Live attenuated SARS-CoV-2 and vaccines using it

Abstract

The present invention relates to a) a polynucleotide encoding a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and / or b) a polynucleotide encoding 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 or consists of at least one sequence portion comprising codon pair deoptimization compared to the SARS-CoV-2 genome, and the polynucleotide further comprises a furin cleavage site modification that results in the loss of a furin cleavage site naturally present in the SARS-CoV-2 genome. The present invention further relates to a live-attenuated SARS-CoV-2 comprising this polynucleotide, a vaccine comprising this live-attenuated SARS-CoV-2, and related methods.

Description

[Technical Field]

[0001] The present invention relates to codon pair deoptimized polynucleotides encoding SARS-CoV-2 proteins, attenuated SARS-CoV-2 comprising such polynucleotides, pharmaceutical compositions comprising such attenuated SARS-CoV-2, vaccination methods for administering the pharmaceutical compositions, vectors comprising such polynucleotides, host cells comprising such polynucleotides, and methods for producing viruses. ... [Background technology]

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 as the causative agent of coronavirus disease 2019 (COVID-19) (Wu et al., 2020; Zhou et al., 2020b). The virus is highly transmissible among humans (Chan et al., 2020). The virus spread rapidly around the world within weeks, and the world is currently battling the COVID-19 pandemic.

[0003] SARS-CoV-2 primarily replicates in the upper respiratory tract (Zou et al., 2020). SARS-CoV-2 infection can cause a wide range of clinical manifestations, from asymptomatic to life-threatening (Chen et al., 2020; Zhou et al., 2020a). Elderly individuals and those with pre-existing conditions are particularly at higher risk of developing more severe illness, including 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 enormous health, psychological, economic, and social burdens. To date (December 2021), over 270 million people have been infected with the virus, of which over 5.3 million have died as a result of the infection (https: / / coronavirus.jhu.edu / map.html) (Dong et al., 2020).

[0004] The unprecedented scale and severity of the COVID-19 pandemic has led to rapid development of new diagnostic tests, therapeutics, and vaccines that can be used to stop the spread of the virus and contain the pandemic. More than 90 vaccines are in clinical trials worldwide, although only a few have reached the final stages of testing (Zimmer et al., 2021). Nearly all vaccines that have been or are being evaluated in clinical trials are inactivated or subunit virus preparations ( Ella et al., 2021 ; Gao et al., 2020 ; Wang et al., 2020 ; Zhang et al., 2021 ), replication-deficient 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 ). 2020; Walsh et al., 2020).

[0005] SARS-CoV-2 is rapidly evolving (Tegally et al., 2021; Faria et al., 2021; Davies et al., 2021). Benefiting from its global spread, the virus continues to adapt to its new hosts and to infection- and vaccine-induced immunity. Over the course of the pandemic, numerous genetic variants have emerged (Tegally et al., 2021; Faria et al., 2021; Davies et al., 2021). Variants with increased infectivity, increased morbidity and mortality, or the ability to evade infection- and vaccine-induced immunity pose an increasing threat to public health. The World Health Organization (WHO) and national health authorities have independently established classification systems to categorize emerging variants into variants of concern (VOI), variants under investigation (VUI), and variants of concern (VOC) based on their risk to public health (see Table 1 in 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)). Furthermore, the WHO recommends that VOI and VOC be displayed using Greek alphabetic characters to facilitate communication with the public. As of August 12, 2021, viruses belonging to the 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) lineages have been classified as VOCs by several health authorities. In countries where these variants have emerged, they have rapidly replaced existing variants and begun to spread worldwide.

[0006] The B.1.1.7 variant, first detected in the UK in December 2020, is 50–100% more transmissible and potentially more lethal than earlier variants, but it does not appear to be prone to evading infection- or vaccination-induced immunity (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 has rapidly become the dominant variant in Europe and the US. The B.1.351 variant, first detected in South Africa in May 2020, is not only more transmissible but also has the ability to reinfect individuals and bypass 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 it shares several key mutations (E484K, K417N / T, N501Y) in the mutant spike glycoprotein. B.1.1.28.1 emerged in Manaus, Brazil in late 2020 (Faria et al., 2021). Like the B.1.351 variant, B.1.1.28.1 can bypass the immunity developed after infection with other viral variants, potentially leading to reinfection (Faria et al.). B.1.1.28.1 is estimated to be 40–140% more infectious, more virulent, and 10–80% more fatal than other variants (Faria et al.). On May 7, 2021, the WHO reclassified the B.1.617.2 variant, first detected in India, as a VOC due to its high transmissibility (WHO). As of August 2021, B.1.617.2 has nearly surpassed B.1.1.7 as the dominant variant in Europe and the United States. According to the WHO, B.1.617.2 is the most dangerous strain globally, and B.1.617.2 has attracted considerable attention for its ability to evade infection and vaccine protection (Dyer et al., 2021). The SARS-CoV-2 variant B.1.1.529 was first detected in Botswana in November 2021. BA.1.1.529 was first detected in Botswana in November 2021. BA.1.1.529 has become the predominant variant circulating worldwide.After the original BA.1 variant, several subspecies of Omicron evolved: BA.2, BA.3, BA.4 and BA.5, with BA.5 dominating the world as of August 2022.

[0007] WO 2021 / 154828 (Patent Document 1) and WO 2023 / 283106 (Patent Document 2) describe modified SARS-CoV-2 coronaviruses that have been recoded, e.g., codon-deoptimized or codon-pair-bias-deoptimized, and are useful for reducing the likelihood or severity of SARS-CoV-2 coronavirus infection, preventing SARS-CoV-2 coronavirus infection, eliciting an immune response, or treating 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. COVI-VAC was developed by introducing 283 silent (point) mutations by recoding a segment of the viral spike protein using synonymous, non-optimized codon pairs (codon deoptimization). Additionally, to enhance the safety of the vaccine strain, a furin cleavage site within the spike protein was deleted from the viral genome.

[0009] Chinese Patent No. 112175913 (Patent Document 3) describes an attenuated strain of SARS-CoV-2 and its application in the prevention and / or treatment of novel coronavirus pneumonia. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2021 / 154828 [Patent Document 2] International Publication No. 2023 / 283106 [Patent Document 3] Chinese Patent No. 112175913 [Non-patent literature]

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[0012] It is an object of the present invention to provide novel SARS-CoV-2 vaccine candidates.

[0013] This objective is achieved using a) a specific polynucleotide encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and, optionally, b) 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 (also referred to as nonstructural protein 15), and 2'-O-methyltransferase (also referred to as nonstructural protein 16). In this context, the polynucleotide comprises at least one sequence portion containing deoptimized codon pairs compared to the corresponding SARS-CoV-2 genome portion, or consists of at least one sequence portion containing deoptimized codon pairs. Furthermore, the polynucleotide comprises a furin cleavage site modification that results in functional loss of the furin cleavage site naturally present in the SARS-CoV-2 genome. Thus, the polynucleotide lacks some genetic information present in naturally occurring SARS-CoV-2.

[0014] The term "modified furin cleavage site," as used herein, refers to a site in the nucleotide sequence of a polynucleotide that corresponds to a site in the SARS-CoV-2 genome that encodes a furin cleavage site, but that has a modification that substantially reduces or eliminates the (furin) cleavage susceptibility of the encoded cleavage site. Examples of such modifications are provided herein, e.g., in the Examples, below. The modified furin cleavage site described herein can be any modification that alters cleavage susceptibility, such as a (partial or complete) deletion, insertion, or substitution of a nucleotide or sequence portion compared to the SARS-CoV-2 genome. In some embodiments, the furin cleavage site modifications described herein 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 compared to the portion of the SARS-CoV-2 sequence encoding the furin cleavage site.

[0015] The term "polynucleotide," as used herein, refers to a molecule comprising multiple nucleotides (e.g., mRNA, RNA, cRNA, cDNA, or DNA). The term typically refers to an oligonucleotide 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 in length. Polynucleotides of the present invention consist essentially of or comprise the nucleic acid sequences described herein. Thus, polynucleotides of the present invention may also comprise additional nucleic acid sequences. The term "polynucleotide" encompasses single-stranded and double-stranded polynucleotides. Furthermore, modified polynucleotides, including chemically modified polynucleotides, artificially modified polynucleotides, or naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides, are also encompassed herein.

[0016] The term "SARS-CoV-2" or "severe acute respiratory syndrome coronavirus 2," as used herein, 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 an alpha, beta, gamma, delta, or omicron variant. In some embodiments, the SARS-CoV-2 omicron variant is at least one SARS-CoV-2 omicron variant lineage, 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 variant comprising at least one mutation selected from the group consisting of del69-70, RSYLTPGD246-253N, N440K, G446V, L452R, Y453F, S477G / N, E484Q, E484K, F490S, N501Y, N501S, D614G, Q677P / H, P681H, P681R, and A701V. In some embodiments, SARS-CoV-2 refers to a SARS-CoV-2 variant containing 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, and n969k. In some embodiments, SARS-CoV-2 refers to a SARS-CoV-2 variant containing at least one mutation selected from the group consisting of L452R, F486V, and R493Q. Any combination of these mutations is possible and is disclosed herein. Specifically, within a SARS-CoV-2 variant, all mutations listed in any of the mutation groups mentioned above can be present simultaneously.Therefore, "SARS-CoV-2 protein" and "SARS-CoV-2 genome" can also be understood as the protein and genome, respectively, of a SARS-CoV-2 variant.

[0017] As used herein, the term "codon" refers to a group of three consecutive nucleotides that specify a particular amino acid, translation start or stop signal, in the coding portion of a polynucleotide, such as a messenger RNA molecule or a coding strand of DNA. Typically, a codon is specific for one amino acid, although in SARS-CoV-2, there are known cases where a codon shares at least one nucleotide with another codon.

[0018] As used herein, the term "codon pair" refers to two consecutive codons.

[0019] As used herein, the term "codon pair deoptimization" (CPD) refers to the "reformulation" of codons or the replacement of codons with other codons that encode the same amino acid, such that the encoded protein is the same but non-optimal 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 consists of increasing the number of underrepresented or non-optimal 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 decreased translation efficiency. In some embodiments, the codon pair deoptimization described herein results in fewer proteins, less virus, reduced pathogenicity, and / or an attenuated virus.

[0020] The polynucleotides of the present invention can be used in virus production or in the context of vaccines. Thus, the polynucleotides of the present invention have a reduced risk of uncontrolled replication during production, transportation, storage, processing and / or administration compared to wild-type sequences.

[0021] Typically, this polynucleotide constitutes part of an attenuated SARS-CoV-2. Compared to the wild-type virus, the attenuated virus causes fewer and / or more severe, or no, symptoms in a host organism after the host organism is confronted (infected) with the attenuated virus. At the same time, the live attenuated virus induces a host immune response against the attenuated virus that is at least partially protective against wild-type virus infection and / or at least one symptom thereof.

[0022] In contrast to most vaccines in development, we have generated an attenuated but replication-competent SARS-CoV-2 vaccine candidate by genetically modifying the SARS-CoV-2 genome via codon pair deoptimization (Coleman et al., 2008). CPD is a viral attenuation strategy that has enabled rapid and highly efficient attenuation of a wide variety of viruses (Broadbent et al., 2016; Coleman et al., 2008; Eschke et al., 2018; Groenke et al., 2020; Khedkar 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 positions of existing synonymous codons in one or more viral genes without altering 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). CPD may result in the over-representation of codons that are normally under-represented. Because the effect of CPD is highly dependent on the genomic sequence being deoptimized, it is not possible to provide precise instructions on how to change the codon positions in the recoded sequence during deoptimization. However, those skilled in the art are aware of methods to identify or predict codon pairs that can be replaced by target sites (e.g., target species or tissues, non-naturally occurring codon pairs) where a polynucleotide is intended to be translated. We provide examples herein of which codon pairs are re-encoded to achieve over-representation of non-naturally occurring codon pairs and de-optimization of codon pairs in polynucleotides.Thus, one skilled in the art can arrive at other polynucleotides according to the present invention from at least the codon pairs of the target site and the means and methods provided herein.

[0023] A polynucleotide is considered non-codon-optimized if at least one codon pair is not optimized relative to the corresponding native sequence. Recoded viruses typically do not produce proteins from the recoded genes as efficiently as their parent and may exhibit defects in reproductive fitness, allowing the host to control wild-type virus infection through innate and adaptive immune responses (Eschke et al., 2018; Groenke et al., 2020; Khedkar et al., 2018; Kunec and Osterrieder, 2016; Mueller et al., 2010; Trimpert et al., 2021b; Wimmer et al., 2009). With preserved antigenic identity and replication capacity, recoded attenuated viruses can fully engage the host immune system and elicit a robust immune response.

[0024] Thus, optimizing codon pairs does not alter the resulting protein. Rather, altering the SARS-CoV-2 genome sequence does not alter the resulting protein. However, it typically reduces translation efficiency and therefore viral replication. Therefore, when attenuated SARS-CoV-2 is used as a vaccine, an immune response is achieved without the risk of pathological viral replication in vaccinated patients. Another possible effect of deoptimizing codon pairs is a CpG-mediated immune response, leading to viral attenuation. The present disclosure is not limited to any particular one of these effects.

[0025] Although live-attenuated SARS-CoV-2 lacking the furin cleavage site (FCS) was significantly attenuated, it elicited a robust humoral immune response and maintained a level of protection against heterologous SARS-CoV-2 challenge comparable to that achieved by live-attenuated virus mutants lacking the FCS and the parental wild-type SARS-CoV-2. Most importantly, however, removal of the FCS completely abolished transmission of the vaccine virus between housed hamsters. These results demonstrate that removing the FCS from live-attenuated SARS-CoV-2 is a promising strategy to further enhance vaccine safety and prevent vaccine infection without compromising vaccine efficacy.

