VACCINES AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to the use of 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, as vaccine boosters. Antigens are displayed on virus-like particles (VLPs) and produce an immune response in vaccinated subjects. The invention also relates to methods of treatment using recombinant VLPs as boosters to treat and/or prevent infection with SARS-CoV-2, and methods of preparation thereof.

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 include 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). 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. SARS-CoV-2 belongs to the Coronaviridae family, a family of positive-sense, singlestranded RNA viruses. Like other coronaviruses, SARS-CoV-2 is characterized by a crownlike (“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. 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 2016112921, WO 2021224451, and WO2022229817. 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, for example, in Fougeroux et al. ((2021) Nat. Commun. 12: 324).

However, the ongoing pandemic and repeated emergence of variants with the ability to escape control by some previously developed vaccines has created a need for more effective prophylactic vaccines against SARS-CoV-2 variants. BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide an effective vaccination regime to help prevent and/or ameliorate SARS-CoV-2 infection and related diseases. The invention provides vaccination regimes and booster vaccines comprising virus-like particles displaying at least one SARS-CoV-2 antigen (referred to herein as “VLP-SARS”). 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 (“S”) protein or a portion thereof. The SARS-CoV-2 antigen may be from the first-identified strain of SARS- CoV-2 (formerly called “Wuhan”) or may be from any variant strain. Antigens are displayed on virus-like particles (VLPs) comprising the RNA bacteriophage AP205 coat protein (“AP205”), in some embodiments by using a peptide tag and binding partner that form an isopeptide bond.

The invention also provides methods of treatment using VLPs displaying SARS-CoV- 2 antigens (“VLP-SARS”) as booster vaccinations to treat and/or prevent infection with the SARS-CoV-2 virus and variants thereof. The invention further provides medical uses of the VLP boosters in the prevention and/or amelioration of COVID-19 symptoms. Methods of immunization with specific combinations of prime and booster vaccinations are also provided.

Particularly, in one aspect the booster vaccination regime of the invention comprises a virus-like particle (VLP) comprising an AP205 protein fused to a peptide tag and further 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 (referred to herein as “VLP-SARS”), for example as described in WO 2016112921, WO 2021224451, and WO2022229817 (each of which is specifically incorporated herein by reference in its entirety). When administered to a subject as a booster vaccine, the VLP-SARS stimulates an immune response that prevents or alleviates symptoms of coronavirus infection in a subject caused by SARS-CoV-2 variants such as, for example, the omicron variant, B.l.1.7 and/or B.1.351.

In yet another aspect, the invention provides a vaccination regime or method of vaccinating a subject to induce a Thl immune response in a subject comprising administering a first (“priming”) vaccination to a subject that stimulates a Thl immune response and administering a second (“booster” vaccination) comprising a VLP-SARS. In this manner, the methods and compositions of the invention can be used to direct the immune response in a subject to a Thl response. In some embodiments, the booster vaccination comprising the VLP-SARS further stimulates the immune response elicited by the first (“priming”) vaccination but does not change the type of immune response; that is, the VLP-SARS booster vaccination does not change the immune response from Thl to Th2.

In yet another aspect, the invention provides a booster vaccine comprising a VLP- SARS that has been treated in whole or part with a nuclease, DNAase, and/or RNAase after assembly of the VLP capsid. The invention accordingly also provides a method of treatment of a subject comprising administering to a subject said VLP-SARS that has been treated in whole or part with a nuclease, DNAase, and/or RNAase. This method stimulates an immune response in the subject to the antigen displayed on the VLP-SARS and is helpful in preventing and/or ameliorating a coronavirus infection, for example an infection by a strain of SARS-CoV-2.

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 VLP-SARS; in this example, the VLP comprises AP205 coat protein fused to a peptide tag and the VLP is coated with SARS-CoV- 2 RBD antigen genetically fused to a binding partner (“catcher”) to produce the assembled VLP-SARS. 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 display of antigen on the VLP-SARS, 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 combinations of prime and boost immunizations were tested for their ability to produce an immune response to the target antigen in a model system. In these experiments, Balb/c mice were first immunized (“primed”) with 5 pg of the VLP-SARS known as ABNCoV2, IxlO ⁸ TCID50 MVA-Spike or 5 pg Spike protein + AddaVax™ adjuvant and boosted on day 21 with 5 pg ABNCoV2. In addition, two groups were first immunized with IxlO ⁸ TCID50 MVA-Spike or 5 pg Spike protein + AddaVax™ adjuvant and boosted on day 21 with the same vaccine candidates. Blood for serum isolation was taken 14 days after boost immunization, and ELISA and ePass (Genscript) was used to assess total RBD-specific IgG titers and RBD-binding antibody titers, respectively. Data from samples taken on day 34 are shown in Figure 2 as Mean ± SEM. These experiments demonstrated that the VLP-SARS “ABNCoV2” induced high RBD-binding antibody levels as assessed by RBD-binding inhibition rate, even without additional adjuvant. Spike protein + AddaVax™ adjuvant (designated “AdVx” in the figure legends) induced intermediate RBD-binding antibody levels and the lowest RBD inhibition rate was detected in mice immunized only with MVA- Spike. Most importantly, both MVA-Spike and Spike + AdVx (AddaVax™ adjuvant) induced RBD-binding antibodies that were boosted by ABNCoV2, as seen by increasing RBD-binding antibody levels.

Figure 3 shows results from experiments described in Example 3. These results demonstrated that homologous vaccination with recombinant MVA expressing a modified SARS-CoV-2 “S” protein (“MVA-Spike”) almost exclusively induced anti-RBD IgG2a antibodies, while Spike + AdVx (Spike protein plus AddaVax™ adjuvant) exclusively drove IgGl production. Mice prime immunized and then boosted with ABNCoV2 exhibited a balanced Thl/Th2 immune response with high titers of both IgGl and IgG2a RBD binding antibodies. Importantly, the SARS-VLP ABNCoV2 did not alter the type of preexisting humoral immune response primed by either MVA-Spike or Spike + AdVx (AddaVax™ adjuvant), but rather boosted the preexisting type of antibody response.

