Virus-like particles for the treatment or prevention of infections caused by coronaviruses - Patents.com

Abstract

The present invention relates to novel virus-like particles (VLPs), pharmaceutical compositions comprising said virus-like particles (VLPs) and methods of using said virus-like particles (VLPs) to prevent or treat infections caused by Coronaviridae viruses. Advantageously, these VLPs can be used as vaccines administered orally or nasally.

Description

[Technical field]

[0001] The present invention relates to novel virus-like particles (VLPs), pharmaceutical compositions comprising said virus-like particles (VLPs) and methods of using said virus-like particles (VLPs) to prevent or treat infections caused by Coronaviridae viruses. Advantageously, these VLPs can be used as vaccines administered orally or nasally. [Background technology]

[0002] Prior to 2019, only six coronaviruses were known to cause disease in humans—human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1) and Middle East respiratory syndrome coronavirus (MERS-CoV).

[0003] In 2019, coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged as a pandemic, claiming more than 3.9 million lives worldwide with approximately 180 million confirmed cases between December 2019 and June 2021. SARS-CoV-2 is a betacoronavirus that has a propensity to cause severe disease in humans, which is the reason for the huge and fast-spreading outbreaks of respiratory infections, including pneumonia, which can be fatal.

[0004] Several different vaccines against COVID-19 were developed in a short time and became available in early 2021. However, due to the rapid emergence of many variants of SARS-CoV-2 worldwide, there is still a need to improve the quality and durability of the immune response induced in the vaccinated population. In particular, there is still a need for methods to generate robust immune responses, such as efficient cellular and / or humoral immune responses, especially mucosal responses.

[0005] Moreover, all vaccines against COVID-19 available today are delivered by injection, raising safety, patient acceptability and morbidity issues, increasing the cost and reducing safety of mass vaccination, especially in resource-poor developing countries. Therefore, there is still a real need for simple methods of vaccination, especially by oral administration. Indeed, oral delivery offers many notable advantages (e.g., simple administration and improved safety) over other routes of administration, especially compared to parenteral vaccines. However, even though the oral route is an ideal means of delivering vaccines, it is also the most challenging due to the numerous barriers posed by the digestive tract. To allow effective immunization with protein vaccines, antigens must be protected, their uptake enhanced, and the immune response activated. Recently, we have shown that orally administered bioengineered virus-like particles (VLPs) displaying model surface antigens such as parasite variable surface proteins (VSPs) and influenza virus hemagglutinin and neuraminidase can generate immune responses that protect mice from influenza infection (Serradell et al., Nat. Commun. 10:361 (2019)). However, to date, no oral vaccine against COVID-19 is available. Summary of the Invention

[0006] The present invention is believed to meet the above needs by providing novel virus-like particles (VLPs) that display on their surface a protozoan variable surface protein (VSP) and a polypeptide antigen of a Coronaviridae virus.

[0007] Unexpectedly, because each VSP / envelope protein combination has unique structural peculiarities that may affect their performance as immunogens, the inventors have shown that such VLPs can be used to induce robust immune responses or to boost previously induced immune responses against Coronaviridae viruses via mucosal routes such as oral or nasal administration. Moreover, and surprisingly, the inventors have shown that this approach can induce mucosal immunity, which is particularly useful for controlling SARS-CoV-2 infection and spread.

[0008] Thus, in one aspect, the present invention provides a virus-like particle (VLP), in particular a retroviral derived virus-like particle, comprising at least: - a variable surface protein (VSP), a VSP-like protein or a fragment thereof of a microorganism selected from the group consisting of species of the genera Giardia, Tetrahymena, Paramecium and Entamoeba, -Viral proteins or fragments of viruses of the Coronaviridae family The present invention relates to virus-like particles (VLPs) that display a vector on their surface, in particular virus-like particles derived from retroviruses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] As used herein, the term "virus-like particle" (VLP) refers to a structure resembling a virus particle that mimics the three-dimensional structure of a virus. A virus-like particle according to the present invention is non-replicative since it lacks all or part of the viral genome, typically and preferably lacks all or part of the replicative and infectious components of the viral genome. The term "non-replicative" as used herein refers to the inability to replicate the genome contained in the VLP or not contained in the VLP. VLPs are generally multiprotein structures composed of one or more viral proteins. VLPs can form (or "self-assemble") upon expression of viral structural proteins under appropriate circumstances. VLPs overcome some of the shortcomings of vaccines prepared using attenuated viruses, since they can be produced without the need for live virus to be present during the production process. A wide variety of VLPs can be prepared. For example, VLPs can be produced that contain single or multiple capsid proteins, either with or without envelope proteins and / or surface glycoproteins. In some cases, VLPs are not enveloped and are assembled by expression of only one major capsid protein. In other cases, VLPs are enveloped and can contain multiple antigenic proteins found in the corresponding native virus. VLPs are typically similar to their native virus counterparts and can display multivalent antigens.

[0010] The presentation of surface glycoproteins in the context of VLPs is advantageous for the induction of neutralizing antibodies against antigenic proteins, such as viral proteins of the Coronaviridae viruses, compared to other forms of antigen presentation, such as soluble antigens not associated with VLPs. Neutralizing antibodies recognize most often tertiary or quaternary structures, which often require the presentation of antigenic proteins, such as envelope glycoproteins, in their native viral conformation. The proper presentation of envelope glycoproteins on the surface of VLPs is largely unpredictable. Some envelope glycoproteins may require their manipulation for better expression, such as exchanging their membrane and / or cytoplasmic tails with those of another viral protein, for example, vesicular stomatitis virus G glycoprotein (VSV-G). However, if the envelope is expressed as a multimer in wild-type viruses, this may affect the structure of the protein, especially its quaternary structure. Furthermore, co-expression of VSPs and envelope glycoproteins on the same membrane can result in interactions that modify the structure of the envelope glycoprotein and thus its ability to trigger neutralizing antibodies (Nabs).