[0026] It is a surprising finding that the combination of independent attenuation mechanisms—FCS modifications, particularly deletions, and codon pair deoptimization and FCS modifications, particularly deletions—does not impair vaccine efficacy. Often, a combination of different attenuation mechanisms results in "overattenuation," resulting in insufficient viral growth or an inadequate immune response. Interestingly, such negative overattenuation effects were not observed in the currently claimed subject matter. In contrast, FCS modifications, particularly deletions, increase in vitro viral growth and the genetic stability of live-attenuated SARS-CoV-2. The latter is particularly important with regard to regulatory requirements, since the greater the stability of live-attenuated SARS-CoV-2, the easier it will be to obtain marketing authorization for a vaccine composed of live-attenuated SARS-CoV-2 and the greater the clinical safety of such a vaccine.

[0027] Unintended spread of vaccine virus from vaccinated to unvaccinated individuals complicates the use of transmissible live attenuated vaccines (LAVs) (Bull et al., 2018; Layman et al., 2021; Nuismer et al., 2018; Pons-Salort et al., 2016). While self-distribution is desirable in some scenarios, particularly when herd immunity in wildlife is sought (Smithson et al., 2019), unregulated circulation of vaccine virus potentially increases the likelihood of reversion to pathogenicity (Bull et al., 2018; Layman et al., 2021; Nuismer et al., 2018). Recombination between different vaccine viruses or between vaccine and wild-type viruses is particularly problematic because it can result in recombinants with enhanced pathogenicity, transmissibility, and 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 urges particular caution in the use of LAVs regarding the potential for irreversible circulation. Furthermore, transmission of attenuated viruses to immunocompromised individuals is a risk associated with the use of transmissible virus vaccines (Kamboj and Sepkowitz, 2007).

[0028] We have previously 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 outperformed adenoviral vectors and intramuscularly administered mRNA vaccines in inducing systemic and mucosal immunity (Nouailles et al., 2022). It was a surprising discovery that this robust immunity against several SARS-CoV-2 variants was not compromised by the introduction of additional FCS modifications, particularly deletions, into the polynucleotides encoding at least some proteins of attenuated SARS-CoV-2. Rather, the immune protection conferred by vaccination with a live attenuated SARS-CoV-2 vaccine composed of the novel polynucleotide construct was as good as that conferred by vaccination with a previously described live attenuated SARS-CoV-2 vaccine, despite the significantly greater biosafety of the novel construct (Trimpert et al., 2021a; Trimpert et al., 2021b).

[0029] SARS-CoV-2 entry 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 then enables actual cell entry by fusion of the viral envelope with the host cell membrane. To enable infection, the spike protein must be activated by cellular proteases. Activation involves proteolytic cleavage of the spike protein approximately in the middle of the protein at the S1 / S2 region, resulting in two subunits, S1 and S2, bound by noncovalent interactions. Cleavage of the spike protein triggers a conformational change that allows the S1 subunit to bind to ACE2 via its receptor-binding domain, triggering the fusion activity of the membrane-anchored S2 subunit.

[0030] Unlike other closely related viruses, SARS-CoV-2 contains a unique polybasic cleavage motif (PRRA↓) at the S1 / S2 site, called the furin cleavage site (FCS). Although several enzymes can cleave the FCS, it is most efficiently cleaved by the cell surface trypsin-like protease, TMPRSS2 (TMPRSS2) (Hoffmann and Pohlmann, 2021). The TMPRSS2 protease determines the viral entry route (Koch et al., 2021). When host cells express TMPRSS2, the virus is activated at the cell surface and rapidly enters the cell via cell fusion in a pH-independent manner. In contrast, in the absence of TMPRSS2, the virus is endocytosed, and viral entry is mediated by the endosomal / lysosomal cysteine ​​protease cathepsin L.

[0031] To prevent infection with the vaccine virus sCPD9, we deleted the FCS from its spike protein. Preclinical studies have shown that removing the FCS renders the mutant virus nontransmissible and strongly attenuated (Johnson et al., 2021; Lau et al., 2020; Peacock et al., 2021; Sasaki et al., 2021a). However, because removing the FCS accelerates viral attenuation, combining the FCS with different attenuation mutations may result in an overly attenuated virus with limited ability to induce potent immunity. Therefore, it is important to compare the infectivity and protective efficacy of LAV candidates lacking the FCS with those that retain the FCS. On the other hand, if removing the FCS does not impair the protective efficacy of the LAV candidate, removing the FCS is desirable because it enhances the safety of the LAV candidate by introducing a second, independent attenuating mutation into the viral genome.

[0032] Furthermore, beyond eliminating infection and enhancing vaccine safety, removing the FCS may offer important practical advantages in the production of SARS-CoV-2 LAVs. During growth in cells that do not express TMPRSS2, such as Vero cells commonly used by vaccine manufacturers, SARS-CoV-2 mutants lacking a functional FCS rapidly become dominant because they outcompete mutants with an intact 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, we found that sCPD9 also rapidly loses its FCS when grown on cells that do not express TMPRSS2. In contrast, removing the FCS improves the genetic stability of vaccine viruses during production and also increases viral titers on TMPRSS2-deficient cell lines.

[0033] 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 nsp15, an endonuclease. In one embodiment, the polynucleotide encodes nsp16, a 2'-O-methyltransferase. In one embodiment, the polynucleotide encodes the spike protein (sometimes referred to as the spike glycoprotein).

[0034] In one embodiment, the polynucleotide encodes at least one of a spike protein and a nonstructural protein.

[0035] In one embodiment, the polynucleotide encodes at least two of the nonstructural proteins. For example, the polynucleotide encodes an endoribonuclease and a 2'-O-methyltransferase. For another example, the polynucleotide encodes nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, and nonstructural protein 11.

[0036] In one embodiment, the modification of the furin cleavage site is a partial or complete deletion of the furin cleavage site naturally occurring in the SARS-CoV-2 genome.

[0037] In one embodiment, the modification of the furin cleavage site is a loss-of-function mutation of the furin cleavage site naturally occurring in the SARS-CoV-2 genome.

[0038] In one embodiment, the modification of the furin cleavage site is at least a partial replacement of the furin cleavage site naturally occurring in the SARS-CoV-2 genome.

[0039] In one embodiment, the SARS-CoV-2 genome is the portion of the genome extending from position 11,000 to position 27,000 of the SARS-CoV-2 genome. For position numbering and definitions of the terms "SARS-CoV-2 genome" and "wild-type SARS-CoV-2," see GenBank accession number MT108784.1 (freely accessible via the website https: / / www.ncbi.nlm.nih.gov / genbank / ), which consists of 29,891 bases or nucleotides. The first of these bases or nucleotides (5'-end) is located at position 1. The last of these bases or nucleotides (3'-end) is located at position 29,891. Those skilled in the art will recognize how to adjust the numbering of the referenced sequences for embodiments in which the SARS-CoV-2 genome is understood as a sequence from a different SARS-CoV-2 variant. In some embodiments, the polynucleotide of the invention is a codon-pair deoptimized sequence of a sequence comprised in the SARS CoV-2 genome section from position 11,000 to position 24,000. In one embodiment, the genome section extends from position 11,500 to position 26,000, particularly from position 11,900 to position 25,500, particularly from position 11,950 to position 25,350, particularly 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 section extends from position 13,900 to position 14,400. In one embodiment, the genome section extends from position 20,300 to position 21,600. In one embodiment, the genome section extends from position 24,300 to position 25,400. These embodiments can be combined in any desired way.

[0040] In one embodiment, at least one sequence portion comprising deoptimized codon pairs has a length in the range of 750 to 2500 nucleotides, particularly in the range of 800 to 2400 nucleotides, particularly in the range of 900 to 2300 nucleotides, particularly in the range of 999 to 2200 nucleotides, particularly in the range of 1000 to 2100 nucleotides, particularly in the range of 1100 to 2000 nucleotides, particularly in the range of 1146 to 1900 nucleotides, particularly in the range of 1200 to 1836 nucleotides, particularly in the range of 1300 to 1800 nucleotides, particularly in the range of 1400 to 1700 nucleotides, particularly in the range of 1500 to 1600 nucleotides.

[0041] In one embodiment, 15% to 40%, particularly 20% to 35%, and particularly 25% to 30% of the nucleotides in at least one sequence portion containing deoptimized codon pairs differ from those in the corresponding (wild-type) SARS-CoV-2 genome. Such wild-type SARS-CoV-2 genomes are the genome sequences of non-artificially modified viral variants or strains, such as strains 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 variants of Omicron subtypes BA.1, BA.2, BA.3, BA.4, and BA.5. These may also be referred to as authentic SARS-CoV-2 genomes or authentic SARS-CoV-2 genome sequences.

[0042] In one embodiment, 200 to 500 nucleotides, particularly 250 to 450 nucleotides, particularly 300 to 400 nucleotides of at least one sequence portion comprising deoptimized codon pairs differ from the corresponding nucleotides (particularly at the same positions) of the SARS-CoV-2 genome.

[0043] In one embodiment, 40% to 70%, particularly 45% to 65%, particularly 50% to 60%, particularly 55% to 62% of the codons in at least one sequence portion comprising deoptimized codon pairs differ from the respective codons in the corresponding SARS-CoV-2 genome.

[0044] In one embodiment, 150 to 400 codons, particularly 200 to 350 codons, particularly 250 to 300 codons of at least one sequence portion comprising codon pair deoptimization differ from the corresponding codons (particularly at the same positions) of the SARS-CoV-2 genome.

[0045] In one embodiment, the at least one sequence portion comprising deoptimized codon pairs comprises a first deoptimized sequence portion and a second deoptimized sequence portion. Both non-optimized sequence portions are separated from each other by a non-optimized sequence portion comprising at least 300 nucleotides, e.g., 300-1000 nucleotides, particularly 400-900 nucleotides, particularly 500-800 nucleotides, and particularly 600-700 nucleotides. By conserving specific portions of the RNA sequence and non-optimizing the flanking portions upstream and downstream of the conserved RNA sequence, particularly effective attenuation of SARS-CoV-2 can be achieved while maintaining the replication capacity of SARS-CoV-2.

[0046] In one embodiment, the first deoptimized sequence portion has a length ranging from 1300 to 1600 nucleotides, particularly ranging from 1400 to 1500 nucleotides, and particularly ranging from 1450 to 1490 nucleotides. At the same time, the second deoptimized sequence portion has a length ranging from 100 to 400 nucleotides, particularly ranging from 200 to 300 nucleotides, and particularly ranging from 350 to 400 nucleotides. The lengths of the first and second deoptimized sequence portions are optionally selected such that other applicable restrictions are met (e.g., the total length of at least one sequence portion comprising codon pair deoptimization is 2000 nucleotides or less). If the length of at least one sequence portion containing codon pair deoptimization must not exceed 2000 nucleotides, and the first and second deoptimized sequence portions are separated by at least 300 nucleotides of the authentic SARS-CoV-2 genome, it is immediately apparent that only the lower threshold of 1300 nucleotides can be combined with the upper threshold of 400 nucleotides for the first and second deoptimized sequence portions to satisfy the maximum length restriction for at least one sequence portion containing codon pair deoptimization. At the same time, the upper threshold of 1600 nucleotides for the first deoptimized sequence portion can be combined with the lower threshold of 100 nucleotides for the second deoptimized sequence portion to satisfy the maximum length of 2000 nucleotides, taking into account the intermediate 300 non-coding nucleotides.

[0047] In one embodiment, the first deoptimized sequence portion is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, and particularly 100% identical to SEQ ID NO: 2. At the same time, the second deoptimized sequence portion is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, and particularly 100% identical to SEQ ID NO: 4.

[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:6.

[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:8.

[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:10.

[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:15.

[0052] 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.

[0053] 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.

[0054] Even though FCS deletions in the SARS-CoV-2 genome have been described in the prior art, it could not be expected that such deletions (or at least FCS modifications), when combined with deoptimization of specific codon pairs present in the sequences described above, would have the effect observed by the inventors. Rather, it is surprising to find that the immune-protective properties of these codon-pair deoptimized sequences are not compromised by combining codon pair deoptimization with FCS modifications, particularly deletions.

[0055] In one embodiment, the modification of the furin cleavage site comprises the deletion of nucleotides encoding the amino acid sequence XRRA (i.e., the furin cleavage site), where X represents P, R, or H. Table 1 below lists specific embodiments of the presently claimed subject matter relating 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 irrelevant which nucleotides are excised from the SARS-CoV-2 genome upon deletion of the furin cleavage site, as long as the furin cleavage site is no longer present in the resulting protein.

[0056] [Table 1]

[0057] In one embodiment, modification, particularly deletion, of the furin cleavage site has the effect that expression of the polynucleotide results in a protein, particularly a spike protein, in which at least 5, particularly 6, particularly at least 7, particularly at least 8, particularly at least 9, particularly at least 10, particularly 5-10, particularly 6-9, particularly 7-8 consecutive amino acids of the naturally expressed protein are replaced with a single amino acid. This can be achieved by off-reading-frame deletion of nucleotides, whereby a single nucleotide of the first triplet combines with two nucleotides of the second triplet to form a novel triplet not present in the native SARS-CoV-2 genome at this genomic position.

[0058] In one embodiment, the single amino acid substituting for the naturally occurring consecutive amino acids is isoleucine.

[0059] In one embodiment, the modification of the furin cleavage site comprises or consists of 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.