Figure 4 shows data from experiments described in Example 4, in which heterologous combinations of various priming immunizations with the VLP-SARS ABNCoV2 were used to analyze the effect of a knockout of TLR7 (“TLR7-K0”) on the potency of VLP-SARS as a booster vaccine. Serum was isolated from the blood of all mice on day 35 after immunization, and RBD-binding (neutralizing) antibodies were measured by ePass assay (Genscript). As shown in Figure 4, prime and boost immunizations with VLP-SARS (also referred to as “homologous prime-boost”) induced the highest amounts of RBD-binding neutralizing antibody titers (Figure 4). Mice with TLR7 deficiency (or “knockout”) and MyD88 deficiency (or “knockout”) exhibited greatly decreased neutralizing antibody titers in response to this vaccine. Moreover, in heterologous combinations of VLP-SARS booster with priming immunizations of MVA-Spike or Spike protein + Alum, a decrease of RBD-binding antibodies was observed in TLR7-K0 (“knockout”) and MyD88-KO mice. Knockout of most TLRs in the MyD88-KO model did not lead to any further decreases in neutralizing antibodies compared to TLR7-K0 mice. Figure 5 shows results of total anti-RBD IgG levels in the experiments described in Example 4, with very similar trends to the neutralizing antibodies shown in Figure 4. Again, a sharp drop of anti-RBD titers was demonstrated in TLR7-K0 (“knockout”) mice and MyD88-KO mice immunized with the VLP-SARS known as ABNCoV2. The same observation was true in mice immunized with combinations of ABNCoV2 with MVA-Spike and Spike protein + Alum.

Figure 6 shows the results from experiments described in Example 5, in which ABNCoV2 was shown to provide benefit as a stand-alone vaccine and as a booster vaccine. Specifically, ABNCoV2 induced long-term antibody responses against the target antigen (RBD) after both prime and boost immunizations. In these experiments, Balb/c mice were immunized on day 0 ("prime immunization") and day 21 ("boost immunization") with 5 pg of ABNCoV2 or IxlO ⁸ TCID50 MVA-mBN500 (homologous immunization groups). In addition, one group was first immunized with 5 pg of ABNCoV2 on day 0 and then with IxlO ⁸ TCID50 MVA-mBN500 on day 21 (heterologous immunization group). Blood for serum was collected on day 41, 62, 90, 120, 181, 240 and 360 after prime immunization. EEISA was performed to determine total RBD-specific Ig titers.

These experiments demonstrated that ABNCoV2 prime/boost immunization induced stronger and long-lasting RBD-specific antibody levels in mice compared to MVA-mBN500. They further demonstrated the effectiveness of ABNCoV2 as a booster vaccine because MVA-mBN500-prime / ABNCoV2 boost-immunized mice exhibited very comparable, long- lasting antibody responses to those of ABNCoV2 prime/boost immunized animals.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides effective vaccination regimes to help prevent and/or ameliorate SARS-CoV-2 infection and related diseases. The invention provides vaccination regimes and booster vaccines comprising virus-like particles displaying at least one SARS- CoV-2 antigen (referred to herein as “VLP-SARS”). A SARS-CoV-2 antigen for use with these virus-like particles in the vaccination regimes and/or boosters of the invention is any antigen that produces an immune response to a SARS-CoV-2 strain or variant, such as, for example, the receptor-binding domain (RBD) of a SARS-CoV-2 spike (“S”) protein or a portion thereof. The SARS-CoV-2 antigens and/or their sequences may be from any strain or variant of SARS-CoV-2, for example, from the first-identified strain of SARS-CoV-2 (earlier known as “Wuhan”) or a variant such as omicron. The SARS-CoV-2 antigens are displayed on virus-like particles (VLPs) to produce VLP-SARS; in some embodiments, these VLPs comprise the RNA bacteriophage AP205 coat protein (“AP205”) and are attached to the antigen(s) using a peptide tag and binding partner, for example, as described in WO 2016112921, WO 2021224451, WO2022229817, and Fougeroux et al. ((2021) Nat. Commun. 12: 324) (each of which is incorporated herein by reference in its entirety).

The invention also provides methods of treatment using the VLP-SARS as booster vaccinations to treat and/or prevent infection with the SARS-CoV-2 virus and variants thereof. The invention further provides medical uses of the VLP-SARS boosters in the prevention and/or amelioration of COVID-19 symptoms. Specific combinations of prime and booster vaccinations are provided. Particularly, in one aspect the booster vaccination regime of the invention comprises a VLP-SARS that 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, for example as described in WO 2016112921, WO 2021224451, WO2022229817, and Fougeroux et al. ((2021) Nat. Commun. 12: 324). In some embodiments, the VLP is ABNCoV2 as described in WO2022229817 (specifically incorporated herein by reference in its entirety) (also described in Fougeroux et al. (ibid.) as “RBDn-CLP”). When administered to a subject as a booster vaccine, the VLP stimulates an immune response that prevents or alleviates symptoms of coronavirus infection caused by SARS-CoV-2 variants such as, for example, the omicron variant, B .1.1.7 and/or B .1.351.

In yet another aspect, the invention provides a vaccination regime or method of vaccinating a subject to induce a Thl immune response in a subject comprising administering a first (“priming”) vaccination to a subject that stimulates a Thl immune response and administering a second (“booster” vaccination) comprising a VLP-SARS. In this manner, the methods and compositions of the invention can be used to direct the immune response in a subject to a Thl response. In some embodiments, the booster vaccination comprising the VLP-SARS further stimulates the immune response elicited by the first (“priming”) vaccination but does not change the type of immune response; that is, the VLP-SARS booster vaccination does not change the immune response from Thl -like to Th2-like.

