Live attenuated SARS-CoV-2 and vaccine prepared from same

Abstract

The present invention relates to a polynucleotide encoding: a) a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein; and / or b) at least one non-structural SARS-CoV-2 protein selected from the group consisting of a non-structural protein 7, a non-structural protein 8, a non-structural protein 9, a non-structural protein 10, a non-structural protein 11, a non-structural protein 12, an endonuclease and a 2 '-O-methyltransferase, wherein the polynucleotide comprises at least one sequence portion comprising a codon pair deoptimization compared to the SARS-CoV-2 genome, or consists of at least one such sequence portion, and wherein the polynucleotide further comprises a furin cleavage site modification that results in a deletion of a furin cleavage site naturally present in the SARS-CoV-2 genome. The present invention also relates to a live attenuated SARS-CoV-2 comprising the polynucleotide, a vaccine comprising the live attenuated SARS-CoV-2, and a related method.

Description

[0001] The present invention relates to a codon pair deoptimized polynucleotide encoding a respiratory syndrome coronavirus 2 (SARS-CoV-2) protein, an attenuated live SARS-CoV-2 comprising such a polynucleotide, a pharmaceutical composition comprising such an attenuated live SARS-CoV-2, a vaccination method for administering the pharmaceutical composition, a vector comprising such a polynucleotide, a host cell comprising such a polynucleotide, and a method for producing a virus.

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as the causative agent of coronavirus disease 2019 (COVID-19) in December 2019 (Wu et al., 2020; Zhou et al., 2020b). The virus is highly transmissible between people (Chan et al., 2020). It spread rapidly throughout the world in about a few weeks, and the world is still fighting the ongoing COVID-19 pandemic.

[0003] SARS-CoV-2 replicates primarily in the upper respiratory tract (Zou et al., 2020). Infection with SARS-CoV-2 can result in a wide range of clinical manifestations, from asymptomatic to life-threatening disease symptoms (Chen et al., 2020; Zhou et al., 2020a). In particular, the elderly and patients with pre-existing conditions are at greater risk of developing more severe disease (e.g., pneumonia, acute respiratory distress syndrome, and multiple organ failure) (Chen et al., 2020; Garg et al., 2020; Zhou et al., 2020a). The ongoing pandemic has imposed a huge health, psychological, economic, and social burden. To date (December 2021), more than 270 million people have been infected with the virus, of which more than 5.3 million have died as a result of infection (https: / / coronavirus.jhu.edu / map.html) (Dong et al., 2020).

[0004] The unprecedented scale and severity of the COVID-19 pandemic has prompted the rapid development of new diagnostic tests, treatments, and vaccines that can be used to curb the spread of the virus and limit the pandemic. Globally, more than 90 vaccines are being tested in clinical trials, but only a few have reached the final stage of testing (Zimmer et al., 2021). Almost all vaccines that have been or are being evaluated in clinical trials are based on inactivated or subunit viral preparations (Ella et al., 2021; Gao et al., 2020; Wang et al., 2020; Zhang et al., 2021), replication-defective viral vectors (Emary et al., 2021; Logunov et al., 2021; Solforosi et al., 2021; Voysey et al., 2021; Zhu et al., 2020) or DNA / RNA molecules (Anderson et al., 2020; Baden et al., 2021; Corbett et al., 2020; Dagan et al., 2021; Jackson et al., 2020; Mulligan et al., 2020; Polack et al., 2020; Sahin et al., 2020; Walsh et al., 2020).

[0005] SARS-CoV-2 is evolving rapidly (Tegally et al., 2021; Faria et al., 2021; Davies et al., 2021). Benefiting from its global presence, the virus continues to adapt to its new hosts and infection or vaccine-induced immunity. During the course of the pandemic, many genetic variants have emerged (Tegally et al., 2021; Faria et al., 2021; Davies et al., 2021). Variants that exhibit increased infectivity, cause higher morbidity and mortality, or have the ability to evade infection or vaccine-induced immunity pose an increased threat to public health. The World Health Organization (WHO) and other national health agencies have independently established a classification system that classifies emerging variants as variants of interest (VOI), variants under investigation (VUI), or variants of concern (VOC) based on their risk to public health (see Table 1 of Trimpert et al. "Live attenuated virus vaccine protects against SARS-CoV-2 variants of concern B.1.1.7 (Alpha) and B.1.351 (Beta)", Science Advances, Vol. 7, No. 49 (2021)). In addition, to simplify communication with the public, WHO recommends that VOI and VOC should also be labeled using letters of the Greek alphabet. As of August 12, 2021, viruses belonging to lineages B.1.1.7 (Alpha), B.1.351 (Beta), B.1.1.28.1 (Gamma), B.1.617.2 (Delta), and most recently B.1.159.1 (Omicron) are classified as VOCs by several health agencies. In the countries where they appeared, these variants quickly replaced pre-existing variants and began to spread globally.

[0006] The B.1.1.7 variant was first detected in the UK in December 2020. It is 50% to 100% more transmissible than earlier variants and may also be more deadly, but has not shown a tendency to escape immunity induced by infection or vaccination (Davies et al., 2021; Volz et al., 2021; Abu-Raddad et al., 2021). The B.1.1.7 variant has been detected in 132 countries and quickly became the dominant variant in Europe and the United States. The B.1.351 variant was first detected in South Africa in May 2020. It is not only more transmissible, but also capable of reinfecting individuals and breaking through vaccine protection (Madhi et al., 2021; Johnson & Johnson; Naveca et al., 2021). The B.1.1.28.1 variant is similar to B.1.351 in that both share some important mutations in the spike glycoprotein (E484K, K41 7N / T, and N501Y). B.1.1.28.1 emerged in Manaus, Brazil in late 2020 (Faria et al., 2021). Similar to the B.1.351 variant, it can cause reinfection because it can bypass immunity that occurs after infection with other viral variants (Faria et al., 2021; Naveca et al., 2021). It is estimated that B.1.1.28.1 is 40% to 140% more transmissible and pathogenic than other variants, and 10% to 80% more lethal (Faria et al., 2021). On May 7, 2021, the WHO reclassified the B.1.617.2 variant, first detected in India, as a VOC because of its high transmissibility (WHO). As of August 2021, B.1.617.2 has largely outcompeted B.1.1.7 and has become the dominant variant in Europe and the United States. According to the WHO, B.1.617.2 is the most dangerous strain worldwide and has attracted considerable attention for its ability to evade infection and vaccine-mediated protection (Dyer et al., 2021). The variant of SARS-CoV-2, B.1.1.529, was first detected in Botswana in November 2021. It has become the dominant variant circulating throughout the world. After the original BA.1 variant, several sub-variants of Omicron have evolved: BA.2, BA.3, BA.4 and BA.5, and as of August 2022, BA.5 is dominant in the world.

[0007] WO 2021 / 154828 A1 and WO 2023 / 283106 A1 describe modified SARS-CoV-2 coronaviruses. These viruses have been recoded, such as codon deoptimization or codon pair bias deoptimization, and can be used to reduce the likelihood or severity of SARS-CoV-2 coronavirus infection, prevent SARS-CoV-2 coronavirus infection, induce an immune response, or treat SARS-CoV-2 coronavirus infection.

[0008] Y. Wang et al., 2021 described a live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine named COVI-VAC, which was developed by recoding a segment of the viral spike protein with synonymous suboptimal codon pairs (codon pair deoptimization), thereby introducing 283 silent (point) mutations. In addition, to increase the safety of the vaccine strain, the furin cleavage site within the spike protein was deleted from the viral genome.

[0009] CN 112175913A describes an attenuated strain of SARS-CoV-2 and its use in preventing and / or treating novel coronavirus pneumonia.

[0010] One object of the present invention is to provide new SARS-CoV-2 vaccine candidates.

[0011] This purpose is achieved with a specific polynucleotide encoding: a) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein; and optionally b) at least one non-structural SARS-CoV-2 protein selected from the following: non-structural protein 7, non-structural protein 8, non-structural protein 9, non-structural protein 10, non-structural protein 11, non-structural protein 12, endoribonuclease (also known as non-structural protein 15) and 2'-O-methyltransferase (also known as non-structural protein 16). In this context, the polynucleotide comprises at least one sequence portion comprising a codon pair deoptimization compared to the corresponding SARS-CoV-2 genome portion or is composed of at least one such sequence portion. In addition, the polynucleotide comprises a furin cleavage site modification, which results in the loss of function of the furin cleavage site naturally present in the SARS-CoV-2 genome. Therefore, the polynucleotide lacks some genetic information present in the naturally occurring SARS-CoV-2.

[0012] The term "furin cleavage site modification" used herein refers to a site in the nucleotide sequence of a polynucleotide, which site corresponds to the site encoding the furin cleavage site in the SARS-CoV-2 genome, but has a modification so that it causes the (furin) cleavage sensitivity of the encoded cleavage site to be significantly reduced or lost. Some examples of such modifications are hereinafter, for example, shown in the embodiments. The furin cleavage site modification described herein can be any modification that changes the cleavage sensitivity, for example, compared with the SARS-CoV-2 genome, (partial or complete) deletion, insertion or replacement of nucleotides or sequence portions. In some embodiments, compared with the sequence portion of the SARS-CoV-2 encoding the furin cleavage site, the furin cleavage site modification described herein comprises (embody) no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23 or no more than 24 nucleotide modifications.

[0013] The term "polynucleotide" as used herein refers to a molecule containing multiple nucleotides (e.g., mRNA, RNA, cRNA, cDNA, or DNA). The term generally refers to an oligonucleotide having a length greater than 200, preferably greater than 300, preferably greater than 400, preferably greater than 500, preferably greater than 600, preferably greater than 700, preferably greater than 800, preferably greater than 900, preferably greater than 998 nucleotide residues. The polynucleotide of the present invention is essentially composed of the nucleic acid sequence described herein, or comprises the above nucleic acid sequence. Therefore, it may also comprise another nucleic acid sequence. The term polynucleotide encompasses single-stranded and double-stranded polynucleotides. In addition, modified polynucleotides are also encompassed herein, including chemically modified polynucleotides, artificially modified polynucleotides, or naturally occurring modified polynucleotides (e.g., glycosylated or methylated polynucleotides).

[0014] As used herein, the term "SARS-CoV-2" or "Severe Acute Respiratory Syndrome Coronavirus 2" refers to any variant classified as SARS-CoV-2. In some embodiments, the SARS-CoV-2 described herein is at least one SARS-CoV-2 variant selected from the group consisting of alpha, beta, gamma, delta, or omega variants. In some embodiments, the SARS-CoV-2 omega variant is at least one SARS-CoV-2 omega sublineage, such as BA.1, BA.2, BA.3, BA.4, or BA.5. In some embodiments, SARS-CoV-2 refers to a SARS-CoV-2 spike mutant comprising at least one mutation selected from the group consisting of del 69-70, RSYLTPGD246-253N, N440K, G446V, L452R, Y453F, S477G / N, E484Q, E484K, F490S, N501Y, N501 S, D614G, Q677P / H, P681H, P681R, and A701V. In some embodiments, SARS-CoV-2 refers to a SARS-CoV-2 variant comprising at least one mutation selected from the group consisting of G142D, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K. In some embodiments, SARS-CoV-2 refers to a SARS-CoV-2 variant comprising at least one mutation selected from the group consisting of L452R, F486V, and R493Q. Any combination of these mutations is possible and disclosed herein. In particular, all mutations listed in any of the aforementioned mutation groups may be present simultaneously in a SARS-CoV-2 variant. Therefore, "SARS-CoV-2 protein" and "SARS-CoV-2 genome" can also be understood as the protein and genome of the SARS-CoV-2 variant, respectively.

[0015] The term "codon" as used herein refers to any group of three consecutive nucleotides in the coding portion of a polynucleotide (e.g., a messenger RNA molecule or a coding strand of DNA) that specifies a specific amino acid, translation start or stop signal. Typically, a codon is specific to one amino acid, however, for SARS-CoV-2, it is known that a codon shares at least one nucleotide with another codon.

[0016] As used herein, the term "codon pair" refers to two consecutive codons.

[0017] The term "codon pair deoptimization" (codon-pair deoptimization, CPD) used herein refers to the "re-formulation" of codons or the replacement of codons by other codons encoding the same amino acids, so that the encoded protein is the same, but suboptimal codon pairs and / or CpG dinucleotides appear. Methods for codon pair deoptimization are known in the art (see, e.g., Coleman et al., 2008, Mueller et al., 2010). In some embodiments, the codon pair deoptimization described herein includes increasing the number of underrepresented or suboptimal codon pairs and CpG dinucleotides in the recoded genome. In some embodiments, the codon pair deoptimization described herein results in increased mRNA decay and / or reduced translation efficiency. In some embodiments, the codon pair deoptimization described herein results in less protein, less virus, reduced virulence and / or attenuated live virus.

[0018] The polynucleotides of the invention can be used in the context of virus production or vaccines. Thus, compared to wild-type sequences, the polynucleotides of the invention emit a reduced risk of uncontrolled replication during production, transport, storage, handling and / or administration.

[0019] Typically, the polynucleotide forms part of a live attenuated SARS-CoV-2. Compared to the wild-type virus, the live attenuated virus causes fewer and / or less severe symptoms, or even no symptoms, in the host organism after the host organism is confronted (infected) with the attenuated virus. At the same time, the live attenuated virus induces an immune response of the host to the attenuated virus, which is at least partially protective against wild-type virus infection and / or at least one symptom thereof.

[0020] Compared to most vaccines under development, the inventors have produced attenuated but replicative SARS-CoV-2 vaccine candidates by genetically modifying the SARS-CoV-2 genome via codon pair deoptimization (Coleman et al., 2008). CPD is a viral attenuation strategy that enables a wide range of viruses to be rapidly and efficiently attenuated (Broadbent et al., 2016; Coleman et al., 2008; Eschke et al., 2018; Groenke et al., 2020; Khekar et al., 2018; Kunec and Osterrieder, 2016; Le Nouen et al., 2014; Mueller et al., 2010; Shen et al., 2015; Trimpert et al., 2021a; Trimpert et al., 2021b). CPD rearranges the position of existing synonymous codons in one or more viral genes without changing the codon bias or amino acid composition of the encoded protein (Eschke et al., 2018; Groenke et al., 2020; Khedkar et al., 2018; Kunec and Osterrieder, 2016; Osterrieder and Kunec, 2018; Trimpert et al., 2021a; Trimpert et al., 2021b). Codons that are naturally underrepresented can be overrepresented by CPD. Since the effect of CPD is highly dependent on the genomic sequence to be deoptimized, it is not possible to give exact instructions on how the codon positions in the recoded sequence should be changed during deoptimization. However, those skilled in the art know how to determine or estimate codon pairs that can be replaced by naturally underrepresented codon pairs at the target site where the polynucleotide is intended to be translated (e.g., in the target species or target tissue). The inventors herein provide some examples of recoding codon pairs to achieve over-representation of naturally under-represented codon pairs and codon pair deoptimization of polynucleotides. Thus, a skilled person can - at least from the codon pairs at the target site and the means and methods provided herein - obtain other polynucleotides according to the present invention.

[0021] A polynucleotide is considered to be codon pair deoptimized if at least one codon pair is deoptimized relative to the corresponding native sequence. Recoded viruses are generally not as efficient as their parents in producing proteins from recoded genes and may show defects in replication fitness, which enables the host to control wild-type virus infection through innate and adaptive immune responses (Eschke et al., 2018; Groenke et al., 2020; Khekar et al., 2018; Kunec and Osterrieder, 2016; Mueller et al., 2010; Trimpert et al., 2021b; Wimmer et al., 2009). Conserved antigenic properties and replication potential enable recoded attenuated viruses to fully engage the host's immune system and elicit a strong immune response.

[0022] Therefore, by codon pair deoptimization, the resulting protein is not changed. In contrast, although the genomic sequence of SARS-CoV-2 is changed, the resulting protein remains the same. However, the translation efficiency is generally reduced, so that viral replication is also reduced. When attenuated live SARS-CoV-2 is used as a vaccine, this leads to an immune response without the risk of pathological viral replication in patients receiving the vaccine. Another possible effect of codon pair deoptimization is a CpG-mediated immune response, which leads to viral attenuation. The present disclosure is not limited to a specific one of these effects.

[0023] Live attenuated SARS-CoV-2 lacking the furin cleavage site (FCS) was severely attenuated but elicited a strong humoral immune response and maintained a similar level of protection to live attenuated virus variants with an intact FCS and parental wild-type SARS-CoV-2 in heterologous SARS-CoV-2 challenge. Most importantly, however, removal of the FCS completely abolished transmission of the vaccine virus between co-housed hamsters. These results suggest that removal of the FCS from live attenuated SARS-CoV-2 is a promising strategy to further improve vaccine safety and prevent transmission without compromising vaccine efficacy.