[0011] As used herein, the term "variable surface protein", "VSP protein" or "VSP" refers to a polypeptide that covers the entire surface of the Giardia parasite and is the major antigen recognized by the host immune system. VSP proteins are cysteine-rich proteins with multiple "CXXC" motifs (X is any amino acid) with several specific features including the presence of CXC motifs, Giardia-specific zinc finger motifs, and GGCY motifs in some VSPs (Nash, Mol. Microbiol. 45:585-590 (2002); Adam et al., BMC Genomics 10:424 (2010)). More precisely, VSP proteins are type 1 integral membrane proteins that vary in size from 20 to 200 kDa, with a variable amino-terminal cysteine-rich region (the extracellular domain that corresponds to the host / parasite interface and confers resistance to proteolytic digestion and low pH to the protein) and a conserved carboxy-terminal region that contains a hydrophobic transmembrane region and a short cytosolic tail (CRGKA) that contains only 5 amino acids that are not detected by the immune system. Only one VSP protein is expressed on the surface of each parasite at any given time (Nash, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 352:1369-1375 (1997)). Giardia VSPs, more specifically the extracellular domain of Giardia VSPs, contain multiple CXXC motifs separated by several amino acids, preferably 3-20 amino acids, more specifically 5-8 amino acids. Thus, in some embodiments, the VSP is a fragment, analog or derivative of a VSP or VSP-like protein from Giardia, and the VSP comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 CXXC motifs.In some embodiments, the VSP comprises at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, about 90, or at least about 100 CXXC motifs from a Giardia VSP.

[0012] Within the context of the present invention, the terms "variable surface protein", "VSP protein" or "VSP proteins" include any Giardia VSP protein, in particular any VSP of the complete repertoire of Giardia lamblia. In fact, Giardia parasites encode a repertoire of about 200 genes encoding VSPs of assemblage A (see, for example, Morrison et al., Science 317:1921-1926 (2010); Adam et al., BMC Genomics 10:424 (2010)), and two reports from Svard's group described the VSP repertoires of isolates from VSP assemblages B and E (Jerlstrom-Hultqvist et al., BMC Genomics 11:543 (2010); Franzen et al., PLoS Pathog. 5(8):cl000560 (2009)). The extracellular domains of VSPs allow the parasite to survive the harsh environment of the upper small intestine. VSPs are highly resistant to variable pH (reactivity to conformational epitopes by monoclonal antibodies raised against specific VSPs is invariant at pH 2-12) and to digestion by trypsin and several other proteases. Moreover, VSPs remain attached to the intestinal mucosa (Rivero et al., Nat. Med. 16(5):551-7(2010)).

[0013] It should be further noted that polypeptides containing at least one CXXC motif (C represents a cysteine ​​residue and X represents any amino acid residue), such as Giardia VSP or other microbial VSP-like proteins, can also be generated in vitro by genetic engineering and produced in heterologous systems. Thus, chemically or cell-produced polypeptides are encompassed, including polypeptides with amino acid mutations not found in wild-type parasites (e.g., variants of Giardia VSP). Thus, VSPs can be prepared by any procedure well known in the art, such as solid-phase synthesis, liquid-phase synthesis, or genetic engineering.

[0014] The VSPs used in the present invention can be chemically modified. Chemical modifications can be aimed at obtaining VSPs with increased protection against enzymatic degradation in vivo and / or increased ability to cross membrane barriers, thereby increasing the half-life of the VSPs and maintaining or improving the biological activity of the VSPs. Any chemical modification known in the art can be used in accordance with the present invention to modify the VSPs. Such chemical modifications include, but are not limited to, the following: (a) modifications to the N-terminus and / or C-terminus of the VSP protein, such as, for example, N-terminal acylation (preferably acetylation) or deamination, or modification of the C-terminal carboxyl group to an amide or alcohol group; (b) modifications at the amide bond between two amino acids: acylation (preferably acetylation) or alkylation (preferably methylation) at the nitrogen atom or alpha carbon of the amide bond linking the two amino acids; (c) A modification at the alpha carbon of the amide bond linking two amino acids, such as, for example, acylation (preferably acetylation) or alkylation (preferably methylation) at the alpha carbon of the amide bond linking two amino acids. (d) a change in chirality, for example, by replacing one or more naturally occurring amino acids (L-enantiomers) with the corresponding D-enantiomers; (e) retroinversion, in which one or more naturally occurring amino acids (L-enantiomers) are replaced by the corresponding D-enantiomers, together with an inversion of the amino acid chain (from C-terminus to N-terminus); and / or (f) Azapeptides in which one or more alpha carbons are replaced by nitrogen atoms.

[0015] The term "variable surface protein", "VSP protein" or "VSP" as used herein includes "VSP" proteins from Giardia, homologs (e.g., orthologs and paralogs) of VSPs and VSP-like proteins found in other organisms, as well as fragments, variants and derivatives thereof. The term "homolog" as used in reference to a VSP protein or VSP-encoding gene of a first family or species refers to a separate VSP protein or VSP-encoding gene of a second family or species that is determined by functional, structural or genomic analysis to be a VSP protein or VSP-encoding gene of the second family or species that corresponds to the original VSP protein or VSP-encoding gene of the first family or species. As used herein, the term "homolog" refers to any VSP protein or VSP-encoding gene that is related to a reference VSP protein or VSP-encoding gene inherited from a common ancestral DNA sequence. The term homolog includes both orthologs and paralogs. The term "ortholog" refers to VSP homologs of different species that have evolved from a common ancestral gene by speciation. Typically, orthologs retain the same or similar function despite differences (mutations) in their primary structure. The term "paralog" refers to VSP homologs within the same species that evolved by genetic duplication of a common ancestral gene. In many cases, paralogs exhibit related (but not necessarily identical) functions. To the extent that a particular species has evolved multiple related genes from an ancestral DNA sequence shared with another species, the term ortholog can encompass the term paralog. In most cases, homologs have functional, structural or genomic similarity. Techniques are known that allow easy cloning of enzyme or gene homologs using gene probes and PCs. The identity of the cloned sequence as a homolog can be confirmed using functional assays and / or by genomic mapping of the gene.

[0016] A "VSP-like protein" or a fragment thereof may be selected from among polypeptides derived from microorganisms other than Giardia that share sequence homology and biochemical properties with Giardia, in particular polypeptides containing multiple CXXC motifs, preferably multiple CXXC motifs separated by 5-8 amino acids. Indeed, by alignment of the extracellular domain (query) of a Giardia VSP sequence with other VSP-like molecule sequences, the presence of multiple CXXC motifs, especially separated by 5-8 amino acids, was observed in proteins belonging to species of Paramecium, Tetrahymena and Entamoeba. Thus, representative fragments of the primary sequences of the Entamoeba species surface kinase and the Paramecium and Tetrahymena species surface proteins predict a conserved domain containing a CXXC motif in a VSP-like structure (compared to the Giardia VSP responsible for resistance to pH, temperature and proteolytic digestion).