In yet another aspect, the invention provides a booster vaccine comprising a VLP- SARS in which the VLP component has been treated with a nuclease and/or a DNAase and/or an RNAase after assembly of the VLP capsid. The invention accordingly also provides a method of treatment of a subject comprising administering to a subject said VLP- SARS that has been treated in part or as an assembled VLP-SARS with a nuclease and/or a DNAase and/or an RNAase. This method of treatment stimulates an immune response in the subject to the antigen displayed on the VLP-SARS without exposing the subject to excess bacterial RNA or other nucleic acid matter that may have accumulated on the VLP-SARS during the production process.

In most embodiments, the vaccination regimes and booster vaccines of the invention are administered to a subject without a squalene adjuvant or other adjuvant included in the vaccine (also sometimes referred to as a pharmaceutical composition). These embodiments without adjuvant or free of adjuvant provide several advantages. For example, customarily, squalene adjuvants are prepared from shark liver oil. The omittance of those adjuvants helps avoid exploiting and killing sharks and thus has desirable environmental effects. Moreover, the source material of the squalene adjuvants (i.e., sharks and shark liver oil) is limited. This aspect may become relevant for example in case of a high demand for those adjuvants, for example, during another pandemic or new “wave” of SARS-CoV-2 variants. However, in some embodiments, the vaccination regimes and booster vaccines of the invention are administered to a subject along with an adjuvant such as, for example, Alum, which is known in the art.

It has been shown that VLPs displaying SARS-CoV-2 antigens induced neutralizing antibodies not only against the originally identified strain of SARS-CoV-2 (“Wuhan”) but also against variants (“mutants”) that developed and emerged during the COVID-19 pandemic (see, for example, Fougeroux et al. (2021) Nat. Commun. 12: 324). 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 these VLP vaccines is very beneficial 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. The methods and compositions of the invention expand the protections offered against SARS-CoV-2 variants currently known and that will arise in the future. VLPs

In embodiments of the invention, VLPs display or are linked to a peptide of interest that is a SARS-CoV-2 antigen, as further discussed elsewhere herein. By “VLP” i.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” or “capsids.” 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.

In some embodiments, the structure of the VLP is provided by self-assembly of particle-forming proteins, such as, for example, the AP205 protein, known in the art and described in U.S. Pat. No. 7,138,252. In some embodiments, a particle-forming protein is fused to a peptide tag and an antigen (z.e., 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 (z.e.,VLPs) 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 (diagrammed in Figure 1). Methods for designing and producing suitable VLPs displaying SARS-CoV-2 antigens are known in the art, for example, as discussed in WO 2016112921, WO 2021224451, WO2022229817, and Fougeroux et al. ((2021) Nat. Commun. 12: 324).

In some embodiments of the invention, the capsid or particle or VLP is treated after assembly of the capsid with a nuclease, RNAase, and/or DNAase to remove excess bacterial and other RNA and/or DNA from the VLP preparation. Suitable nucleases for this purpose are known in the art and are commercially available, for example, such as Benzonase® endonuclease (Merck, Burlington, MA, USA). Thus, in some embodiments, the invention provides a pharmaceutical composition such as a VLP-SARS vaccine comprising a VLP component that was treated with a nuclease, RNAase, and/or DNAase following capsid assembly. The treatment with nuclease, RNAase, and/or DNAase may occur at any point following capsid assembly, for example, immediately following capsid assembly; during a step in which VLP component and the antigen component are combined by allowing formation of isopeptide bonds; following combination of the VLP and the antigen component by any other suitable process; or prior to formulation into a pharmaceutical composition for administration to a subject. In this manner, the invention also provides a pharmaceutical composition comprising a VLP-SARS that was treated with a nuclease, RNAase, and/or DNAase and comprises a VLP component and/or a VLP displaying a SARS-CoV-2 antigen. In some embodiments, the VLP displaying a SARS-CoV-2 antigen is ABNCoV2 (described in Fougeroux et al. ((2021) Nat. Commun. 12: 324, as “RBDn-CLP”)).

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 particleforming 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. 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, for example, as described in WO 2016112921, WO 2021224451, WO2022229817, and Fougeroux et al. ((2021) Nat. Commun. 12: 324).

In some embodiments, these 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 VLP-SARS is suitable for use in the compositions and methods of the invention. 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 capsid or VLP protein 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 these constructs, for example, to enhance binding properties of the fusion proteins or assembly of the final VLP-SARS product. The antigens 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, for display on the VLP. SARS-CoV-2 antigens

VLPs for use in the compositions and methods of the invention display or are linked to a peptide of interest that is a SARS-CoV-2 antigen to produce a “VLP-SARS.” 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 (“S”) 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 (“S”) 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 as the SARS-CoV-2 antigen. 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-SARS, 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 (e.g., portions of the “S” and/or “N” proteins of SARS-CoV-2). In such fusion proteins, at least one portion can be from a pail 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 “wild-type” or “Wuhan” strain. Numerous variant strains of SARS-CoV-2 have been identified as of September 2022 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 SARS-CoV-2 variant strains and nucleotide sequences encoding them are readily obtained by one of skill in the art and adapted for use in the VLP-SARS of the invention. In some embodiments, VLP-SARS display more than one different antigen, e.g., RBD antigens from more than one strain or variant of SARS-CoV-2 are displayed on VLP components, and/or more than one fragment or portion of a SARS-CoV-2 protein from one strain or from more than one strain of SARS-CoV-2 is displayed on VLP components to produce a mixture of VLP-SARS.

