mL). Challenge control group (E) entered the study at day 112. For groups B and D, a booster immunization was given at week 14. Intramuscular injections were given in the muscle of the left upper leg. Subjects were monitored for adverse effects following immunization and during the study.

Subjects were challenged with virus at week 20 and monitored for antibody, cytokine, and chemokine responses. For viral challenge, all subjects were exposed via combined intranasal and intratracheal route to 10⁵ TCID50 SARS-CoV-2 (CoV isolate BetaCoV/ German/ BavPatl/ 2020 p.4, European Virus Archive, Berlin, Germany). The p4 low passage stock was prepared on Vero E6 cells; supernatant was collected and stored at -80 C, and titer determined using a 50% tissue culture infective dose assay (TCID50 assay) on Vero E6 cells, at 1 x 10⁵ TCIDso/mL. For inoculation, virus was diluted to 2 x 10⁴ TCID50 per mL, and each animal received 0.25 mL in each nostril and 4.5 mL intratracheally, for a total calculated dose of 1 x 10⁵ TCID50.

Two weeks after infection, all challenged animals from groups B and E were euthanized and full necropsies were performed. Animals from groups A, C, and D were euthanized during the third week after infection and tissue samples were collected for analysis.

All four vaccinated groups showed a significant reduction in total amount of replicating virus relative to the control group in bronchoalveolar lavage (“BAL”) samples, measured as both virus copies/mL and amount of virus RNA (Figure 3). Measurements of samples from nose and throat were also taken; throat samples showed decreases in replicating virus from some vaccinated groups (data not shown). For data shown in the right-hand panel, p-values were obtained using the Mann-Whitney test. RBD-specific antibodies

Serum samples obtained pre-immunization and throughout the course of the study were tested for the presence of antibodies to SARS-CoV-2 spike protein (RBD) using an ELISA (Figure 2); positive human plasma samples were included in the assay for comparison (data not shown). Antibody levels from groups A, B, and C were higher at week 2 than human convalescent serum antibody levels, and at week 16, anti -RBD IgG antibody levels were highest in group B subjects (Figure 2, left panel; note the graphs use a log scale). As shown in Figure 2, antibodies were highest in Day 16 samples from the high dose group without MF59® adjuvant, followed by the low dose group without MF59® adjuvant, followed by the low dose group with MF59® adjuvant, followed by the high dose group with MF59® adjuvant.

Virus neutralizing antibodies

Neutralization assays were performed as follows. Human plasma from SeraCare and NHP serum samples from week 4 were heat inactivated by incubating at 56°C for 30 minutes. Two-fold serial dilutions were prepared in media (DMEM + 2% FCS + 1% Pen/Strep + L- Glutamine). Sera were mixed with SARS-CoV-2 at a final titer of 200 TCID50/90pL and incubated at 4°C overnight. Control samples included one including SARS-CoV-2 but lacking serum and another control lacking both serum and virus.

Virus/serum mixtures were then added to 2 x 10⁴ VeroE6-hTMPRSS2 cells seeded in flat-bottom 96-well plates 24 hours earlier, and these mixtures were then incubated for 72 hours in an incubator at 37°C in the presence of 5% C02 and humidification. Cells were then fixed in 5% formalin and stained with crystal violet; cytopathic effect (CPE) was evaluated using a light microscope. Based on dilution curves obtained, Plaque Reduction Neutralization Titer 50 values (PRNT50 titers) were approximated using commercially available software (GraphPad) (Figure 2, right panel).

Conclusions

Data from this Non-human Primate (“NHP”) study showed that the VLP vaccine was immunogenic and protected the subjects against SARS-CoV-2. Antibodies were induced at levels comparable to those in human convalescent subjects by either a single intramuscular administration of adjuvanted VLP vaccine or the higher dose of vaccine tested without adjuvant; however, a second administration of non-adjuvanted VLP vaccine produced titers more than 50-fold higher. Antibodies were durable for at least three to four months. Immune responses were not boosted by the challenge with SARS-CoV-2 virus. SARS-CoV-2 virus load in lung was significantly reduced in all vaccinated groups compared to non-vaccinated controls, but was reduced most effectively in the group that received 2 administrations of the high dose vaccine (100 pg).