[0017] In the present invention, the terms "Giardia", "Tetrahymena", "Paramecium" and "Entamoeba" refer to genera of microorganisms, each of which includes various species that appear to infect different hosts. For example, the genus Giardia includes, but is not limited to, Giardia lamblia (also known as Giardia intestinalis or Giardia duodenalis), Giardia enterica, Giardia muris, Giardia simondi, Giardia bovis, Giardia canis, Giardia cati, and Giardia microti.

[0018] In one embodiment, the present invention relates to a VLP as defined above, wherein the microorganism is selected from the group consisting of Giardia lamblia, Tetrahymena thermophila, Paramecium tetraurelia and Entamoeba histolytica.

[0019] In one embodiment, the microorganism is Giardia lamblia. In another embodiment, the microorganism is Tetrahymena thermophila. In another embodiment, the microorganism is Entamoeba histolytica. In another embodiment, the microorganism is Paramecium tetraurelia.

[0020] As used herein, the term "fragment of a protein" generally encompasses any polypeptide comprising at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 consecutive or non-consecutive amino acids of any specified protein or polypeptide, regardless of whether the term refers to a VSP or a viral protein. When the term "fragment of a protein" is applied to an antigen according to the present invention, such a fragment should be capable of inducing the production of antibodies or the stimulation of T cells in vivo. Thus, a fragment of a protein should comprise at least one immunogenic epitope. When the term "fragment of a protein" is applied in accordance with the present invention to VSPs or VSP-like proteins of other microorganisms, such fragments should be able to retain the ability to attach to cells, in particular mucosal cells, more particularly intestinal epithelial cells, and ultimately to induce an immune response by themselves.

[0021] In a preferred embodiment, the VSP is the extracellular domain of Giardia VSP or a fragment thereof (as the extracellular domain is the amino-terminal cysteine-rich region containing multiple CXXC motifs of Giardia VSP protein). In fact, the extracellular domain of Giardia VSP is a domain that is resistant to pH, temperature and proteolytic digestion. Thus, in another preferred embodiment, the polypeptide according to the present invention comprises only the extracellular domain of Giardia VSP or a fragment thereof. Thus, in such an embodiment, the transmembrane region and cytoplasmic tail of Giardia VSP are excluded. It should be noted that the peptide signal may also be removed.

[0022] In one embodiment, the present invention relates to a VLP as defined above, wherein the VSP, VSP-like protein or fragment thereof is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ 9, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO 4, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, 99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123 and SEQ ID NO:124.

[0023] In a particular embodiment, the present invention relates to a VLP as defined above, wherein the VSP, VSP-like protein or fragment thereof comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0024] In the present invention, the VSP, VSP-like protein or fragment thereof is exposed on the surface of the VLP. In this regard, the VSP, VSP-like protein or fragment thereof may be exposed through binding to various structures such as an envelope protein or a fragment thereof, a synthetic linker, or through a chemical or enzymatic reaction involving an antibody.

[0025] In one embodiment, the VLP displays an envelope protein comprising at least a portion of the transmembrane domain of the envelope protein fused to a VSP, a VSP-like protein or a fragment thereof.

[0026] In one embodiment, the VLP displays a viral envelope protein, such as an envelope protein from vesicular stomatitis virus (VSV), or a fragment thereof (e.g., the transmembrane (TM) region and / or the cytoplasmic tail (CT) of the VSV G glycoprotein), fused to a VSP, VSP-like protein, or a fragment thereof exposed on the surface of the VLP, e.g., by genetic or chemical fusion with a VSV envelope protein or a fragment thereof.

[0027] In one embodiment, the present invention relates to a VLP as defined above, in which a VSP, a VSP-like protein or a fragment thereof, preferably the extracellular domain of a VSP, is fused to the transmembrane domain of the VSV-G glycoprotein.

[0028] In one embodiment, the VSP, VSP-like protein or fragment thereof comprises or consists of SEQ ID NO:125. [Table 2]

[0029] In one embodiment, the present invention relates to a VLP as defined above, in which a viral protein or a fragment thereof of a virus of the Coronaviridae family is fused to the transmembrane domain of the VSV-G glycoprotein.

[0030] In another particular embodiment, the envelope may be functionalized, which allows any selected molecule of interest to be bound to the envelope through covalent or non-covalent interactions.The functionalized envelope may include a linker, which allows any selected molecule of interest to be (specifically) bound.As an example, the envelope may include an avidin or biotin moiety, which allows the specific binding of the molecule to the avidin or biotin moiety.

[0031] Expression of the capsid protein Gag in eukaryotic cells is sufficient to generate genome-free retroviral particles. In one embodiment, the VLP comprises a retroviral or lentiviral Gag protein, such as a murine leukemia virus (MLV) or HIV Gag protein, or a fragment thereof, and even more preferably a modified retroviral Gag protein.

[0032] In the present invention, the VLP displays (ie, comprises on the outer surface of the VLP) a viral protein or fragment thereof from a Coronaviridae virus.

[0033] Indeed, the VLPs of the present invention facilitate the correct delivery of antigenic proteins from Coronaviridae viruses into the mucosa without extensive degradation in the gastrointestinal tract. The viral proteins (or fragments thereof) are used as antigens to induce immune responses against Coronaviridae viruses. Coronaviridae viruses include the subfamily Letovirinae and the subfamily Orthocronavirinae. The subfamily Letovirinae includes the alpaletovirus. The subfamily Orthocronavirinae includes the alphacoronavirus, the betacoronavirus, the deltacoronavirus and the gammacoronavirus. For example, the coronavirus may be selected from the group consisting of severe acute respiratory syndrome (SARS) coronavirus, including SARS-CoV and SARS-CoV-2 (also known as COVID-19), Middle East Respiratory Syndrome-related (MERS) coronavirus, human coronavirus OC43 (HCoV-OC43), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), and variants thereof.

[0034] In one embodiment, the Coronaviridae virus is a coronavirus, preferably selected from the group consisting of SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1 and variants thereof.

[0035] In the most preferred embodiment, the coronavirus is SARS-CoV-2 or a mutant thereof.

[0036] SARS-CoV-2 has many mutations. Most changes have little or no effect on the properties of the virus. However, some changes may affect the properties of the virus, such as how easily the virus spreads, the severity of associated disease, or the performance of therapeutics, diagnostic tools, or other public health and social measures.

[0037] In one embodiment, the variant of SARS-CoV-2 is selected from the group including alpha, beta, gamma, delta, epsilon, zeta, eta, theta, iota and kappa variants named by the World Health Organization (WHO).