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. A “stimulated immune response” or increased immune response in a subject that has been vaccinated using the compositions and/or methods of the invention may comprise an increase in any feature or component of an immune response known in the art, e.g., an increase in neutralizing antibodies and/or T cell responses, and/or production of cytokines such as, for example, IL-12, TNF-a, and/or IFN-y. An immune response is deemed to be “stimulated or “increased” if there is a measurable or statistically significant increase in any feature or component of the immune response following vaccination in comparison to the same feature or component prior to vaccination (with either or both of a prime or boosting vaccination).

VLPs displaying antigens and methods of preparation

Compositions of the invention or for use in the methods of the invention can comprise a particle-forming protein or capsid-forming protein linked or fused to a peptide tag and a SARS-CoV-2 antigen linked or fused to a binding partner, wherein the peptide tag and binding partner are capable of interacting (e.g., 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 included in the invention 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 tag 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, 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 SARS-CoV-2 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 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 (i.e., VLP-SARS) displaying antigens from non-coupled particle-forming protein and antigen components. The assembled VLP-SARS 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. The assembly of the components and/or success of the final preparation or formulation of the VLP-SARS vaccine can be assessed by in vivo assays of immunogenicity or by in vitro assays showing that the components have bound to each other. Exemplary methods are known in the art and also described, for example, in Fougeroux et al. ((2021) Nat. Commun. 12: 324) and WO2022229817.

In some embodiments, methods of the invention include treating the particle-forming protein and/or the antigen preparation with a nuclease and/or RNAase and/or DNAase. In such embodiments, the step of treating the particle-forming protein with the nuclease and/or RNAase and/or DNAase occurs after formation of the capsid or particle (e.g., the VLP), after assembly of the capsid or particle with the antigen so that the antigen is displayed on the particle (e.g., after assembly of the VLPs displaying antigen), and/or as part of formulating the VLP-SARS into a pharmaceutical composition for use as a vaccine.

While the invention is not bound by any particular mechanism of operation, it is thought that bacterial RNA and/or DNA, possibly enclosed within the capsid or VLP component, contributes to the immunogenicity of the VLP components and of VLP-SARS (see, e.g., Example 4 of the instant application). Also, it is believed that bacterial RNA and/or DNA contributes to capsid or VLP formation during the methods of production described herein. However, for formulation into a pharmaceutical composition to be used for administration to subjects, it is desirable that extraneous bacterial material including RNA and DNA be removed from the composition. Accordingly, provided herein are methods and compositions in which the VLP components and/or assembled VLP-SARS are treated with nuclease, RNAase, and/or DNAase to remove these superfluous bacterial products, but such treatment steps should occur after assembly of the VLP (capsid) component so that proper assembly is not disrupted.

Methods of Treatment

The vaccination regimes and booster vaccines of the invention can be used in prophylactic treatment of any subject (including animal subjects) in which they produce an immune response, including, for example, a human subject or patient. VLP-based COVID- 19 vaccines have been shown to be safe and effective (see, e.g., Prates-Syed et al. (2021) Vaccines 9:1409, although it should be noted that Prates-Syed Figure 5F incorrectly summarizes the structure of ABNCoV2). ABNCoV2, a VLP-based vaccine, is currently the subject of a Phase III clinical trial (see Trial NCT05329220 at ClinicalTrials dot gov). In 2022, data from a Phase II trial of non-adjuvanted ABNCoV2 were announced and showed that this vaccine induced a significant boost to the neutralizing antibodies against the Omicron variant in the majority of subjects with a fold increase in the same range as previously reported for the original (“Wuhan”) SARS-CoV2 variant. While the neutralizing antibody titers against Omicron were the lowest when compared to all other variants previously reported (Wuhan, Alpha, Beta and Delta), they were boosted to levels associated with a high level of protection across both dose groups, 50pg and lOOpg. These data followed the announcement of results from the ABNCoV2 Phase II trial in December 2021 and February 2022, demonstrating that a single vaccination with 50pg or lOOpg ABNCoV2 can boost neutralizing antibodies to levels reported to be highly efficacious (>90%) against SARS-CoV2, irrespective of the type of vaccine previously received (mRNA or adenovirusbased) or the initial level of neutralizing antibody titers before booster vaccination with ABNCoV2. Accordingly, methods of treatment incorporating the methods described in these clinical trials are included in the present invention.

Stimulation of Thl response

In yet another aspect, the invention provides a vaccination regime or method of vaccinating a subject comprising administering a first (“priming”) vaccination to a subject that stimulates a Thl immune response and administering a second (“booster” vaccination) comprising a VLP displaying a SARS-CoV-2 antigen (i.e., a “VLP-SARS). As demonstrated in the working examples herein, the VLP-SARS booster vaccination further stimulates the immune response elicited by the first (“priming”) vaccination but does not change the type of immune response; that is, the VLP-SARS booster vaccination does not change the immune response from Thl to Th2 or vice versa. In this manner, the invention provides compositions and methods for inducing and stimulating a Thl immune response to SARS-CoV-2 in a subject. In some embodiments, a subject is identified as having a Th2 immune response to SARS-CoV-2 and is vaccinated with a first immunization and a VLP-SARS booster to try to stimulate a Thl immune response. In some embodiments, a subject has previously been vaccinated against SARS-CoV-2 or has been infected with SARS-CoV-2 but now is seronegative for anti-SARS-CoV-2 antibodies or has experienced a decrease in such antibodies; in these embodiments, a subject can be vaccinated with a “first” or priming immunization that stimulates a Thl response (e.g., MVA-Spike) and a VLP-SARS booster that further stimulates the immune response induced by the first immunization. In such embodiments, the invention provides a method of optionally identifying a subject as having a Th2 immune response and comprising the steps of: (a) administering a priming immunization of a recombinant MVA encoding a SARS-CoV-2 antigen such as, for example, an S protein (“Spike”) or portion thereof; and (b) administering a boosting immunization of VLP-SARS (e.g., ABNCoV2).