EXAMPLE 3: Neutralization of SARS-CoV-2 variants by serum from VLP vaccinated non-human primates

The VLP vaccine described above (ABNCoV2) was evaluated for its ability to induce SARS-CoV-2 wild-type strain (Wuhan) specific as well as SARS-CoV-2 variant-specific neutralizing antibodies in vaccinated NHPs.

Six rhesus macaques were immunized intramuscularly with 100 pg VLPs with no adjuvant (in 0.4 mL PBS). A booster immunization was given at week 14. Two weeks after the booster immunization blood was drawn and serum was obtained.

Neutralization assays of the NHP serum samples were carried out as described above (see Example 2). Briefly, sera were mixed with SARS-CoV-2 wild-type strain (Wuhan) (group 1), SARS-CoV-2 variant B.1.1.7 (group 2), or SARS-CoV-2 variant B.1.351 (group

3)·

Control samples were included in the assays as described above. PRNT50 titers were determined as described above and results are shown in Figure 4.

As demonstrated in Figure 4, vaccination of NHP with the VLP vaccine elicited neutralizing antibodies against SARS-CoV-2 wild-type strain (Wuhan), SARS-CoV-2 variant B.1.1.7 and SARS-CoV-2 variant B.1.351 at comparable amounts.

In continuing experiments, additional data was collected from NHPs. For example, data presented in Figure 5 further shows that neutralizing antibodies in serum from these non human primates were induced at comparable levels following vaccination with the VLP vaccine described in Example 1 (ABNCoV2) in different doses. The SARS-CoV-2 variants or strains against which the antibodies were tested included the Wuhan wild-type strain or variant as well as the variants designated B.1.1.7 (“Alpha”), B.1.351 (“Beta”), and Bl.617.2 (“Delta”). Cross-neutralization of variants by NHP serum was observed following immunization with ABNCoV2 at both the high dose (100 pg, data points and bars on left side of graph) and low dose (15pg, data points and bars on right side of graph). This data demonstrated that immunization of NHPs with ABNCoV2 induced neutralizing antibodies against other SARS-CoV-2 variants.

Conclusion

As shown in Figure 4, the VLP vaccine ABNCoV2 induced neutralizing antibodies in NHPs not only against the wild-type strain of SARS-CoV-2 originally detected in Wuhan, China, but also without statistical difference (P = 0.41) similarly against two variants, namely the “British variant” (B.1.1.7, also referred to as “Alpha”) and the “South- African variant”

(B.1.351, also referred to as “Beta”). These results imply that the VLP vaccine is capable of conferring protection against an infection with at least the two tested variants equally well as against the wild-type strain. Additional data shown in Figure 5 supported these results and extended them to a third tested variant (B 1.617.2, also referred to as “Delta”) and to a lower dose level of 15 pg of ABNCoV2 (data points and bars shown on right side of Figure 5).

In addition, antibodies in the NHPs were durable within the monitored 3 -month timeframe and levels of neutralizing antibodies were >50-fold higher than those measured in human convalescent samples. These high-level neutralizing antibodies translated into efficacy against challenge, with no viral load detected by PCR in the majority of animals vaccinated with the human dose of 100 pg ABNCoV2 and a significant reduction even in the 15 pg ABNCoV2 vaccination group, compared to non-vaccinated controls that all harbored a high number of viral copies in their lungs.

EXAMPLE 4: Human clinical trials showed that a VLP vaccine displaying RBD antigen is highly immunogenic

Phase 1 human clinical trial: In a Phase 1 human clinical trial investigating a dose response of the ABNCoV2 vaccine in seronegative individuals, the vaccine was well tolerated across all dose groups. Neutralization titers against the SARS-CoV-2 “Wuhan” strain were up to 12-fold higher than the titers in Human Convalescent Sera (HCS), and were comparable for all SARS-CoV-2 Variants Of Concern (VOCs), including the “Delta” variant (Bl.617.2).