[0038] In one embodiment, the present invention relates to a VLP as defined above, wherein the viral proteins are the structural proteins of SARS-CoV-2: spike (S) protein, membrane (M) protein, envelope (E) protein and nucleocapsid (N) protein.

[0039] The SARS-CoV-2 spike glycoprotein (or S protein) mediates viral attachment to the host cell surface receptor ACE2 and facilitates viral entry by assisting in fusion between the viral and host cell membranes. It is the most exposed and immunogenic viral protein. The SARS-CoV-2 spike glycoprotein is a trimeric macromolecule with two furin-like protease cleavage sites. One of these sites is at the interface between the S1 and S2 subunits, which has polybasic residues characteristic of SARS-CoV-2. The other cleavage site is located within the S2 subunit.

[0040] The SARS-CoV-2 membrane glycoprotein (or M protein) is conserved across β-coronaviruses. The SARS-CoV-2 membrane glycoprotein (or M protein) has a triple transmembrane domain and facilitates the installation of the spike.

[0041] The SARS-CoV-2 envelope protein (or E protein) is conserved across β-coronaviruses. The SARS-CoV-2 envelope protein (or E protein) is a small integral membrane protein that pentamerizes to form ion pores across the membrane, called viroporins. The SARS-CoV-2 envelope protein (or E protein) is essential for virus assembly and release.

[0042] The SARS-CoV-2 nucleocapsid protein (or N protein) is expressed during the early stages of infection and is the most abundant protein. The SARS-CoV-2 nucleocapsid protein (or N protein) forms the ribonucleoprotein core by binding to the viral RNA. The SARS-CoV-2 nucleocapsid protein (or N protein) helps the RNA enter the cell and interact with cellular components.

[0043] In one embodiment, the invention relates to a VLP as defined above, wherein the viral protein is selected from the group consisting of the S protein, the M protein, the E protein and the N protein, preferably the S protein.

[0044] In a non-limiting example, the present invention provides a method for producing a composition comprising: -S protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 131, - the M protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 132, and -N protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 133 Regarding the VLP defined above. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0045] In one embodiment, the VLP of the invention comprises: - Giardia lamblia VSPs, VSP-like proteins or fragments thereof, and -SARS-CoV-2 S protein or fragments thereof is displayed on its surface.

[0046] In one embodiment, the present invention relates to a VLP as defined above, in which a viral protein of a Coronaviridae virus is glycosylated.

[0047] In one aspect, the invention relates to a VLP as defined above, wherein the viral protein of a Coronaviridae virus is the S protein of SARS-CoV-2, said S protein being glycosylated.

[0048] Modulation of glycosylation is often exploited by pathogens for immune evasion. Masking epitopes on protein immunogens with "self" glycans can also shield the underlying protein surface from humoral immune surveillance. Applying glycosylation to viral antigens is an efficient strategy to improve the elicitation of broadly neutralizing antibodies that incorporate glycans in the antibody's binding epitope.

[0049] In one embodiment, the present invention relates to a VLP as defined above, in which a viral protein of the Coronaviridae family is stabilized in a prefusion conformation.

[0050] In one aspect, the invention relates to a VLP as defined above, wherein the viral protein of a Coronaviridae virus is the S protein of SARS-CoV-2, said S protein being stabilized in a prefusion conformation.

[0051] Viral proteins in prefusion conformation are in a conformational state before the viral envelope fuses with the membrane of the cell that the virus infects. Prefusion stabilized viral proteins are often better immunogens than their wild-type counterparts. In addition, prefusion stabilization tends to increase the recombinant expression of viral fusion glycoproteins, possibly by preventing misfolding resulting from the tendency to adopt a more stable postfusion conformation.

[0052] In one embodiment, the VLP of the invention displays at least two different viral proteins on its surface, preferably the S and M proteins.

[0053] In one embodiment, the VLP of the invention displays at least three different viral proteins on its surface, preferably the S, M and N proteins.

[0054] In one embodiment, the VLP of the invention further comprises a Gag protein fused at its carboxy terminus to a further antigen from a Coronaviridae virus. In one embodiment, the VLP of the invention further displays an N protein or a fragment thereof fused to a Gag protein. In a particular embodiment, the first (aa 5-102) and second half (aa 242-353) of N are fused to a Gag protein. In one embodiment, the VLP of the invention further comprises a Gag protein fused at its carboxy terminus to an N protein or a fragment thereof from a Coronaviridae virus, preferably from SARS-CoV-2. This provides an additional superior T cell response against the fused N protein or a fragment thereof.

[0055] In a preferred embodiment, the VLP of the invention comprises: - Giardia lamblia VSPs, VSP-like proteins or fragments thereof, - the S protein of SARS-CoV-2 or a fragment thereof, preferably stabilized in the prefusion conformation, and - The N protein of SARS-CoV-2 or a fragment thereof, optionally fused to the Gag protein is displayed on its surface.

[0056] In a preferred embodiment, the VLP of the invention comprises: - Giardia lamblia VSPs, VSP-like proteins or fragments thereof, - the S protein of SARS-CoV-2 or a fragment thereof, preferably stabilized in the prefusion conformation; - the M protein of SARS-CoV-2 or a fragment thereof, and - The N protein of SARS-CoV-2 or a fragment thereof, optionally fused to the Gag protein is displayed on its surface.

[0057] All aspects and features given above for the VLPs of the invention apply mutatis mutandis to other aspects of the invention described below which involve said VLPs.

[0058] In another aspect, the present invention relates to a pharmaceutical composition comprising as an active ingredient a VLP as defined above.

[0059] In one embodiment, the pharmaceutical composition of the invention is a vaccine.

[0060] In one embodiment, the pharmaceutical composition of the present invention further comprises at least one pharma- ceutically acceptable excipient.

[0061] "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse allergic or other untoward reactions when administered to a mammal, particularly a human, as appropriate. A pharmaceutically acceptable excipient refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0062] Such pharma- ceutically acceptable excipients include, but are not limited to, water, salts, buffers, adjuvants, carriers, or other substances desirable for improving the effectiveness of the composition. Examples of pharmaceutical materials suitable for use in preparing pharmaceutical compositions are provided in a number of sources, including REMINGTON'S PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co., (1990)).

[0063] In addition, the pharmaceutical composition may contain additional adjuvants or immunogenic or biologically active agents.For example, the pharmaceutical composition may further contain an adjuvant selected from aluminum, particularly aluminum phosphate, AS03, MF59 and AS04.