In some embodiments, a patient is identified as being SARS-CoV-2 seronegative and/or having decreased amounts of SARS-CoV-2 antibodies and then is treated by administering a priming immunization of an MVA-Spike and a boosting immunization of VLP-SARS. In some embodiments, a patient is identified as potentially in need of stimulation of an immune response (because, for example, time has passed since they were exposed to SARS-CoV-2 and/or since they were previously vaccinated for SARS-CoV-2 and/or they are infected with SARS-CoV-2); in such embodiments, the patient is is treated by (a) administering a priming immunization of a recombinant MVA expressing a SARS-CoV-2 antigen (e.g., “MVA-Spike” and (b) a boosting immunization of a VLP-SARS (such as, for example, ABNCoV2). In some embodiments described above, the MVA-Spike expresses a modified Spike protein such as, for example, the MVA known as MVA-mBN500, described in detail in WO2021250219. In some of the embodiments above, the VLP-SARS is ABNCoV2, described in detail in WO2022229817. These prime/boost immunizations comprising MVA-Spike and a VLP-SARS induce a Thl response, for example, as measured by a statistically significant increase in IL- 12, IL-2, TNF-a, and/or IFN-y, and/or a preponderance of IgGl antibodies (in humans). Assays for measuring such cells and/or cytokines are known in the art.

In the methods of the invention with priming and boosting immunizations, these immunizations can be given several days or several weeks apart, or may be given several months apart, but generally are given no more than 2 months apart. 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.”

Methods and compositions of the invention 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 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 VLP-SARS 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 that stimulate an immune response can also be shown to 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 method or composition 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., VLP-SARS) to be administered to a subject in a vaccination or immunization 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 VLP-SARS to be administered to a subject 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 SARS- CoV-2 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 (VLP-SARS) may be used.

It will be understood that serum titers of neutralizing antibodies in a patient will increase following administration of VLP-SARS because the immune response is stimulated, but in subjects with measurable pre-existing levels of antibodies and/or neutralizing antibodies (that is, subjects who have a high baseline antibody titer), the increase in antibodies and/or neutralizing antibodies following administration of VLP-SARS may appear to be lower than the increase observed in subjects 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 VLP-SARS so long as the increase is statistically significant, even if the increase is as little as 2-fold. 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 subject comprising administering VLP-SARS to the subject, and VLP-SARS can be administered to subjects 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 subject, the serum antibody titers are measured at least or about one week after administration of the VLP-SARS or at least or about two weeks after administration of the VLP-SARS.

Some subjects 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 VLP-SARS 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 VLP-SARS produces an increase in anti-SARS-CoV-2 antibodies and/or neutralizing antibodies in a subject 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 VLP -SARS 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 or is free of adjuvant, such as, for example, Alum, 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, Alum, 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 VLP-SARS

In some embodiments, the method of producing a VLP-SARS 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 (see, e.g., Fougeroux et al. ((2021) Nat. Commun. 12: 324)). The particle-forming protein may be any of those listed herein, including, for example, a capsid protein that is AP205.

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 et al. (2012) Biotech. Adv. 30: 613-28). The AP205 component can be expressed in and prepared from E. coli cultures (see, e.g., Thrane et 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 (see, e.g., Fougeroux et al. ((2021) Nat. Commun. 12: 324)).

Assays

The following assays, in addition to the assays and methods employed in the examples herein and similar assays known in the art, are readily performed by one of skill in the art and can be used to assess or evaluate the immune response of a subject to the compositions and methods of the invention.

Antigen-specific antibodies

Serum samples are obtained pre-immunization and at appropriate intervals following immunization and tested for the presence of antibodies to the SARS-CoV-2 antigen (e.g., spike protein or RBD) using an ELISA. Stimulation of an immune response by the immunization is indicated by a statistically significant increase in antigen- specific and/or neutralizing antibodies in the subject.

Virus neutralizing antibodies

Neutralization assays can be performed as follows, but many similar assays are known in the art and readily adapted and performed by one of skill in the art. Human plasma samples from before and after immunization are heat inactivated by incubating at 56°C for 30 minutes. Two-fold serial dilutions are prepared in media (DMEM + 2% FCS + 1% Pen/Strep + L-Glutamine). Sera are mixed with SARS-CoV-2 at a final titer of 200 TCID50/90pL and incubated at 4°C overnight. Control samples included one with SARS-CoV-2 but lacking serum and another control lacking both serum and virus.

Virus/serum mixtures are then added to 2 x 10 ⁴ VeroE6-hTMPRSS2 cells seeded in flat-bottom 96-well plates 24 hours earlier, and these mixtures are then incubated for 72 hours in an incubator at 37°C in the presence of 5% CO2 and humidification. Cells are then fixed in 5% formalin and stained with crystal violet; cytopathic effect (CPE) is evaluated using a light microscope. Based on dilution curves obtained, Plaque Reduction Neutralization Titer 50 values (PRNT50 titers) are approximated using commercially available software (e.g., GraphPad).

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’ or “without” 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 an ingredient (for example, adjuvant or squalene) means that the composition of the present invention does not contain the ingredient.

"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.”

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.

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. 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, Alum (aluminum hydroxide gel, e.g., Alhydrogel® adjuvant, InvivoGen, San Diego, CA, USA), 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 a suitable 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, VLP-SARS (z.e., assembled VLPs displaying SARS-CoV-2 antigen(s)) are administered in a first vaccination and later after a certain period of time are administered in a second vaccination and/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 means that the active agent (z.e., VLPs) used in the first vaccination are the same as those used in a second or further vaccinations, and “heterologous” prime-boost means that the vaccine that is first administered to a subject is different from the vaccine that is subsequently administered to a patient, for example, in a second or boosting immunization or vaccination. In some methods of the instant invention, VLP-SARS are used as booster vaccinations to further stimulate the immune response of the subject to the antigen(s) displayed on the VLP.