The Phase 1 trial (“COUGH- 1”) assessed five different dose levels of the ABNCoV2 vaccine. Doses of ABNCoV2 from 6 pg to 70 pg were administered to human patients in a two-dose schedule. These human patients were previously unvaccinated and were seronegative. At ABNCoV2 dose levels from 6 pg to 25 pg, administration with and without the MF59® adjuvant (Novartis, Cambridge, MA, USA) was tested. Humoral and cellular responses (B cells and T cells) were measured along with other indicators of safety and immunogenicity for all 45 enrolled patients.

Results from the Phase 1 trial demonstrated that ABNCoV2 was well tolerated across all dose groups, with no observed difference in adverse event profile after the first and second vaccination. One patient reported a serious adverse event of a basal cell carcinoma located near the administration site of the vaccine (ABNCoV26 pg plus MF59® adjuvant). This event was reported as possibly related to the vaccine in the patient, who was a carrier of albinism and had a previous history of melanoma and basal cell carcinoma.

Administration of ABNCoV2 produced a dose-dependent increase in neutralizing antibodies at doses up to 25 pg, reaching a plateau at higher doses (Figure 6). Increases in antibody titers following administration of ABNCoV2 were up to 12-fold higher than titers seen in human convalescent sera samples (HCS). Data shown in Figure 6 are from patients in the Phase 1 clinical trial, 14 days after the second dose of non-adjuvanted ABNCoV2.

Administration of ABNCoV2 induced high levels of neutralizing antibodies against SARS-CoV-2 in human patients (Figure 7). PRNT50 titers were determined 14 days after the second ABNCoV2 dose of 25 pg was administered to patients (data points on left side of graph) in comparison to human convalescent samples (HCS, data points on right side of graph) showed higher levels of antibodies and strong cross neutralization of SARS-CoV-2 variants of concern (“VOC”), including for the Delta variant (B 1.617.2). No reduction in neutralization capacity was seen for ABNCoV2 against the Alpha or Delta variants, and only a 2.2-fold reduction of neutralization capacity against the Beta variant was observed. In contrast, the currently approved Comimaty® vaccine was reported to show a more than 10- fold reduction in neutralization against the Beta variant (see, e.g., Lustig el al. (July 2021) Euro Surveill. 26: 2100557; Planas et al. (July 2021) Nature 596: 276-280).

(Geometric mean titers with geometric standard deviations are also shown in Figure 7. PRNT50 is the concentration of serum needed in a Plaque Reduction Neutralization Test to reduce the number of plaques by 50% compared to the serum free virus and is a measurement of the titer of virus-neutralizing antibody in serum; methods for determining this value are well known in the art. Phase 2 human clinical trial: In a Phase 2 trial investigating ABNCoV2 dose response in a single administration of vaccine to seropositive individuals, initial results showed that a 100pg dose of ABNCoV2 promoted a strong immunostimulatory effect, increasing the existing levels of SARS-CoV-2 neutralizing antibodies against the Wuhan variant by 2-40-fold, depending on the initial levels of antibodies. Similar fold increases were also observed for all SARS-CoV-2 VOCs (Wuhan, Alpha, Beta, and Delta). No related serious adverse events were reported.

The Phase 2 trial involved healthy adult human patients in three groups:

Group 1: 28 patients who were seronegative for SARS-CoV-2 antibodies at screening and were vaccinated with two doses of 100 pg ABNCoV2

Group 2: 103 patients who were seropositive for SARS-CoV-2 antibodies at screening (with a prior SARS-CoV-2 vaccination or previously infected with SARS-CoV-2) and were vaccinated with one dose of 100 pg ABNCoV2

Group 3: 66 patients who were seropositive for SARS-CoV-2 antibodies at screening (with a prior SARS-CoV-2 vaccination or previously infected) and were vaccinated with one dose of 50 pg ABNCoV2

This Phase 2 trial was designed to evaluate the safety, tolerability, and immunogenicity of the vaccine in adult human patients, and particularly its effectiveness when administered to patients who had previously been vaccinated with another SARS-CoV- 2 vaccine (i.e., to evaluate ABNCoV2 when administered as a “booster”).