[0024] It was very unexpected to find that attenuation by modification (especially deletion) of FCS and combined attenuation by codon pair deoptimization and modification (especially deletion) of FCS, but independent attenuation mechanisms, did not compromise vaccine efficacy. In many cases, "over-attenuation" can be observed by combining different attenuation effects, resulting in insufficient viral growth or insufficient immune response. Interestingly, no such negative over-attenuation was observed for the subject matter currently claimed. In contrast, modification (especially deletion) of FCS resulted in increased in vitro viral growth and improved genetic stability of attenuated live SARS-CoV-2. The latter is particularly important in terms of regulatory requirements, because the higher the stability of the attenuated live SARS-CoV-2, the easier it is for a vaccine containing the attenuated live SARS-CoV-2 to obtain a marketing license, and the higher the medical safety of such a vaccine.

[0025] Unintentional transmission of vaccine viruses from vaccinated individuals to unvaccinated individuals is a factor that can complicate the use of transmissible live attenuated vaccines (LAV) (Bull et al., 2018; Layman et al., 2021; Nuismer et al., 2018; Pons-Salort et al., 2016). Although self-dissemination is desirable in some cases, especially when herd immunity is sought in wildlife (Smithson et al., 2019), uncontrolled circulation of vaccine viruses may increase the likelihood of virulence reversion (Bull et al., 2018; Layman et al., 2021; Nuismer et al., 2018). Recombination between different vaccine viruses or between vaccines and field viruses is particularly problematic because it can produce recombinants with increased virulence, transmissibility, or immune evasion (Burns et al., 2014; Combelas et al., 2011; Lee et al., 2012; Ming et al., 2020). The rapid evolution of SARS-CoV-2 prompts extreme caution in the use of LAVs because they may circulate irreversibly. In addition, transmission of attenuated viruses to immunocompromised individuals is a danger associated with the use of transmissible virus vaccines (Kamboj and Sepkowitz, 2007).

[0026] The inventors have found that sCPD9 confers robust immunity against several SARS-CoV-2 variants in a COVID-19 hamster model (Trimpert et al., 2021a; Trimpert et al., 2021b). Most importantly, sCPD9 outperforms intramuscularly administered adenoviral vectors and mRNA vaccines in the ability to induce systemic and mucosal immunity (Nouailles et al., 2022). It was a very unexpected discovery that this robust immunity against several SARS-CoV-2 variants was not compromised by additionally introducing modifications (particularly deletions) of FCS into polynucleotides encoding at least some proteins of attenuated live SARS-CoV-2. On the contrary, the immune protection conferred by vaccination with attenuated live SARS-CoV-2 containing the new polynucleotide construct is as good as the immune protection conferred by vaccination with previously described attenuated live SARS-CoV-2 (Trimpert et al., 2021a; Trimpert et al., 2021b), but the biosafety of the new construct is significantly higher.

[0027] The entry of SARS-CoV-2 into host cells is mediated by its major surface protein, the spike protein. The spike protein initiates infection by binding to its cellular receptor, angiotensin-converting enzyme 2 (ACE2), and enables actual cell entry through fusion between the viral envelope and the host cell membrane. To enable infection, the spike protein must be activated by cellular proteases. Activation involves proteolytic cleavage of the spike protein near the midpoint of the protein at the S1 / S2 site to produce two subunits, S1 and S2, which are held together by non-covalent interactions. Cleavage of the spike protein induces a conformational change that enables the S1 subunit to bind to ACE2 through its receptor binding domain and triggers the fusion activity of the membrane-anchored S2 subunit.

[0028] Unlike other closely related viruses, SARS-CoV-2 contains a unique polytomous cleavage motif (PRRA↓) at the S1 / S2 site, which is called the furin cleavage site (FCS). Although several enzymes can cut FCS, it is most effectively cut by transmembrane protease serine 2 (TMPRSS S2) (cell surface trypsin-like protease) (Hoffmann and Pohlmann, 2021). The TMPRSS2 protease determines the entry pathway of the virus (Koch et al., 2021). When host cells express TMPRSS2, the virus is activated at the cell surface and rapidly enters the cell through cell fusion in a pH-independent manner. In contrast, if TMPRSS2 is not present, the virus is endocytosed and viral entry is mediated by cathepsin L (endosome / lysosome cysteine ​​protease).

[0029] In order to prevent the spread of vaccine virus sCPD9, the inventors deleted FCS from its spike protein. Preclinical studies have shown that removing FCS renders mutant viruses non-transmissible and strongly attenuated (Johnson et al., 2021; Lau et al., 2020; Peacock et al., 2021; Sasaki et al., 2021a). However, since removing FCS can enhance viral attenuation, combining it with different attenuation mutations can result in over-attenuated viruses with limited ability to induce strong immunity. Therefore, it is important to compare the transmissibility and protective efficacy of LAV candidates lacking FCS with those with intact FCS. On the other hand, if removing FCS does not impair the protective effect of LAV candidates, then removing FCS is desirable because it improves the safety of LAV candidates by introducing a second and independent attenuation mutation into the viral genome.

[0030] In addition, in addition to eliminating transmission and improving vaccine safety, removing FCS has potentially important practical advantages for the production of SARS-CoV-2 LAV. During proliferation in cells that do not express TMPRSS2 (such as Vero cells commonly used by vaccine manufacturers), SARS-CoV-2 variants lacking functional FCS quickly become dominant because they outperform variants with complete FCS (Davidson et al., 2020; Klimstra et al., 2020; Lau et al., 2020; Liu et al., 2020; Ogando et al., 2020; Sasaki et al., 2021b; Wong et al., 2021). Consistent with these reports, the inventors found that sCPD9 also rapidly lost its FCS when it proliferated on cells that do not express TMPRSS2. In contrast, removing FCS improves the genetic stability of vaccine viruses during production and also increases viral titers on TMPRSS2-deficient cell lines.

[0031] In one embodiment, the polynucleotide encodes nonstructural protein 7. In one embodiment, the polynucleotide encodes nonstructural protein 8. In one embodiment, the polynucleotide encodes nonstructural protein 9. In one embodiment, the polynucleotide encodes nonstructural protein 10. In one embodiment, the polynucleotide encodes nonstructural protein 11. In one embodiment, the polynucleotide encodes nonstructural protein 12. In one embodiment, the polynucleotide encodes endonuclease nsp15. In one embodiment, the polynucleotide encodes 2'-O-methyltransferase nsp16. In one embodiment, the polynucleotide encodes spike protein (sometimes also referred to as spike glycoprotein).

[0032] In one embodiment, the polynucleotide encodes the spike protein and at least one non-structural protein.

[0033] In one embodiment, the polynucleotide encodes at least two of the nonstructural proteins. As an example, in one embodiment, the polynucleotide encodes an endoribonuclease and a 2'-O-methyltransferase. As another example, in one embodiment, the polynucleotide encodes nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, and nonstructural protein 11.

[0034] In one embodiment, the furin cleavage site modification is a partial or complete deletion of the furin cleavage site naturally present in the SARS-CoV-2 genome.

[0035] In one embodiment, the furin cleavage site modification is a loss-of-function mutation of the furin cleavage site naturally present in the SARS-CoV-2 genome.

[0036] In one embodiment, the furin cleavage site modification is at least a partial replacement of a furin cleavage site naturally present in the SARS-CoV-2 genome.

[0037] In one embodiment, the SARS-CoV-2 genome is a portion of the genome extending from position 11,000 to position 27,000 of the SARS-CoV-2 genome. For the position numbering and definition of the terms "SARS-CoV-2 genome" and "wild-type SARS-CoV-2", reference is made to GeneBank Accession No. MT108784.1 (available free of charge via the website https: / / www.ncbi.nlm.nih.gov / genbank / ), which contains 29,891 bases or nucleotides. The first of these bases or nucleotides (at the 5' end) is located at position 1. The last of these bases or nucleotides (at the 3' end) is located at position 29,891. The skilled person knows how to adjust the numbering of the reference sequence of the embodiment, wherein the SARS-CoV-2 genome is understood to be a sequence from different SARS-CoV-2 variants. In some embodiments, the polynucleotide of the present invention is a codon pair deoptimization sequence of a sequence contained in a portion of the SARSCoV-2 genome from position 11,000 to position 24,000. In one embodiment, the genome portion extends from the 11th,500th to the 26th,000th, particularly from the 11th,900th to the 25th,500th, particularly from the 11th,950th to the 25th,350th, particularly from the 12th,000th to the 24th,000th. In one embodiment, the genome portion extends from the 11th,950th to the 14th,400th. In one embodiment, the genome portion extends from the 11th,900th to the 13th,500th. In one embodiment, the genome portion extends from the 13th,900th to the 14th,400th. In one embodiment, the genome portion extends from the 20th,300th to the 21st,600th. In one embodiment, the genome portion extends from the 24th,300th to the 25th,400th. These embodiments can be combined in any desired manner.

[0038] In one embodiment, the length of at least one sequence portion comprising codon pair deoptimization is in the range of 750 nucleotides to 2500 nucleotides, in particular 800 nucleotides to 2400 nucleotides, in particular 900 nucleotides to 2300 nucleotides, in particular 999 nucleotides to 2200 nucleotides, in particular 1000 nucleotides to 2100 nucleotides, in particular 1100 nucleotides to 2000 nucleotides, in particular 1146 nucleotides to 1900 nucleotides, in particular 1200 nucleotides to 1836 nucleotides, in particular 1300 nucleotides to 1800 nucleotides, in particular 1400 nucleotides to 1700 nucleotides, in particular 1500 nucleotides to 1600 nucleotides.

[0039] In one embodiment, 15% to 40%, in particular 20% to 35%, and in particular 25% to 30% of the nucleotides of at least one sequence portion comprising codon pair deoptimization are different from the nucleotides of the corresponding (wild-type) SARS-CoV-2 genome. Such a wild-type SARS-CoV-2 genome is a genome sequence of a non-artificially modified virus variant or lineage, such as lineage B.1.1.7 (alpha), B.1.351 (beta), B.1.1.28.1 (gamma), B.1.617.2 (delta) or B.1.159.1 (Omicron), including any subvariants, such as Omicron subvariants BA.1, BA.2, BA.3, BA.4, BA.5. It can also be represented as a true SARS-CoV-2 genome or a true SARS-CoV-2 genome sequence.

[0040] In one embodiment, 200 to 500 nucleotides, particularly 250 to 450 nucleotides, in particular 300 to 400 nucleotides of at least one sequence portion comprising codon pair deoptimization differ from (particularly identically positioned) nucleotides of the corresponding SARS-CoV-2 genome.

[0041] In one embodiment, 40% to 70%, particularly 45% to 65%, particularly 50% to 60%, particularly 55% to 62% of the codons of at least one sequence portion comprising codon pair deoptimization differ from the corresponding codons of the corresponding SARS-CoV-2 genome.

[0042] In one embodiment, 150 to 400 codons, particularly 200 to 350 codons, in particular 250 to 300 codons of at least one sequence portion comprising codon pair deoptimization are different from (particularly identically positioned) codons of the corresponding SARS-CoV-2 genome.

[0043] In one embodiment, at least one sequence portion comprising a codon pair deoptimization comprises a first deoptimized sequence portion and a second deoptimized sequence portion. The two deoptimized sequence portions are separated from each other by a non-deoptimized sequence portion, the non-deoptimized sequence portion comprising at least 300 nucleotides, for example 300 to 1000 nucleotides, in particular 400 to 900 nucleotides, in particular 500 to 800 nucleotides, in particular 600 to 700 nucleotides. By preserving specific portions of the RNA sequence and by deoptimizing the flanking portions upstream and downstream of the conserved RNA sequence, a particularly high efficacy in attenuating SARS-CoV-2 is achieved while maintaining the replication capacity of SARS-CoV-2.

[0044] In one embodiment, the length of the first deoptimized sequence portion is in the range of 1300 nucleotides to 1600 nucleotides, in particular 1400 nucleotides to 1500 nucleotides, in particular 1450 nucleotides to 1490 nucleotides. At the same time, the length of the second deoptimized sequence portion is in the range of 100 nucleotides to 400 nucleotides, in particular 200 nucleotides to 300 nucleotides, in particular 350 nucleotides to 400 nucleotides. If desired, the lengths of the first deoptimized sequence portion and the second deoptimized sequence portion are selected so that other applicable restrictions are met (e.g., the total length of at least one sequence portion including codon pair deoptimization does not exceed 2000 nucleotides). If the length of the at least one sequence portion comprising a codon pair deoptimization does not exceed 2000 nucleotides, taking into account that the first deoptimized sequence portion and the second deoptimized sequence portion are separated by at least 300 nucleotides of the authentic SARS-CoV-2 genome, it is immediately obvious that a lower threshold of only 1300 nucleotides can be combined with a higher threshold of 400 nucleotides for the first and second deoptimized sequence portions to meet the restriction on the maximum length of the at least one sequence portion comprising a codon pair deoptimization. At the same time, taking into account the intervening 300 non-recoded nucleotides, a higher threshold of 1600 nucleotides for the first deoptimized sequence portion can be combined with a lower threshold of 100 nucleotides for the second deoptimized sequence portion to meet the maximum length of 2000 nucleotides.

[0045] In one embodiment, the first deoptimized sequence portion is at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identical to SEQ ID NO. 2. At the same time, the second deoptimized sequence portion is at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identical to SEQ ID NO. 4.

[0046] In one embodiment, the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO.6.

[0047] In one embodiment, the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO.8.

[0048] In one embodiment, the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO.10.

[0049] In one embodiment, the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO.15.

[0050] In one embodiment, the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO.16.

[0051] In one embodiment, the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO.17.

[0052] Although FCS deletions in the SARS-CoV-2 genome are described in the prior art, it cannot be assumed that such deletions have the effects observed by the inventors when such deletions (or at least FCS modifications) are combined with specific codon pair deoptimizations present in the above sequences. Quite unexpectedly, by combining codon pair deoptimization with modification (particularly deletion) of FCS, the immunoprotective properties of these codon pair deoptimization sequences were not compromised.

[0053] In one embodiment, the furin cleavage site modification comprises the deletion of nucleotides encoding the amino acid sequence XRRA (i.e., furin cleavage site), wherein X represents P, R, or H. Table 1 below lists some specific embodiments of the presently claimed subject matter, which relate to different amino acid sequences that are deleted in the expressed protein due to the deletion of the furin cleavage site. It should be noted that it is not very relevant which nucleotides are excised from the SARS-CoV-2 genome by the deletion of the furin cleavage site, as long as the furin cleavage site is no longer present in the resulting protein. Table 1: Some embodiments of amino acids deleted due to deletion of the furin cleavage site.

[0054] In one embodiment, the furin cleavage site is modified, particularly deleted, so that the expression of the polynucleotide produces a protein, particularly a spike protein, wherein at least or exactly 5, particularly at least or exactly 6, particularly at least or exactly 7, particularly at least or exactly 8, particularly at least or exactly 9, particularly at least or exactly 10, particularly 5 to 10, particularly 6 to 9, particularly 7 to 8 consecutive amino acids of the naturally expressed protein are replaced by a single amino acid. This can be achieved by deletions outside the reading frame of the nucleotides. By such deletions outside the reading frame, a single nucleotide of the first triplet is combined with two nucleotides of the second triplet to form a new triplet that is not present in the natural SARS-CoV-2 genome at the genomic position.

[0055] In one embodiment, the single amino acid replacing the naturally expressed contiguous amino acids is isoleucine.

[0056] In one embodiment, the furin cleavage site modification consists of or comprises a deletion of a nucleic acid sequence as defined by SEQ ID NO. 18 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO. 18.

[0057] In one aspect, the present invention relates to attenuated SARS-CoV-2. The attenuated SARS-CoV-2 comprises a partially recoded genomic RNA sequence, i.e., a partially recoded genomic viral sequence. The partially recoded genomic RNA sequence is a codon pair deoptimization sequence encoding a spike protein and / or a specific non-structural protein (nsp). The non-structural protein is selected from non-structural protein 7, non-structural protein 8, non-structural protein 9, non-structural protein 10, non-structural protein 11 (a small protein with only 13 amino acids), non-structural protein 12 (also known as RNA-dependent RNA polymerase), non-structural protein 15 (endoribonuclease) and non-structural protein 16 (2'-O-methyltransferase) of attenuated SARS-CoV-2. The attenuated SARS-CoV-2 also comprises a furin cleavage site modification that causes the furin cleavage site to lose its function. The furin cleavage site is naturally present in the SARS-CoV-2 genome. As a result, the spike protein of the live attenuated SARS-CoV-2 does not contain a functional furin cleavage site, while the unmodified SARS-CoV-2 contains such a furin cleavage site.

[0058] In one embodiment, the partially recoded genomic RNA sequence encodes nonstructural protein 12. In one embodiment, the partially recoded genomic sequence encodes a spike protein (sometimes also referred to as a spike glycoprotein).