Whether a vaccination is a “booster” vaccination as used herein depends on the timing and effect of the vaccination on the immune response of the subject. Generally, as used in the art, a “booster” vaccine is a second or subsequent dose of vaccine any time after a first dose of vaccine is administered to a naive subject. Because the SARS-CoV-2 pandemic has existed worldwide for more than 2 years to date, many people have already received at least one dose of a SARS-CoV-2 vaccine, yet it has been shown that the amount of circulating antibody in the blood or serum of previously vaccinated subjects decreases over time such that additional stimulation of the immune response is desirable to protect against SARS-CoV- 2 infection and/or disease. In some embodiments of the methods and compositions of the invention, by “booster” is intended that a dose of vaccine is administered in conjunction with a “priming” vaccination which is administered to a subject who may or may not have received previous SARS-CoV-2 vaccination(s) of any type, and that the increase in immune response resulting from the priming vaccination is still present when the booster is administered to the subject. In some embodiments of the methods and compositions of the invention, a subject is given a set of vaccinations comprising a first SARS-CoV-2 vaccination (referred to herein as the “prime” or “priming” vaccination) and a second vaccination that is an VLP-SARS vaccine (referred to herein as the “boost” or “booster” vaccination) while the subject’s immune response is still increased as a result of the prime vaccination, in comparison to the immune response prior to the prime vaccination (e.g., as indicated by the titer of neutralizing antibodies in the serum of the subject). Generally, in the methods of the invention, the first and second (i.e., “prime” and “boost”) vaccinations are given several weeks apart, for example, two weeks apart, at least 2 weeks apart, between 2-3 weeks apart, at least 3 weeks apart, between 3-4 weeks apart, at least 4 weeks apart, between 4-5 weeks apart, at least 5 weeks apart, 5-6 weeks apart, or at least 6 weeks apart, but no more than 7, 8, or 9 weeks apart.

In some embodiments of the invention, a VLP-SARS vaccine that has been prepared with nuclease, DNAase, and/or RNAase as described elsewhere herein is administered as a booster when it is administered to a subject who has previously received a SARS-CoV-2 vaccine at any time, even if it is more than 2 months, more than 3 months, or more than a year prior to the administration of the nuclease-treated booster. These embodiments are suitable for administration to any patient for whom an increase in neutralizing antibody titer is desirable regardless of vaccination or infection history.

By “increased immune response” or “stimulating immune response” is intended that a statistically significant increase in neutralizing antibodies is observed in a subject following vaccination; preferably antibodies are measured around one or two weeks after immunization (with either priming or booster immunization, as appropriate) and preferably measurements are made with the SARS-CoV-2 strain or strains corresponding to the antigen or antigen sequence used in the vaccine.

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.), 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 al., 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 1 is a virus-like particle (VLP) displaying a receptor binding domain (RBD) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) for use in the prevention or preventive amelioration of symptoms of coronavirus disease 19 (COVID-19), wherein the VLP displaying the RBD (VLP-SARS) is used to boost an immune response previously induced with a recombinant Modified Vaccinia Virus Ankara encoding a SARS-CoV-2 spike protein (MVA-Spike) or with a SARS-CoV-2 spike protein (Spike protein).

Item 2 is the VLP-SARS for use of item 1, comprising an AP205 protein fused to a peptide tag.

Item 3 is the VLP-SARS for use of item 1 or 2, wherein said VLP-SARS is ABN- CoV-2.

Item 4 is a method of preparing a pharmaceutical composition, comprising the step of treating a VLP-SARS preparation with a nuclease, RNAase, or DNAase, preferably Benzonase®, after the VLP component is assembled.

Item 5 is the method of item 4, wherein the VLP component is not treated with a nuclease, RNAase, or DNAase, prior to assembly of the VLP component.

Item 6 is a pharmaceutical composition prepared according to the method of item 4 or 5.

Item 7 is a pharmaceutical composition comprising a VLP component that was treated with a nuclease, RNAase, or DNAase following capsid assembly. Item 8 is the pharmaceutical composition of item 7, wherein said VLP component comprises an AP205 protein fused to a peptide tag.

Item 9 is the pharmaceutical composition of item 7 or 8, wherein the VLP component is a part of ABN-CoV-2.

Item 10 is a pharmaceutical composition of anyone of items 6 to 9 for use in the prevention or preventive amelioration of symptoms of COVID-19, wherein the pharmaceutical composition is used to booster an immune response previously induced with an MVA-Spike or a Spike protein.

Item 11 is a VLP-SARS for use of anyone of items 1 to 3, or a pharmaceutical composition for use of claim 10, wherein the immune response is boosted from 6 to 10, preferably from 7 to 9, more preferably 8 weeks after the immune response has been induced.

Item 12 is the VLP-SARS for use of item 11, or the pharmaceutical composition for use of item 11, wherein the immune response previously induced is a Th 1 immune response.

Item 13 is the VLP-SARS for use of item 11 or 12, or the pharmaceutical composition for use of item 11 or 12, wherein the immune response boosted by the VLP-SARS is also a Thl immune response.

Item 14 is the VLP-SARS for use of anyone of items 11 to 13, or the pharmaceutical composition for use of anyone of items 11 to 13, wherein the boosted immune response induces RBD neutralizing antibodies.

Item 15 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 (VLP-SARS), that has been treated following assembly of the VLP with a nuclease, DNAase, and/or RNAase so that superfluous bacterial nucleic acid material remaining after the production process has been removed from the VLP and/or VLP-SARS.