Results for the Group 2 seropositive patients (n=103) showed that ABNCoV2 (lOOpg) provided a strong boosting effect, increasing the existing levels of SARS-CoV-2 neutralizing antibodies against the Wuhan strain by 2-40-fold depending on the initial levels of antibodies. A similar fold increase was observed for all SARS-CoV-2 variants tested (Wuhan, Alpha, Beta and Delta) following the booster vaccination with ABNCoV2. No related serious adverse events were reported in the trial.

Figure 8 shows neutralizing antibody titer increases in patients in the Phase 2 trial who were initially seropositive. Antibody titers are shown at week 0 (“baseline”), week 1, and week 2 for the overall population (leftmost panel) and for three groups of patients stratified by baseline antibody level (left to right) against the SARS-CoV-2 Wuhan strain. Data is shown for patients in the 100 pg dose group (NT = neutralization titer; LLOQ = lower limit of quantitation). Figure 9 shows neutralizing antibody responses from human patients in the Phase 2 trial. Neutralizing antibody response is shown for seropositive subjects at week 2 for SARS- CoV-2 strains Alpha, Beta, and Wuhan. The three bars shown in Figure 9 for each strain indicate the percentage of subjects with at least a 2-fold increase (left-most bar), at least a 4- fold increase (middle bar), and at least a 6-fold increase (right-most bar). The upper left quadrant of Figure 9 shows overall results, while the other three quadrants show results for patients grouped by relationship of baseline NT (neutralization titer) to LLOQ (lower limit of quantitation).

These results demonstrate the ability of the ABNCoV2 vaccine to induce outstanding levels of neutralizing antibodies.

EXAMPLE 5: Phase III trial will be conducted using a non-inferiority design comparing ABNCoV2 immunogenicitv to a licensed boost vaccine

A Phase 3 trial is to demonstrate non-inferiority of the ABNCoV2-mediated boost response in terms of GMT of neutralizing antibodies to the SARS-CoV-2 Wuhan strain, as compared to a licensed mRNA-based boost vaccine, in a cohort with a homologous previous vaccination regimen at least six months before. Secondary objectives include analysis of neutralizing antibodies against Variants of Concern (those relevant at the time of analysis) and extent of boost response in various cohorts based on other primary vaccination regimens than the primary endpoint cohort. Exploratory objectives include T cell responses (IFNY and IL-4), analysis of RBD-binding antibodies, and duration of humoral boost responses.

Each subject will receive one dose of ABNCoV2 vaccine (active treatment groups) or 1 dose of a comparator vaccine (control groups); subjects will include a wide age-range including older adults. The trial will include various cohorts defined by the previously administered number and type of SARS-CoV-2 vaccine administrations, such as, for example, subjects with a previous homologous 2-dose regimen at least 6 months before screening, subjects with heterologous (or “mix & match”) regimens at least 3 months before screening, or subjects who already had a first boost administration at least 3 months ago and are eligible for a further boost at time of screening. Sequences in the sequence listing:

SEQ ID NO:l = AP205 amino acid sequence (wt, UniProt Q9AZ42)

Met Ala Asn Lys Pro Met Gin Pro He Thr Ser Thr Ala Asn Lys He Val Trp Ser Asp Pro Thr Arg Leu Ser Thr Thr Phe Ser Ala Ser Leu Leu Arg Gin Arg Val Lys Val Gly He Ala Glu Leu Asn Asn Val Ser Gly Gin Tyr Val Ser Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly Cys Ala Asp Ala Cys Val He Met Pro Asn Glu Asn Gin Ser He Arg Thr Val He Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu Lys Ala Glu Trp Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe Ala Ser Gly Asn Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala Ala He Val Ser Ser Asp Thr Thr Ala

SEQ ID NO:2 = AP205 amino acid sequence (containing Pro5Thr mutation; see US Pat. No. 7138252)