[0059] In one embodiment, the partially recoded genomic RNA sequence comprises at least two of the nonstructural proteins. As an example, in one embodiment, the partially recoded genomic RNA sequence encodes an endoribonuclease and a 2'-O-methyltransferase. As another example, in one embodiment, the partially recoded genomic RNA sequence encodes nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, and nonstructural protein 11.

[0060] In one embodiment, the partially recoded genomic RNA sequence is located in the portion of the genome extending from position 11,000 to position 27,000 of the attenuated SARS-CoV-2 genome. According to the GenBank accession number MT108784.1, the genome of the wild-type SARS-CoV-2 contains 29,891 bases or nucleotides. The genome of the attenuated SARS-CoV-2 is essentially of similar length. The difference is that the length of the polyA tail at the 3' end determined by sequencing is eight adenine nucleotides longer than that of the wild-type SARS-CoV-2. It should be noted that there is still some uncertainty in determining the length of the polyA tail by sequencing. Therefore, the polyA tail in the wild-type sequence may be longer or shorter than shown in the sequence according to the GenBank accession number MT108784.1. Similarly, the polyA tail in the attenuated SARS-CoV-2 sequence may be longer or shorter than currently determined. Another difference is that the attenuated SARS-CoV-2 lacks at least 12 nucleotides, which encode the furin protease cleavage site in the wild-type SARS-CoV-2.

[0061] The first of the 29,891 bases or nucleotides of the wild-type SARS-CoV-2 genome (at the 5' end) is located at position 1. The last of these bases or nucleotides (at the 3' end) is located at position 29,891. In one embodiment, the genome portion extends from position 11,500 to position 26,000, in particular from position 11,900 to position 25,500, in particular from position 11,950 to position 25,350, in particular from position 12,000 to position 24,000. In one embodiment, the genome portion extends from position 11,950 to position 14,400. In one embodiment, the genome portion extends from position 11,900 to position 13,500. In one embodiment, the genome portion extends from position 13,900 to position 14,400. In one embodiment, the portion of the genome extends from position 20,300 to position 21,600. In one embodiment, the portion of the genome extends from position 24,300 to position 25,400. These embodiments may be combined in any desired manner.

[0062] In one embodiment, the length of the partially recoded genomic RNA sequence is in the range of 750 nucleotides to 2500 nucleotides, in particular 800 nucleotides to 2400 nucleotides, in particular 900 nucleotides to 2300 nucleotides, in particular 999 nucleotides to 2200 nucleotides, in particular 1000 nucleotides to 2100 nucleotides, in particular 1100 nucleotides to 2000 nucleotides, in particular 1146 nucleotides to 1900 nucleotides, in particular 1200 nucleotides to 1836 nucleotides, in particular 1300 nucleotides to 1800 nucleotides, in particular 1400 nucleotides to 1700 nucleotides, in particular 1500 nucleotides to 1600 nucleotides.

[0063] In one embodiment, 15% to 40%, in particular 20% to 35%, and in particular 25% to 30% of the nucleotides of the partially recoded genomic RNA sequence are different from the nucleotides of the corresponding wild-type genomic RNA sequence. Such a wild-type genomic RNA sequence is a genomic viral sequence of a non-artificially modified viral variant or lineage, such as lineage B.1.1.7 (Alpha), B.1.351 (Beta), B.1.1.28.1 (Gamma), B.1.617.2 (Delta) or B.1.159.1 (Omicron). It can also be expressed as a true SARS-CoV-2 genomic RNA sequence.

[0064] In one embodiment, 200 to 500 nucleotides, particularly 250 to 450 nucleotides, in particular 300 to 400 nucleotides of the partially recoded genomic RNA sequence differ from the identically positioned nucleotides of the corresponding wild-type viral genomic RNA sequence.

[0065] In one embodiment, 40% to 70%, in particular 45% to 65%, in particular 50% to 60%, in particular 55% to 62% of the codons of the partially recoded genomic RNA sequence (i.e. the three nucleotides encoding a specific amino acid in each case) differ from the corresponding codons of the corresponding wild-type viral genomic RNA sequence.

[0066] In one embodiment, 150 to 400 codons, particularly 200 to 350 codons, in particular 250 to 300 codons of the partially recoded genomic RNA sequence differ from the codons of the same location of the corresponding wild-type viral genomic RNA sequence.

[0067] In one embodiment, the partially recoded genomic RNA sequence comprises a first recoded portion and a second recoded portion. The two recoded portions are separated from each other by a non-recoded genomic portion comprising at least 300 nucleotides, such as 300 to 1000 nucleotides, particularly 400 to 900 nucleotides, particularly 500 to 800 nucleotides, particularly 600 to 700 nucleotides. By preserving specific portions of the RNA sequence and by recoding the flanking portions upstream and downstream of the conserved RNA sequence, a particularly high efficacy in attenuating SARS-CoV-2 is achieved while maintaining the overall viability of SARS-CoV-2.

[0068] In one embodiment, the length of the first recoded portion is in the range of 1300 nucleotides to 1600 nucleotides, in particular 1400 nucleotides to 1500 nucleotides, in particular 1450 nucleotides to 1490 nucleotides. At the same time, the length of the second recoded portion is in the range of 100 nucleotides to 400 nucleotides, in particular 200 nucleotides to 300 nucleotides, in particular 350 nucleotides to 400 nucleotides. If desired, the lengths of the first recoded portion and the second recoded portion are selected so that other applicable restrictions are met (e.g. the total length of the recoded genomic RNA sequence does not exceed 2000 nucleotides). If the length of the partially recoded genomic RNA sequence does not exceed 2000 nucleotides, it is immediately obvious that a lower threshold of only 1300 nucleotides for the first and second recoded portions can be combined with a higher threshold of 400 nucleotides to meet the restriction on the maximum length of the partially recoded genomic RNA sequence, taking into account that the first recoded portion and the second recoded portion are separated by at least 300 nucleotides of the authentic SARS-CoV-2 genome sequence. At the same time, taking into account the middle 300 non-recoded nucleotides, the higher threshold of 1600 nucleotides for the first recoded portion can be combined with the lower threshold of 100 nucleotides for the second recoded portion to meet the maximum length of 2000 nucleotides.

[0069] In one embodiment, the first recoded portion is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO. 2. At the same time, the second recoded portion is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO. 4.

[0070] In one embodiment, the partially recoded genomic RNA sequence is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO.6.

[0071] In one embodiment, the partially recoded genomic RNA sequence is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO.8.

[0072] In one embodiment, the partially recoded genomic RNA sequence is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO.10.

[0073] The phrase "% identity" or "having a percentage (%) sequence identity" relative to a reference sequence is defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to those in a reference sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0074] The skilled person knows that the sequence as exemplified herein can be changed to a certain percentage, and does not change or does not change biological function, for example attenuation or the protein of coding substantially.Therefore, the skilled person using the means and methods described herein can modify the codon pair according to the teaching of the present invention.For example, the codon pair can be replaced with the deoptimization codon of sequence described herein similarly attenuation and / or the synonymous version of natural underrepresentation.Therefore, the deoptimization of similar degree can be realized by replacing the codon in the positional range described herein.

[0075] The present invention is based at least in part on the discovery that codon inversion within the range of positions described herein is particularly useful for attenuating the SARS-CoV-2 virus while fully maintaining the immunogenicity and replication capacity of the virus. It is also based on the discovery that furin cleavage site modifications that result in loss of function of the furin cleavage site naturally present in the SARS-CoV-2 genome further attenuate SARS-CoV-2 while fully maintaining its immunogenicity.

[0076] The recoded sequences and their locations in the SARS-CoV-2 genome are summarized in Table 2 below. Table 2: Summary of recoded RNA sequences. pp = polyprotein nsp = nonstructural protein

[0077] It should be noted that WT6A / CPD6A encodes only nine nucleotides (3 amino acids) of nsp12 (RNA-dependent RNA polymerase, RdRp (RNA-dependent RNA polymerase)). The majority of this protein is encoded by WT6B / CPD6B.

[0078] The middle part of fragment WT6 is not recoded in fragment CPD6 because it contains a conservative regulatory RNA sequence that is essential for viral replication. Fragment WT6 encodes a -1 ribosomal frameshift element (so-called RNA pseudoknot structure). This structure promotes ribosomal frameshifting, and during this process, the reading frame of translation changes at the junction between open reading frame (open reading frame, ORF) 1a and 1b. During this process, a single nucleotide in the slippery sequence (slippery sequence) located downstream of the RNA pseudoknot structure is read twice by the translating ribosome, and the reading frame is moved -1 nucleotide (the ribosome slides one nucleotide backward at the slippery sequence). Usually, the translation of ORF1a terminates at the stop codon of ORF1a. However, when -1 ribosomal frameshifting occurs, the translation of ORF1a continues directly to ORF1b, and produces polyprotein (polyprotein, pp) 1ab. Thus, the CPD6A sequence is translated into both polyproteins 1a and 1ab, but the CPD6B sequence is only translated into polyprotein 1ab.

[0079] The differences between the CPD sequences and the underlying wild-type sequence are summarized in Table 3 below. Table 3: Summary of differences between CPDs and wild-type sequences.

[0080] It should be noted that the primary structure of the protein encoded by the CPD RNA sequence is identical to that of the wild-type (original) RNA sequence. As explained above, CPD does not change the primary structure of the resulting protein. This is summarized in Table 4. Table 4: Summary of protein sequences.

[0081] In one embodiment, by recoding the sequence parts CPD6A, CPD6B, sCPD9 and sCPD10, different fragments of the SARS-CoV-2 genome are generated, which form part of the attenuated live SARS-CoV-2. These fragments are listed in Table 5 below. Table 5: Genome segments of SARS-CoV-2 containing recoded sequence portions.

[0082] In one aspect, the present invention relates to a live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a polynucleotide according to the above description.

[0083] In one embodiment, the attenuated live SARS-CoV-2 has a nucleic acid sequence that is at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO. 19. In addition to the codon pair deoptimization region, the attenuated live SARS-CoV-2 comprises a 24-nucleotide deletion of the furin protease cleavage site in the spike protein gene, resulting in the amino acid sequence NSPRRARSV (SEQ ID NO. 32) containing the furin protease cleavage site in the spike glycoprotein being replaced by the amino acid isoleucine.

[0084] In one embodiment, SARS-CoV-2 has a nucleic acid sequence that is at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO. 20. In addition to the codon pair deoptimization region, the attenuated live SARS-CoV-2 also comprises a 24-nucleotide deletion of the furin protease cleavage site in the spike protein gene, resulting in the amino acid sequence NSPRRARSV (SEQ ID NO. 32) containing the furin protease cleavage site in the spike glycoprotein being replaced by the amino acid isoleucine.

[0085] In one aspect, the present invention relates to a pharmaceutical composition comprising a live attenuated SARS-CoV-2 according to any of the previous descriptions. Such a pharmaceutical composition may also contain auxiliary substances such as adjuvants, for example for enhancing the patient's immune response. Suitable adjuvants are potassium alum; aluminum hydroxide; aluminum phosphate; calcium phosphate hydroxide; hydroxyaluminum phosphate sulfate; paraffin oil; propolis; inactivated bacteria of the species Bordetella pertussis or Mycobacterium bovis; plant saponins from Quillaja, soybeans and / or Polygala senega; cytokines IL-1, IL-2 and / or IL-12; and Freund's complete adjuvant.

[0086] In one aspect, the present invention relates to the further medical use of such pharmaceutical composition as a vaccine.

[0087] In one aspect, the present invention relates to the further medical use of such a pharmaceutical composition as a vaccine for immunosuppressed individuals, in particular for individuals receiving glucocorticoid therapy, such as dexamethasone therapy.

[0088] In one aspect, the invention relates to a method for preparing a vaccine from such a pharmaceutical composition.

[0089] In one aspect, the present invention relates to a method for vaccinating a human or animal patient in need thereof. The animal patient is in particular a non-human mammal, such as a rodent, canine, feline or mustelid. The method comprises the step of administering to the patient a pharmaceutical composition according to the previous description.

[0090] In one embodiment, the human or animal patient is an immunosuppressed patient, particularly a patient receiving glucocorticoid therapy, such as dexamethasone therapy.

[0091] In one embodiment, administration is by intranasal administration, oral administration, parenteral administration such as subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intravenous infusion or intraperitoneal infusion. Intranasal or oral administration is particularly suitable. Through these administration routes, the attenuated live SARS-CoV-2 is presented to the patient to be vaccinated in the same or similar manner as natural exposure to the virus.

[0092] In one embodiment, vaccination is performed by administering 1×10 3 Up to 1×10 8 focus-forming unit (FFU), especially 1×104 Up to 1×10 7 FFU, especially 1×10 5 Up to 1×10 6 The dosage is carried out by administering the pharmaceutical composition at a dose of 100 FFU. The dosage is selected so that the pharmaceutical composition is well tolerated by the patient, but causes an immune response that protects the patient from SARS-CoV-2 infection or a severe course of infection with SARS-CoV-2. In one embodiment, the dosage is one of the minimum protective dose and the highest tolerable dose, or between the minimum protective dose and the highest tolerable dose.

[0093] A variety of factors can influence the dosage for a particular application. For example, the frequency of administration, duration of treatment, preventive or therapeutic purpose, use of multiple therapeutic agents, route of administration, previous treatment, the patient's clinical history, the judgment of the attending physician, and the severity of the disease, disorder and / or condition can influence the desired dosage to be administered.

[0094] As with dosage, a variety of factors can influence the actual frequency of administration for a particular application. For example, dosage, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the disease, disorder and / or condition may require increased or decreased frequency of administration.

[0095] In some cases, the effective duration of administration of the pharmaceutical compositions of the invention (and any additional therapeutic agents) can be any duration that reduces the severity or occurrence of symptoms of the disease, disorder, and / or condition to be treated without causing significant toxicity to the subject. Various factors can affect the actual effective duration for a particular treatment. For example, the effective duration can vary with the frequency of administration, effective amount, use of multiple therapeutic agents, route of administration, and severity of the disease, disorder, and / or condition being treated.

[0096] In one embodiment, the pharmaceutical preparation is administered to the patient at least twice, wherein the second administration is separated from the first administration by a first time period. In this context, the first time period is in the range of 2 weeks to 36 months, in particular 3 weeks to 30 months, in particular 4 weeks to 24 months, in particular 5 weeks to 21 months, in particular 6 weeks to 18 months, in particular 7 weeks to 15 months, in particular 8 weeks to 12 months, in particular 9 weeks to 10 months, in particular 10 weeks to 8 months, in particular 12 weeks to 6 months, in particular 13 weeks to 4 months.

[0097] In one embodiment, the administration of the pharmaceutical preparation to the patient is offset in time from the administration of different vaccines (e.g., such as a carrier-based vaccine, an mRNA-based vaccine, a protein-based vaccine) to the patient, i.e., after or before the patient is vaccinated with different vaccines. In this context, the administration of the pharmaceutical composition is offset from the administration of different vaccines by a second time period. In this context, the second time period is in the range of 2 weeks to 36 months, particularly 3 weeks to 30 months, particularly 4 weeks to 24 months, particularly 5 weeks to 21 months, particularly 6 weeks to 18 months, particularly 7 weeks to 15 months, particularly 8 weeks to 12 months, particularly 9 weeks to 10 months, particularly 10 weeks to 8 months, particularly 12 weeks to 6 months, particularly 13 weeks to 4 months.

[0098] In one aspect, the invention relates to a vector comprising a polynucleotide according to the above description.

[0099] The term "vector" as used herein refers to a nucleic acid molecule capable of transferring or transporting itself and / or another nucleic acid molecule to a cell. The transferred nucleic acid is usually connected to a carrier nucleic acid molecule, i.e., inserted into the carrier nucleic acid molecule. The vector may be included in a sequence that directs autonomous replication in the cell, or may include a sequence that is sufficient to allow integration into the host cell DNA. In some embodiments, the vector described herein is selected from the following vectors: plasmids (e.g., DNA plasmids or RNA plasmids), shuttle vectors, transposons, cosmids, artificial chromosomes (e.g., bacteria, yeast, humans), and viral vectors.

[0100] In some embodiments, the vectors described herein are used in combination with at least one transfection enhancing agent, such as a transfection enhancing agent selected from the group consisting of oligonucleotides, lipoplexes, polymers, polyplexes, dendrimers, inorganic nanoparticles, and cell penetrating peptides.

[0101] In one aspect, the invention relates to a host cell comprising a polynucleotide according to the above description.

[0102] The term "host cell" as used herein refers to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived from primary transformed cells regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the original transformed cell are included herein.

[0103] In one embodiment, the host cell described herein comprises at least one cell type selected from the group consisting of Chinese hamster ovary (CHO), Vero, Vero E6, Vero TMPRSS, MRC 5, Per.C6, PMK, WI-38, and baby hamster kidney fibroblasts (BHK cells).

[0104] In one aspect, the present invention relates to a method for producing a virus. The method comprises the steps of: a) culturing a host cell according to the preceding paragraph; and b) isolating the virus, wherein the virus is a live attenuated SARS-CoV-2.