[0060] In one aspect, the present invention relates to a live-attenuated SARS-CoV-2. The live-attenuated SARS-CoV-2 comprises a partially encoded genomic RNA sequence, i.e., a partially encoded genomic viral sequence. The partially encoded genomic RNA sequence is a codon-pair-optimized sequence encoding the spike protein and / or specific nonstructural proteins (nsp). The nonstructural proteins are selected from the group consisting of live-attenuated SARS-CoV-2 nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, nonstructural protein 11 (a small protein only 13 amino acids long), nonstructural protein 12 (also known as RNA-dependent RNA polymerase), nonstructural protein 15 (endoribonuclease), and nonstructural protein 16 (2'-O-methyltransferase). The live-attenuated SARS-CoV-2 further comprises a modified furin cleavage site, resulting in functional loss of the furin cleavage site. This furin cleavage site is naturally present in the SARS-CoV-2 genome. As a result, the spike protein of live-attenuated SARS-CoV-2 does not contain a functional furin cleavage site, whereas non-recombinant SARS-CoV-2 does contain such a furin cleavage site.

[0061] In one embodiment, the partially encoded genomic RNA sequence encodes nonstructural protein 12. In one embodiment, the partially encoded genomic sequence encodes spike protein (sometimes referred to as spike glycoprotein).

[0062] In one embodiment, the partially encoded genomic RNA sequence includes at least two of the nonstructural proteins. For example, the partially encoded genomic RNA sequence encodes an endoribonuclease and a 2'-O-methyltransferase. For another example, the partially encoded genomic RNA sequence encodes nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, and nonstructural protein 11.

[0063] In one embodiment, the partially encoded genomic RNA sequence is present in the genome portion extending from position 11,000 to position 27,000 of the live-attenuated SARS-CoV-2 genome. According to GenBank accession number MT108784.1, the wild-type SARS-CoV-2 genome contains 29,891 bases or nucleotides. The genomes of the live-attenuated SARS-CoV-2 are essentially similar in length. The difference is the length of the 3' poly(A) tail, which sequencing has revealed to be 8 adenine nucleotides longer than that of wild-type SARS-CoV-2. It should be noted that there remains uncertainty in determining the length of the poly(A) tail by sequencing. Therefore, the poly(A) tail of the wild-type sequence may be longer or shorter than indicated by the sequence under GenBank accession number MT108784.1. Similarly, the poly(A) tail of the live-attenuated SARS-CoV-2 sequence may be longer or shorter than currently determined. A further difference is that the live-attenuated SARS-CoV-2 lacks at least 12 nucleotides that encode the furin cleavage site of wild-type SARS-CoV-2.

[0064] The first base of the 29,891 bases (5' end) of the genome of wild-type SARS-CoV-2 is located at position 1. The last of these bases or nucleotides (3' end) is located at position 29,891. In one embodiment, the genome portion extends from position 11,500 to 26,000, particularly from position 11,900 to 25,500, particularly from position 11,950 to 25,350, particularly from position 12,000 to 24,000. In one embodiment, the genome portion extends from position 11,950 to 14,400. In one embodiment, the genome section extends from position 11,900 to position 13,500. In one embodiment, the genome section extends from position 13,900 to position 14,400. In one embodiment, the genome section extends from position 20,300 to position 21,600. In one embodiment, the genome section extends from position 24,300 to position 25,400. These embodiments can be combined in any desired way.

[0065] In one embodiment, the partially encoded genomic RNA sequence has a length in the range of 750 to 2500 nucleotides, particularly in the range of 800 to 2400 nucleotides, particularly in the range of 900 to 2300 nucleotides, particularly in the range of 999 to 2200 nucleotides, particularly in the range of 1000 to 2100 nucleotides, particularly in the range of 1100 to 2000 nucleotides, particularly in the range of 1146 to 1900 nucleotides, particularly in the range of 1200 to 1836 nucleotides, particularly in the range of 1300 to 1800 nucleotides, particularly in the range of 1400 to 1700 nucleotides, particularly in the range of 1500 to 1600 nucleotides.

[0066] In one embodiment, 15% to 40%, particularly 20% to 35%, particularly 25% to 30% of the nucleotides of the partially encoded genomic RNA sequence differ from the nucleotides of the corresponding wild-type genomic RNA sequence. Such wild-type genomic RNA sequences are genomic viral sequences of non-artificially modified virus variants or strains, such as strains 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). Such wild-type genomic RNA sequences may also be referred to as SARS-CoV-2 genomic RNA sequences.

[0067] In one embodiment, 200 to 500 nucleotides, particularly 250 to 450 nucleotides, especially 300 to 400 nucleotides of the partially encoded genomic RNA sequence differ from the nucleotides at the same positions of the corresponding wild-type viral genomic RNA sequence.

[0068] In one embodiment, 40% to 70%, particularly 45% to 65%, particularly 50% to 60%, particularly 55% to 62% of the codons (i.e., the three nucleotides in each case that code for a specific amino acid) of the partially encoded genomic RNA sequence differ from the respective codons of the corresponding wild-type viral genomic RNA sequence.

[0069] In one embodiment, between 150 and 400 codons, particularly between 200 and 350 codons, especially between 250 and 300 codons, of the partially encoded genomic RNA sequence differ from the codons at the same positions of the corresponding wild-type viral genomic RNA sequence.

[0070] In one embodiment, the partially encoded genomic RNA sequence comprises a first encoded portion and a second encoded portion, both of which are separated from each other by a non-recoded genomic portion comprising at least 300 nucleotides, e.g., 300-1000 nucleotides, particularly 400-900 nucleotides, particularly 500-800 nucleotides, and particularly 600-700 nucleotides. By conserving specific portions of the RNA sequence and recoding adjacent portions upstream and downstream of the conserved RNA sequence, particularly effective attenuation of SARS-CoV-2 can be achieved while maintaining the general viability of SARS-CoV-2.

[0071] In one embodiment, the first encoded portion has a length ranging from 1300 to 1600 nucleotides, particularly from 1400 to 1500 nucleotides, and particularly from 1450 to 1490 nucleotides. At the same time, the second encoded portion has a length ranging from 100 to 400 nucleotides, particularly from 200 to 300 nucleotides, and particularly from 350 to 400 nucleotides. The lengths of the first and second recoded portions are optionally selected to satisfy other applicable constraints (such as a total length of the recoded genomic RNA sequence of 2000 nucleotides or less). If the length of a partially encoded genomic RNA sequence must not exceed 2000 nucleotides, considering that the first and second encoded portions are separated by at least 300 nucleotides of the authentic SARS-CoV-2 genome sequence, it is immediately clear that the lower threshold of 1300 nucleotides and the upper threshold of 400 nucleotides for the first and second encoded portions can only be combined to meet the maximum length limit of the partially encoded genomic RNA sequence. At the same time, the upper threshold of 1600 nucleotides for the first encoded portion can be combined with the lower threshold of 100 nucleotides for the second encoded portion, taking into account the intermediate 300 non-coding nucleotides, thereby meeting the maximum length of 2000 nucleotides.

[0072] In one embodiment, the first coding 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 coding 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.

[0073] In one embodiment, the partially encoded 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.

[0074] In one embodiment, the partially encoded 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.

[0075] In one embodiment, the partially encoded 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.

[0076] The term "% identical" or "% sequence identity" with respect to a reference sequence is defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to the nucleotides or amino acid residues in the reference sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum % sequence identity, and not considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining % amino acid sequence identity can be achieved in a variety of ways within the skill of those 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 the algorithms required to achieve maximum alignment across the entire length of the sequences being compared.

[0077] Those skilled in the art will recognize that the sequences exemplified herein can be changed to a certain extent without substantially changing or altering the biological function of the protein, such as attenuation or encoding.Therefore, those skilled in the art using the means and methods described herein can modify codon pairs according to the teachings of the present invention.For example, codon pairs can be replaced with attenuated and / or non-naturally occurring synonymous versions, similar to the deoptimized codon pairs of the sequences described herein.Therefore, by replacing codon pairs in the position ranges described herein, the same degree of deoptimization can be achieved.

[0078] The present invention is based, at least in part, on the discovery that codon deoptimization of the range of positions described herein is particularly useful for attenuating SARS-CoV-2 virus while substantially maintaining immunogenicity and viral replication capacity. Furthermore, it is based on the discovery that furin cleavage site modifications that result in the functional loss of a furin cleavage site naturally present in the SARS-CoV-2 genome further attenuate SARS-CoV-2 while substantially maintaining immunogenicity.

[0079] The individual recoded sequences and their locations in the SARS-CoV-2 genome are summarized in Table 2 below.

[0080] [Table 2]

[0081] It should be noted that WT6A / CPD6A encodes only 9 nucleotides (3 amino acids) of nsp12 (RNA-dependent RNA polymerase, RdRp), the majority of which is encoded by WT6B / CPD6B.

[0082] The central portion of the WT6 fragment was not encoded by the CPD6 fragment because it contains a conserved regulatory RNA sequence essential for viral replication. The WT6 fragment encodes a -1 ribosomal frameshift element, a so-called RNA pseudoknot structure. This structure promotes ribosomal frameshifting, a process in which the translational reading frame changes at the junction of open reading frames (ORFs) 1a and 1b. During this process, one nucleotide in the slippery sequence located downstream of the RNA pseudoknot structure is read twice by the translating ribosome, resulting in a -1 nucleotide shift in the reading frame (the ribosome slides back one nucleotide along the slippery sequence). In most cases, translation of ORF1a terminates at its stop codon. However, if a -1 ribosomal frameshift occurs, translation of ORF1a continues uninterrupted until ORF1b is reached, resulting in the production of polyprotein (pp)1ab. Thus, the CPD6A sequence is translated into both polyproteins 1a and 1ab, whereas the CPD6B sequence is translated only into polyprotein 1ab.

[0083] The differences between the CPD sequence and the underlying wild-type sequence are summarized in Table 3 below.

[0084] [Table 3]

[0085] 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, the CPD does not alter the primary structure of the resulting protein. This is summarized in Table 4.

[0086] [Table 4]

[0087] By recoding sequence portions CPD6A, CPD6B, sCPD9, and sCPD10, different fragments of the SARS-CoV-2 genome that form part of the attenuated SARS-CoV-2 in the embodiments were generated, and these fragments are shown in Table 5 below.

[0088] [Table 5]

[0089] 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.

[0090] In one embodiment, the attenuated 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 non-codon pair optimized region, this attenuated SARS-CoV-2 comprises a 24 nucleotide deletion of the furin cleavage site in the spike protein gene, in which the amino acid sequence NSPRR ARSV (SEQ ID NO: 32) comprising the furin cleavage site is substituted for the amino acid isoleucine in the spike glycoprotein.

[0091] In one embodiment, the 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 non-codon pair optimized region, this attenuated SARS-CoV-2 comprises a 24 nucleotide deletion of the furin cleavage site of the spike protein gene, substituting the amino acid sequence NSPRRARSV (SEQ ID NO: 32) comprising the furin cleavage site of the spike protein with the amino acid isoleucine.

[0092] In one aspect, the present invention relates to a pharmaceutical composition comprising a live attenuated SARS-CoV-2 virus as described in any of the preceding paragraphs. Such a pharmaceutical composition may further comprise an auxiliary substance, such as an adjuvant, to enhance the patient's immune response. Suitable adjuvants include potassium alum; aluminum hydroxide; aluminum phosphate; calcium hydroxide phosphate; aluminum sulfate hydroxyphosphate; paraffin oil; propolis; killed bacteria of the species Bordetella pertussis or Mycobacterium bovis; plant saponins from Quillaja, soybean, and / or Polygala senega; cytokines IL-1, IL-2, and / or IL-12; and Freund's complete adjuvant.

[0093] In one aspect, the present invention relates to the further medical use of such pharmaceutical compositions as vaccines.

[0094] In one aspect, the present invention relates to the further medical use of such pharmaceutical compositions as vaccines for immunosuppressed individuals, particularly those receiving glucocorticoid treatment such as dexamethasone treatment.

[0095] In one aspect, the invention relates to a method for preparing a vaccine from such a pharmaceutical composition.

[0096] In one aspect, the present invention relates to a method of vaccinating a human or animal patient in need thereof, the animal patient being in particular a non-human mammal such as a rodent, dog, cat or mustelid, which method comprises administering to the patient a pharmaceutical composition according to the above description.

[0097] In one embodiment, the human or animal patient is an immunosuppressed patient, particularly a patient receiving glucocorticoid treatment, such as dexamethasone treatment.

[0098] In one embodiment, administration is by parenteral administration, such as intranasal administration, oral administration, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intravenous infusion, or intraperitoneal infusion. Nasal or oral administration is particularly suitable. These administration routes present the live attenuated SARS-CoV-2 to the recipient's body in a manner similar to or similar to natural exposure to the virus.

[0099] In one embodiment, vaccination is * 10 3 From 1 * 10 8 During FFU, especially 1 * 10 4 From 1 * 10 7 During FFU, especially 1 * 10 5 From 1 * 10 6 This is accomplished by administering a dose of a pharmaceutical composition containing live attenuated SARS-CoV-2 for a period of FFU. The dose is selected so that the pharmaceutical composition is well tolerated by the patient but induces an immune response that protects the patient against infection or a severe course of infection with SARS-CoV-2. In one embodiment, the dose is one of the minimum protective dose and the maximum tolerated dose, or is between the minimum protective dose and the maximum tolerated dose.

[0100] Various factors can influence the dosage used for a particular application, including frequency of administration, duration of treatment, prophylactic or therapeutic intent, use of multiple therapeutic agents, route of administration, previous treatments, the patient's clinical history, the attending physician's discretion, and the severity of the disease, disorder, and / or condition, which may influence the amount that needs to be administered.

[0101] As with the dosage, various factors can influence the actual frequency of administration used for a particular application, e.g., dosage, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the disease, disorder, and / or condition may require more or less frequent administration.