Met Ala Asn Lys Thr Met Gin Pro lie Thr Ser Thr Ala Asn Lys lie Val Trp Ser Asp Pro Thr Arg Leu Ser Thr Thr Phe Ser Ala Ser Leu Leu Arg Gin Arg Val Lys Val Gly He Ala Glu Leu Asn Asn Val Ser Gly Gin Tyr Val Ser Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly Cys Ala Asp Ala Cys Val He Met Pro Asn Glu Asn Gin Ser He Arg Thr Val He Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu Lys Ala Glu Trp Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe Ala Ser Gly Asn Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala Ala He Val Ser Ser Asp Thr Thr Ala

SEQ ID NO:3 = peptide tag DNA sequence ggtaatccgctgattgtgatggtgaatgataccaccaaagtgaaa

SEQ ID NO:4 = peptide tag amino acid sequence

Gly Asn Pro Leu He Val Met Val Asn Asp Thr Thr Lys Val Lys

SEQ ID NO:5 = peptide binding partner DNA sequence attgataccatgagcggtctgagcggtgaaaccggtcagagcggtaataccaccattgaagaggatagcaccacacatgtgaaattca gcaaacgcgatagcaacggcaaagaactggcaggcgcaatgattgaactgcgtaatctgagtggtcagaccattcagagctgggtta gtgatggcaccgttaaagatttttatctgatgcctggcacctatcagtttgttgaaaccgcagcaccggaaggttatgagctggcagcac cgattacctttaccgttcaggataacggcgaagttattattcagggccgcctgacacgtggcgatgttcatatt

SEQ ID NO:6 = peptide binding partner amino acid sequence

Phe Ser Met Gly He Asp Thr Met Ser Gly Leu Ser Gly Glu Thr Gly Gin Ser Gly Asn Thr Thr He Glu Glu Asp Ser Thr Thr His Val Lys Phe Ser Lys Arg Asp Ser Asn Gly Lys Glu Leu Ala Gly Ala Met He Glu Leu Arg Asn Leu Ser Gly Gin Thr He Gin Ser Trp Val Ser Asp Gly Thr Val Lys Asp Phe Tyr Leu Met Pro Gly Thr Tyr Gin Phe Val Glu Thr Ala Ala Pro Glu Gly Tyr Glu Leu Ala Ala Pro He Thr Phe Thr Val Gin Asp Asn Gly Glu Val He He Gin Gly Arg Leu Thr Arg Gly Asp Val His He

SEQ ID NO:7 = amino acid sequence of SARS-CoV-2 full-length S protein (YP_009724390.1, SARS-CoV-2 isolate Wuhan-Hu-1, NC_045512.2)