[0105] All embodiments of the polynucleotides can be combined in any desired manner and can be transferred to live attenuated SARS-CoV-2, pharmaceutical compositions, uses thereof, methods of vaccinating patients, vectors, host cells, and methods of producing viruses, alone or in any combination. All embodiments of live attenuated SARS-CoV-2 can be combined in any desired manner and can be transferred to polynucleotides, pharmaceutical compositions, uses thereof, methods of vaccinating patients, vectors, host cells, and methods of producing viruses, alone or in any combination. Similarly, all embodiments of pharmaceutical compositions can be combined in any desired manner and can be transferred to polynucleotides, live attenuated SARS-CoV-2, uses of pharmaceutical compositions, methods of vaccinating patients, vectors, host cells, and methods of producing viruses, alone or in any combination. All embodiments of the uses of pharmaceutical preparations can be combined in any desired manner and can be transferred to polynucleotides, live attenuated SARS-CoV-2, pharmaceutical preparations, methods of vaccinating patients, vectors, host cells, and methods of producing viruses, alone or in any combination. Finally, all embodiments of the method for vaccinating a patient can be combined in any desired manner and can be transferred to polynucleotides, live attenuated SARS-CoV-2, pharmaceutical preparations, uses of pharmaceutical preparations, vectors, host cells, and methods of producing viruses, alone or in any combination.

[0106] Further details of aspects of the present invention will be described below with reference to exemplary embodiments and accompanying drawings. In the drawings:

[0107] Figures 1A to 1D Schematic depiction of the native structure and exemplary recoding of the SARS-CoV-2 genome;

[0108] Figure 2 The growth kinetics of sCPD9-ΔFCS and sCPD9 SARS-CoV-2 in Vero E6 cells are shown;

[0109] Figures 3A to 3E Virological and histopathological findings in exposed hamsters are shown;

[0110] Figure 4A and 4B Clinical parameters of infected Syrian hamsters are shown;

[0111] Figures 5A to 5E Shown are clinical, virological, pathological, and serological findings in hamsters following challenge infection with the SARS-CoV-2 delta variant;

[0112] Figures 6A to 6I The setup, results, and biological background of experiments related to in vivo and in vitro co-infection with sCPD9-ΔFCS and BA.5 are shown;

[0113] Figures 7A to 7J The setup and results of experiments showing the effect of immunosuppression on the safety, efficacy and spread of sCPD9-ΔFCS;

[0114] Figures 8A to 8F Virological results of contact hamsters vaccinated with sCPD9-ΔFCS for B.1 and BA.5 infection are shown. Fig. 8E and 8F The following descriptions of the symbols used are for Figures 8A to 8D It is also effective;

[0115] Figures 9A to 9D Systemic and mucosal immunity of vaccinated contact animals was shown, with Fig. 9C and 9D The following descriptions of the symbols used are for Fig.9A and 9B It is also effective;

[0116] Figures 10A to 10C Clinical and virological outcomes of primary contacts of vaccinated B.1 shedders are shown. Fig. 10C The symbols used are shown next to the Fig. 10A and 10B It is also effective;

[0117] Figures 11A to 11C Clinical and virological outcomes of primary contacts of vaccinated B.5 transmitters are shown, with Fig. 11C The description of the symbols used shown below is also valid for Figures 1 1A and 1 1B; and

[0118] Figures 12A to 12C Shows systemic and mucosal immunity in naive animals, where Fig. 12C The following descriptions of the symbols used are for Fig. 12A and 12B It is also effective. Some exemplary embodiments

[0119] The inventors generated a series of recoded SARS-CoV-2 mutants, characterized them in cultured cells and also in vivo using Syrian and Roborovski hamster models. A single dose of intranasal immunization with live attenuated virus in a robust small animal model of COVID-19 was demonstrated to elicit a strong immune response and provide complete protection against SARS-CoV-2 challenge. Vaccine design

[0120] The inventors aim to generate attenuated SARS-CoV-2 vaccine candidates by large-scale recoding of the SARS-CoV-2 genome by CPD (Eschke et al., 2018; Groenke et al., 2020; Khedkar et al., 2018; Kunec and Osterrieder, 2016). To achieve viral attenuation in humans, the inventors recoded the genome of SARS-CoV-2 with the most underrepresented codon pairs in human genes (Groenke et al., 2020). Genetically modified SARS-CoV-2 mutants were generated using a recently established SARS-CoV-2 reverse genetics system (Thi NhuThao et al., 2020) ( Figures 1A to 1D ). The system relies on 12 subgenomic fragments of the SARS-CoV-2 genome, which are assembled into a single yeast / bacterial artificial chromosome (YAC / BAC) by transformation-associated recombination (TAR) cloning in Saccharomyces cerevisiae (Noskov et al., 2002). The subgenomic fragments are about 3000bp long, and adjacent fragments overlap each other by about 300bp to enable assembly of SARS-CoV-2 infectious clones by homologous recombination. The applied vaccine design and development are described in more detail in Trimpert et al., 2021a and Trimpert et al., 2021b.

[0121] In this context, Figures 1A to 1D The structure and recoding of the SARS-CoV-2 genome are shown.

[0122] Figure 1A The SARS-CoV-2 genome is shown to be a single-stranded, positive-sense RNA molecule of approximately 30,000 nucleotides (nt) encoding 11 typical ORFs. "3CL-Pro" indicates 3C-like protease; "RdRp" indicates RNA-dependent RNA polymerase; "ExoN" indicates 3' to 5' exoribonuclease; "EndoRNAse" indicates endoribonuclease; and "2'-O-MT" indicates 2'-O-ribose methyltransferase.

[0123] like Figure 1B As shown, following infection, ORF 1a / 1ab is directly translated and cleaved into 15 proteins of the replication-transcription complex.

[0124] like Figure 1C As shown, recoded SARS-CoV-2 mutants were constructed using a recently established SARS-CoV-2 reverse genetics system consisting of 12 subgenomic segments. Segments 1, 11, and 12 were not recoded. Dark gray boxes represent recoded sequences CPD2 to 10, and light gray boxes represent parental, non-recoded sequences in each segment 2 to 10. The frameshift element contained in segment 6 and the transcription regulatory sequence (TRS) of the spike gene in segment 9 were excluded from the recoding process (the light gray box exists between the two dark gray boxes in CPD6 and CPD9).

[0125] like Figure 1D As shown, dark grey boxes indicate the recoded sequences sCPD3 to 5 and sCPD8 to 10 in different subgenomic fragments.

[0126] To maintain full compatibility with available reverse genetics systems, the inventors recoded only the SARS-CoV-2 sequences that were not present in the overlapping portions of the subgenomic fragments (approximately 2,500 bp in each recoded fragment) ( Figure 1A To ID). This design enabled the inventors to generate a wide variety of SARS-CoV-2 mutants carrying single or multiple recoded segments.

[0127] The inventors recoded nine segments (segments 2 to 10) of the SARS-CoV-2 reverse genetics system. Relatively short segments 1 and 12 of 591 and 1,812 bp, respectively, and segment 11 containing many short ORFs were excluded from recoding. In order to ensure that the mutant virus has replication ability, two genomic regions containing essential cis-acting RNA elements were excluded from recoding: the frameshift element carried by segment 6 and the transcriptional regulatory sequence (TRS) of the spike gene in segment 9. In addition, the first 500 bp of ORF 1a located in segment 2 were not recoded. Materials and Methods Study design and animal husbandry

[0128] This exemplary embodiment aims to determine the effect of removing the furin cleavage site (FCS) of sCPD9 on the spread of vaccine viruses, their immunogenicity, protective efficacy and inter-host transmission. Compared with WT (B.1) SARS-CoV-2 and sCPD9 SARS-CoV-2, the transmissibility of sCPD9-ΔFCS (i.e., SARS-CoV-2 with sCPD9 sequence and furin cleavage site deletion) was studied. In addition, the potential recombination between the SARS-CoV-2 variant Omicron BA.5 and the vaccine candidate sCPD9-ΔFCS virus was evaluated in a co-infection experiment. In addition, the safety and transmissibility of the sCPD9-ΔFCS vaccine candidate were studied in immunosuppressed hamsters.

[0129] Syrian hamsters (Mesocricetus auratus strain RjHan: AURA) were purchased from Janvier Labs and housed in pairs in individual ventilated cages. Food and water were provided ad libitum, and the cages were filled with nesting material. The room temperature was maintained at a constant range of 22°C to 24°C, and the relative humidity was 40% to 55%. Before the start of the experiment, the animals were adapted to the housing conditions for seven days.

[0130] Thirty-six 10-week-old male and female Syrian hamsters were used to evaluate the transmission of B.1, sCPD9, and sCPD9-ΔFCS viruses from infected to naive animals.

[0131] Half of each animal received 1×10 5FFU WT (B.1), sCPD9 or sCPD9-ΔFCS. Immediately after WT infection or sCPD9 / sCPD9-ΔFCS vaccination, hamsters were kept in separate cages and reunited with their uninfected / unvaccinated conspecifics one day after infection / vaccination. The weight of all hamsters was recorded daily, and clinical status was checked twice a day. From 1 to 6 dpc (days after contact), oral swabs were collected from contact hamsters every day. Contact animals were euthanized on 6 dpc to assess viral loads in the upper and lower respiratory tracts, signs of pneumonia, and seroconversion.

[0132] WT-infected and sCPD9 / sCPD9-ΔFCS-vaccinated animals were treated with 1×10 5 The hamsters were challenged with 1 PFU of SARS-CoV-2 delta variant. In addition, for comparison purposes, a group of 6 uninfected / unvaccinated hamsters were challenged with delta variant. Hamsters were then euthanized at 2 and 5 dpch (days post-challenge) to collect blood, trachea and lungs for virology, serology and histopathology analysis.

[0133] To investigate the possibility of recombination between the SARS-CoV-2 variant Omicron BA.5 and sCPD9-ΔFCS, twelve 4-week-old female Syrian hamsters were used for co-infection experiments. After acclimation, six hamsters were co-infected with 1 × 10 4 FFU of sCPD9-ΔFCS and 1×10 4 The SARS-CoV-2 variant Omicron BA.5 was inoculated with 100 FFU of SARS-CoV-2. The infection was induced under general anesthesia as described below. Subsequently, the infected animals were isolated in separate cages to prevent accidental transmission of the viral inoculum to the original animals. After one day, they were co-housed with their original partners again. Body weights and oral swabs were collected from all animals every day to screen for viral transmission. Clinical status was monitored twice a day. After 6 days of co-rearing, the animals were euthanized to collect blood, trachea and lungs for virological analysis.

[0134] To evaluate the safety and transmissibility of the vaccine in immunosuppressed animals, twelve 4-week-old female Syrian hamsters were immunocompromised by daily subcutaneous injections of dexamethasone (2 mg / kg). On the third day of immunosuppression, 1×10 50 μg of the vaccine were administered intranasally under general anesthesia as described below. 4Six hamsters were vaccinated with 100 FFU of sCPD9-ΔFCS vaccine. Vaccinated animals were kept in separate cages for 24 hours and then re-housed with unvaccinated and immunosuppressed individuals of the same species. During the co-feeding period, oral swabs were collected from all hamsters every day to detect potential vaccine virus transmission. Contact animals were euthanized 6 days after contact (dpc), while vaccinated hamsters were killed 21 days after vaccination (dpv). Blood, trachea and lungs were collected for serological, virological and histopathological analysis. cell

[0135] Minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 IU / ml penicillin G and 100 μg / ml streptomycin was used to culture Vero E6 (ATCC CRL-1586) and WHO VeroRCB 10-87 cells. For Vero E6-TMPRSS2 cells (NIBSC 100978), the culture medium additionally contained 1000 μg / ml geneticin (G418) to select cells expressing TMPRSS2. The cells were maintained at 37°C and 5% CO2. Virus

[0136] SARS-CoV-2 variants B.1 (B.1, BetaCoV / Munich / ChVir984 / 2020, hCoV-19 / Germany / BY-ChVir-929 / 2020, EPI ISL 406862), Delta (B.1.617.2, Human, 2021, Germany exIndia, 20A / 452R, EVAg: 009V-04187), and SARS-CoV-2 mutants B.1-ΔFCS, sCPD9, and sCPD9-ΔFCS were cultured on Vero E6-TMPRSS2 cells. SARS-CoV-2 variants Omicron BA.1 (BA.1.18, hCoV-19 / Germany / BE-ChVir26335 / 2021, EPI_ISL_7019047) and BA.5 (BE.1.1, hCoV-19 / Germany / SH-ChVir29057_V34 / 2022, EPI_ISL_16221625) were propagated on CaLu-3 cells. BAC-derived SARS-CoV-2 variant B.1 (GenBank: MT108784) was grown on Vero E6 cells and used for growth kinetics and plaque size assays. The titer of the virus stock was determined by plaque assay on Vero E6 cells and the virus was stored at -80°C before experimental infection. Ethical Statement

[0137] In vivo and in vitro experiments were performed at the Institut für Virologie, Freie The experiments were performed in a biosafety level three (BSL-3) laboratory in Berlin, Germany. Animal work was performed in accordance with institutional, national, and international guidelines for the care and humane use of animals and authorized by the Landesamt für Gesundheit und Soziales, Berlin (license number 0086 / 20). Infection / vaccination

[0138] Hamsters were infected or vaccinated under general anesthesia (0.15 mg / kg medetomidine, 2.0 mg / kg midazolam, and 2.5 mg / kg butorphanol). 5 FFU B.1 (wild type), sCPD9 or sCPD9-ΔFCS were diluted in 60 μl MEM and applied intranasally. Mock-vaccinated individuals received 60 μl plain MEM without virus. 21 days after vaccination or primary infection, 1×10 5 The challenge infection was carried out with 100 FFU of SARS-CoV-2 delta variant. RNA extraction and reverse transcription quantitative PCR (RT-qPCR)

[0139] The genome copies in the oropharyngeal swabs were quantified, and 2.5 mg of lung tissue was homogenized in a bead mill (Analytic Jena). For RNA extraction, the innuPREP viral DNA / RNA kit (Analytic Jena, Jena, Germany) was used according to the manufacturer's instructions. For RT-qPCR, the NEB Luna Universal probe one-step RT-qPCR kit (New England Biolabs) was used. Using primers and probes as reported by Corman et al. (Corman et al., 2020), the following cycling conditions were applied on a qTower G3 cycler (Analytic Jena): 10 minutes of reverse transcription at 55°C, 3 minutes of enzyme activation at 94°C, and 40 cycles of 15 seconds at 94°C and 30 seconds at 58°C. Plaque assay

[0140] In order to quantify the virus with replication ability, 10 times serial dilutions of 50 mg homogenized lung tissue were prepared and plated on Vero E6 cells grown in 12-well plates. After incubating the cells for 2.5 hours at 37 ° C and 5% CO2, the cells were covered with 1.5% sodium carboxymethylcellulose (Sigma Aldrich) diluted in complete growth medium. In order to fix the cells 72 hours after infection, 4% formaldehyde solution (pH 6.5) buffered with PBS was used. After staining with 0.75% methylene blue (aqueous solution), the plaque forming units in each well were counted. Neutralization test

[0141] Serum samples from all hamsters were tested for neutralization ability against SARS-CoV-2 (B.1). In addition, serum from challenge infected animals (0, 2, 5dpch) was tested for neutralization activity against SARS-CoV-2 delta variant (B.1.617). To this end, serum was inactivated at 56°C for 30 minutes. Subsequently, two-fold serial dilutions (1:8 to 1:1024) were prepared in 96-well plates, and 200PFU SARS-CoV-2 diluted in MEM (1% FBS, 1% P / S) were added to all wells. After incubation at 37°C for 1 hour, the dilutions were plated on subconfluent Vero E6 cells in 96-well cell culture plates and incubated for another 72 hours. Finally, the plates were fixed with 4% formaldehyde solution and stained with 0.75% methylene blue (aqueous solution). Wells without virus-induced cytopathic effect were considered neutralized and reported as the titer of the corresponding serum. Positive and negative controls were included in all plates. Histopathology

[0142] After the animals were sacrificed, the left lung lobe was carefully removed and fixed in 4% formaldehyde solution for 48 hours. Subsequently, the tissue was embedded in paraffin and cut at 2 μm thickness to stain it with hematoxylin and eosin (H&E). Lung preparation and pneumonia scoring were performed with standardized procedures as described by Osterrieder et al. 2020. Growth dynamics

[0143] Confluent WHO Vero RCB 10-87 cells in T25 flasks were infected with B.1 (BetaCoV / Munich / ChVir984 / 2020, B.1, EPI_ISL_406862), B.1-ΔFCS, sCPD9 or sCPD9-ΔFCS at an MOI of 0.01. The virus was diluted in a final volume of 5 ml of complete cell culture medium and added to each flask. After infection, supernatants were collected at 24, 48, 72 and 96 hours after infection and subjected to a freeze-thaw cycle. Ten-fold dilutions were prepared and plated on confluent Vero E6 cells seeded in 12-well plates. After 1.25 hours, the cells were covered with MEM containing 1.5% carboxymethylcellulose and fixed with 4% formaldehyde solution after 48 hours. For plaque visualization, immunofluorescence staining was performed as described (Trimpert et al., 2021b). Co-culture of sCPD9-ΔFCS and Omicron BA.5

[0144] CaLu-3 cells were seeded in T25 flasks and grown to a density of 90%. Prior to co-infection with 100 FFU of SARS-CoV-2 variant Omicron BA.5 and 1,000 FFU of sCPD9-ΔFCS, the cell culture medium was replaced with 5 ml DMEM / F12 1:1 containing 10% FBS, 100 IU / ml penicillin G, 100 μg / ml streptomycin, and 1% non-essential amino acids. After 72 hours, the supernatant was harvested and clarified by centrifugation at 5,000 rpm for 15 minutes. Subsequently, 1% of the supernatant was transferred to previously uninfected CaLu-3 cells. The assay was performed in triplicate and continued for a total of 10 passages. Sequencing

[0145] After RNA was extracted from the cell culture supernatant as described above, libraries were prepared and sequenced using Illumina technology (Illumina). For library preparation, of Ultra TM II RNA Library Preparation Kit (New England Biolabs). This method relies on standard library preparation steps for Illumina sequencing, such as end repair, adapter ligation, and PCR enrichment. of The enriched sequencing libraries were quantified using a library quantification kit (New England Biolabs) and then pooled and sequenced on an Illumina Miseq system (Illumina).