[0102] In some cases, the effective period for administering the pharmaceutical composition of the present invention (and any additional therapeutic agents) can be any period 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. Several factors can affect the actual effective period used for a particular treatment. For example, the effective period can vary depending on the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of the disease, disorder, and / or condition to be treated.

[0103] In one embodiment, the pharmaceutical formulation is administered to the patient at least twice, wherein the second administration is separated from the first administration by a first period of time, in this context the first period of time is in the range of 2 weeks to 36 months, particularly in the range of 3 weeks to 30 months, particularly in the range of 4 weeks to 24 months, particularly in the range of 5 weeks to 21 months, particularly in the range of 6 weeks to 18 months, particularly in the range of 7 weeks to 15 months, particularly in the range of 8 weeks to 12 months, particularly in the range of 9 weeks to 10 months, particularly in the range of 10 weeks to 8 months, particularly in the range of 12 weeks to 6 months, particularly in the range of 13 weeks to 4 months.

[0104] In one embodiment, the pharmaceutical formulation is administered to a patient after or before administering a different vaccine (e.g., a vector-based vaccine, an mRNA-based vaccine, a protein-based vaccine, etc.), i.e., after or before vaccinating the patient with a different vaccine, with a time offset. In this context, the administration of the pharmaceutical composition is offset by a second period relative to the administration of the different vaccine. In this context, the second period is in the range of 2 weeks to 36 months, particularly in the range of 3 weeks to 30 months, particularly in the range of 4 weeks to 24 months, particularly in the range of 5 weeks to 21 months, particularly in the range of 6 weeks to 18 months, particularly in the range of 7 weeks to 15 months, particularly in the range of 8 weeks to 12 months, particularly in the range of 9 weeks to 10 months, particularly in the range of 10 weeks to 8 months, particularly in the range of 12 weeks to 6 months, particularly in the range of 13 weeks to 4 months.

[0105] In one aspect, the present invention relates to a vector comprising a polynucleotide according to the above description.

[0106] As used herein, the term "vector" refers to a nucleic acid molecule that can transfer or transport itself and / or another nucleic acid molecule into a cell. The transferred nucleic acid is generally linked, i.e., inserted, into the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication within the cell, or may contain sequences sufficient to allow integration into the host cell DNA. In some embodiments, the vector described herein is a vector selected from the group consisting of a plasmid (e.g., a DNA plasmid or an RNA plasmid), a shuttle vector, a transposon, a cosmid, an artificial chromosome (e.g., bacterial, yeast, human), and a viral vector.

[0107] In some embodiments, the vectors described herein are used in combination with at least one transfection enhancer, e.g., a transfection enhancer selected from the group consisting of oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides.

[0108] In one aspect, the present invention relates to a host cell comprising a polynucleotide according to the above description.

[0109] As used herein, the term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. As used herein, mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included.

[0110] In embodiments, the host cells described herein comprise at least one cell type selected from the group of Chinese hamster ovary (CHO), Vero, Vero E6, Vero TMPRSS, MRC 5, Per.C6, PMK, WI-38, baby hamster kidney fibroblasts (BHK cells).

[0111] In one aspect, the present invention relates to a method for producing a virus, the method comprising 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.

[0112] All embodiments of the polynucleotides can be combined in any desired manner and transferred individually or in any combination into attenuated SARS-CoV-2 organisms, pharmaceutical compositions, uses thereof, methods of vaccinating patients, vectors, host cells, and methods of producing virus. All embodiments of the attenuated SARS-CoV-2 can be combined in any desired manner and transferred individually or in any combination into polynucleotides, pharmaceutical compositions, uses thereof, methods of vaccinating patients, vectors, host cells, and methods of producing virus. Similarly, all embodiments of the pharmaceutical compositions can be combined in any manner and transferred individually or in any combination into polynucleotides, attenuated SARS-CoV-2, uses of pharmaceutical compositions, methods of vaccinating patients, vectors, host cells, and methods of producing virus. All embodiments of the use of the pharmaceutical preparation can be combined in any desired way and can be transferred individually or in any combination to the polynucleotide, the attenuated SARS-CoV-2, the pharmaceutical preparation, the method of vaccinating a patient, the vector, the host cell, and the method of producing the virus. Finally, all embodiments of the method of vaccinating a patient can be combined in any desired way and can be transferred individually or in any combination to the polynucleotide, the attenuated SARS-CoV-2, the pharmaceutical preparation, the use of the pharmaceutical preparation, the vector, the host cell, and the method of producing the virus. [Brief explanation of the drawings]

[0113] Further details of aspects of the invention will now be described with reference to exemplary embodiments and the accompanying drawings, in which:

[0114] [Figure 1A] Schematic representation of the native structure and exemplary recoding of the SARS-CoV-2 genome. [Figure 1B] Schematic representation of the native structure and exemplary recoding of the SARS-CoV-2 genome. [Figure 1C] Schematic representation of the native structure and exemplary recoding of the SARS-CoV-2 genome. [Figure 1D] Schematic representation of the native structure and exemplary recoding of the SARS-CoV-2 genome. [Figure 2] Figure 1 shows the growth kinetics of sCPD9-ΔFCS and sCPD9 SARS-CoV-2 in Vero E6 cells. [Figure 3A] Virological and histopathological findings in contact hamsters are shown. [Figure 3B] Virological and histopathological findings in contact hamsters are shown. [Figure 3C] Virological and histopathological findings in contact hamsters are shown. [Figure 3D] Virological and histopathological findings in contact hamsters are shown. [Figure 3E] Virological and histopathological findings in contact hamsters are shown. [Figure 4A] Clinical parameters of infected Syrian hamsters are shown. [Figure 4B] Clinical parameters of infected Syrian hamsters are shown. [Figure 5A] Clinical, virological, pathological, and serological outcomes in hamsters after challenge with the SARS-CoV-2 delta variant are shown. [Figure 5B] Clinical, virological, pathological, and serological outcomes in hamsters after challenge with the SARS-CoV-2 delta variant are shown. [Figure 5C] Clinical, virological, pathological, and serological outcomes in hamsters after challenge with the SARS-CoV-2 delta variant are shown. [Figure 5D] Clinical, virological, pathological, and serological outcomes in hamsters after challenge with the SARS-CoV-2 delta variant are shown. [Figure 5E]Clinical, virological, pathological, and serological outcomes in hamsters after challenge with the SARS-CoV-2 delta variant are shown. [Figure 6A] The experimental setup, results, and biological background of in vivo and in vitro coinfection of sCPD9-ΔFCS and BA.5 are presented. [Figure 6B] The experimental setup, results, and biological background of in vivo and in vitro coinfection of sCPD9-ΔFCS and BA.5 are presented. [Figure 6C] The experimental setup, results, and biological background of in vivo and in vitro coinfection of sCPD9-ΔFCS and BA.5 are presented. [Figure 6D] The experimental setup, results, and biological background of in vivo and in vitro coinfection of sCPD9-ΔFCS and BA.5 are presented. [Figure 6E] The experimental setup, results, and biological background of in vivo and in vitro coinfection of sCPD9-ΔFCS and BA.5 are presented. [Figure 6F] The experimental setup, results, and biological background of in vivo and in vitro coinfection of sCPD9-ΔFCS and BA.5 are presented. [Figure 6G] The experimental setup, results, and biological background of in vivo and in vitro coinfection of sCPD9-ΔFCS and BA.5 are presented. [Figure 6H] The experimental setup, results, and biological background of in vivo and in vitro coinfection of sCPD9-ΔFCS and BA.5 are presented. [Figure 6I] The experimental setup, results, and biological background of in vivo and in vitro coinfection of sCPD9-ΔFCS and BA.5 are presented. [Figure 7A] Experimental setup and results are shown regarding the effect of immunosuppression on the safety, efficacy, and spread of sCPD9-ΔFCS. [Figure 7B] Experimental setup and results are shown regarding the effect of immunosuppression on the safety, efficacy, and spread of sCPD9-ΔFCS. [Figure 7C] Experimental setup and results are shown regarding the effect of immunosuppression on the safety, efficacy, and spread of sCPD9-ΔFCS. [Figure 7D] Experimental setup and results are shown regarding the effect of immunosuppression on the safety, efficacy, and spread of sCPD9-ΔFCS. [Figure 7E] Experimental setup and results are shown regarding the effect of immunosuppression on the safety, efficacy, and spread of sCPD9-ΔFCS. [Figure 7F] Experimental setup and results are shown regarding the effect of immunosuppression on the safety, efficacy, and spread of sCPD9-ΔFCS. [Figure 7G] Experimental setup and results are shown regarding the effect of immunosuppression on the safety, efficacy, and spread of sCPD9-ΔFCS. [Figure 7H] Experimental setup and results are shown regarding the effect of immunosuppression on the safety, efficacy, and spread of sCPD9-ΔFCS. [Figure 7I] Experimental setup and results are shown regarding the effect of immunosuppression on the safety, efficacy, and spread of sCPD9-ΔFCS. [Figure 7J] Experimental setup and results are shown regarding the effect of immunosuppression on the safety, efficacy, and spread of sCPD9-ΔFCS. [Figure 8A] Figure 8 shows the virological results of sCPD9-ΔFCS vaccinated contact hamsters for B.1 and BA.5 infections. The legends used below Figures 8E and 8F also apply to Figures 8A-8D. [Figure 8B] Figure 8 shows the virological results of sCPD9-ΔFCS vaccinated contact hamsters for B.1 and BA.5 infections. The legends used below Figures 8E and 8F also apply to Figures 8A-8D. [Figure 8C]Figure 8 shows the virological results of sCPD9-ΔFCS vaccinated contact hamsters for B.1 and BA.5 infections. The legends used below Figures 8E and 8F also apply to Figures 8A-8D. [Figure 8D] Figure 8 shows the virological results of sCPD9-ΔFCS vaccinated contact hamsters for B.1 and BA.5 infections. The legends used below Figures 8E and 8F also apply to Figures 8A-8D. [Figures 8E-8F] Figure 8 shows the virological results of sCPD9-ΔFCS vaccinated contact hamsters for B.1 and BA.5 infections. The legends used below Figures 8E and 8F also apply to Figures 8A-8D. [Figure 9A] 9C and 9D show systemic and mucosal immunity in vaccinated contact animals. The legends used below FIGS. 9C and 9D are also valid for FIGS. 9A and 9B. [Figure 9B] 9C and 9D show systemic and mucosal immunity in vaccinated contact animals. The legends used below FIGS. 9C and 9D are also valid for FIGS. 9A and 9B. [Figure 9C-9D] 9C and 9D show systemic and mucosal immunity in vaccinated contact animals. The legends used below FIGS. 9C and 9D are also valid for FIGS. 9A and 9B. [Figures 10A-10B] The clinical and virological outcomes of naive contacts of vaccinated B.1 shedding individuals are shown; the notation used following Figure 10C is also valid for Figures 10A and 10B. [Figure 10C] The clinical and virological outcomes of naive contacts of vaccinated B.1 shedding individuals are shown; the notation used following Figure 10C is also valid for Figures 10A and 10B. [Figures 11A-11B] The clinical and virological outcomes of naive contacts of vaccinated B.5 shedding individuals are shown; the notation explanations given below Figure 11C also apply to Figures 11A and 11B. [Figure 11C] shows the clinical and virological outcomes of naive contacts of vaccinated B.5 shedding individuals; the explanation of the symbols used below Figure 11C also applies to Figures 11A and 11B. [Figure 12A] 12C shows systemic and mucosal immunity in naive contact animals. The legend below FIG. 12C also applies to FIGS. 12A and 12B. [Figure 12B] Figures 12A to 12C show systemic and mucosal immunity in naive contact animals, and the legend below Figure 12C is valid for Figures 12A and 12B. [Figure 12C] 12C is a diagram showing systemic and mucosal immunity in naive contact animals; the explanation of the symbols used below FIG. 12C is also valid for FIGS. 12A and 12B. DETAILED DESCRIPTION OF THE INVENTION

[0115] We generated a series of encoded SARS-CoV-2 mutants and characterized them in cell culture and in vivo using Syrian hamster and Roborovski hamster models. We demonstrated that a single intranasal immunization with live-attenuated virus elicited a strong immune response and provided complete protection against SARS-CoV-2 challenge in a robust small animal model of COVID-19.

[0116] (Vaccine Design) Our goal was to generate an attenuated SARS-CoV-2 vaccine candidate through extensive recoding of the SARS-CoV-2 genome using CPD (Eschke et al., 2018; Groenke et al., 2020; Khedkar et al., 2018; Kunec and Osterrieder, 2016). To achieve viral attenuation in humans, we recoded the genome of SARS-CoV-2 with the codon pairs least frequently expressed in human genes (Groenke et al., 2020). Genetically engineered SARS-CoV-2 mutants were generated using a recently established reverse genetics system for SARS-CoV-2 (Thi Nhu Thao et al., 2020) (Figures 1A to 1D). The system relies on 12 subgenomic fragments of the SARS-CoV-2 genome, 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 approximately 3000 bp in length, with adjacent fragments overlapping by approximately 300 bp to allow for the construction of SARS-CoV-2 infectious clones by homologous recombination. The design and development of the applied vaccine are detailed in Trimpert et al., 2021a and Trimpert et al., 2021b.

[0117] In this regard, Figures 1A to 1D show the structure and recoding of the SARS-CoV-2 genome.

[0118] Figure 1A shows that the SARS-CoV-2 genome is a single-stranded, positive-sense RNA molecule of approximately 30,000 nucleotides (nt) encoding 11 canonical ORFs. "3CL-Pro" indicates 3C-like proteinase, "RdRp" indicates RNA-dependent RNA polymerase, "ExoN" indicates 3'-to-5' exoribonuclease, "EndoRNAse" indicates endoribonuclease, and "2'-O-MT" indicates 2'-O-ribose methyltransferase.