Item 16 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; that has been treated during preparation with a nuclease, DNAase, and/or RNAase, 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.l.1.7, B.1.351, B l.617.2, and/or omicron. In some embodiments, this immune response is a measurable increase in anti-SARS-CoV-2 antibodies and/or neutralizing antibodies in the subject following immunization, for example one or two weeks following immunization.

Item 17 is a pharmaceutical composition, or a vaccine, comprising a VLP-SARS, optionally further comprising a pharmaceutically acceptable carrier or excipient.

Item 18 is the pharmaceutical composition of item 17, or the vaccine of item 17, that does not contain an adjuvant.

Various headings and subsections are used and delineated throughout this application; such headings and organization are intended to assist the reader but are not to be construed as limiting the meaning of a portion of the description or limiting the ability of different sections to modify each other.

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 VLP-SARS vaccines

Design, expression, and purification of recombinant protein.

VLP-SARS were constructed essentially as described in Fougeroux et al. ((2021) Nat. Commun. 12: 324). Briefly, the RBD coding region of the SARS-CoV-2 sequence was genetically fused with a 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 (0.1m2, 10 kDa MWCO, PAEE) mounted in a SIUS-ES filter holder atop a SIUS-ES 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. 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 E. coli cells (New England Biolabas) according to manufacturer’s protocols and purified as described below for the VLP vaccines.

Formulation and purification of the VLP-SARS vaccines.

Tag-VLP and RBD antigen were mixed in a 1:1 molar ratio in IxPBS, 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 complexes of RBD and VLP. The RBD-Catcher (or

“binding partner”) coupling efficiency was calculated as percentage conjugation (z.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 antigen- VLP (referred to generally herein as “VLP-SARS”) was purified by dialysis (cutoff 1000 kDa) in a IxPBS with 5% (v/v) glycerol for immunization studies or 400 mM xylitol for quality assessment.

Quality assessment

Purified VLP-SARS as described above were 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 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 particle population and the percent polydispersity (%Pd) was calculated by Wyatt DYNAMICS software (v7.10.0.21, US).

Example 2: VLP-SARS comprising the RBD is an efficient booster of induced antigenbinding antibody responses

A VLP-SARS comprising an RBD as described above was previously evaluated for its ability to induce an immune response in non-human primates (NHPs) (rhesus macaques) and for its ability to protect vaccinated NHPs from infection with and/or symptoms produced by SARS-CoV-2. The VLPs used in these experiments comprised: (1) the RBD of the SARS-CoV-2 spike protein genetically fused at the N-terminus to a binding partner; and (2) a peptide tag 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).

This VLP-SARS (“ABNCoV2”) was further explored for use in combination with other SARS-CoV-2 vaccines to produce an immune response to SARS-CoV-2. Specifically, exemplary vaccines used in these experiments were “MVA-Spike” and “Spike protein.” “MVA-Spike” is a recombinant modified vaccinia virus Ankara also known as MVA- mBN500 and encoding a modified, prefusion stabilized SARS-CoV-2 full length protein (described in detail in WO2021250219). “Spike protein” is the full-length, wild-type S protein of SARS-CoV-2.

In these experiments, Balb/c mice were prime/boost immunized on days 0 and 21; mice were primed with 5 pg ABNCoV2, IxlO ⁸ TCID50 MVA-Spike or 5 pg Spike protein + AddaVax™ adjuvant (InvivoGen, San Diego, CA, USA) and boosted on day 21 with 5 pg ABNCoV2. In addition, two groups were prime immunized with IxlO ⁸ TCID50 MVA-Spike or 5 pg Spike protein + AddaVax™ adjuvant and boosted on day 21 with the same vaccine candidates. Blood for serum isolation was taken 14 days after boost immunization.

ELISA and ePass (Genscript) was used to assess total RBD-specific IgG titers and RBD-binding antibody titers, respectively. For determination of RBD-binding antibody titers, sera are diluted in 8 (eight) 1:3 dilution steps with a starting dilution of 1:100. Diluted samples are pre-incubated with HRP-labelled RBD before incubation on a ACE2-coated plate. Titers are calculated at 50% RBD binding inhibition compared to negative control. Data from samples taken on day 34 are shown in Figure 2 as Mean ± SEM.

These experiments demonstrated that the RBD-VLP “ABNCoV2” induced high RBD-binding antibody levels as assessed by RBD-binding inhibition rate, even without additional adjuvant. Spike protein + AdVx induced intermediate RBD-binding antibody levels and the lowest RBD inhibition rate was detected in mice immunized only with MVA- Spike. Most importantly, both MVA-Spike and Spike + AdVx-induced RBD-binding antibodies were boosted by ABNCoV2, as seen by increasing RBD-binding antibody levels.

Example 3: RBD-VLP boost immunization does not change the anti-RBD IgG subclasses induced by prime immunization

Mice were immunized as described in Example 2. ELISA was performed on RBD- coated plates with a starting dilution of 1:300 to determine anti-RBD IgGl or IgG2 titers using appropriate anti-Mouse IgG antibodies. Arbitrary titers were calculated via a 4PL-fit curve with an intercept at OD = 0.3. Data are shown in Figure 3 as Mean ± SEM.

The type of IgG subclasses produced in response to infection or vaccines are driven by the initial immune response, especially by cytokines, that lead to a class-switch in activated B cells. For example, IL-4 produced by type 2 T helper cells (Th2) in response to protein antigens can promote antibodies of the IgGl class (in mice). Antiviral Thl responses can cause elevated IFNg levels that may lead to increased switch of B cells to produce IgG2a in mice (McHeyzer-Williams et al. (2005) Ann. Rev. Immunol. 23: 487-513).