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gin Cys Val Asn Leu Thr Thr Arg Thr Gin Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gin Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala He His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn He He Arg Gly Trp He Phe Gly Thr Thr Leu Asp Ser Lys Thr Gin Ser Leu Leu He Val Asn Asn Ala Thr Asn Val Val He Lys Val Cys Glu Phe Gin Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gin Pro Phe Leu Met Asp Leu Glu Gly Lys Gin Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn He Asp Gly Tyr Phe Lys lie Tyr Ser Lys His Thr Pro He Asn Leu Val Arg Asp Leu Pro Gin Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro He Gly He Asn He Thr Arg Phe Gin Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gin Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr He Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly He Tyr Gin Thr Ser Asn Phe Arg Val Gin Pro Thr Glu Ser He Val Arg Phe Pro Asn He Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg He Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val He Arg Gly Asp Glu Val Arg Gin He Ala Pro Gly Gin Thr Gly Lys He Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val He Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp He Ser Thr Glu He Tyr Gin Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly Phe Gin Pro Thr Asn Gly Val Gly Tyr Gin Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gin Gin Phe Gly Arg Asp He Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gin Thr Leu Glu He Leu Asp He Thr Pro Cys Ser Phe Gly Gly Val Ser Val He Thr Pro Gly Thr Asn Thr Ser Asn Gin Val Ala Val Leu Tyr Gin Asp Val Asn Cys Thr Glu Val Pro Val Ala He His Ala Asp Gin Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gin Thr Arg Ala Gly Cys Leu He Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp He Pro He Gly Ala Gly He Cys Ala Ser Tyr Gin Thr Gin Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Gin Ser He He Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser He Ala He Pro Thr Asn Phe Thr He Ser Val Thr Thr Glu He Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr He Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gin Tyr Gly Ser Phe Cys Thr Gin Leu Asn Arg Ala Leu Thr Gly He Ala Val Glu Gin Asp Lys Asn Thr Gin Glu Val Phe Ala Gin Val Lys Gin He Tyr Lys Thr Pro Pro He Lys Asp Phe Gly Gly Phe Asn Phe Ser Gin He Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe He Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe He Lys Gin Tyr Gly Asp Cys Leu Gly Asp He Ala Ala Arg Asp Leu He Cys Ala Gin Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met He Ala Gin Tyr Thr Ser Ala Leu Leu Ala Gly Thr He Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gin He Pro Phe Ala Met Gin Met Ala Tyr Arg Phe Asn Gly He Gly Val Thr Gin Asn Val Leu Tyr Glu Asn Gin Lys Leu He Ala Asn Gin Phe Asn Ser Ala He Gly Lys He Gin Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gin Asp Val Val Asn Gin Asn Ala Gin Ala Leu Asn Thr Leu Val Lys Gin Leu Ser Ser Asn Phe Gly Ala He Ser Ser Val Leu Asn Asp He Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gin He Asp Arg Leu He Thr Gly Arg Leu Gin Ser Leu Gin Thr Tyr Val Thr Gin Gin Leu He Arg Ala Ala Glu He Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gin Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gin Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gin Glu Lys Asn Phe Thr Thr Ala Pro Ala He Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gin Arg Asn Phe Tyr Glu Pro Gin He He Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val He Gly He Val Asn Asn Thr Val Tyr Asp Pro Leu Gin Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp He Ser Gly He Asn Ala Ser Val Val Asn He Gin Lys Glu He Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu He Asp Leu Gin Glu Leu Gly Lys Tyr Glu Gin Tyr He Lys Trp Pro Trp Tyr He Trp Leu Gly Phe lie Ala Gly Leu He Ala He Val Met Val Thr He Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr

SEQ ID NO: 8 = linker (amino acid sequence) GSGTAGGGSGS

SEQ ID NO: 9 = cVLP-AP205 subunit monomer

GNPLIVMVNDTTKVKGSGTAGGGSGSANKPMQPITSTANKIVWSDPTRLSTTFSASL LRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAEN LATLKAEWETHKRNVDTLF ASGNAGLGFLDPTAAIV S SDTT A

SEQ ID NO: 10 = peptide binding partner amino acid sequence

GIDTMS GL S GET GQ S GNTTIEED S TTH VKF SKRD SN GKEL AGAMIELRNL S GQTIQ S W VSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTVQDNGEVIIQGRLTRGDVHI

SEQ ID NO: 11 = linker sequence GSGS

SEQ ID NO: 12 = C-tag sequence EPEA

SEQ ID NO: 13 = RBD antigen sequence

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCV I AWN SNNLD SK V GGN YNYL YRLFRK SNLKPFERDIS TEI Y Q AGS TPCN GVEGFNC YF PLQ S YGF QPTNGV GY QP YRV VVL SFELLH AP AT V C GPKK S TNL VKNKC VNFNFN GL TGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCS

SEQ ID NO: 14 = Catcher-RBD antigen sequence

GIDTMS GL S GET GQ S GNTTIEED S TTH VKF SKRD SN GKEL AGAMIELRNL SGQTIQ S W

VSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTVQDNGEVIIQGRLTRGDVHIG

SGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSAS

FSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFT

GCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN

CYFPLQS Y GF QPTNGVGY QPYRVVVLSFELLHAP AT VCGPKKSTNLVKNKC VNFNF

NGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSGSGSEPEA

Claims

CLAIMS We claim:

1. A virus-like particle (VLP) comprising: a. an AP205 protein fused to a peptide tag having the amino acid sequence set forth in SEQ ID NO:4, or a derivative thereof; and b. a SARS-CoV-2 antigen fused to a peptide binding partner having the amino acid sequence set forth in SEQ ID NO:6, or a derivative thereof, whereby the SARS-CoV-2 antigen is displayed on the surface of said VLP, that when administered to a subject as a vaccine stimulates the production of antibodies against said SARS-CoV-2 antigen.