[0064] However, an advantageous feature of the present invention is the high immunogenicity of the composition even in the absence of an adjuvant, so in other embodiments, the pharmaceutical composition lacks an adjuvant. The absence of an adjuvant also minimizes the occurrence of undesirable inflammatory T cell responses, which represent a safety concern in vaccination.

[0065] The pharmaceutical composition of the present invention is preferably formulated for oral or mucosal administration. The dose used for oral or mucosal administration can be adapted as a function of various parameters, in particular as a function of the type of pathology involved, or as a function of the desired duration of treatment. The pharmaceutical composition can be administered to any suitable mucosa, including oral administration (through the mucosa of the digestive system), nasal administration, vaginal administration, sublingual administration, ocular administration, rectal administration, urinal administration, intramammary administration, pulmonary administration, auricular administration (i.e. through the ear) and buccal administration, preferably buccal administration or sublingual administration (oral mucosa administration). In a preferred embodiment, the pharmaceutical composition is formulated for oral or nasal administration.

[0066] Once formulated, the pharmaceutical composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.The formulation is easily administered in various dosage forms, such as tablets or other solid types for oral or mucosal administration, sustained release capsules; and any other forms currently in use.Thus, the pharmaceutical composition can be in the form of sprays, aerosols, mixtures, suspensions, dispersions, emulsions, gels, pastes, syrups, creams, ointments, implants (ear, eye, skin, nose, rectum and vagina), intramammary preparations, vagitories, suppositories or uteritories).In some embodiments, the use of liposomes is contemplated.The formation and use of liposomes are known to those skilled in the art.

[0067] More specifically, the pharmaceutical compositions are formulated such that the VLPs, and in particular the viral proteins displayed on their surface, are optionally resistant to enzymatic and chemical degradation in the upper gastrointestinal tract.

[0068] In another aspect, the present invention relates to a VLP as defined above for use as a medicament.

[0069] The present invention also relates to a method for the prevention and / or treatment of a viral infection in a subject in need thereof, comprising administering to said subject an effective amount of a VLP as defined above.

[0070] The present invention also relates to the use of a VLP as defined above in the manufacture of a medicament.

[0071] As used herein, the term "subject" refers to mammals such as rodents, cats, dogs, and primates. Preferably, a subject according to the present invention is a human.

[0072] In the context of the present invention, the term "treat" or "treatment" as used herein means to reverse, alleviate, inhibit the progression of, or prevent the disease or infection to which such term applies, or one or more symptoms of such disease or infection.

[0073] Within the meaning of the present invention, the term "prevent" or "prevention" in relation to a disease or infection is intended to mean a reduction in the risk of occurrence of said disease or infection.

[0074] By "effective amount" is intended the minimum amount of VLP required to confer a therapeutic or prophylactic benefit to a subject. For example, an effective amount for a mammal is an amount that induces, ameliorates, or otherwise causes an improvement in infectious disease symptoms, disease progression, or a physiological condition associated with an infectious disease or resistance to succumbing to an infectious disease.

[0075] In another aspect, the present invention relates to a VLP as defined above for use in the treatment or prevention of infections with viruses of the Coronaviridae family.

[0076] The present invention also relates to a method for the prevention and / or treatment of an infection with a virus of the Coronaviridae family in a subject in need thereof, comprising administering to said subject an effective amount of a VLP as defined above.

[0077] The present invention also relates to the use of a VLP as defined above in the manufacture of a medicament for the prevention and / or treatment of an infection with a virus of the Coronaviridae family.

[0078] In another aspect, the present invention relates to a VLP as defined above for use as a vaccine against infections caused by viruses of the Coronaviridae family.

[0079] The present invention also relates to a method for vaccination (or immunization) of a subject against a virus of the Coronaviridae family, comprising the step of administering to said subject an effective amount of a VLP as defined above.

[0080] The present invention also relates to the use of a VLP as defined above in the manufacture of a vaccine against a virus of the Coronaviridae family.

[0081] In a preferred embodiment, the pharmaceutical composition is administered by a mucosal route.

[0082] In one embodiment, the VLPs of the invention are administered orally or intranasally to the patient.

[0083] In one embodiment, the VLPs of the invention are administered by subcutaneous or intramuscular injection as a booster to patients / animals who have previously received a vaccine against a virus of the Coronaviridae family, preferably an adenovirus-based vaccine.

[0084] In one embodiment, the VLPs of the invention are administered to the patient twice.

[0085] In one embodiment, the VLPs of the invention are administered to the patient three or more times.

[0086] In one embodiment, the patient / animal receives a first administration of a VLP of the invention and a second administration of a VLP of the invention, both administrations separated by at least 1 week, preferably at least 2 weeks, more preferably at least 3-4 weeks.

[0087] In one embodiment, VLPs are administered to a patient to induce production of immunoglobulin A (IgA) by mucosal surfaces of the aerodigestive tract, particularly in the bronchi and alveoli. IgA production can be measured by detecting IgA in bronchoalveolar lavage fluid, as shown in the examples.

[0088] The following figures and examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Although the invention has been described with reference to specific embodiments of the invention, it should be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, method, method step or steps to the objective, spirit and scope of the invention. All such modifications are intended to be within the scope of the appended claims. [Brief description of the drawings]

[0089] [Figure 1] Immunogenicity of different mutants of spike in mice. Swt: S wild type of SARS-CoV-2 (original Wuhan strain Acc.No.NC_045512 and its mutant D614G). Sst1: S artificial subtype 1 (K986P / V987P, 682RRAR-685GSAS(modFurinCS)). Sst2: S artificial subtype 2 (T791C / A879C, 682RRAR-685GSAS(modFurinCS)). Sst3: S artificial subtype 3 (S884C / A893C, 682RRAR-685GSAS(modFurinCS)). Sst4: S artificial subtype 4 (G885C / Q913C, 682RRAR-685GSAS(modFurinCS)). Sst5: S artificial subtype 5 (S884C / Q913C, 682RRAR-685GSAS(modFurinCS)). [Diagram 2] Serum antibody responses to oral administration of different vaccine formulations in hamsters. [Diagram 3] Serum antibody responses to intramuscular administration of different vaccine formulations in hamsters. [Figure 4] Bronchoalveolar lavage IgA responses. [Diagram 5] Neutralizing antibodies against SARS-CoV-2 invasion. [Figure 6] Bronchoalveolar IgA after oral boost in intramuscularly vaccinated hamsters. [Figure 7] Vaccinated hamsters challenged with SARS-CoV-2. EXAMPLES

[0090] example To design and evaluate a thermostable, orally administered, enveloped virus-like particle (e-VLP) vaccine against SARS-CoV-2, the following experiments were performed.