[0146] The generated Illumina sequencing data were processed with Trimmomatic v.0.39 (Bolger et al., 2014) and mapped against the BA.5 (NCBI accession number: ON249995) and sCPD9-ΔFCS genome reference (GenBank: MZ064545.1) (Trimpert et al., 2021b) using the Burrows-Wheeler aligner v.0.7.17 (Li and Durbin, 2009). Mapping statistics were generated using Samtools v1.10 (Danecek et al., 2021) and the alignment was visualized using IGV v2.9.4 (Robinson et al., 2011) for Linux. To detect single-nucleotide polymorphisms (SNPs), the Bayesian genetic variant detector Freebayes (arXiv: 1207.3907 [q-bio.GN] 2012) was used. All SNPs with a minimum mapping quality of 5, a minimum count of 3, and a minimum score of 0.1 were initially considered. SNPs detected between the starting BA.5 isolates used in these studies and the BA.5 reference were removed from further analysis because they existed before these experiments. A table containing the removed SNPs is provided. The consensus sequence for each sample was obtained using BCFtools (Danecek et al., 2021).

[0147] In view of the slight differences in the genome structure between BA.5 and sCPD9-ΔFCS and the high entropy between the sample and the sCPD9-ΔFCS reference, direct sequence comparison with sCPD9-ΔFCS is inefficient. To facilitate this process, a full sample consensus sequence containing all detected SNPs was created on the backbone of the BA.5 reference (most similar to all samples) using SNP sites (Page et al., 2016) and BCFtools. The full sample consensus was compared with both BA.5 and sCPD9-ΔFCS references using EMBOSS extender (Myers and Miller, 1988), and a new SNP table was created using SNP sites to verify the position and identity of all detected SNPs relative to these two references. All SNPs that appeared between the BA.5 initial isolate and the sCPD9-ΔFCS reference and therefore did not appear during the co-infection experiment were removed from the analysis. result Depletion of FCS modestly increases viral titers on TMPRSS-2-negative cells

[0148] In order to improve the genetic stability of sCPD9 and reduce its transmissibility, the inventors have produced an sCPD9 derivative named sCPD9-ΔFCS, which lacks FCS in the spike protein. The sCPD9-ΔFCS mutant is designed to contain the same deletion in the spike protein that occurs naturally during serial passage on cultured Vero E6 cells, called "Bristol deletion" (Davidson et al., 2020). The length of the introduced deletion is 24 nucleotides. It removes 9 amino acids "NSPRRARSV" from the spike protein, including the entire FCS, and instead introduces isoleucine as a new amino acid at the same position (Davidson et al., 2020). Therefore, a total of 8 amino acids are removed from the spike protein.

[0149] It has been widely reported that SARS-CoV-2 virus variants lacking FCS have a clear growth advantage on different Vero cell lines compared to viruses with intact FCS. Based on these results, it was found that removal of FCS slightly increased the titer of sCPD9-ΔFCS virus after propagation on Vero cells. Since WHO RCB 10-87 Vero cells are widely used for vaccine production, the ability to produce slightly higher peak virus titers more quickly on these specific cells has great practical significance because it reduces the cost of vaccine production ( Figure 2 ). In this context, Figure 2 Growth kinetics of sCPD9-ΔFCS and sCPD9 on WHO Vero RCB 10-87 cells are shown as mean ± standard deviation (SD). Time points for harvesting supernatants are shown in hours post infection (hpi). FCS deletion prevents transmission of sCPD9-ΔFCS to unvaccinated contacts

[0150] To study the ability of sCPD9 and sCPD9-ΔFCS to transmit to unvaccinated contact animals, Syrian hamsters were infected with sCPD9, sCPD9-ΔFCS or parental SARS-CoV-2 (WT) on day 0. 24 hours after infection, each infected hamster was placed in a separate ventilated cage with an unvaccinated hamster. On the first day of this cohabitation, oral swabs of all naive animals in contact with WT-infected animals were strongly positive for SARS-CoV-2 RNA. On days 1 to 3 of cohabitation, all naive animals in contact with sCPD9-vaccinated subjects were infected with the virus, and the viral replication process in their upper airways was similar (although delayed) compared with WT-infected animals. In contrast, during the first 7 days of cohabitation (which was the duration of the observation period), naive animals in contact with sCPD9-ΔFCS-vaccinated subjects did not become positive for SARS-CoV-2 RNA ( Figure 3A ). In this context, Figure 3A The genomic RNA (gRNA) copies found in oral swabs collected daily (1 to 6 dpc) from contact hamsters are shown (expressed as mean ± SD). Two-way ANOVA and Tukey's multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) were performed. In this figure and all figures, the non-filled circle or square (the first bar from the left) represents sCPD9-ΔFCS, the light gray circle or square (the second bar from the left) represents sCPD9, and the dark gray circle or square (the third bar from the left) represents B.1 (WT SARS-CoV-2).

[0151] Natural transmission of WT SARS-CoV-2 in contact animals caused the expected COVID-19-like pneumonia, which was confirmed by histological examination on day 7 of cohousing ( Figure 3B ). Consistent with previous findings, inflammatory changes in the lungs were greatly attenuated in animals infected with sCPD9. In addition, the lungs of animals housed with animals vaccinated with sCPD9-ΔFCS showed almost no signs of inflammation. sCPD9-ΔFCS is highly attenuated in Syrian hamsters

[0152] All hamsters remained clinically healthy following infection with sCPD9 or sCPD9-ΔFCS, whereas WT-infected animals showed expected mild to moderate signs of illness, such as labored breathing and significant weight loss, during the first week after infection ( Figure 4A ). In this context, Figure 4AShown are the percentage body weight loss after the initial vaccination / infection shown for each group until the challenge-infection time point of 21 dpi / dpv (dpi = days post infection, dpv = days post vaccination).

[0153] However, in the absence of other visible signs of disease, animals infected with sCPD9 showed a slight trend towards decreased body weight in the week following vaccination, whereas animals vaccinated with sCPD9-ΔFCS showed relatively stable body weight.

[0154] Although all contact animals exposed to sCPD9- or WT-infected animals became infected with the corresponding viruses, clinical signs of disease and weight loss only occurred in animals infected with the WT virus ( Figure 4B , showing the percentage of body weight development of each group of contact animals during the co-housing period). Figure 4A and Figure 4B Both show violin plots with quartiles and median (truncated).

[0155] Similarly, severe histological signs of lung inflammation were seen only in contact animals infected with WT virus at day 7 after exposure ( Figure 3B , showing the number of gRNA copies recovered from oropharyngeal swabs and homogenized lungs and the detection of replication-competent virus in lung tissue; the detection limit is marked with a dashed line). Kruskal-Wallis test and Dunn's multiple comparison test were performed to compare the FIG. 3B to FIG. 3D The data shown in were statistically evaluated (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001).

[0156] Figure 3C Replication-competent virus detected in lung tissue is shown. Again, the limit of detection is marked with a dotted line.

[0157] Figure 3D Shown are histopathology scores, including percentage of lung consolidation area; lung inflammation scores with pneumonia severity, neutrophil, lymphocyte and macrophage influx, bronchial epithelial necrosis, bronchitis, alveolar epithelial necrosis, perivascular lymphocyte cuffs, and type II pneumocyte hyperplasia; and edema scores accounting for perivascular and alveolar edema.

[0158] Figure 3E Neutralizing activity against SARS-CoV-2 B.1 WT of sera taken from contact hamsters on day 6 after contact is shown.

[0159] Only mild inflammatory changes appeared in the lungs of contact animals infected with sCPD9, whereas inflammatory changes were further reduced or disappeared in the lungs of contact animals infected with sCPD9-ΔFCS without virus infection. FCS deficiency does not reduce vaccine efficacy

[0160] On day 21 after infection, all infected animals were challenged with the pathogenic SARS-CoV-2 delta variant. None of the challenged animals developed clinical signs of disease or exhibited significant weight loss (which was observed in unvaccinated control animals) ( Figure 5A Body weight changes are shown, controlled until the analysis time point 2+5 dpch, and expressed as percentage / group). In this and all other figures, the medium grey squares (fourth bar from the left) represent mock-vaccinated animals.

[0161] As expected, protection against challenge virus replication was comparable in all three infection groups. All groups showed high viral RNA loads in the upper respiratory tract on day 2 post-challenge ( Figure 5B , showing gRNA quantification in oropharyngeal swabs and lungs and infectious viral particles detected in homogenized lung tissue). Figures 5B to 5E In the figure, the dashed line indicates the detection limit. In addition, two-way ANOVA and Tukey's multiple comparison test were used to compare the Figures 5B to 5E Data shown in the table were statistically analyzed (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). However, by day 5 after challenge, the high viral RNA loads observed in all study groups had decreased to levels close to the limit of detection. On day 2 after challenge, lower respiratory tract protection was more pronounced, with significantly reduced viral RNA loads in the lungs of infected animals and the lowest levels of replication-competent virus ( Figure 5B By day 5 post-challenge, RNA loads were close to or below the limit of detection, and no replicating virus was present in the lungs of any challenged subject.

[0162] Overall, virological parameters indicated high protective efficacy of sCPD9 and sCPD9-ΔFCS vaccine candidates and were comparable to those of WT virus.

[0163] The protective efficacy of sCPD9 and sCPD9-ΔFCS vaccine viruses was determined by examining lung histopathology of uninfected and sCPD9-, sCPD9-ΔFCS-, and WT-infected hamsters on days 2 and 5 after challenge infection ( Figure 5C , showing infectious viral particles detected in homogenized lung tissue, and Figure 5D, showing the percentage of lung area that was consolidated by SARS-CoV-2 infection; illustrating the following lung inflammation scores: pneumonia severity, influx of neutrophils, lymphocytes and macrophages, bronchial epithelial necrosis, bronchitis, alveolar epithelial necrosis, perivascular lymphocyte cuffing and type II pneumocyte hyperplasia; scoring of pulmonary edema based on the extent of perivascular and alveolar edema). Here, it is evident that WT-infected animals were more susceptible to inflammation and showed a trend toward increased immune cell influx compared to animals infected with sCPD9 or sCPD9-ΔFCS viruses or unvaccinated animals. This improved by day 5 after challenge, indicating that the immune system of WT-infected animals experienced a transient overreaction shortly after challenge, causing the lung damage observed on day 2 after challenge. Importantly, all animals showed excellent and comparable protection against COVID-19-like pneumonia at all time points examined after challenge.

[0164] Finally, humoral immune responses against SARS-CoV-2 WT and delta variants were compared in infected animals before and at days 2 or 5 after challenge ( Figure 5E , showing the neutralizing activity of hamster sera taken at day 0 (before challenge), 2 and 5 after SARS-CoV-2 Delta challenge against B.1 and Delta variants; the detection range was from dilution 1:8 to 1:1024). The results showed that vaccination / infection with sCPD9, sCPD9-ΔFCS or WT virus induced comparable levels of neutralizing antibodies at all time points tested. sCPD9-ΔFCS is slightly more attenuated than sCPD9, but has equivalent protective effects

[0165] The results shown here show that removing FCS from sCPD9 produces a non-transmissible and highly attenuated virus in the Syrian hamster model. Despite the high level of attenuation, the sCPD9-ΔFCS virus showed equivalent protection against the SARS-CoV-2 variant Delta as the parental sCPD9 virus with an intact FCS in the spike protein. In terms of virological, serological and histological parameters, both sCPD9 and sCPD9-ΔFCS vaccine viruses can provide excellent and comparable protection against viral replication and disease. Overall, the protection conferred by the two vaccine viruses is comparable to that induced by infection with WT virus. It is worth noting that during the first day after the attack, animals infected with WT showed stronger signs of inflammation in the lungs.

[0166] Thorough histological examination showed that vaccination with sCPD9-ΔFCS or sCPD9 had another significant advantage over vaccination with WT virus with respect to subsequent virus challenge infection. Previous WT virus infection and subsequent challenge with SARS-CoV-2 resulted in significant alveolar bronchiolysis in the tested animals. In contrast, no alveolar bronchiolysis was observed in animals challenged with SARS-CoV-2 after previous vaccination with sCPD9-ΔFCS or sCPD9. Benefits of FCS removal for large-scale production of LAV SARS-CoV-2 vaccines

[0167] Many reports have shown that FCS impairs the replication of SARS-CoV-2 in TMPRSS2-deficient cells (e.g., Vero or Vero-E6 cells), as SARS-CoV-2 variants lacking a functional FCS quickly become dominant after passage in TMPRSS2-deficient cells (Davidson et al., 2020; Klimstra et al., 2020; Lau et al., 2020; Liu et al., 2020; Ogando et al., 2020; Sasaki et al., 2021b; Wong et al., 2021). These results suggest that mutant viruses lacking a functional FCS have a strong selective advantage for replication in cells that do not express TMPRSS2. The results shown here confirm these observations, as the sCPD9-ΔFCS mutant replicated at higher titers than the original sCPD9 virus in Vero cells.

[0168] Therefore, removing FCS from SARS-CoV-2-LAV has two advantages for LAV vaccine production. Since most vaccine manufacturers use Vero cells to produce LAV vaccines, increasing viral titers also helps reduce production costs, but more importantly, it helps purify a sufficient number of infectious virus particles required for a single vaccination.

[0169] The second major advantage of removing the FCS is that it greatly improves the genetic stability of LAV vaccine candidates. One of the prerequisites for the use of LAV vaccines in humans is that the vaccine virus population has a high degree of genetic homogeneity. Since SARS-CoV-2 rapidly loses its FCS at the S1 / S2 boundary after passage into Vero cells, removing the FCS eliminates the genetic instability problem and the vaccine virus can be produced with high genetic homogeneity. In vivo coinfection does not produce viral recombinants

[0170] To test the potential recombination between the vaccine and circulating field virus in vivo, Syrian hamsters were treated with equal amounts of sCPD9-ΔFCS and the Omicron variant BA.5. (1× 10 4 FFU / animal). After 24 hours, infected animals were co-housed with naive hamsters to assess host-to-host transmission. Oral swabs were collected daily; all animals were euthanized after 6 days of co-housing. Fig. 6A A schematic diagram of the experimental design is shown.

[0171] Figure 6B Shown are changes in animal body weights following co-infection or contact with co-infected animals. Infected animals showed a wider distribution of body weights compared to contacts, with mild, transient weight loss occurring in some individuals.

[0172] Figure 6C It is shown that no replication-competent virus was detected in the lungs of experimentally infected animals at day 7 after infection (circles: co-infected animals; triangles: contact animals).For this analysis, infectious virus particles were detected in homogenized lung tissue.

[0173] Fig.6D Figures 4 to 5 show the use of the targeted assay in oral swabs from co-infected hamsters (upper or left) and contact hamsters (lower or right). Fig.6D ), oropharyngeal swab( Fig. 6E ) and lung tissue ( Fig. 6F ) detected in the virus gRNA copies: SARS-CoV-2E gene (envelope) present in both viruses (results in FIG. 6D to FIG. 6G ), or a sequence that is unique to two different viruses—the spike gene of the Omicron BA.5 virus (results in Fig.6D 6G), or the re-encoded sCPD9 region of the sCPD9-ΔFCS virus (results in Figures 6D to 6G represented by triangles). Figures 6C to 6G The dashed line in the figure indicates the detection limit.