[0119] As shown in Fig. 1B , after infection, ORF 1a / 1ab is directly translated and cleaved into 15 proteins of the replication-transcription complex.

[0120] As shown in Figure 1C, a SARS-CoV-2 mutant consisting of 12 subgenomic fragments was constructed using a recently established SARS-CoV-2 reverse genetics system. Fragments 1, 11, and 12 were not recoded. The dark gray boxes indicate the recoded sequences CPD2-10, and the light gray boxes indicate the parental non-recoded sequences in each fragment 2-10. The frameshifting element contained in fragment 6 and the transcriptional regulatory sequence (TRS) of the spike gene contained in fragment 9 were excluded from the recoding process (light gray box located between the two dark gray boxes of CPD6 and CPD9).

[0121] As shown in Figure 1D, the dark grey boxes indicate the recoded sequences of sCPD3-5 and sCPD8-10 in different subgenomic fragments.

[0122] To maintain full compatibility with available reverse genetics systems, we encoded only SARS-CoV-2 sequences not present in the overlapping subgenomic fragments (approximately 2,500 bp in each coding fragment) (Figures 1A to 1D). This design allowed us to generate a wide variety of SARS-CoV-2 mutants with single or multiple coding fragments.

[0123] We recoded nine fragments (fragments 2-10) of the SARS-CoV-2 reverse genetics system. Fragments 1 and 12 were relatively short, at 591 and 1,812 bp, respectively. Fragment 11, which contains many short ORFs, was excluded from recoding. To ensure that the mutant virus was replication-competent, two genomic regions containing essential cis-acting RNA elements were excluded from recoding: the frameshifting element in fragment 6 and the transcriptional regulatory sequence (TRS) of the spike gene in fragment 9. Additionally, the first 500 bp of ORF 1a, located in fragment 2, was not recoded.

[0124] Materials and Methods (Study design and animal husbandry) This exemplary embodiment aimed to determine the effects of deleting the furin cleavage site (FCS) of sCPD9 on vaccine virus transmission, immunogenicity, protective efficacy, and host-to-host spread. The transmissibility of sCPD9-ΔFCS (i.e., SARS-CoV-2 lacking the sCPD9 sequence and furin cleavage site) was examined in comparison with wild-type (WT) SARS-CoV-2 and sCPD9 SARS-CoV-2. Furthermore, potential recombination between the SARS-CoV-2 mutant Omicron BA.5 and the vaccine candidate sCPD9-ΔFCS virus was evaluated in a coinfection experiment. Furthermore, the safety and infectivity of the sCPD9-ΔFCS vaccine candidate were examined in immunosuppressed hamsters.

[0125] Syrian hamsters (Mesocricetus auratus; breed RjHan:AURA) were purchased from Janvier Labs and housed in pairs in individually ventilated cages. Food and water were available ad libitum, and cages contained nesting material. Room temperature was maintained at 22–24°C and relative humidity at 40–55°C. Animals were allowed to acclimate to the housing environment for 7 days before the start of the experiment.

[0126] Thirty-six 10-week-old Syrian hamsters of each sex were used to assess transmission of B.1, sCPD9, and sCPD9-ΔFCS viruses from infected to naive animals.

[0127] Half of each animal had 1 * 10 5 Hamsters were intranasally administered either FFU WT (B.1), sCPD9, or sCPD9-ΔFCS. Immediately after WT infection or sCPD9 / sCPD9-ΔFCS vaccination, they were housed in individual cages and reunited with uninfected / unvaccinated siblings 1 day after infection / vaccination. Body weights were recorded daily for all hamsters, and their clinical status was checked twice daily. Oral swabs were collected daily from exposed hamsters between 1 and 6 dpc (days after exposure). Exposed animals were euthanized on day 6 and assessed for viral load in the upper and lower respiratory tract, signs of pneumonia, and seroconversion.

[0128] WT-infected and sCPD9 / sCPD9-ΔFCS-vaccinated animals were cultured at 1 day post-infection / vaccination. * 10 5 Hamsters were challenged with the PFU SARS-CoV-2 delta mutant. Six uninfected / unvaccinated hamsters were also challenged with the delta mutant for comparison. Hamsters were then euthanized 2 and 5 days later, and blood, trachea, and lung samples were collected for virological, serological, and histopathological analyses.

[0129] To investigate the possibility of recombination between the SARS-CoV-2 variant Omicron BA.5 and sCPD9-ΔFCS, we performed a coinfection experiment using 12 4-week-old female Syrian hamsters. After acclimation, six hamsters were infected with 1 × 10 FFU of sCPD9-ΔFCS and 1 × 10 4 The animals were co-infected intranasally with full-fledged units (FFU) of the SARS-CoV-2 variant Omicron BA.5. Infection was performed under general anesthesia. Infected animals were then isolated in separate cages to prevent accidental virus transmission to naive animals. After 6 days, infected animals were rehoused with naive partners. Body weights and oral swabs were collected daily from all animals to screen for viral infection. Clinical status was monitored twice daily. After 6 days of cohabitation, animals were euthanized, and blood, trachea, and lung samples were collected for virological analysis.

[0130] To evaluate the safety and infectivity of the vaccine in immunosuppressed animals, 12 4-week-old female Syrian hamsters were immunosuppressed by daily subcutaneous injections of dexamethasone (2 mg / kg). On the third day of immunosuppression, 6 hamsters were injected with 1 × 10 4 Hamsters were vaccinated with FFU sCPD9-ΔFCS by intranasal application under general anesthesia, as described below. Vaccinated animals were housed in individual cages for 24 hours and then reunited with their unvaccinated and immunosuppressed housemates. During the cohabitation period, oral swabs were collected daily from all hamsters to detect possible vaccine virus infection. Contact animals were euthanized 6 days post-contact (dpc), and vaccinated hamsters were sacrificed 21 days post-vaccination (dpv). Blood, tracheas, and lungs were collected for serological, virological, and histopathological analyses.

[0131] (cell) Minimum 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 Vero RCB 10-87 cells. For VeroE6-TMPRSS2 cells (NIBSC 100978), the medium was supplemented with 1000 μg / ml geneticin (G418) to select for cells expressing TMPRSS2. Cells were maintained at 37°C and 5% CO2.

[0132] (virus) SARS-CoV-2 mutants 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 ex India, 20A / 452R, EVAg: 009V-04187), and SARS-CoV-2 mutants B.1-ΔFCS, sCPD9, and sCPD9-ΔFCS were cultured in 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 propagated on Vero E6 cells and used for growth kinetics and plaque size assays. Viral stock titers were determined by plaque assays on Vero E6 cells, and the virus was stored at -80°C before experimental infection.

[0133] (Ethics statement) In vivo and in vitro experiments were performed in a biosafety level 3 (BSL-3) laboratory at the Institut fur Virologie, Free University Berlin, Germany. Animal experiments were performed in accordance with institutional, national, and international animal care and humane use guidelines and were approved by the Landesamt fur Gesundheit und Soziales Berlin (permit number 0086 / 20).

[0134] (Infectious diseases / vaccinations) Hamsters were infected or vaccinated under general anesthesia (0.15 mg / kg medetomidine, 2.0 mg / kg midazolam, 2.5 mg / kg butorphanol). * 10 5FFU B.1 (wild type), sCPD9, or sCPD9-ΔFCS was diluted in 60 μl of MEM and administered intranasally. Mock-vaccinated mice were administered 60 μl of plain MEM without virus. 21 days after vaccination or primary infection, the mice were vaccinated for 1 hour, as with the primary infection. * 10 5 A challenge infection with the FFU SARS-CoV-2 Delta variant was performed.

[0135] (RNA extraction and reverse transcription quantitative PCR (RT-qPCR)) Genome copies were quantified from oropharyngeal swabs and 2.5 mg of lung tissue homogenized with a bead mill (Analytic Jena). RNA was extracted using the innuPREP Virus DNA / RNA Kit (Analytic Jena, Jena, Germany). RT-qPCR was performed using the NEB Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs). Cycling conditions were 5510 min for reverse transcription, followed by 943 min, 9415 s, and 5830 s for enzyme activation, repeated 40 times, using primers and probes according to Corman et al. (Corman et al., 2020) (Corman et al., 2020) in a qTower G3 cycler (Analytic Jena).

[0136] (Plaque assay) To quantify replication-competent virus, 10-fold serial dilutions of 50 mg of homogenized lung tissue were prepared and plated onto Vero E6 cells cultured in 12-well plates. Cells were incubated at 37°C and 5% CO2 for 2.5 hours and then overlaid with 1.5% sodium carboxymethylcellulose (Sigma-Aldrich) diluted in complete growth medium. 72 hours postinfection, cells were fixed using a PBS-buffered 4% formaldehyde solution (pH 6.5). Plaque-forming units were counted per well after staining with 0.75% methylene blue (aqueous solution).

[0137] (Neutralization test) All hamster serum samples were tested for neutralizing activity against SARS-CoV-2 (B.1). Additionally, sera from challenged animals (0, 2, and 5 days post-transplant) were tested for neutralizing activity against the SARS-CoV-2 delta mutant (B.1.617). For this purpose, sera were inactivated for 5630 min. Then, two-fold serial dilutions (1:8 to 1:1024) were prepared in 96-well plates, and 200 PFU of SARS-CoV-2 diluted in MEM (1% FBS, 1% P / S) was added to all wells. After 371 h of incubation, the dilutions were plated onto subconfluent Vero E6 cells in 96-well cell culture plates and incubated for an additional 72 h. Finally, the plates were fixed with 4% formaldehyde solution and stained with 0.75% methylene blue (aqueous). Wells without viral cytotoxicity were considered neutralized and reported as the titer for each serum. Positive and negative controls were included on every plate.

[0138] (Histopathology) After sacrificing the animals, the left lung lobe was carefully removed and fixed in 4% formaldehyde for 48 hours. The tissue was then embedded in paraffin and sectioned at 2 μm thickness for staining with hematoxylin and eosin (H&E). Lung preparation and pneumonia scoring were performed according to the standardized procedures described by Osterrieder et al. (2020).

[0139] (growth kinetics) Confluent T25 flasks of WHO Vero RCB 10-87 cells were infected with either B.1-βCoV / Munich / ChVir984 / 2020, B.1, EPI_ISL_406862, B.1-ΔFCS, sCPD9, or sCPD9-ΔFCS at an MOI of 0.01. Virus was diluted in a final volume of 5 ml of complete cell culture medium and added to each flask. Supernatants were harvested at 24, 48, 72, and 96 hours postinfection and subjected to one freeze-thaw cycle. Tenfold dilutions were prepared and plated onto confluent Vero E6 cells seeded in 12-well plates. Cells were overlaid with MEM containing 1.5% carboxymethylcellulose after 1.25 hours and fixed with 4% formaldehyde after 48 hours. For visualization of plaques, immunofluorescence staining was performed as described ( Trimpert et al., 2021b ).

[0140] (Co-culture of sCPD9-ΔFCS and Omicron BA.5) CaLu-3 cells were seeded in T25 flasks and grown to 90% confluency. Prior to coinfection with 100 FFU of SARS-CoV-2 variant Omicron BA.5 and 1,000 FFU of sCPD9-ΔFCS, the cell culture medium was changed to 5 ml of 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 collected and clarified by centrifugation at 5,000 rpm for 15 minutes. A % of the supernatant was then transferred to uninfected CaLu-3 cells. Assays were performed in triplicate and continued for a total of 10 passages.

[0141] (sequence) After RNA extraction from cell culture supernatants as described above, libraries were prepared and sequenced using Illumina technology (Illumina). The NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (New England Biolabs) was used for library preparation. This approach relies on standard library preparation steps for Illumina sequencing, including end repair, adapter ligation, and PCR enrichment. Quantification of the enriched sequencing libraries was performed using the NEBNext® Library Quant Kit for Illumina® (New England Biolabs). The libraries were then pooled and sequenced on an Illumina Miseq System (Illumina).

[0142] The generated Illumina sequencing data were processed with Trimmomatic v.0.39 (Bolger et al., 2014) and mapped to the BA.5 (NCBI accession number: ON249995) and sCPD9-ΔFCS genome references (GenBank: MZ064545.1) (Trimpert et al., 2021b) using Burrows-Wheeler aligner v.0.7.17 (Li and Durbin, 2009). Mapping statistics were generated using Samtools v1.10 (Danecek et al., 2021), and alignments were visualized using IGV v2.9.4 for Linux (Robinson et al., 2011). For single nucleotide polymorphism (SNP) detection, we used the Bayesian gene variation detector Freebayes (arXiv:1207.3907 [q-bio.GN] 2012). All SNPs with a minimum mapping quality of 5, a minimum count of 3, and a minimum fraction of 0.1 were initially considered. SNPs detected between the starting BA.5 isolate and the BA.5 reference strain used in these studies were removed from further analysis because they were present prior to these experiments. A table containing the removed SNPs is provided. Consensus sequences for each sample were obtained using BCFtools (Danecek et al., 2021).

[0143] Direct sequence comparison with sCPD9-ΔFCS is not efficient given the subtle differences in the genome structure between BA.5 and sCPD9-ΔFCS, as well as the high entropy between the sample and the sCPD9-ΔFCS reference. To facilitate this process, a sample-wide 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. This sample-wide consensus was aligned to both the BA.5 and sCPD9-ΔFCS references using EMBOSS Stretcher (Myers and Miller, 1988). A new SNP table was created using SNP-sites, and the location and identity of all detected SNPs were verified against both references. Any SNPs that occurred between the BA.5 isolate and the sCPD9-ΔFCS reference were excluded from the analysis.