[0091] e-VLPs are particularly suitable for inducing robust neutralizing immune responses. Indeed, e-VLPs have the same lipid membrane as the cells from which they are derived. Similarly, the viral envelope proteins expressed by the VLPs have the same conformation as the viral envelope proteins have on the lipid membrane of the infected cells, and the same conformation as the virus itself. Many of the neutralizing antibodies (Nabs) target conformational structures, making e-VLPs particularly suitable for Nab induction. Furthermore, e-VLPs can be utilized with variable surface proteins (VSPs) from the intestinal parasite Giardia lamblia, which confers resistance to e-VLPs after oral administration.

[0092] We generated and tested e-VLPs decorated with VSPs expressing various forms of the S protein, with or without M expression (VSP-e-VLPs). Unexpectedly, we found that VSP-e-VLPs expressing pre-fusion stabilized forms of S and M elicited robust mucosal Nabs against SARS-CoV-2 in mice and hamsters, which translated into complete protection from viral challenge. Such vaccines could be part of the armamentarium against SARS-CoV-2 in prime-boost vaccine strategies or as a boost to existing vaccines based on platforms that do not allow efficient homologous boosting (such as adenoviral platforms) and mRNA-based vaccines that are highly efficient against severe forms of the disease but do not result in infection.

[0093] material and method Virus. SARS-CoV-2 isolates were grown in VeroE6 cells at 37 °C in Opti-MEM I (Invitrogen) containing 0.3% bovine serum albumin (BSA) and 1 μg L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin per mL. All experiments with SARS-CoV-2 were performed in a biosafety level 3 (BSL3) containment laboratory at CIDIE CONICET (Argentina) or in an enhanced BSL3 containment laboratory at the Sorbonne University (France).

[0094] Experimental animals. For immunization and challenge experiments, group sizes were selected based on previous experience, and littermates of the same sex were randomly assigned. The number of animals for each experiment and all procedures was in accordance with the protocol approved by the Institutional Committee for Care and Use of Experimental Animals. Five female and five male SPF golden Syrian hamsters aged 1 month per group were used in the challenge experiments, and five female and five male golden Syrian hamsters aged 6 months per group were used in the immunization studies. For the challenge experiments, 10 hamsters per group were administered 10 mg of 10 ... 5 Animals were inoculated with PFU of SARS-CoV-2 (in 100 μL) or PBS (mock). Baseline body weights were measured before infection and body weights were monitored for 28 days. Animals were not harmed during blood collection.

[0095] Neutralization assay. Sera collected 14 dpi from intranasally inoculated hamsters were tested for virus-neutralizing antibody titers by microneutralization assay in Vero E6 cells. Briefly, 50-fold serially diluted (1:50 to 1:5000) serum samples were mixed with 100 TCID50 of SARS-CoV-2 virus and incubated for 1 h at 37 °C. The mixture was then added to Vero E6 cells and further incubated for 72 h at 37 °C. Neutralizing antibody titers were defined as the highest dilution that inhibited 50% of the cytotoxic effect.

[0096] VLP expression plasmids. For pGag, the cDNA sequence encoding the Gag capsid protein of MLV Gag without its C-terminal Pol sequence (Uniprot: P0DOG8.1) was obtained by enzymatic digestion from the plasmid pBL36-HCV and cloned into the phCMV expression vector. For the SARS-CoV-2 spike protein mutants, the cDNA sequence was also cloned into the phCMV expression vector. All plasmids were verified by sequencing.

[0097] Generation, production, purification and validation of VLPs. VLPs were produced by transient transfection of either HEK293 or HEK293-1267 cells with pGag, pGag-N1, pGag-N2, pS and its mutants, and pM plasmid DNA using PEI as a transfection reagent. Cells were transfected with 70 μg total DNA per flask at 70% confluence in T175 flasks at a PEI:DNA mass ratio of 3:1. VLP-containing supernatants were harvested 72 hours after transfection, filtered through a 0.45 μm pore membrane, concentrated 20-fold in a centrifugal filter device (Centricon® Plus-70-100K, Millipore), and purified by ultracentrifugation through a 20% sucrose cushion in a SW41T Beckman rotor (25,000 rpm, 4 hours, 4° C.). The pellet was resuspended in sterile TNE buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1 mM EDTA). Protein was measured using the Bradford method. For Western blotting, proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes, followed by incubation with specific primary antibodies. Secondary antibodies conjugated with alkaline phosphatase were used and detected by BCIP / NBT substrate.

[0098] Immunization. Golden Syrian hamsters were fasted for 4 hours and then orally immunized with 2 doses of 100 μg of different VLPs administered weekly. For subcutaneous immunization, 2 doses of 10 μg of different VLPs administered weekly. Animals in the negative control group (naive) received oral immunization with vehicle only. During immunization, animals were not anesthetized.

[0099] Fluid Collection. Blood was collected weekly from the retro-orbital sinus of the hamsters and serum was separated and stored at -80° C. Bronchoalveolar lavage (BAL) was collected via the trachea by insufflation-aspiration of 1 mL PBS containing protease inhibitors.

[0100] Enzyme-linked immunosorbent assay (ELISA) test. The levels of IgG and IgA antibodies against spike protein were determined by ELISA by sensitizing the plates with homogenates of killed whole virus produced in vitro. Quantification of spike was performed using purified proteins: human coronavirus HCoV-229E spike protein (S1+S2 ECD) (Sino Biological, Inc. 40605-V08B) and SARS-CoV-2 spike protein (Active Trimer) R&D Cat. #10549-CV). The following secondary antibodies were used: Mouse anti-hamster IgG cocktail, clones: G94-56, G70-204 (BD Biosciences, Cat. #554009); Mouse anti-hamster IgM, clone: ​​G188-9, (BD Biosciences Cat. #554035); Hamster immunoglobulin A (IgA) ELISA kit (MyBiosources Cat. #MBS029668). Mouse monoclonal (H6) anti-SARS-CoV-2 spike glycoprotein (Abcam. Cat. no. ab273169).