[0174] like Fig.6D It is shown that low levels of replication-competent virus were detected in the lung tissues of 3 contact hamsters on day 6 of co-housing.

[0175] RT-qPCR of daily oral swab samples from infected hamsters showed that sCPD9-ΔFCS was detectable only within the first 3 days after infection, whereas BA.5 ( Fig.6D ).

[0176] In contrast, small numbers of sCPD9-specific gRNA copies were found in the more sensitive oropharyngeal swabs ( Fig. 6E sCPD9-specific gRNA was not found in swabs and lung samples collected from contact animals, whereas BA.5-specific RNA was detected in large quantities in daily swabs, oropharyngeal swabs, and lungs on day 2 after contact ( Fig.6D to F). This demonstrates the transmission of BA.5, whereas sCPD9-ΔFCS remained non-transmissible under co-infection conditions. Therefore, no recombination events that could restore FCS in the vaccine virus and thus enable transmission of the vaccine virus were observed in the chosen experimental setting. In vitro co-culture did not reveal significant recombination events

[0177] Figure 6G Replication of Omicron BA.5 and sCPD9-ΔFCS in CaLu-3 cells is shown. CaLu-3 cells were infected with 1,000 FFU of sCPD9-ΔFCS and 100 FFU of BA.5, and after 72 hours of incubation, 1% of the supernatant was used as the inoculum for the next viral passage (n=3). The resulting viral population was serially passaged 10 times on CaLu-3 cells to evaluate recombination events between the vaccine and the field virus. RNA was extracted from the cell culture supernatant of each passage. A qPCR assay targeting a conserved region within the SARS-CoV-2E gene was used to assess the total SARS-CoV-2 gRNA content, while an assay targeting the FCS region in the BA.5 viral spike gene and the genetically recoded sCPD9 region was used to distinguish the vaccine from the field virus. Consistent with the in vivo data, the qPCR results showed that the BA.5 virus surpassed sCPD9-ΔFCS within one passage. sCPD9-specific RNA levels dropped to levels near the detection limit at passage 1, whereas BA.5 maintained replication, producing high levels of gRNA across the experimental range ( Figure 6G ). This suggests that sCPD9-ΔFCS has a considerable growth disadvantage in cell culture, which limits the possibility of vaccine recombination with wild-type virus in the same replication compartment. Therefore, the risk of recombination between sCPD9-ΔFCS and wild-type virus is reduced.

[0178] To confirm the absence of highly adaptive recombinants, total RNA sequencing was performed on cell culture supernatants from different passages and replicates of the co-cultured viruses. Sequencing analysis showed that, starting from passage 1, all sequences above the detection threshold originated exclusively from the BA.5 virus. No evidence for the presence of sequences of sCPD9-ΔFCS origin was found in any of the analyses performed. Although some de-novo mutations seemed to be selected, which could indicate adaptation to cell culture, the emergence of sCPD9-ΔFCS / BA.5 recombinants with a selective advantage over the BA.5 virus in cultured human cells could be excluded in the experimental setup performed ( Figure 6H , I).

[0179] Figure 6H SNPs identified in passages 1, 2, 3, 6, 7, and 10 of the co-infection experiment and their corresponding positions within the BA.5 reference genome are shown. The figure shows the SNPs identified in the three replicates containing the most SNPs in each passage, regardless of the passage conditions. Only SNPs identified in >10% of the sequence reads are depicted. ( Fig.6I Shown are all unique SNPs (with >10% read support) that emerged during the co-infection experiment compared to both the BA.5 and sCPD9-ΔFCS genomic reference sequences. sCPD9-ΔFCS is also safe and immunogenic in immunosuppressed hamsters

[0180] Fig. 7A A schematic overview of the experimental setup is shown. Starting 3 days before vaccination or contact, Syrian hamsters were immunosuppressed by daily subcutaneous injection of dexamethasone (Dex) at a dose of 2 mg / kg. After 3 days of treatment, hamsters were vaccinated with sCPD9-ΔFCS to allow determination of the effect of immunosuppression on vaccine safety and humoral immune responses to vaccination. Subsequently, 24 hours after vaccination, vaccinated hamsters were co-housed with naive and immunosuppressed contact animals. Oral swabs were collected from all hamsters daily. Contacts were euthanized 6 days after contact (dpc), while vaccinated hamsters were euthanized 21 days after vaccination (dpv).

[0181] Figure 7B Shown are weight changes in immunosuppressed animals after vaccination or contact with vaccinated hamsters. After vaccination, immunosuppressed hamsters showed stable weight and absence of clinical disease ( Figure 7B ).exist Figures 7B to 7J In the figure, triangles represent immunosuppressed contact hamsters, whereas squares represent immunosuppressed hamsters vaccinated with sCPD9-ΔFCS vaccine.

[0182] SARS-CoV-2 RNA was detectable in oral swabs up to 8 days after vaccination, with highest levels observed on days 1 and 2 ( Figure 7C Viral gRNA copies in oral swabs are shown). After 8 days, no viral RNA was detected in oral and oropharyngeal swabs ( Figure 7C , D). Low levels of gRNA were detected in lung tissue on day 21 after vaccination, indicating prolonged viral replication in the lower respiratory tract compared with the upper respiratory tract ( Fig.7D gRNA copies in oropharyngeal swabs and lung tissue are shown). However, no replication-competent virus could be recovered from lung samples collected at this time point ( Fig. 7E The number of replication-competent viruses in lung tissue is shown).

[0183] Figure 7C Neutralizing activity of sera collected from immunocompetent animals (IC; indicated by circles) and immunosuppressed animals vaccinated with sCPD9-ΔFCS at 21 days after vaccination (dpv) against SARS-CoV-2 variant B.1 (upper limit of detection = 1:1,024). Significance was tested using the Mann-Whitney test (p < 0.05). These serum neutralization assays enable determination of the humoral immune response of immunosuppressed animals 21 days after receiving a single dose of sCPD9-ΔFCS. Trendingly, serum neutralization titers were lower in immunosuppressed animals compared to immunocompetent animals that received the same vaccination. However, despite immunosuppression, all hamsters developed a substantial humoral response to vaccination ( Figure 7F In addition, lung histopathology was evaluated and showed no evidence of pneumonia in immunosuppressed hamsters following vaccination with sCPD9-ΔFCS ( Figure 7G to J). Overall, sCPD9-ΔFCS remained safe and immunogenic in animals treated with high-dose glucocorticoids.

[0184] Figures 7C to 7F The dashed line in the figure indicates the detection limit. sCPD9-ΔFCS is not transmissible between immunosuppressed hamsters

[0185] Next, it was determined whether the sCPD9-ΔFCS virus could be transmitted between immunosuppressed animals. To test this, the six immunosuppressed and sCPD9-ΔFCS-vaccinated animals described above were contacted with six dexamethasone-treated and immunologically naive hamsters and co-housed for 6 consecutive days ( Fig. 7A During the entire cohabitation period, no clinical signs of illness or significant weight loss were observed ( Figure 7BAlthough vaccine virus RNA has been detected in oral swabs from vaccinated individuals ( Figure 7C ), but no transmission of vaccine virus was observed, as evidenced by the absence of detectable viral RNA in both the upper and lower respiratory tracts of immunosuppressed contact animals ( Fig.7D , E). sCPD9-ΔFCS is safe in immunosuppressed animals and does not show recombination with circulating SARS-CoV-2 variants

[0186] Infection experiments in dexamethasone-treated animals showed that sCPD9-ΔFCS was also safe for individuals undergoing glucocorticoid treatment. Glucocorticoids are well known for their immunosuppressive effects and are commonly used to treat airway inflammation, making them particularly relevant immunosuppressive treatments in the context of COVID-19. Dexamethasone treatment has previously been shown to exert a strong immunosuppressive effect and enhance SARS-CoV-2 replication in Syrian hamsters (Wyler, 2022). Despite this, it was found that under dexamethasone treatment, the replication of the vaccine virus sCPD9-ΔFCS remained moderate, with RNA levels decreasing toward the limit of detection within a week after vaccination. Dexamethasone-treated hamsters showed neither clinical signs of disease nor any obvious pathological state. In addition, in dexamethasone-treated animals, the vaccine virus remained non-transmissible and induced a fairly strong humoral immune response. These results demonstrate the safety and efficacy of sCPD9-ΔFCS in immunosuppressed patients, especially in individuals receiving glucocorticoid treatment. Based on preliminary evaluation of initial data, such effects on humoral immune responses, safety and efficacy in immunosuppressed patients could not be observed to the same extent following vaccination with sCPD9 that still contained FCS.

[0187] Both in vitro and in vivo co-infection experiments showed that the LAV virus sCPD9-ΔFCS was rapidly and consistently outcompeted by the Omicron BA.5 field isolate, indicating a strong selective disadvantage for the attenuated vaccine virus. This limits the replication of the vaccine and the field virus in the same host or host compartment, thereby limiting the potential for recombination events between the two viruses. Although recombination events between the two viruses cannot be formally excluded in the chosen experimental setup, sequence analysis made it possible to confirm that no recombinants acquired a selective advantage over the BA.5 variant in the co-infection experiments. In addition, the in vivo co-infection experiments showed that co-infection of the vaccine with the field virus did not lead to increased virulence and did not produce transmissible vaccine viruses. Additional experiments demonstrated the superiority of sCPD9-ΔFCS

[0188] Additional experiments demonstrating the superiority of sCPD9-ΔFCS are explained in more detail below. Materials and methods Study Design

[0189] The aim of this study was to compare the protection provided by two different types of vaccines against the transmission of SARS-CoV-2. In two different trials, the extent of transmission of SARS-CoV-2 from infected transmitter hamsters to vaccinated animals and from vaccinated and subsequently infected transmitters to naive contact hamsters was investigated. For this purpose, hamsters were randomly assigned to groups of 12 animals. The experimental design of the two studies included three groups according to a prime-boost regimen, in which the hamsters were vaccinated on day 0 and day 21, respectively. The three groups received two doses of sCPD9-ΔFCS (10 4 FFU) (intranasal), 5 μg mRNA vaccine BNT162b2 ( Pfizer-BioNTech) (intramuscular) or two doses of mock vaccination.

[0190] In the first experiment, vaccinated hamsters were housed with infected transmitter animals two weeks after receiving a booster vaccination. 5 FFU of SARS-CoV-2 variant B.1 or Omicron subvariant BA.5 infected the transmitter hamsters. For the second experiment, two weeks after the second vaccination, animals previously vaccinated with primary-boost vaccination were infected with SARS-CoV-2 variant B.1 or Omicron subvariant BA.5. Subsequently, 24 hours after infection, the latter were co-raised with the original contact hamsters. In both experiments, the hamsters were housed for 6 days. During this period, oral mucosal swabs were collected daily to determine the viral load in the upper airways of the transmitters and contact hamsters. In addition, body weight and clinical status were also assessed. On the 6th day after contact, all hamsters were euthanized to collect blood, nasal washes, lungs and skulls for virology, serology and histopathology. cell

[0191] Vero E6 (ATCC CRL-1586) and Vero E6-TMPRSS2 (NIBSC 100978) cells were cultured in minimal essential medium (MEM) containing 10% fetal bovine serum (PAN Biotech), 100 IU / ml penicillin G and 100 mg / ml streptomycin (Carl Roth). To ensure selection of cells expressing TMPRSS2, the culture medium for Vero E6-TMPRSS2 cells also contained an additional 1000 μg / ml of geneticin (G418). CaLu-3 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing 20% ​​fetal bovine serum (PAN Biotech), 1% non-essential amino acids, 100 IU / ml penicillin G and 100 mg / ml streptomycin (Carl Roth). All cells were cultured at 37°C and 5% CO2. Virus

[0192] The ancestral SARS-CoV-2 variant B.1 (B.1, hCoV-19 / Germany / BY-ChVir-929 / 2020, EPI_ISL_406862) propagated on Vero E6 cells and the Omicron subvariant BA.5 (BE.1.1, hCoV-19 / Germany / SH-ChVir29057_V34 / 2022, EPI_ISL_16221625) grown on CaLu-3 cells were used to infect transmitter hamsters. In addition, the delta variant B.1.617.2 (B.1.617.2, human, 2021, Germany ex India, 20A / 452R, EVAg: 009V-04187) propagated on Vero E6-TMPRSS2 cells and the subvariant BA.1 (BA.1.18, hCoV-19 / Germany / BE-ChVir26335 / 2021, EPI_ISL_7019047) grown on CaLu-3 cells were used for serum neutralization assays. Prior to infection experiments, plaque assays were performed on Vero E6 cells to determine the titers of all virus stocks. The vials were stored at -80°C. Ethical Statement

[0193] Animal work was performed in accordance with all applicable national and international regulations and with approval from the national regulatory authority, Landesamt für Gesundheit und Soziales, Berlin, Germany (permit number 0086 / 20). All in vitro and animal experiments were performed at the Institut für Virologie, Freie Berlin certified BSL-3 laboratory. Animal husbandry

[0194] Syrian hamsters (Mesocricetus auratus; breed RjHan: AURA) aged 5 to 7 weeks were purchased from Janvier Laboratories. They were placed in individually ventilated cages (IVCs; Tecniplast) equipped with nesting material in groups of 2 to 3 animals. Prior to vaccination, the animals were adapted to the housing conditions for seven days. The hamsters had free access to water and food at all times. During all experiments, cage temperature and relative humidity were monitored and ranged from 22°C to 24°C and 40% and 55%. Vaccine preparation and vaccination

[0195] The live attenuated vaccine candidate sCPD9-ΔFCS was propagated on Vero E6-TMPRSS2 cells. The titer was determined by plaque assay on VeroE6 cells. Prior to vaccination, the stock solution was adjusted to a final titer of 2 × 10 5 FFU / ml. Under general anesthesia (0.15 mg / kg medetomidine, 2.0 mg / kg midazolam and 2.5 mg / kg butorphanol), the cells were 10 4 FFU / animal were vaccinated intranasally.

[0196] BNT162b2 It was prepared according to the manufacturer's instructions. The final concentration of mRNA was diluted to 50 μg / ml instead of the 100 μg / ml recommended for humans. Dilutions were prepared with 0.9% NaCl sterile water immediately before vaccination and administered intramuscularly at a dose of 5 μg / hamster.

[0197] Animals assigned to the mock group received minimal essential medium (MEM) intranasally under general anesthesia. Nasal wash

[0198] To obtain nasal washes from all animals, a cannula was used to puncture the skull just median to the nasal septum. Subsequently, a pipette tip was inserted and 200 μl of PBS was applied. The wash was drawn over the nostrils and the wash process was repeated twice. Approximately 150 μl of sample was collected from each animal. RNA isolation and RT-qPCR

[0199] In preparation for RNA extraction, 25 mg of lung tissue was homogenized in a bead mill (Analytik Jena). RNA was isolated from buccal swabs, oral swabs, and lung tissue using the innuPREP Viral DNA / RNA Kit (Analytik Jena, Jena, Germany) as recommended by the manufacturer. For detection of SARS-CoV-2 RNA, reverse transcription quantitative PCR (RT-qPCR) was performed using the NEB Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) on a qTower G3 cycler (Analytik Jena) under the following cycling conditions: reverse transcription at 55°C for 10 min, enzyme activation at 94°C for 3 min, and 40 cycles of 94°C for 15 s and 58°C for 30 s (Corman et al., 2020). Plaque assay and indirect immunofluorescence staining

[0200] Replicating virus was determined in 50 mg of lung tissue. For quantification, lung samples were homogenized in a bead mill (AnalytikJena), serially diluted in MEM, and plated on 12-well plates containing confluent Vero E6 cells. After 2.5 hours at 37°C and 5% CO2, the inoculum was removed and the cells were incubated with 2X Eagle's minimal essential medium (EMEM; Lonza) containing 1.5% microcrystalline cellulose and sodium carboxymethylcellulose (Vivapur 611p; JRS Pharma). TM BioWhittaker TM ) medium. 72 hours after infection, the plates were fixed with 4% PBS-buffered formaldehyde.