[0144] (result) Deletion of FCS moderately increases viral titers in TMPRSS-2-negative cells. To increase the genetic stability of sCPD9 and reduce its transmissibility, we generated an sCPD9 derivative (designated sCPD9-ΔFCS) lacking the FCS in the spike protein. The sCPD9-ΔFCS mutant was engineered to contain a deletion, called the "Bristol deletion," identical to the deletion of the spike protein that naturally occurs during serial passage in cultured Vero E6 cells (Davidson et al., 2020). The introduced deletion is 24 nucleotides long. It removes nine amino acids, "NSPRRARSV," from the spike protein, including the entire FCS, and instead introduces a novel amino acid, isoleucine, at the same position (Davidson et al., 2020). This removes a total of eight amino acids from the spike protein.

[0145] It has been widely reported that FCS-deficient SARS-CoV-2 virus mutants have a clear advantage in growth on different Vero cell lines compared with viruses that lack the FCS. In line with these results, we found that FCS removal slightly increased the titer of sCPD9-ΔFCS virus grown on Vero cells. Because WHO RCB 10-87 Vero cells are widely used for vaccine production, the ability to generate slightly higher peak virus titers more quickly on these specific cells is of great practical importance, as it reduces the cost of vaccine production (Figure 2). In this regard, Figure 2 shows the mean (± standard deviation (SD)) growth kinetics of sCPD9-ΔFCS and sCPD9 on WHO Vero RCB 10-87 cells. Supernatants were harvested at indicated hours post-infection (hpi).

[0146] Deletion of the FCS prevents sCPD9-ΔFCS from infecting unvaccinated contacts. To examine the ability of sCPD9 and sCPD9-ΔFCS to infect unvaccinated contact animals, Syrian hamsters were infected with sCPD9, sCPD9-ΔFCS, or parental SARS-CoV-2 (WT) on day 0. Twenty-four hours after infection, each infected hamster was placed in an individual ventilated cage with an unvaccinated hamster. Already on the first day of cohabitation, oral swabs from all naive animals in contact with WT-infected animals were strongly positive for SARS-CoV-2 RNA. All naive animals in contact with sCPD9-vaccinated subjects contracted the virus on days 1–3 of cohabitation, and showed a similar, albeit delayed, course of viral replication in the upper respiratory tract compared to WT-infected animals. In contrast, naive animals in contact with sCPD9-ΔFCS-vaccinated individuals did not become SARS-CoV-2 RNA positive during the first 7 days of cohabitation, the observation period (Figure 3A). Figure 3A shows the mean ± SD of genomic RNA (gRNA) copies detected in buccal swabs collected daily (1–6 dpc) from contact hamsters. Two-way analysis of variance and Tukey's multiple comparison test were performed ( * p<0.05, ** p < 0.01,*** p<0.001, *** p<0.0001). In this and all figures, the open circles or squares (first bar from the left) represent sCPD9-ΔFCS, the light grey circles or squares (second bar from the left) represent sCPD9, and the dark grey circles or squares (third bar from the left) represent B.1 (WT SARS-CoV-2).

[0147] Natural infection with wild-type SARS-CoV-2 caused the expected COVID-19-like pneumonia in contact animals, as evidenced by histological examination on day 7 of cohabitation (Figure 3B). Consistent with previous findings, lung inflammatory changes were significantly attenuated in animals infected with sCPD9. Furthermore, the lungs of animals cohabited with sCPD9-ΔFCS-vaccinated animals showed little signs of inflammation.

[0148] (Syrian hamster sCPD9-ΔFCS is highly attenuated) All hamsters remained clinically healthy after infection with sCPD9 or sCPD9-ΔFCS, but WT-infected animals showed the expected mild to moderate signs of disease, such as forced breathing and significant weight loss, during the first week postinfection (Figure 4A). In this context, Figure 4A shows the percentage weight loss by group after primary vaccination / infection and up to the challenge infection time point, 21 dpi / dpv (dpi = days postinfection, dpv = days postvaccination).

[0149] However, in the absence of other visible signs of illness, sCPD9-infected animals showed a slight tendency to lose weight, whereas sCPD9-ΔFCS-vaccinated animals showed relatively stable weights 1 week after vaccination.

[0150] All contact animals exposed to sCPD9- or WT-infected animals were infected with the respective viruses; however, clinical signs of disease and weight loss occurred only in animals infected with the WT virus (Figure 4B, % weight gain of contact animals over the cohabitation period, by group). Both Figures 4A and 4B are truncated violin plots with quartiles and medians.

[0151] Similarly, severe histological signs of lung inflammation on day 7 post-contact were present only in contact animals infected with WT virus (Figure 3B, showing gRNA copy numbers recovered from oropharyngeal swabs and homogenized lungs, and the number of replication-competent viruses detected in lung tissue; the limit of detection is indicated by the dotted line). Data depicted in Figure 3B to 3D were statistically evaluated by Kruskal-Wallis test and Dunn's multiple comparison test ( * p < 0.05, ** p<0.01, *** p<0.001, *** p<0.0001).

[0152] Figure 3C shows replication-competent virus detected in lung tissue, with the limit of detection again indicated by the dotted line.

[0153] Figure 3D shows histopathological scoring, including a pneumonia score indicating the severity of pneumonia, and an edema score indicating the influx of neutrophils, lymphocytes, and macrophages, bronchial epithelial necrosis, bronchitis, alveolar epithelial necrosis, perivascular lymphocytic cuff, pneumocyte type II hyperplasia, perivascular edema, and alveolar edema.

[0154] Figure 3E shows the neutralizing activity of sera collected from hamsters 6 days post-exposure against SARS-CoV-2 B.1 WT.

[0155] Contact animals infected with sCPD9 showed only mild inflammatory changes in the lungs, whereas these changes were further reduced or absent in the lungs of contact animals infected with sCPD9-ΔFCS that were not infected with the virus.

[0156] (Deletion of FCS does not reduce vaccine efficacy) All infected animals were challenged with a pathogenic SARS-CoV-2 delta mutant on day 21 post-infection. None of the challenged animals developed clinical signs of disease or exhibited the significant weight loss observed in unvaccinated control animals (Figure 5A, showing controlled weight changes up to analysis time point 2 + 5 dpch, expressed as percentages by group). In this and all other figures, the medium gray box (fourth bar from the left) represents mock-vaccinated animals.

[0157] As expected, protection against challenge virus replication was comparable in all three infected groups. Two days after challenge, high concentrations of viral RNA were detected in the upper respiratory tract in all groups (Figure 5B, showing quantification of gRNA in oropharyngeal swabs and infectious viral particles detected in lungs and homogenized lung tissue). In Figures 5B–5E, dotted lines indicate the limits of detection. Furthermore, the data shown in Figures 5B–5E were statistically analyzed by two-way ANOVA and Tukey's multiple comparison test (Figure 5C). * p<0.05, ** p<0.01, *** p<0.001, *** p<0.0001). However, the high viral RNA loads observed decreased to levels near the limit of detection by day 5 postchallenge in all study groups. Protection in the lower respiratory tract was more pronounced on day 2 postchallenge, with significantly lower viral RNA loads and minimal levels of replication-competent virus in the lungs of infected animals (Figure 5B). By day 5 postchallenge, RNA loads were near or below the limit of detection, and no replicating virus was present in the lungs of challenged subjects.

[0158] Overall, virological parameters confirmed the high protective efficacy of the sCPD9 and sCPD9-ΔFCS vaccine candidates, comparable to the WT virus.

[0159] The protective efficacy of the sCPD9 and sCPD9-ΔFCS vaccine viruses was determined by examining lung histopathology in uninfected, sCPD9-, sCPD9-ΔFCS-, and WT-infected hamsters on days 2 and 5 postchallenge (Figure 5C shows infectious virus particles detected in homogenized lung tissue, and Figure 5D shows the percentage of lung area enhanced by SARS-CoV-2 infection). Pneumonia scores were assessed based on the severity of pneumonia, including neutrophil, lymphocyte, and macrophage influx, bronchial epithelial necrosis, bronchitis, alveolar epithelial necrosis, perivascular lymphocytic excretion, and alveolar type II hyperplasia. Pulmonary edema was scored based on the degree of perivascular and alveolar edema. Here, we found that WT-infected animals were more prone to developing inflammation and exhibited increased immune cell influx compared with animals infected with sCPD9 or sCPD9-ΔFCS viruses and unvaccinated animals. This situation improved by day 5 post-challenge, suggesting that the immune system of WT-infected animals transiently overreacted immediately after challenge, leading to the lung injury observed on day 2 post-challenge. Importantly, all animals demonstrated good and comparable protection against COVID-19-like pneumonia at all post-challenge time points examined.

[0160] Finally, we compared humoral immune responses to SARS-CoV-2 WT and Delta mutants in infected animals before challenge and on days 2 or 5 after challenge (Figure 5E, showing neutralizing activity against B.1 and Delta mutants in hamster sera collected on days 0 (prechallenge), 2, and 5 after SARS-CoV-2 Delta challenge; detection range reached from dilutions of 1:8 to 1:1024). Vaccination / infection with sCPD9, sCPD9-ΔFCS, or WT virus induced similar levels of neutralizing antibodies at all time points tested.

[0161] sCPD9-ΔFCS is slightly less potent than sCPD9, but its protective effect is comparable. The results presented here demonstrate that removal of the FCS from sCPD9 results in a nontransmissible, highly attenuated virus in a Syrian hamster model. Despite the high degree of attenuation, the sCPD9-ΔFCS virus provided protection against SARS-CoV-2 variant delta equivalent to that of the parental sCPD9 virus, which has an intact FCS in the spike protein. Both the sCPD9 and sCPD9-ΔFCS vaccine viruses demonstrated excellent and comparable protection against viral replication and disease across virological, serological, and histological parameters. Overall, both vaccine viruses provided protection comparable to that induced by WT virus infection. Surprisingly, animals infected with the WT virus showed signs of intense lung inflammation for the first few days after challenge.

[0162] Extensive histological examination revealed that vaccination with sCPD9-ΔFCS or sCPD9 was superior to vaccination with WT virus for subsequent viral challenge. Experimental animals infected with WT virus and subsequently challenged with SARS-CoV-2 exhibited significant alveolar broncholysis. In contrast, no alveolar-tracheal dissection was observed in animals vaccinated with sCPD9-ΔFCS or sCPD9 and subsequently challenged with SARS-CoV-2.

[0163] (Advantages of FCS removal in large-scale production of LAV SARS-CoV-2 vaccines) Many reports have shown that FCS inhibits SARS-CoV-2 replication in TMPRSS2-deficient cells, such as Vero and Vero-E6 cells, because SARS-CoV-2 mutants lacking a functional FCS rapidly become dominant when passaged 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 presented here support these observations, as the sCPD9-ΔFCS mutant replicated to higher titers than the original sCPD9 virus in Vero cells.

[0164] Thus, removing FCS from SARS-CoV-2-LAV has two advantages for LAV vaccine production: because most vaccine manufacturers use Vero cells for LAV vaccine production, increasing viral titers reduces production costs and, more importantly, facilitates the purification of sufficient numbers of infectious viral particles for a single vaccination.

[0165] A second major benefit of removing the FCS is that it significantly improves the genetic stability of LAV vaccine candidates. One of the prerequisites for using LAV vaccines in humans is a high genetic homogeneity of the vaccine virus population. Because SARS-CoV-2 rapidly loses its FCS at the S1 / S2 boundary after passage in Vero cells, removing the FCS eliminates the problem of genetic instability and allows for the production of vaccine viruses with high genetic homogeneity.

[0166] (In vivo coinfection does not result in viral recombinants) To test for possible recombination between the vaccine and circulating field viruses in vivo, Syrian hamsters were infected with equal doses of sCPD9-ΔFCS and the omicron mutant BA.5 (1 × 104 Animals were infected with 100 FFU / animal. After 24 hours, infected animals were housed with naive contact hamsters to assess host-to-host transmission. Oral swabs were collected daily; all animals were euthanized after 6 days of co-housing. Figure 6A is a schematic diagram of the experimental design.

[0167] Figure 6B shows the changes in animal weight after coinfection or contact with coinfected animals. Compared with contacts, infected animals showed a wider range of weights, with some individuals experiencing mild, transient weight loss.

[0168] Figure 6C shows that no replication-competent virus was detected in the lungs of experimentally infected animals (circles: coinfected animals, triangles: contact animals) on day 7 postinfection. For this analysis, infectious virus particles were detected in homogenized lung tissue.

[0169] Figures 6D-E show viral gRNA copies detected in oral swabs (Figure 6D), oropharyngeal swabs (Figure 6E), and lung tissue (Figure 6F) from co-infected hamsters (top or left panels) and contact hamsters (bottom or right panels). These were obtained using assays targeting either the E gene (envelope) of SARS-CoV-2, present in both viruses (shown as circles in Figures 6D-G), or sequences specifically present in two different viruses (the spike gene of the omicron BA virus, shown as squares in Figures 6D-G), or the sCPD9 region of the sCPD9-ΔFCS virus (shown as triangles in Figures 6D-G). The dotted lines in Figures 6C-G indicate the limits of detection.

[0170] As shown in Fig. 6D, low levels of replication-competent virus were detected in the lung tissues of the three contact hamsters on day 6 of cohabitation.

[0171] When RT-qPCR was performed on daily oral swab samples from infected hamsters, sCPD9-ΔFCS was detected only during the first 3 days postinfection, whereas BA.5 was detected throughout the experimental period ( Fig. 6D ).