[0101] Statistical analysis. For in vitro proteolysis densitometry experiments, unpaired two-tailed Student's t-test was used. Prism (GraphPad Software) was used to perform one-way or two-way ANOVA on the data sets with Tukey's multiple comparison test or Bonferroni post-hoc test, respectively. All figures show the mean ± standard error of the mean. Statistically significant differences are indicated in each graph as *p<0.05, **p<0.01 and ***p<0.001, ns=not significant.

[0102] Example I - Immunogenicity in mice of different mutants of S. Experiments were performed with the spike protein (S) of SARS-CoV-2. Several mutants of the spike protein (or a subtype called "Sst") to stabilize the receptor binding domain (RBD) in an open conformation and the S protein stabilized in the prefusion state were designed. Several point mutations were used: Cys-molecular clamp, furin cleavage site removal and Pro substitution. VLPs were then produced as described and verified for correct composition. e-VLPs were orally administered to Balb / c mice and serum and BAL Ig levels were determined by ELISA (Figure 1).

[0103] Notably, the spike mutant lacking the furin cleavage site (modFurinCS) and carrying two Pro substitutions was the most efficient in eliciting high levels of antibodies. Identical experiments performed in Ob / Ob, Db / Db and aged mice of both sexes showed similar results (not shown), indicating that oral administration of VSP-e-VLPs did not vary between groups, regardless of the underlying condition, sex or age of the mice. Therefore, the most effective mutant of S, namely Sst1, was selected for inclusion in e-VLPs for immunization and challenge experiments in the golden Syrian hamster model.

[0104] Example II - Antibody responses to oral administration of different vaccine formulations The C-terminal cytoplasmic tail (CT) of SARS-CoV-2 S is known to be important for proper glycosylation of the S glycoprotein. For this reason, it was hypothesized that differential glycosylation of S should affect efficient Nab generation, as numerous glycosylation sites exist near the receptor binding domain (RDB). For this reason, the CT was modified (modCT) to abolish endoplasmic reticulum (ER) retention. In addition, the envelope membrane protein M of SARS-CoV-2 was incorporated into the e-VLPs. Indeed, M is known to retain S in the ER to improve the first steps of glycosylation, and then remains attached to the CT of S during its movement throughout the Golgi apparatus, where final glycosylation is achieved. Although that was the main reason for including M in the e-VLPs, subsequent reports showed specific T cell responses against several epitopes of this protein in patients who had recovered after contracting Covid-19. Therefore, incorporation of M into the e-VLP envelope may benefit not only proper glycosylation of S, but also the production of stronger cellular responses against the virus. Therefore, e-VSPs with / without M and with / without modified CT were generated. e-VLPs for oral administration with Giardia-derived VSP incorporated on the VLP surface and e-VLPs for intramuscular injection without VSP were also produced to compare both immunization routes.

[0105] Orally administered formulations showed that the absence of Giardia VSPs decorating the different e-VLPs avoided any immune response, most likely due to the disruption of VLPs along the upper small intestine (Figures 2A and 2B). Furthermore, modification of the CT of S appeared to be detrimental to the induction of either serum IgG or IgA produced in animals compared to S with wild-type CT. However, when M was incorporated into the VSP-e-VLPs, IgG and IgA serum antibody titers were increased.

[0106] Example III - Antibody responses to intramuscular administration of different vaccine formulations. VSP-e-VLPs administered intramuscularly (im) to hamsters induced high levels of both Igs in serum, and the presence of Giardia VSPs on the e-VLPs promoted higher levels of antibodies than plain e-VLPs (Figure 3). As expected, serum IgG levels were higher than IgA levels. Important variations between individual animals were observed for all intramuscularly administered formulations. However, these values ​​were acceptable when compared to values ​​obtained with other vaccine formulations or in plasma from convalescent patients.

[0107] The differences observed between the same formulations administered either orally or intramuscularly in these animals suggest that although the oral route is expected to show a higher degree of variation between animals, it is not as variable as between the different formulations and may be explained by the type of Ig generated. Notably, given that intramuscular administration was performed in the absence of added adjuvants, the high immunogenicity of VSP-e-VLPs can be explained by the adjuvant properties of VSP, which have been demonstrated to activate TLR-4. The lower immunogenicity of e-VLPs lacking VSP may be solely due to the particulate properties of VLPs and repeated exposure of the antigen on the surface of the VLPs.

[0108] These results clearly demonstrate that the presence of VSP on the surface of e-VLPs is essential to avoid particle disruption when administered orally, but that plain e-VSP generates a substantial response when administered by injection in the absence of adjuvant, a response that is strongly increased in the presence of VSP.

[0109] Example IV - Bronchoalveolar lavage IgA response When analyzing the presence of IgA in animals immunized orally or by injection of the formulation, it was found that animals immunized intramuscularly had consistently lower levels of IgA titers compared to orally administered animals, where only the e-VLP version containing VSP, M and stabilized S showed high titers of IgA in bronchoalveolar lavage fluid (Figure 4). Together, these results confirm the high immunogenicity of the e-VLP formulation and the crucial importance of the VSP to protect and immunoactivate the particles for oral administration.

[0110] Example V - Neutralizing antibodies against SARS-CoV-2 entry Furthermore, IgA present in both orally and intramuscularly administered selected e-VLPs demonstrated high levels of Nabs in the BAL, and these levels were increased when VSP and M were included in the particles (Figure 5). Notably, both wild-type and stabilized S proteins were capable of generating Nabs of comparable titers, a phenomenon already observed across different commercial vaccines administered to humans.

[0111] Example VI - Oral boost after intramuscular administration VSP-e-VLPs are thermostable and can even withstand several freeze / thaw cycles, making them highly suitable for use in regions of the world where refrigeration supplies for vaccine injections are in short supply. Indeed, even though most countries have already launched large-scale vaccination campaigns, infections with SARS-CoV-2 continue in most parts of the world due to complications posed by the high level of refrigeration of current vaccines, the lack of sterilizing immunity of intramuscularly administered vaccines, or the need for trained personnel for intramuscular administration.

[0112] For these reasons, an oral booster was applied to animals previously vaccinated by injection. In these animals, a third dose of a formulation containing VSP, stabilized S and M induced a six-fold increase in the level of IgA in the BAL compared to those given only two doses intramuscularly (Figure 6).

[0113] Example VII - SARS-CoV-2 attack Finally, animals immunized with selected VSP-e-VLP formulations were challenged with SARS-CoV-2 to determine the response of hamsters. In this case, only formulations of e-VLPs with stabilized S and M present on the particles, with or without VSP pseudotyping, were tested.