2. The VLP of claim 1, wherein said peptide tag has the sequence set forth in SEQ ID NO:4.

3. The VLP of claim 1, wherein said peptide binding partner has the sequence set forth in SEQ ID NO:6.

4. The VLP of claim 1, wherein said AP205 has the sequence set forth in SEQ ID NO:l or SEQ ID NO:2.

5. The VLP of claim 1, wherein said antigen has the sequence set forth in SEQ ID NO:7, or is a fragment thereof.

6. The VLP of claim 5, wherein said antigen comprises amino acids 319-591 of SEQ ID NO:7.

7. The VLP of claim 1 that comprises an isopeptide bond between said binding partner and said peptide tag.

8. A virus-like particle (VLP) comprising: i. an AP205 protein fused to a peptide tag; and ii. a SARS-CoV-2 antigen comprising the wild-type (Wuhan) spike protein Receptor Binding Domain (RBD), whereby the SARS-CoV-2 antigen is displayed on the surface of said VLP; and that when administered to a subject as a vaccine stimulates the production of antibodies against said SARS-CoV-2 antigen.

9. The VLP of claim 8, wherein said peptide tag has the sequence set forth in SEQ ID NO:4.

10. The VLP of claim 8, wherein said AP205 has the sequence set forth in SEQ ID NO:l or SEQ ID NO:2.

11. The VLP of claim 8, wherein said antigen has the sequence set forth in SEQ ID NO:7, or is a fragment thereof.

12. The VLP of claim 8, wherein said antigen comprises amino acids 319-591 of SEQ ID NO:7.

13. A vaccine comprising the VLP of claim 1 in an aqueous solution that contains no squalene.

14. A method of treating a subject to prevent or ameliorate symptoms of a coronavirus infection, preferably coronavirus disease 19 (COVID-19), comprising the step of administering the vaccine of claim 13 to a subject.

15. The method of claim 14, wherein the step of administering the vaccine to a subject produces an immune response in said subject comprising an increase in antibodies against said SARS-CoV-2 antigen and/or neutralizing antibodies against a SARS-CoV-2 variant.

16. The method of claim 15, wherein the SARS-CoV-2 variant is B.1.1.7,

B.1.351, and/or B 1.617.2.

17. The method of claim 14, wherein the step of administering the vaccine produces an immune response in said subject that prevents or ameliorates symptoms of infection with a strain of SARS-CoV-2 that is not the SARS-CoV-2 wild-type strain (Wuhan).

18. A VLP of claim 1 for use in the prevention or treatment of a coronavirus infection, preferably coronavirus disease 19 (COVID-19), wherein the coronavirus infection is caused by SARS-CoV-2, preferably SARS-CoV-2 wild-type strain (Wuhan) or a variant thereof.

19. The VLP for use of claim 18, wherein the SARS-CoV-2 variant is B.1.1.7,

B.1.351, and/or B 1.617.2.

20. The VLP for use of claim 1 for use in the prevention or treatment of a coronavirus infection, wherein the coronavirus infection is caused by a SARS-CoV-2 variant that is not the SARS-CoV-2 wild-type strain (Wuhan).

21. A subunit monomer of a virus-like particle (VLP) comprising: a peptide tag having the sequence set forth in SEQ ID NO:4, a linker having the sequence set forth in SEQ ID NO:8, and an AP205 protein comprising the sequence set forth in SEQ ID NO: 1 but missing the initial methionine.

22. An RBD-antigen component comprising: a binding partner having the sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 10, a linker having the sequence set forth in SEQ ID NO: 11, the SARS-CoV-2 antigen sequence set forth in SEQ ID NO: 13, a linker having the sequence set forth in SEQ ID NO: 11, and a C-tag having the sequence set forth in SEQ ID NO: 12.

23. A VLP comprising a subunit monomer of claim 21 and an RBD-antigen component of claim 22.

24. A vaccine comprising the VLP of claim 23.

25. A method of treating a subject to prevent or ameliorate symptoms of a coronavirus infection, preferably coronavirus disease 19 (COVID-19), comprising the step of administering the vaccine of claim 24 to a subject.