[0172] In contrast, low levels of sCPD9-specific gRNA copies were detected in more sensitive oropharyngeal swabs (Figure 6E). Although sCPD9-specific gRNA was not detected in swabs or lung samples collected from contact animals, BA.5-specific RNA was abundantly detected in daily swabs from day 2 post-contact onward, as well as in oropharyngeal swabs and lung samples (Figure 6D-F). This indicates that sCPD9-ΔFCS remained noninfectious under co-infection conditions, whereas BA.5 became infected. Therefore, recombination events that would restore the vaccine virus's FCS and thereby enable vaccine virus infection were not observed in the selected experimental setup.

[0173] (In vitro co-culture did not suggest any significant recombination events.) Figure 6G shows the replication of Omicron BA.5 and sCPD9-ΔFCS in CaLu-3 cells. CaLu-3 cells were infected with 1,000 FFU of sCPD9-ΔFCS and 100 FFU of BA.5. After 72 hours of incubation, 1% of the supernatant was used as the inoculum for the next virus passage (n=3). The resulting virus population was passaged 10 times in CaLu-3 cells to assess recombination events between the vaccine and field viruses. RNA was extracted from the cell culture supernatant at 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, and an assay targeting the FCS region within the spike gene of the BA.5 virus and the genetically encoded sCPD9 region was used to distinguish between vaccine and field viruses. Consistent with the in vivo data, qPCR results showed that sCPD9-ΔFCS was overtaken by the BA.5 virus within one passage. Although sCPD9-specific RNA levels declined to near the limit of detection by passage 1, BA.5 maintained replication, resulting in high levels of gRNA detected throughout the entire experimental range (Figure 6G). This suggests that sCPD9-ΔFCS is at a significant growth disadvantage in cell culture, limiting the possibility of recombination between vaccine and field viruses within the same replication compartment. As a result, the risk of recombination between sCPD9-ΔFCS and wild-type virus was reduced.

[0174] To confirm the absence of highly fit recombinants, we performed total RNA sequencing of cell culture supernatants obtained from different passages and co-cultured viral replication. Sequence analysis showed that, from passage 1 onward, all sequences above the detection threshold were derived exclusively from the BA.5 virus. Neither analysis found evidence for the presence of sequences derived from sCPD9-ΔFCS. Although some de novo mutations appear to have been selected, likely indicating 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 can be excluded with the experimental setup we performed (Figure 6H,I).

[0175] Figure 6H shows the SNPs identified at passages 1, 2, 3, 6, 7, and 10 of the coinfection experiment and their respective locations within the BA.5 reference genome. This panel displays the SNPs identified in the three replicates that had the most SNPs in each passage, regardless of the passaging conditions. Only SNPs identified with 10% or more sequence reads are depicted. (Figure 6I shows all unique SNPs (with 10% or more read support) that appeared during the coinfection experiment compared to both the BA.5 and sCPD9-ΔFCS genome reference sequences.)

[0176] (sCPD9-ΔFCS is safe and immunogenic in immunosuppressed hamsters) Figure 7A shows a schematic of the experimental setup. Syrian hamsters were immunosuppressed by daily subcutaneous injection of dexamethasone (Dex) at a dose of 2 mg / kg starting 3 days before vaccination or exposure. Three days after administration, the hamsters were vaccinated with sCPD9-ΔFCS to examine the safety of the vaccine and the effect of immunosuppression on the humoral immune response to vaccination. Vaccinated hamsters were then housed with naive and immunosuppressed contact animals 24 hours after vaccination. Daily oral swabs were collected from all hamsters. Contact hamsters were euthanized at 6 days post-exposure (dpc), and vaccinated hamsters were euthanized at 21 days post-vaccination (dpv).

[0177] Figure 7B shows the changes in body weight of immunosuppressed animals after vaccination or contact with vaccinated hamsters. After vaccination, the immunosuppressed hamsters showed stable body weight and no clinical disease was observed (Figure 7B). In Figures 7B-7J, triangles indicate immunosuppressed contact hamsters, and squares indicate immunosuppressed hamsters vaccinated with sCPD9-ΔFCS.

[0178] SARS-CoV-2 RNA was detectable in oral swabs up to 8 days after vaccination, with the highest levels observed on days 1 and 2 (Figure 7C shows viral gRNA copies in oral swabs). After 8 days, viral RNA was undetectable in oral and oropharyngeal swabs (Figures 7C, D). Low levels of gRNA were detected in lung tissue 21 days after vaccination, suggesting prolonged viral replication in the lower respiratory tract compared with the upper respiratory tract (Figure 7D shows gRNA copies in oropharyngeal swabs and lung tissue). However, no replication-competent virus was recovered from lung samples collected at that time point (Figure 7E shows the number of replication-competent viruses in lung tissue).

[0179] Figure 7C shows the neutralizing activity of sera collected 21 days post-vaccination (dpv) from sCPD9-ΔFCS-vaccinated immunodeficient animals (IC; indicated by circles) and sCPD9-ΔFCS-vaccinated immunosuppressed animals 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 allowed us to measure humoral responses in immunosuppressed animals 21 days after a single dose of sCPD9-ΔFCS. Serum neutralization titers tended to be lower in immunosuppressed animals compared with immunodeficient animals receiving the same vaccine. Nevertheless, despite immunosuppression, all hamsters developed substantial humoral responses to vaccination (Figure 7F). Furthermore, lung histopathology was evaluated, and no evidence of pneumonia was observed in immunosuppressed hamsters after vaccination with sCPD9-ΔFCS (Figure 7G-J). Overall, sCPD9-ΔFCS remains safe and immunogenic in animals undergoing high-dose glucocorticoid treatment.

[0180] The dotted lines in Figures 7C to 7F indicate the detection limits.

[0181] sCPD9-ΔFCS does not spread between immunosuppressed hamsters. Next, we investigated whether the sCPD9-ΔFCS virus could be transmitted between immunosuppressed animals. To test this, six immunosuppressed animals vaccinated with the sCPD9-ΔFCS vaccine described above were placed in contact with six dexamethasone-treated, immunologically naive hamsters for six consecutive days (Figure 7A). During the cohabitation period, no clinical signs of illness or significant weight loss were observed (Figure 7B). Although vaccine virus RNA was detected in oral swabs from vaccinated animals (Figure 7C), no transmission of vaccine virus was observed, as evidenced by the absence of detectable viral RNA in the upper and lower respiratory tracts of immunosuppressed contact animals (Figure 7D, E).

[0182] sCPD9-ΔFCS is safe in immunosuppressed animals and does not recombine with circulating SARS-CoV-2 variants. Infection experiments in dexamethasone-treated animals demonstrated that sCPD9-ΔFCS was safe even in individuals undergoing glucocorticoid therapy. Glucocorticoids are well known for their immunosuppressive properties and are frequently used to treat airway inflammation, making immunosuppressive therapy particularly relevant in the context of COVID-19. It has previously been demonstrated that dexamethasone administration exerts a potent immunosuppressive effect and increases SARS-CoV-2 replication in Syrian hamsters (Wyler, 2022). Nevertheless, replication of the vaccine virus sCPD9-ΔFCS remained moderate even under dexamethasone treatment, and RNA levels were found to decrease to the limit of detection within one week after vaccination. Dexamethasone-treated hamsters showed no clinical symptoms of disease or obvious pathological findings. Furthermore, the vaccine virus remained nontransmissible and induced a significant humoral immune response in dexamethasone-treated animals. These results demonstrate the safety and efficacy of sCPD9-ΔFCS in immunosuppressed patients, particularly those receiving glucocorticoid therapy. Preliminary evaluation of early data indicates that the efficacy, safety, and efficacy of this humoral immune response in immunosuppressed patients is not observed to the same extent when vaccinated with sCPD9 alone, which constitutes the FCS.

[0183] In vitro and in vivo coinfection experiments demonstrated that the LAV virus sCPD9-ΔFCS rapidly and consistently outcompeted the Omicron BA.5 field isolate, indicating a strong selective disadvantage for the attenuated vaccine virus. This restricts the vaccine virus and the field virus from replicating in the same host or host compartment, thereby limiting the possibility of recombination events between the two viruses. While the experimental setup we chose cannot formally exclude recombination events between the two viruses, sequence analysis confirmed that no recombinants were selectively superior to the BA.5 mutant in coinfection experiments. Furthermore, in vivo coinfection experiments demonstrated that coinfection with the vaccine virus and the field virus did not result in increased virulence or yield infectious vaccine viruses.

[0184] (Additional experiment demonstrating the superiority of sCPD9-ΔFCS) Below, we describe in more detail additional experiments demonstrating the superiority of sCPD9-ΔFCS.

[0185] Materials and Methods (Research Design) The objective of this study was to compare the protective efficacy of two vaccines against SARS-CoV-2 infection. The extent of SARS-CoV-2 infection was investigated in two separate studies: from infected shed hamsters to vaccinated animals, and from vaccinated infected shed hamsters to naive contact hamsters. For this purpose, hamsters were randomly assigned to groups of 12 animals. The experimental design for both studies included three groups that followed a prime-boost schedule, in which hamsters were vaccinated on days 0 and 21, respectively. The three groups received two intranasal doses of sCPD9-ΔFCS (104 FFU), two intramuscular doses of 5 μg of the mRNA vaccine BNT162b2 (Comirnaty®, Pfizer-BioNTech), or two mock vaccinations.

[0186] In the first study, vaccinated hamsters were housed with infected shedding animals two weeks after the booster vaccination. 24 hours before cohabitation, the shedding hamsters were infected intranasally with 105 FFU of either SARS-CoV-2 variant B.1 or Omicron variant BA.5. In the second study, animals previously vaccinated with a prime-boost vaccine were infected with either SARS-CoV-2 variant B.1 or Omicron variant BA.5 two weeks after the second vaccination. The latter was then housed with uninfected hamsters 24 hours after infection. In both studies, the hamsters were housed for six days. During this period, oral mucosal swabs were collected daily, and viral loads in the upper respiratory tracts of shedding and contact hamsters were measured. In addition, weight and clinical condition were assessed. Six days after exposure, all hamsters were euthanized, and blood, nasal washes, lungs, and skull samples were collected for virological, serological, and histopathological examination.

[0187] (cell) Vero E6 (ATCC CRL-1586) and Vero E6-TMPRSS2 (NIBSC 100978) cells were cultured in minimum 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 TMPRSS2-expressing cells, the medium for Vero E6-TMPRSS2 cells additionally contained 1000 μg / ml geneticin (G418). CaLu-3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 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 in 5% CO2.

[0188] (virus) The SARS-CoV-2 ancestral variant B.1 (B.1, hCoV-19 / Germany / BY-ChVir-929 / 2020, EPI_ISL_406862) was propagated in Vero E6 cells, and the omicron subvariant BA.5 (BE.1.1, hCoV-19 / Germany / SH-ChVir29057_V34 / 2022, EPI_ISL_16221625) was propagated in CaLu-3 cells and used to infect decidual hamsters. Additionally, serum neutralization assays were performed using the delta variant B.1.617.2 (B.1.617.2, human, 2021, Germany, originating from India, 20A / 452R, EVAg: 009V-04187) grown in Vero E6-TMPRSS2 cells and the omicron subvariant BA.1 (BA.1.18, hCoV-19 / Germany / BE-ChVir26335 / 2021, EPI_ISL_7019047) grown in CaLu-3 cells. Plaque assays were performed on Vero E6 cells to measure the titer of all virus stocks prior to infection experiments. Vials were stored at -80°C.

[0189] (Ethics statement) Animal experiments were performed in compliance with all applicable national and international regulations and with the approval of the Landesamt fur Gesundheit und Soziales, Berlin, Germany (permit number 0086 / 20). All in vitro and animal experiments were performed in a certified BSL-3 laboratory at the Institut fur Virologie, Free University of Berlin, Germany.

[0190] (Livestock) Syrian hamsters (Mesocricetus auratus; breed RjHan:AURA) were purchased from Janvier Labs at 5–7 weeks of age. They were housed in groups of 2–3 in individually ventilated cages (IVCs; Tecniplast) with nesting material. Hamsters were allowed to acclimate to the environment for 7 days prior to vaccination. They had free access to water and food at all times. The cage temperature was 22–24°C and the relative humidity was 40–55% throughout the experiment.

[0191] (Vaccine preparation and administration) The live vaccine candidate, sCPD9-ΔFCS, was propagated in Vero E6-TMPRSS2 cells. Titers were determined by plaque assay using Vero E6 cells. Prior to vaccination, stocks were grown to a final titer of 2 × 10 5 FFU / ml. 10 per animal 4 Nasal vaccination with FFU was performed under general anesthesia (0.15 mg / kg medetomidine, 2.0 mg / kg midazolam, 2.5 mg / kg butorphanol).

[0192] BNT162b2 (Comirnaty®) was prepared according to the manufacturer's instructions. The final mRNA concentration was diluted to 50 μg / ml, rather than the 100 μg / ml recommended for human use. Dilutions were made in 0.9% NaCl sterile water immediately before vaccination, and 5 μg per hamster was administered intramuscularly.

[0193] Animals assigned to the sham group were administered minimum essential medium (MEM) intranasally under general anesthesia.

[0194] (nasal wash) To obtain nasal washes for all animals, a cannula was inserted into the skull anterior to the nasal septum. A pipette tip was then inserted and 200 μl of PBS was applied. The wash was collected into the nostril, and the wash procedure was repeated twice. Approximately 150 μl of sample was collected per animal.

[0195] (RNA isolation and RT-qPCR) In preparation for RNA extraction, 25 mg of lung tissue was homogenized in a bead mill (Analytik Jena). RNA was isolated from oral swabs, oral swabs, and lung tissue using the innuPREP Virus DNA / RNA Kit (Analytik Jena, Jena, Germany) according to the manufacturer's recommendations. To detect SARS-CoV-2 RNA, reverse transcription quantitative PCR (RT-qPCR) was performed in a qTower G3 cycler (Analytik Jena) using the NEB Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) with the following cycling conditions: reverse transcription at 55°C for 10 minutes, enzyme activation at 94°C for 3 minutes, followed by 40 cycles of 94°C for 15 seconds and 58°C for 30 seconds (Corman et al., 2020).