[0201] For indirect immunofluorescence staining, cells were permeabilized with 0.1% Triton X-100 and blocked with 3% BSA diluted in PBS for 30 minutes. After the plate was washed with PBS, a polyclonal anti-SARS coronavirus nucleocapsid primary antibody (Invitrogen) was added for 1 hour, followed by goat anti-rabbit IgG-AlexaFluor 488 secondary antibody (Invitrogen) for 45 minutes. In order to determine the titer, plaques were counted using an inverted fluorescence microscope (Axiovert S100, Zeiss). Serum neutralization assay

[0202] Neutralizing activity against SARS-CoV-2 variant B.1 and Omicron subvariant BA.5 was determined in all hamster sera (0 days after attack, 6dpc). In addition, neutralizing capacity against delta variant and Omicron subvariant BA.1 was tested in serum samples on day 6. Two-fold serial dilutions (1:8 to 1:1,024) of complement-inactivated (56°C for 30 minutes) hamster sera were prepared in 96-well plates. 200FFU SARS-CoV-2 diluted in MEM was applied to each well and incubated at 37°C for 1 hour. Subsequently, the dilutions were plated on Vero E6 cells cultured in 96-well plates and incubated at 37°C for 72 hours (B.1, delta) or 96 hours (Omicron BA.1, BA.5). Afterwards, the cells were fixed with PBS-buffered formaldehyde (4%, pH 6.5) and stained with methylene blue (0.75% aqueous solution). Neutralization was considered effective in wells that did not show cytopathic effect. The last neutralization well was reported as the titer. Positive and negative controls were included in all plates. For the purpose of plotting the results, samples with no neutralizing activity were set to a titer of 1:4. Enzyme-linked immunosorbent assay (ELISA)

[0203] SARS-CoV-2 specific IgG levels against the spike protein of B.1 and BA.5 variants as well as the nucleocapsid protein and ORF3a protein were measured in hamster serum using in-house ELISA. A transparent 96-well plate with a flat bottom (MEDISORP, Thermo Fisher Scientific, catalog number: MW96F) was coated with 5 μl of purified, recombinant, His-tagged SARS-CoV-2 antigens: the spike protein of the B.1 variant (D614G) (Acro Biosystems, catalog number: SPN-C52H3), the spike protein of the BA.5.5 variant (GenBank accession: QHD43416, Acro Biosystems, catalog number: SPN-C522p), the nucleocapsid protein (GenBank accession: QHD43423, Ray Biotech, catalog number: 230-01104) and the ORF3a protein (Thermo Fisher Scientific, catalog number: RP-87667). Antigen was diluted to a final concentration of 20 μg / ml in 1×PBS. In addition, each well was supplemented with 45 μl of coating buffer (50 mM Na2CO3, 50 mM NaHCO3, pH 9.6). After the plate was incubated at 4°C for 12 hours, the plate was washed 4 times with washing buffer (0.05% Tween 20 in 1×PBS) and blocked for 1 hour with blocking buffer (1×PBS, 1% BSA, 10% FCS). Serum samples were diluted 1:100 in dilution buffer (1×PBS, 2% BSA, 0.1% Tween 20) and plated in duplicate with 50 μl / well. The plate was then incubated at room temperature for 2 hours, followed by another washing step. Then, 50 μl of secondary antibody (horseradish peroxidase (HRP)-conjugated polyclonal goat anti-hamster IgG (H+L) antibody (ThermoFisher Scientific, catalog number: 10537453)) diluted 1:1000 in 1× PBS was added to each well. After incubation for 1 hour at room temperature, the plate was washed again and 50 μl of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB; TCI chemicals, catalog number: T3854) was added to each well. After 15 minutes, the reaction was stopped with 1M H2SO4. The optical density was measured at 450nm and 570nm using a SpectraMax Plus 384 plate reader (Molecular Devices).

[0204] In addition, SARS-CoV-2-specific IgA levels against the spike protein of the B.1 and BA.5 variants were measured in nasal washes following the above protocol with slight modifications. Nasal washes were diluted 1:50 in dilution buffer and plated in duplicate. For detection of IgA, polyclonal HRP-conjugated rabbit anti-hamster IgA (Brookwood Biomedical, catalog number: sab3003a) was diluted 1:750 and used as a secondary antibody. After incubation for 1 hour at room temperature, the plates were washed and 50 μl of 1-Step TM Ultra TMB ELISA substrate solution (Thermo Fisher Scientific, catalog number: 34028) was used to stop the reaction after incubation at room temperature for 20 minutes. Histopathology and immunohistochemistry

[0205] For histopathological analysis, the left lung lobe and peeled skull were fixed in a PBS-buffered formaldehyde solution (4%) for 48 hours. The skull was rinsed under tap water for 30 minutes and decalcified in a buffered EDTA solution (pH = 7.0) at 65°C for 3 days. The skull was trimmed to obtain anterior sections at the tip of the first triangular ruga of the hard palate and more caudal sections from the level of the first molar. 2 μm thick sections were cut from conventional formalin-fixed, paraffin-embedded samples and stained with hematoxylin and eosin or prepared for immunohistochemistry. Histopathological analysis of lung sections was performed as described (Osterrieder et al., 2020). The presence of lymphocytes, granulocytes, necrosis, epithelial flattening, and ciliary loss in nasal sections were scored as 0 = less than 5% of the epithelium affected, 1 = 5% to 40% of the epithelium affected, 2 = 41% to 80% of the epithelium affected, or 3 = more than 80% of the epithelium affected. In addition, airway exudates were characterized. For immunohistochemical analysis, nasal sections were dewaxed in xylene and rehydrated in decreasing concentrations of ethanol. Endogenous peroxidase was blocked with H2O2. Antigen retrieval was achieved by microwave treatment of sections at 600W for 12 minutes in 750ml buffered citric acid (containing 1% Triton X 100 (Roth)). Monoclonal mouse anti-SARS-CoV-2 nucleocapsid protein primary antibody (Sino Biological, dilution: 1:500) was incubated overnight at 4°C. For universal negative controls, sections were incubated with unrelated purified mouse IgG (BioGenex) instead of anti-SARS-CoV-2 antibodies. Nonspecific binding was blocked with 20% goat serum for 30 minutes. After washing with PBS / Triton buffer, a secondary antibody (goat anti-mouse IgG (Vector Laboratories, diluted 1:200)) was applied and incubated for 30 minutes. After 8 minutes of signal enhancement with the Vectastain Elite ABC kit (Vector Laboratories), the signal was developed with diaminobenzidine tetrahydrochloride (Merck). Hematoxylin was used as a counterstain. For histopathological evaluation, an Olympus BX41 microscope equipped with a DP80 microscope digital camera (Olympus) and cellSens TM imaging software version 1.18 (Olympus Soft Imaging Solutions) was used. Automatic digitization was driven using an Aperio CS2 slice scanner (Leica Biosystems). Micrographs were generated with image Scope software (Leica Biosystems).Adobe Photoshop or GIMP software is used to generate the chart panel. LAV vaccine prevents clinical symptoms after natural transmission of SARS-CoV-2 B.1 and BA.5

[0206] Syrian hamsters received two doses of sCPD9-ΔFCS, BNT162b2 or mock vaccine at 3-week intervals (day 0 and day 21). After another 14 days (day 35 after vaccination), hamsters were exposed to transmitter animals that had been infected with ancestral virus variant B.1 or Omicron BA.5 variant one day before. The animals were housed for 6 days and closely monitored for disease symptoms and viral load in the upper airway.

[0207] Fig. 8A and 8B B.1 spreaders and contact animals ( Fig. 8A ) and BA.5 Transmitters and contact animals ( Figure 8B ). Violin plots (truncated) show weight, group median, and quartiles of vaccinated contacts (n=6). Weights of transmitters (n=3) are presented as medians. All three groups of transmitter animals infected with the ancestral SARS-CoV-2 variant B.1 showed moderate weight loss and typical clinical signs of COVID-19-like pneumonia ( Fig. 8A ). As expected, vector animals infected with the BA.5 variant (Wolter et al. 2022, Uraki et al. 2022) showed less pronounced weight loss, with some variability between infection groups ( Figure 8B ).

[0208] sCPD9-ΔFCS and mRNA vaccines effectively prevented weight loss in vaccinated contact animals exposed to both B.1 or BA.5 transmitters ( Fig. 8A and 8B As expected, mock-vaccinated animals exposed to B.1 vectors showed a gradual loss of body weight starting on day 3 after exposure ( Fig. 8A At the same time, mock-vaccinated animals exposed to BA.5 transmitters did not experience weight loss, which may be due to the lower pathogenicity of the BA.5 variant and the resulting milder course of disease observed in infected animals ( Figure 8B ). LAV alone prevents transmission of SARS-CoV-2 B.1 and BA.5

[0209] To monitor viral replication and spread, oral swabs were collected daily during co-housing of infected and contact animals. In addition, oral swabs and lungs were obtained on day 6 post-contact (dpc) to quantify viral RNA levels and replicating virus. Viral gRNA copies and replicating virus in oropharyngeal swabs and lung tissues were quantified as B.1 infection ( Fig. 8E ) and BA.5 infection ( Figure 8F ) and their respective vaccinated c contacts. Figures 8C to 8F Results for vaccinated contact animals (n=6) are presented as medians and ranges, with symbols representing individual values. For transmitter hamsters (n=3), medians are shown. Parametric statistics were performed on log-transformed data. Figure 8C and 8D Ordinary two-way ANOVA and Tukey's multiple comparison test were performed. Fig. 8E and 8F Ordinary one-way ANOVA and Tukey's multiple comparison test were performed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0210] Infected transmitter animals showed high levels of SARS-CoV-2 RNA, which gradually decreased by day 6. Overall, viral loads were higher in B.1 transmitters compared with BA.5 transmitters ( Figure 8C and 8D ).

[0211] Consistent with the observed weight loss, oral swabs from mock-vaccinated contacts exposed to B.1 transmitters had high levels of SARS-CoV-2 RNA starting at 2 dpc ( Figure 8C ). Although mRNA vaccination prevented weight loss, it did not protect against B.1 infection, as demonstrated by persistently high SARS-CoV-2 RNA levels in oral swabs after day 2. However, mRNA vaccination resulted in reduced viral RNA levels compared with mock vaccination. In contrast, contacts vaccinated with sCPD9-ΔFCS exhibited minimal SARS-CoV-2 RNA levels that fluctuated around the limit of detection, indicating repeated exposure to the virus but without signs of productive infection in the upper airways ( Figure 8C ).

[0212] Similar results were obtained in vaccinated hamsters exposed to BA.5-infected transmitters. SARS-CoV-2 RNA levels in oral swabs from mRNA- and mock-vaccinated animals peaked at 4 to 5 dpc ( Fig.8D ), while the viral RNA levels in hamsters vaccinated with sCPD9-ΔFCS were close to the detection limit, indicating effective prevention of BA.5 infection ( Fig.8D ).

[0213] Consistent with these findings, both mRNA- and mock-vaccinated hamsters exposed to B.1 or BA.5 transmitters showed high SARS-CoV-2 RNA levels in oral swabs and lungs at 6 dpc ( Fig. 8E and 8F In addition, replication-competent virus was detected in the lung tissue of mock-vaccinated animals exposed to B.1 transmitters, most of which had cleared the infection by then ( Fig. 8E Despite slightly lower levels of viral RNA, replicating virus was found in the lungs of two mRNA-vaccinated hamsters and three mock-vaccinated hamsters exposed to BA.5 vectors, reflecting a delay in peak viral replication compared with B.1 infection ( Figure 8F ).

[0214] Lung lesions were mostly absent in animals vaccinated with sCPD9-ΔFCS and were less reduced in animals vaccinated with mRNA vaccines exposed to B.1 transmitters. Hamsters experimentally infected with B.1 virus or infected with B.1 after mock vaccination developed typical COVID-19-like pneumonia lesions. Specifically, infected animals developed prominent patchy bronchointerstitial pneumonia with necrotizing suppurative bronchitis and bronchiolitis, alveolar type II epithelial hyperplasia, vascular endotheliitis, diffuse alveolar damage, and perivascular and alveolar edema. Histopathological analysis showed that tissue changes, immune cell infiltration, and edema were significantly reduced in animals vaccinated with sCPD9-ΔFCS and mRNA vaccines. Inflammatory lesions were mild in all animals infected with Omicron BA.5 and were most prominent in experimentally infected transmitter hamsters. However, mock-vaccinated contacts still developed mild pneumonia. Protection in mRNA-vaccinated hamsters was slightly less effective. In contrast, animals vaccinated with sCPD9-ΔFCS that were exposed to B.1 or BA.5 transmitters showed no substantial evidence of pneumonia, indicating highly effective protection provided by the vaccine. Strong humoral immune responses accompany the protective efficacy of LAV

[0215] To assess humoral immunity, the neutralization capacity of sera collected at 6 dpc against SARS-CoV-2 variants B.1, Delta, BA.1, and BA.5 was evaluated. In addition, enzyme-linked immunosorbent assays (ELISAs) were performed using sera and nasal washes collected at 6 dpc.

[0216] The results are Figures 9A to 9D In this context, Fig.9A The neutralization capacity of hamster sera taken from vaccinated animals in contact with B.1 or BA.5 transmitters against SARS-CoV-2 variants B.1, BA.5, Delta, and BA.1 is shown (upper limit of detection = 1:1,024, lower limit of detection is indicated by a dotted line). The results are presented as mean ± SEM, where symbols represent individual values. Fig. 9B Shown are SARS-specific IgG levels against B.1 spike, BA.5 spike, nucleocapsid, and ORF3a in sera collected from vaccinated contacts at 6 days post-exposure (dpc). Fig. 9C Shown are SARS-specific IgA levels against B.1 and BA.5 spikes in nasal washes obtained from vaccinated contacts at 6 dpc. Fig. 9B and 9C Results are shown as optical density (OD) readout at 450 nm. Box plots represent 25th to 75th percentiles, with the center line representing the median and whiskers from minimum to maximum. Symbols represent individual values.

[0217] 9A to 9C The results were statistically evaluated by Kruskal-Wallis test and Dunn's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Fig.9D Results were obtained by semiquantitative scoring of SARS-CoV-2 N protein immunohistochemistry (IHC) signals in the nasal epithelium of vaccinated contacts. Scores of inflammatory changes in the nasal epithelium (including lymphocytes, neutrophil influx, necrosis of olfactory and respiratory epithelial cells, apoptosis, loss of cilia, and flattened epithelial cells) are presented as medians and ranges. Symbols represent individual values. Ordinary one-way ANOVA and Tukey's multiple comparison test were performed. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0218] As expected, vaccination with sCPD9-ΔFCS elicited robust neutralizing antibody production, with comparable levels in groups exposed to different challenge viruses ( Fig.9A). This finding, combined with the virological results, suggests that mucosal immunity induced by sCPD9-ΔFCS vaccination prevents the replication of naturally circulating viruses. However, brief exposure to viral antigens resulted in a slight increase in neutralizing activity. In contrast, animals that were mock-vaccinated showed increased serum neutralization capacity against the specific challenge virus. A similar trend was observed in animals that were vaccinated with mRNA vaccines, but the difference was less pronounced ( Fig.9A ). As expected, specific sera neutralizing capacity was similar in all transmitter animals and was determined by the challenge virus.

[0219] ELISA showed that the levels of anti-B.1 spike and anti-BA.5 spike IgG antibodies were comparable in hamsters vaccinated with sCPD9-ΔFCS and mRNA vaccines ( Fig. 9B As expected, nucleocapsid and ORF3a-specific antibodies were only present in animals vaccinated with sCPD9-ΔFCS, highlighting the broad immunity provided by LAV ( Fig. 9B Not surprisingly, IgG levels in transmitter animals were relatively uniform and strongly affected by the challenge virus. Mucosal immunity was investigated by measuring SARS-CoV-2-specific IgA levels in nasal washes obtained at 6 dpc ( Fig. 9C ). Regardless of the challenge virus, animals vaccinated with sCPD9-ΔFCS showed similar levels of anti-B.1 spike and anti-BA.5 spike IgA antibodies ( Fig. 9C ). In contrast, only mRNA-vaccinated hamsters exposed to B.1 carriers produced appreciable IgA levels. mRNA-vaccinated animals exposed to BA.5 carriers and both mock-vaccinated groups lacked measurable mucosal IgA responses. The absence of IgA in the nasal washes of mRNA-vaccinated hamsters in contact with BA.5 carriers suggests that mRNA vaccination conferred only limited mucosal immunity prior to viral exposure. However, exposure to the homologous B.1 variant resulted in a significant induction of mucosal IgA antibodies ( Fig. 9C ). Transmitter animals showed lower IgA levels, which corresponded to the virus used for infection. Preventing mucosal infections with SARS-CoV-2 B.1 and BA.5 with LAV vaccination

[0220] To further evaluate vaccination-induced protection in the upper airways, the presence of SARS-CoV-2 nucleocapsids and histological signs of infection and inflammation in the nasal epithelium were evaluated at 6 dpc. Although SARS-CoV-2-positive cells were absent in the nasal respiratory and olfactory epithelium of contacts vaccinated with sCPD9-ΔFCS, abundant antigen was detected in mRNA- and mock-vaccinated animals ( Fig.9D ).