[0114] The results showed that oral immunization with VSP-e-VLPs prevented animals from losing weight for 2 weeks after infection, similar to animals immunized by injection and then boosted orally. In contrast, control animals lost weight during that period and recovered shortly thereafter, as reported for experimental infections in hamsters. An intermediate situation could be observed in animals immunized only intramuscularly (Figure 7).

[0115] VIII-Conclusion These experimental results show for the first time that it is possible to co-express SARS-CoV-2 envelope proteins on e-VLPs with Giardia VSPs, such as to generate NAbs against SARS-CoV-2 after oral administration. Whereas plain e-VLPs did not generate Ab responses, VSP-e-VLPs generated Ab responses in the range of responses to intramuscular administration. This is noteworthy because it indicates that VSPs protect the SARS-CoV-2 Env protein from degradation while maintaining the proper conformation of the protein.

[0116] Moreover, these results also demonstrate the dual properties of VSPs, which not only provide protection from degradation but also have a strong adjuvant effect. Indeed, when the vaccines were administered intramuscularly, VSP-e-VLPs always yielded higher titers of antibodies than their regular e-VLP counterparts. Of note, SARS-CoV-2 VLPs were recently reported to generate good Nab responses after intramuscular administration, but there have been no reports of IgA at mucosal sites.

[0117] In addition to its ease, oral administration is also known to have the advantage of inducing better mucosal immunity. This is indeed the case here, with BAL IgA also detectable only after oral administration, although plasma levels are high. This is a clear advantage for a vaccine against SARS-CoV-2, since it should reduce viral transmission. In this line, SARS-CoV-2 was still detected in the BAL of vaccinated macaques that otherwise appeared to be protected from infection.

[0118] In this study, we did not test specific T cell responses. However, e-VLPs are known to robustly induce such responses, and indeed, using VSP-HA-VLPs, potent CTLs were able to kill HA-expressing tumor cells (Serradell et al., Nat. Commun. 10:361 (2019)). Furthermore, it is well known that the IgG and IgA responses here are T cell dependent, and therefore a good antibody response attests to a good T cell response.

[0119] Along these lines, it has previously been shown that fusion of a viral peptide to GAG, the retroviral protein precursor that induces the formation and release of viral particles / VLPs, allows the generation of a more potent T cell response against this peptide. Fusion of large fragments or SARS-CoV-2 N structural proteins, or stretches of immunodominant peptides to Gag, would clearly be a means to further enhance the immunogenicity of VSP-e-VLPs.

[0120] SARS-CoV-2 VSP-e-VLPs could be used as a stand-alone vaccine, possibly using a prime-boost scheme of administration. VSP-e-VLPs are heat stable and retain their properties at room temperature, which could be particularly advantageous for vaccination in countries that would benefit from a heat stable vaccine. VSP-e-VLPs could also be used as a booster for other vaccine designs.

[0121] In this line, the duration of protection conferred by currently used vaccines is still unclear. Follow-up studies of infected patients indicate that, at least for some patients, the persistence of Nab and the duration of protection may be several months. These, in addition to the emergence of viral variants, may make it necessary to periodically boost the immune response of vaccinees. For some vaccine designs, especially those based on adenoviral vectors, re-administration of the same vector may be less efficient due to the immune response generated against the vector. For these reasons, boosting with VSP-e-VLPs may be of particular interest. For other vaccine designs, orally administered vaccines may be better tolerated, especially if repeated administration is required in other years.

[0122] Taken together, given the issues specific to each vaccine design (thermostability, side effects, lack of mucosal immunity induction, immunogenicity to the vector, among other drawbacks), the availability of multiple vaccines against SARS-CoV-2 guarantees a better chance of containing the pandemic.

[0123] Consequently, a heat-stable, orally administered e-VLP vaccine would be a valuable addition to the armamentarium against this virus.

Claims

1. 1. A virus-like particle (VLP) comprising at least: - a variable surface protein (VSP), a VSP-like protein or a fragment thereof of a microorganism selected in the group consisting of Giardia spp., Tetrahymena spp., Paramecium spp. and Entamoeba spp., - Viral proteins or fragments thereof of Coronaviridae viruses A virus-like particle (VLP) that displays the above on its surface.

2. 2. The VLP of claim 1, wherein the microorganism is selected from the group consisting of Giardia lamblia, Tetrahymena thermophila, Paramecium tetraurelia, and Entamoeba histolytica.

3. The VLP according to claim 1 or 2, wherein the VSP, VSP-like protein or fragment thereof, preferably the extracellular domain of the VSP, is fused to the transmembrane domain of the vesicular stomatitis virus G protein (VSV-G).

4. The VLP of any one of claims 1 to 3, wherein the Coronaviridae virus is a coronavirus, preferably selected from the group consisting of SARS-CoV-1, SARS-CoV-2, MERS-CoV and mutants thereof.

5. The VLP according to any one of claims 1 to 4, wherein said viral protein is selected from the group consisting of spike (S) protein, membrane (M) protein, envelope (E) protein and nucleocapsid (N) protein, preferably S protein.

6. The VLP, - Giardia lamblia VSP, VSP-like proteins or fragments thereof, - SARS-CoV-2 S protein or a fragment thereof The VLP of any one of claims 1 to 5, which displays on its surface.

7. The VLP of any one of claims 1 to 6, further comprising a Gag protein fused at its carboxy terminus to the N protein or a fragment thereof from a Coronaviridae virus, preferably from SARS-CoV-2.

8. The VLP of any one of claims 1 to 7, wherein the S protein is stabilized before fusion.

9. The VLP according to any one of claims 1 to 8, wherein said VLP displays at least two different viral proteins on its surface, preferably an S protein and an M protein.

10. A VLP according to any one of claims 1 to 9 for use as a medicament.

11. A VLP according to any one of claims 1 to 9 for use in the treatment or prevention of infections with viruses from the Coronaviridae family.

12. The VLP according to any one of claims 1 to 9 for use as a vaccine against infections with viruses from the Coronaviridae family.

13. The VLP for use according to any one of claims 10 to 12, wherein said VLP is administered orally or intranasally to a human / animal patient.

14. The VLP for use according to any one of claims 10 to 13, wherein said VLP is administered as a booster to a patient / animal who has previously been vaccinated against a virus from the Coronaviridae family by subcutaneous or intramuscular injection.

15. A pharmaceutical composition comprising the VLP of any one of claims 1 to 9.

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