To analyze the type of IgG response induced or boosted by ABNCoV2, treatments were designed to elicit a Thl response (MVA-Spike coding for SARS-CoV2 Spike protein) or a Th2 response (Spike protein + AddaVax™ adjuvant). Balb/c mice were prime/boost immunized on days 0 and 21 with 5 pg ABNCoV2, IxlO ⁶ TCID50 MVA-Spike or 5 pg Spike protein + AddaVax™ and boosted on day 21 with 5 pg ABNCoV2. In addition, two groups were prime immunized with IxlO ⁶ TCID50 MVA-Spike or 5 pg Spike protein + Alum (Alhydrogel® adjuvant, InvivoGen, San Diego, CA, USA) and boosted on day 21 with 5 pg ABNCoV2. Serum from all groups was harvested at final day 35 and was analyzed by ELISA for RBD binding immunoglobulin (Ig) IgG subclasses IgGl and IgG2a (see Figure 3). Homologous vaccination with MVA-Spike almost exclusively induced anti-RBD IgG2a antibodies while Spike + AddaVax™ adjuvant exclusively drove IgGl production. Interestingly, mice prime immunized and boosted with ABNCoV2 exhibited a balanced Thl/Th2 response with high titers of both IgGl and IgG2a RBD binding antibodies. Importantly, ABNCoV2 did not alter the type of preexisting humoral immune response primed by either MVA-Spike or Spike + AdVx but rather boosted the preexisting type of antibody response.

Example 4: RBD-binding neutralizing antibodies induced by RBD-VLP immunization are severely reduced in TLR7-KO mice

C57BL/6 and knockout mice were prime immunized with 5 pg ABNCoV2, IxlO ⁶ TCID50 MVA-Spike or 5 pg Spike protein + Alum (Alhydrogel® adjuvant, InvivoGen, San Diego, CA, USA) and boosted on day 21 with 5 pg ABNCoV2. Blood for serum isolation was taken 14 days after boost immunization. For determination of RBD-binding antibody titers, sera were diluted in 8 (eight) 1:3 dilution steps with a starting dilution of 1:100. Diluted samples were pre-incubated with HRP-labelled RBD before incubation on a ACE2- coated plate. Titers were calculated at 50 % RBD binding inhibition compared to negative control. Data are shown in Figure 4 as Mean ± SEM.

In these experiments, mice were prime immunized at day 0 with ABNCoV2 and different other vaccines against SARS-CoV-2. MVA-Spike encoding Spike and Spike protein + Alum were used as controls for vaccines that should not depend on TLR7 signaling. Predictions for effects of the MyD88 knockout (“KO”) were uncertain because while it has been reported that MVA interacts with cells via TLR9 (see, e.g., Samuelsson et al. (2008) J. Clin. Invest. 118: 1776-84), it has also been reported that responses to MVA can be TLR- independent (Waibler et al. (2007) J. Virol. 81: 12102-10).

Heterologous combinations of various vaccines with ABNCoV2 were used to analyze the effect of a knockout of TLR7 (“TLR7-KO”) on the potency of ABNCoV2 as a booster vaccine. Serum was isolated from the blood of all mice on day 35 after immunization. RBD- binding (=neutralizing) antibodies were measured by ePass assay (Genscript). Prime and boost immunizations with ABNCoV2 induced the highest amounts of RBD-binding neutralizing antibody titers (Figure 4). Importantly, TLR7 deficiency and MyD88 deficiency greatly decreased neutralizing antibody titers for this vaccine. Moreover, in heterologous combinations of ABNCoV2 with MVA-Spike or Spike protein + Alum, a decrease of RBD- binding antibodies in TLR7-K0 and MyD88-KO mice was noticed. Knockout of most TLRs in the MyD88-KO model did not lead to any further decreases in neutralizing antibodies compared to TLR7-K0 mice.

To determine total anti-RBD Ig antibody titers, ELISA was performed on the day 35 serum samples using RBD-coated plates with a starting dilution of 1:300. Arbitrary titers were calculated via a 4PL-fit curve with an intercept at OD = 0.3. Data are shown in Figure 5 as Mean ± SEM. Total anti-RBD IgG levels showed very similar trends to the neutralizing antibodies. Again, a sharp drop of anti-RBD titers was demonstrated in TLR7-KO mice and MyD88-KO mice immunized with ABNCoV2. The same observation was true in mice immunized with combinations of ABNCoV2 with MVA-Spike and Spike protein + Alum.

Example 5: ABNCoV2 induces long-lasting RBD-specific antibody responses after homologous prime/boost immunization and as a booster

Balb/c mice were prime immunized on day 0 with 5 pg ABNCoV2 or IxlO ⁸ TCID50 MVA-mBN500. On day 21 they were boost immunized either with the same vaccine (homologous immunization groups) or with ABNCoV2 (heterologous immunization group). For the boost immunizations, the dose of the vaccines was the same as prime immunizations. As a control group, mice received TBS via IM injection. Serum was harvested on days 41, 62, 90, 120, 181, 240 and 360. ELISA was performed on RBD-coated plates using serum samples with a starting dilution of either 1:100 or 1:300, to determine RBD binding immunoglobulin (anti-RBD Ig) titers. Arbitrary titers were calculated via a 4PL-fit curve with an intercept at OD = 0.3. Data are indicated in Figure 6 as Mean ± SEM.

These results showed that prime/boost ABNCoV2 immunization and ABNCoV2 prime / MVA-mBN500 boost immunization induced comparable RBD specific antibody titers, which was higher than MVA-mBN500 prime/boost immunization. A decrease in antibody titers in ABNCoV2 boost immunized animals was observed between day 41 and day 90. Thereafter antibody titers seemed to remain stable and long-lasting, demonstrating the benefit of ABNCoV2 as a stand-alone vaccine as well as a booster.