[0221] Consistent with the immunohistochemical results, immune cell influx and inflammatory lesions were observed only in the olfactory and respiratory epithelia of animals vaccinated with mRNA and mock vaccines, with reduced inflammation in hamsters vaccinated with mRNA. Notably, inflammatory lesions were less pronounced in animals exposed to the BA.5 vector ( Fig.9D ). Consistent with the expected rapid clearance of SARS-CoV-2 (Sia et al., 2020), at 7 days post-infection (dpi), transmitter hamsters showed only a small number of infected epithelial cells and mild signs of inflammation. Interrupting transmission of SARS-CoV-2 B.1 to primary contacts with LAV vaccination

[0222] The effect of vaccination on limiting the spread of the virus from vaccinated and experimentally infected hamsters to naive animals was also examined. Syrian hamsters were given two doses of sCPD9-ΔFCS or BNT162b2, administered 21 days apart (day 0 and day 21). After 14 days (day 35), hamsters were infected with SARS-CoV-2 B.1 or Omicron BA.5. 24 hours after infection, infected animals were housed with naive contacts for 6 days while monitoring their clinical status and body weight. Oral swabs were collected daily and lung samples were obtained at 6dpc.

[0223] Figures 10A to 10C (Regarding infection with SARS-CoV-2 B.1) and Figures 11A to 11C The results of these experiments are shown (for infection with SARS-CoV-2 Omicron BA.5).

[0224] Fig. 10A The percentage of weight loss from vaccinated and B.1 infected transmitters and naive contact animals is shown. Violin plots (truncated) show weights of naive contacts (n=6) as group median and quartiles. Weights of transmitters (n=3) are shown as median. Fig. 10B Viral gRNA copies in oral swabs from vaccinated and B.1 infected transmitter hamsters and naive contacts are shown. Ordinary two-way ANOVA and Tukey's multiple comparison test were performed. Fig. 10C Viral gRNA copies in oropharyngeal swabs and 2.5 mg of homogenized lung tissue collected at termination are shown. Replicating virus in homogenized lung tissue was quantified as focus forming units (FFU). Fig. 10B and 10C Results for naive animals (n=6) are presented as medians and ranges. Symbols represent individual values. Results for vaccinated and superinfected hamsters are presented as medians. Fig. 10B and 10CIn the figure, parametric statistics are performed on the logarithmically transformed data. The dashed line indicates the detection limit. Fig. 10C Ordinary one-way ANOVA and Tukey's multiple comparison test were performed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0225] Fig.11A The percentage of weight loss of vaccinated and BA.5 infected transmitter animals and naive contacts is shown. The violin plot (truncated) shows the weight of naive contacts (n=6) as group median and quartiles. The weight of transmitters (n=3) is shown as median. Figure 1 1B shows the viral gRNA copies in daily oral swabs of BA.5-infected transmitters and naive contact hamsters after vaccination and challenge infection. Ordinary two-way ANOVA and Tukey multiple comparison test were performed. Fig. 11C The gRNA copy numbers detected in oropharyngeal swabs and 2.5 mg lung tissue obtained at termination are shown. Replication-competent virus in 50 mg homogenized lung was quantified as focus forming units (FFU). Fig. 11B and 11C Results for naive animals (n=6) are presented as medians and ranges, with symbols representing individual values. Results for vaccinated and challenge-infected transmitters (n=3) are presented as medians. Fig. 11B and 11C In the figure, parametric statistics are performed on the log-transformed data. The dashed line indicates the detection limit. Fig. 11C Ordinary one-way ANOVA and Tukey's multiple comparison test were performed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0226] Both vaccines effectively prevented weight loss, whereas mock-vaccinated animals lost weight after B.1 infection ( Fig. 10A ). Virological results demonstrated the strong protective efficacy of the sCPD9-ΔFCS vaccine. The body weight of naive contacts co-housed with sCPD9-ΔFCS-vaccinated and B.1-infected transmitters remained stable. In contrast, contacts of mock- or mRNA-vaccinated and SARS-CoV-2-infected transmitters lost weight starting from 2 and 4 dpc, respectively ( Fig. 10A ), and their oral swabs showed high levels of SARS-CoV-2 RNA. However, contacts of mRNA-vaccinated transmitters showed slightly delayed viral replication kinetics compared with contacts of mock-vaccinated animals ( Fig. 10BThe presence of high RNA levels in the lungs and the detection of replication-competent virus further indicated that all naive animals exposed to mRNA- or mock-vaccinated and B.1-infected hamsters were infected with infection ( Fig. 10C ). In contrast, contacts of sCPD9-ΔFCS-vaccinated animals and B.1-infected animals remained negative for SARS-CoV-2 RNA in the upper and lower airways, and no replicating virus was detected in the lower airways. These results strongly suggest that vaccination with sCPD9-ΔFCS confers highly effective protection against onward transmission of B.1 virus to original contacts. ( Fig. 10B and 10C ).

[0227] Consistent with these observations, hamsters infected with B.1 infection from vaccinated and challenged animals displayed typical signs of COVID-19 pneumonia, including necrotizing suppurative bronchitis and bronchiolitis, alveolar type II epithelial hyperplasia, vascular endotheliitis, diffuse alveolar damage, and perivascular and alveolar edema. However, immune cell influx and edema were reduced in hamsters co-housed with mRNA-vaccinated animals compared with their mock-vaccinated counterparts. Importantly, none of the hamsters in contact with sCPD9-ΔFCS-vaccinated and B.1-infected animals displayed signs of pneumonia. LAV provides superior protection against Omicron BA.5 infection and onward spread

[0228] Due to its attenuated effect on Syrian hamsters, no significant weight loss was observed in hamsters infected with Omicron BA.5 ( Fig.11A ). Animals vaccinated with sCPD9-ΔFCS and superinfected with Omicron BA.5 cleared infection within 48 h, whereas mRNA vaccination only slightly reduced viral loads in the upper and lower respiratory tracts compared with mock vaccination. Importantly, SARS-CoV-2 RNA was still detected in oral swabs of mRNA-vaccinated hamsters until 5 dpc ( Fig. 11B and 11C ). Histopathological examination of vaccinated and B.5-infected hamsters demonstrated the efficacy of both vaccines, however the histopathological changes observed in B.5-infected animals were generally milder than those observed in B.1-infected animals, suggesting that the overall pathology of this variant is milder compared to the original virus.

[0229] No weight loss was observed in the naive groups exposed to vaccinated and BA.5-infected hamsters ( Fig.11A). However, mRNA vaccination failed to prevent continued transmission of BA.5 virus. From 3 dpc onwards, all animals in contact with mRNA- or mock-vaccinated transmitters had comparable SARS-CoV-2 RNA loads ( Fig. 11B In addition, high SARS-CoV-2 RNA levels were found in swabs and lung samples collected from these animals at termination ( Fig. 11C ). In contrast, sCPD9-ΔFCS vaccination greatly reduced transmission to naive contacts, with only one animal becoming infectious around 2 dpc. A second naive animal in the same cage tested positive at 6 dpc, indicating secondary transmission ( Fig. 11B ). At termination, both hamsters tested positive for SARS-CoV-2 RNA in oral swabs and lungs, but only one animal had replicating virus at termination. Meanwhile, replicating virus was present in three contacts of both mRNA- and mock-vaccinated transmitters ( Fig. 11C ).

[0230] Histopathological findings were less pronounced in contacts exposed to vaccinated and BA.5-infected hamsters. Contacts of mock- or mRNA-vaccinated hamsters exhibited mild to moderate pneumonia with increased immune cell influx but no lung consolidation. Naive contacts of sCPD9-ΔFCS-vaccinated animals showed no signs or mild lung inflammation, reflecting their infection status. LAV-induced humoral and mucosal immunity reduces onward transmission of SARS-CoV-2

[0231] exist Figures 12A to 12C The results of the propagation experiment are shown in . Fig. 12A Concentrations of neutralizing antibodies against SARS-CoV-2 variants B.1, BA.5, Delta, and BA.1 in sera from naive hamsters in contact with vaccinated and B.1 or BA.5 infected transmitters are shown. The lower limit of detection is indicated by a dotted line, and the upper limit of detection = 1:1,024. Results are presented as mean ± SEM. Fig. 12B Shown are SARS-specific IgG levels against B.1 spike, BA.5 spike, nucleocapsid, and ORF3a in sera from primary contacts collected at 6 dpc. Fig. 12C Shown are SARS-specific IgA levels against B.1 spike and BA.5 spike in nasal washes obtained at termination. Fig. 12B and 12C The results shown in are expressed as optical density (OD) readout at 450 nm. The box plots represent the 25th to 75th percentiles, with the center line representing the median, the whiskers from the minimum to the maximum value, and the individual values ​​represented by symbols. Figures 12A to 12CThe data shown in were subjected to Kruskal-Wallis test and Dunn's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0232] Naive animals in contact with transmitters vaccinated and infected with sCPD9-ΔFCS did not show seroconversion, whereas contacts of transmitters vaccinated with mRNA or mock and superinfected showed seroconversion dependent on the challenge virus ( Fig. 12A and 12B ). As expected, animals vaccinated with sCPD9-ΔFCS showed a broad and strong humoral immune response before infection, while hamsters vaccinated with mRNA vaccine only generated their humoral response against the B.1 spike protein. Neutralizing antibody titers against BA.5 were detected only in animals vaccinated with sCPD9-ΔFCS vaccine. Repeated infection enhanced the antibody response in all groups.

[0233] Consistent with the virological results, only one contact vaccinated with sCPD9-ΔFCS showed seroconversion. Antibodies against SARS-CoV-2 B.1, Delta, and BA.1 were not detected in any serum samples obtained from contact animals of BA.5 transmitters. In addition, antibodies targeting B.1 S, N, and ORF3a proteins were not detected by ELISA ( Fig. 12B ). Overall, only sCPD9-ΔFCS vaccination induced broad humoral immunity and effectively reduced the transmission of BA.5 to primary contacts.

[0234] Nasal washes from sCPD9-ΔFCS-vaccinated and infected hamsters showed high IgA levels against B.1 and Omicron BA.5 spikes, regardless of the challenge virus. Meanwhile, IgA levels were low or absent in mRNA- and mock-vaccinated and superinfected hamsters, indicating the superior mucosal immunity provided by intranasal vaccination. At 6 dpc, naive contacts had no IgA antibodies, but there was a slight trend toward IgA development in contacts of mRNA- and mock-vaccinated and infected animals, which is consistent with the virological and serological results ( Fig. 12C ).

[0235] Naive contacts of mRNA-vaccinated transmitters were infected with both SARS-CoV-2 variants and showed abundant nucleocapsid expression in the nasal epithelium. Contacts of mock-vaccinated and B.1-challenged hamsters had fewer SARS-CoV-2-positive cells in the nasal epithelium than contacts of mRNA-vaccinated animals, consistent with viral RNA levels in oral swabs at 6 dpc.

[0236] Consistent with previous observations, the nasal epithelium of sCPD9-ΔFCS-vaccinated and B.1-infected primary contacts of the transmitter was free of SARS-CoV-2 nucleoprotein, except for a single hamster infected with BA.5 infectious disease. Consistent with the immunohistochemical results, varying degrees of inflammation and immune cell recruitment were detected in all hamsters infected with infectious disease of either variant, indicating the effectiveness of sCPD9-ΔFCS in preventing viral transmission. Histological results showed that the infection was completely cleared in all vaccinated and infected groups. Summary and discussion

[0237] Currently used LAVs present the entire antigenic repertoire of the virus in the respiratory mucosa, especially triggering the formation of tissue-resident memory T cells (TRM cells), which are specialized subsets of T cells that remain stable in specific tissues (such as the respiratory mucosa) after initial infection or vaccination (Schenkel and Masopust, 2014; Lavelle and Ward, 2022). These cells provide the first line of defense against reinfection by rapidly recognizing and responding to pathogens that re-enter the tissue at the surface of the body (Lavelle and Ward, 2022; Nouailles et al., 2022). In the context of SARS-CoV-2, TRM cells in the respiratory mucosa play a key role in initiating a rapid immune response after viral exposure. When encountering a virus, TRM cells can rapidly activate and release antiviral cytokines, recruit other immune cells to the site of infection, and directly eliminate cells infected with the virus. Their ability to reside in the respiratory mucosa allows them to more effectively survey and respond to local viral threats. By establishing this local immune surveillance network, TRM cells contribute to early control of viral replication and limit viral spread within the respiratory tract. Together with neutralizing (IgA) antibodies, TRM cells contribute to a comprehensive defense against SARS-CoV-2, effectively targeting the virus at its entry and initial replication sites, thereby reducing the likelihood of respiratory infection and transmission (Nouailles et al., 2023).

[0238] The effectiveness of the attenuated virus sCPD9-ΔFCS and the mRNA vaccine BNT1 62b2, both encoding the original form of the SARS-CoV-2 spike protein, in controlling the spread of SARS-CoV-2 was compared. The superior ability of the attenuated virus to prevent or significantly reduce viral transmission was demonstrated. Importantly, this was true even for BA.5, an evolved, highly transmissible, and strongly immune-evading SARS-CoV-2 variant. The emergence of the Omicron variant, which carries a large number of amino acid changes in its spike protein (Wolter et al., 2022; Madhi et al., 2022), has prompted the development of bivalent mRNA vaccines. Compared with monovalent vaccines, these vaccines contain spike proteins of the ancestral B.1 variant and the BA.4 / BA.5 variant, providing superior protection against the Omicron variant (Link-Gelles et al., 2022; Wang et al., 2023). However, it is becoming increasingly clear that SARS-CoV-2 transmission is not controlled or is not adequately controlled by intramuscular spike-based vaccines (Franco-Paredes, 2022).

[0239] Current data suggest that, in the case of SARS-CoV-2, mucosal vaccines have the ability to effectively prevent or reduce viral infection and onward transmission. In addition, they apparently do not require frequent updating of viral antigens, as B.1-based vaccines provide highly effective protection against the BA.5 variant, which is antigenically very different from the B.1 variant. It could be demonstrated that administration of two consecutive doses of sCPD9 effectively boosted immunity. This suggests that pre-existing immunity, such as that conferred by a previous SARS-CoV-2 infection, does not preclude the effectiveness of the mucosal vaccines tested here. On the contrary, regular boosting of existing immunity by mucosal vaccines could provide an important strategy for long-term control of SARS-CoV-2.

[0240] In summary, the results presented here highlight the importance and benefit of developing mucosal vaccines to enhance control of not only SARS-CoV-2 but potentially other respiratory viruses as well. Reducing viral shedding could limit and slow the spread and evolution of respiratory RNA viruses. 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Claims

1. A polynucleotide encoding a) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein; and b) optionally at least one nonstructural SARS-CoV-2 protein selected from the group consisting of nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, nonstructural protein 11, nonstructural protein 12, endoribonuclease and 2'-O-methyltransferase, wherein the polynucleotide comprises at least one sequence portion comprising a codon pair deoptimization compared to the SARS-CoV-2 genome, The polynucleotide further comprises a furin cleavage site modification, wherein the furin cleavage site modification results in the deletion of the furin cleavage site naturally present in the SARS-CoV-2 genome.

2. The polynucleotide of claim 1, wherein the furin cleavage site modification is at least a partial deletion of a furin cleavage site naturally present in the SARS-CoV-2 genome.

3. The polynucleotide of any one of claims 1 and 2, wherein the polynucleotide comprises a nucleic acid sequence defined by SEQ ID NO. 6 or a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO.

6.

4. The polynucleotide of any one of claims 1 and 2, wherein the polynucleotide comprises a nucleic acid sequence defined by SEQ ID NO. 8 or a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO.

8.

5. The polynucleotide of any one of claims 1 and 2, wherein the polynucleotide comprises a nucleic acid sequence defined by SEQ ID NO.10, a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO.10, a nucleic acid sequence defined by SEQ ID NO.15, a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO.15, a nucleic acid sequence defined by SEQ ID NO.16, a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO.16, a nucleic acid sequence defined by SEQ ID NO.17, or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO.

17.

6. The polynucleotide of any one of the preceding claims, wherein the furin cleavage site modification comprises a deletion of nucleotides encoding the amino acid sequence XRRA, wherein X represents P, R or H.

7. The polynucleotide of any of the preceding claims, wherein the furin cleavage site modification is such that expression of the polynucleotide produces a protein in which at least 5 consecutive amino acids of the naturally expressed protein are replaced by a single amino acid.

8. The polynucleotide of any of the preceding claims, wherein the furin cleavage site modification consists of or comprises a deletion of the nucleic acid sequence defined by SEQ ID NO. 18 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO.

18.

9. A live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a polynucleotide according to any one of claims 1 to 8.

10. The live attenuated SARS-CoV-2 according to claim 9, wherein the SARS-CoV-2 has a nucleic acid sequence defined by SEQ ID NO.19, a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO.19, a nucleic acid sequence defined by SEQ ID NO.20, or a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO.

20.

11. A pharmaceutical composition comprising the live attenuated SARS-CoV-2 according to any one of claims 9 to 10.

12. The pharmaceutical composition according to claim 11 for use as a vaccine.

13. A vector comprising the polynucleotide according to any one of claims 1 to 8.

14. A host cell comprising the polynucleotide according to any one of claims 1 to 8.

15. A method for producing a virus, the method comprising the steps of: a) culturing the host cell according to claim 14; and b) isolating the virus, wherein the virus is a live attenuated SARS-CoV-2.

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