[0196] (Plaque assay and indirect immunofluorescence staining) Replicative virus was measured in 50 mg of lung tissue. For quantification, lung samples were homogenized in a bead mill (Analytik Jena), serially diluted in MEM, and plated onto 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 overlaid with 2X Eagle's Minimum Essential Medium (EMEM; Lonza™ BioWhittaker™) containing 1.5% microcrystalline cellulose and sodium carboxymethylcellulose (Vivapur 611p; JRS Pharma). 72 hours postinfection, plates were fixed with 4% PBS-buffered formaldehyde.

[0197] 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 washing the plates with PBS, a primary polyclonal anti-SARS coronavirus nucleocapsid antibody (Invitrogen) was added for 1 hour, followed by a goat anti-rabbit IgG-AlexaFluor 488 secondary antibody (Invitrogen) for 45 minutes. To measure titers, plaques were counted using an inverted fluorescence microscope (Axiovert S100, Zeiss).

[0198] (Serum Neutralization Assay) Neutralizing activity against SARS-CoV-2 variant B.1 and Omicron subvariant BA.5 was tested for all hamster sera (day 0 and 6 dpc). Additionally, neutralizing activity against Delta and Omicron subvariants BA.1 was tested for serum samples on day 6. Two-fold serial dilutions (1:8 to 1:1,024) of complement-inactivated (56°C for 30 min) hamster sera were prepared in 96-well plates. 200 FFU of SARS-CoV-2 diluted in MEM was applied to each well and incubated at 37°C for 1 hour. The dilutions were then plated onto Vero E6 cells cultured in 96-well plates and incubated at 37°C for 72 hours (B.1, Delta) or 96 hours (Omicron). The cells were then 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 showed no cytopathological effect. The final neutralized well was reported as the titer. Positive and negative controls were included on every plate. For plotting the results, samples without neutralizing activity were titered 1:4.

[0199] (Enzyme-linked immunosorbent assay (ELISA)) SARS-CoV-2-specific IgG levels against the spike proteins of the B.1 and BA.5 mutants, as well as the nucleocapsid and ORF3a proteins, were measured in hamster sera using an in-house ELISA. A flat-bottomed, clear 96-well plate (MEDISORP, Thermo Fisher Scientific, catalog number: MW96F) was coated with 5 μl of purified, recombinant, His-tagged SARS-CoV-2 antigens: spike protein of the B.1 variant (D614G) (Acro Biosystems, catalog number: SPN-C52H3), spike protein of the BA.5.5 variant (GenBank: BA.5.5 variant spike protein (GenBank accession: QHD43416, Acro Biosystems, catalog number: SPN-C522p), nucleocapsid protein (GenBank accession: QHD43423, Ray Biotech, catalog number: 230-01104), and ORF3a protein (Thermo Fisher Scientific, Cat. No. RP-87667). The antigen was diluted in 1x PBS to a final concentration of 20 μg / ml. Additionally, 45 μl of coating buffer (50 mM Na2CO3, 50 mM NaHCO3, pH 7.0) was added to each well. 9.6) was added. After incubating the plate at 4°C for 12 hours, the plate was washed four times with washing buffer (0.05% Tween 20 in 1x PBS) and blocked for 1 hour with blocking buffer (1x PBS, 1% BSA, 10% FCS). Serum samples were diluted 1:100 in dilution buffer (1x PBS, 2% BSA, 0.1% Tween 20) and plated in duplicate at 50 μl per well. The plate was then incubated for 2 hours at RT and washed. Next, 50 μl of secondary horseradish peroxidase (HRP)-conjugated polyclonal goat anti-hamster IgG (H+L) antibody (Thermo Fisher Scientific, catalog no. 10537453) diluted 1:1000 in 1x PBS was added to each well.After 1 hour of incubation at RT, 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. The reaction was stopped after 15 minutes with 1 M H2SO4. Optical density was measured at 450 nm and 570 nm using a SpectraMax Plus 384 plate reader (Molecular Devices).

[0200] Additionally, SARS-CoV-2-specific IgA levels against the spike proteins of the B.1 and BA.5 variants were measured in nasal washes according to the protocol described above with minor modifications. Nasal washes were diluted 1:50 in dilution buffer and plated in duplicate. For IgA detection, polyclonal HRP-conjugated rabbit anti-hamster IgA (Brookwood Biomedical, catalog number: sab3003a) was used as the secondary antibody at a dilution of 1:750. After 1 hour of incubation at room temperature, the plate was washed, and 50 μl of 1-Step™ Ultra TMB ELISA substrate solution (Thermo Fisher Scientific, catalog number: 34028) was added to each well. The reaction was stopped after 20 minutes of incubation at room temperature.

[0201] (Histopathy and Immunohistochemistry) For histopathological analysis, the left lung lobe and skinned skull were fixed in PBS-buffered formaldehyde solution (4%) for 48 hours. The skull was washed in tap water for 30 minutes and decalcified in buffered EDTA solution (pH = 7.0) at 65°C for 3 days. The skull was trimmed to obtain a rostral section at the tip of the first triangular ridge of the hard palate and a caudal section at the level of the first molar. 2-μm-thick sections were routinely cut from formalin-fixed, paraffin-embedded specimens and stained with hematoxylin and eosin or prepared for immunohistochemistry. Histopathological analysis of lung sections was performed as described (Osterrieder et al., 2020). Nasal sections were scored for the presence or absence of lymphocytes, granulocytes, necrosis, epithelial flattening, and cilia, with a score of 0 = less than 5% of the epithelium affected, 1 = 5-40% of the epithelium affected, 2 = 41-80% of the epithelium affected, or 3 = 80% or more of the epithelium affected. Airway exudate characteristics were also characterized. For immunohistochemical analysis, nasal sections were delipidated with xylene and rehydrated with lower grade ethanol. Endogenous peroxidase was blocked with H2O(2). Antigen retrieval was performed by heating sections in 750 ml of citrate buffer supplemented with 1% Triton X100 (Roth) in a 600 W microwave for 12 minutes. Primary monoclonal mouse anti-SARS-CoV-2 nucleocapsid protein antibody (Sino Biological, dilution: 1:500) was incubated overnight at 4°C. As a universal negative control, sections were incubated with irrelevant 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. Signal enhancement was performed using the Vectastain Elite ABC Kit (Vector Laboratories) for 8 minutes, followed by development with diaminobenzidine tetrahydrochloride (Merck). Hematoxylin was used as a counterstain.Histopathological evaluation was performed using an Olympus BX41 microscope equipped with a DP80 Microscope Digital Camera (Olympus) and cellSens™ Imaging Software, Version 1.18 (Olympus Soft Imaging Solutions). Automated digitization was performed using an AperioCS2 slide scanner (Leica Biosystems). Photomicrographs were created using image Scope Software (Leica Biosystems). Figure panels were created using Adobe Photoshop or GIMP software.

[0202] The LAV vaccine prevents the development of clinical symptoms after natural infection with SARS-CoV-2B.1 and BA.5. Syrian hamsters were vaccinated twice, 3 weeks apart (days 0 and 21), with sCPD9-ΔFCS, BNT162b2, or mock vaccine. After an additional 14 days (day 35 post-vaccination), the hamsters were exposed to shedding animals that had been infected 1 day earlier with either the ancestral virus variant B.1 or the Omicron BA.5 variant. The animals were housed for 6 days and closely monitored for disease symptoms and viral load in the upper respiratory tract.

[0203] Figures 8A and 8B show the percentage weight loss of B.1 shedding animals versus contact animals (Figure 8A) and BA.5 shedding animals versus contact animals (Figure 8B). Violin plots (truncated) show the weights of vaccinated contact animals (n = 6), group medians, and quartiles. Weights of shedding animals (n = 3) are displayed as medians. All three groups of shedding animals infected with the ancestral SARS-CoV-2 variant B.1 exhibited moderate weight loss and clinical symptoms typical of COVID-19-like pneumonia (Figure 8A). As expected, shedding animals infected with the BA.5 variant (Wolter et al., 2022; Uraki et al., 2022) exhibited less pronounced weight loss, although there was some variability between infection groups (Figure 8B).

[0204] The sCPD9-ΔFCS and mRNA vaccines effectively prevented weight loss in contact-vaccinated animals exposed to both B.1 and BA.5 shedding strains (Figures 8A and 8B). As expected, mock-vaccinated animals exposed to B.1 shedding strains exhibited a progressive loss of body weight beginning on day 3 post-exposure (Figure 8A). In contrast, mock-vaccinated animals exposed to BA.5 shedding strains did not experience weight loss, likely due to the lower virulence of the BA.5 mutant and, consequently, the milder disease course observed in infected animals (Figure 8B).

[0205] (Only LAV protects against SARS-CoV-2 B.1 and BA.5) To monitor viral replication and transmission, oral swabs were collected daily during the cohabitation period between infected and contact animals. Additionally, oral swabs and lung samples were collected 6 days post-contact (dpc) to quantify viral RNA levels and replicated virus. Viral gRNA copies and replicated virus were quantified as focus-forming units (FFU) in oropharyngeal swabs and lung tissue from B.1-infected (Figure 8E) and BA.5-infected (Figure 8F) shedding individuals and their respective vaccinated contacts. In Figures 8C–8F, results for vaccinated contact animals (n = 6) are shown as medians with ranges indicated by symbols. For shedding hamsters (n = 3), medians are shown. Parametric statistics were performed on log-transformed data. For Figures 8C and 8D, a two-way ordinary analysis of variance with Tukey's multiple comparison test was performed. For Figures 8E and 8F, a one-way ordinary analysis of variance with Tukey's multiple comparison test was performed. * p<0.05, ** p<0.01, *** p<0.001, *** p<0.0001.

[0206] Infected shedding animals showed high levels of SARS-CoV-2 RNA, which gradually decreased over day 6. In general, viral loads were higher in B.1 shedding animals compared to BA.5 shedding animals (Figures 8C and 8D).

[0207] Consistent with the observed weight loss, oral swabs from mock-vaccinated individuals exposed to B.1 shedding individuals showed high levels of SARS-CoV-2 RNA from 2 days after vaccination (Figure 8C). Although mRNA vaccination prevented weight loss, it did not provide protection against B.1 infection, as evidenced by persistently high SARS-CoV-2 RNA levels in oral swabs after day 2. Nevertheless, mRNA vaccination reduced viral RNA levels compared with mock vaccination. In contrast, contacts vaccinated with the sCPD9-ΔFCS vaccine showed minimal SARS-CoV-2 RNA levels that fluctuated around the limit of detection, suggesting repeated exposure to the virus without signs of productive infection in the upper respiratory tract (Figure 8C).

[0208] Similar results were obtained in vaccinated hamsters exposed to BA.5-infected shedders. SARS-CoV-2 RNA levels in oral swabs from mRNA-vaccinated and mock-vaccinated animals peaked between 4 and 5 days post-vaccination (Figure 8D). However, viral RNA levels in sCPD9-ΔFCS-vaccinated hamsters were near the limit of detection, suggesting effective protection against BA.5 infection (Figure 8D).

[0209] Consistent with these findings, mRNA-vaccinated and mock-vaccinated hamsters exposed to B.1 or BA.5 shedding agents exhibited high SARS-CoV-2 RNA levels in oral swabs and lungs at 6 days post-transplant (dpc) (Figures 8E and 8F). Furthermore, replication-competent virus was detected in lung tissue from mock-vaccinated animals exposed to B.1 shedding agents (Figure 8E). Despite slightly lower viral RNA levels, replicating virus was detected in the lungs of two mRNA-vaccinated and three mock-vaccinated hamsters exposed to BA.5 shedding agents, reflecting a delayed peak in viral replication compared to B.1 infection (Figure 8F).

[0210] Lung lesions were minimal in sCPD9-ΔFCS-vaccinated animals and were less severely attenuated in mRNA-vaccinated animals exposed to B.1 shedding individuals. Hamsters experimentally infected with B.1 virus or those infected with B.1 after mock vaccination developed lesions typical of COVID-19-like pneumonia. Specifically, infected animals exhibited necrotizing pustular bronchitis and bronchitis, proliferation of alveolar type II epithelium, vascular endotheliitis, diffuse alveolar damage, and prominent patchy bronchointerstitial pneumonia with perivascular and alveolar edema. Histopathological analysis confirmed significant reductions in tissue changes, immune cell infiltration, and edema in sCPD9-ΔFCS-vaccinated and mRNA-vaccinated animals. Inflammatory damage was mild in all animals infected with Omicron BA.5 and was most pronounced in experimentally infected shedding hamsters. However, mock-vaccinated contacts still developed mild pneumonia. Hamsters vaccinated with the mRNA vaccine showed slightly lower protection. In contrast, animals vaccinated with sCPD9-ΔFCS vaccine exposed to B.1 or BA.5 shedding agents did not develop substantial evidence of pneumonia, confirming highly effective protection from this vaccine.

[0211] The protective effect of LAV is accompanied by a strong humoral immune response. To assess humoral immunity, we assessed the neutralizing capacity of sera collected at 6 days of age against SARS-CoV-2 variants B.1, Delta, BA.1, and BA.5. Additionally, enzyme-linked immunosorbent assays (ELISAs) were performed using sera and nasal washes collected at 6 days of age.

[0212] The results are shown in Figures 9A-9D. Figure 9A shows the neutralization potency of hamster sera collected from vaccinated animals that had contact with either B.1 or BA.5 shedding individuals against SARS-CoV-2 variants B.1, BA.5, Delta, and BA.1 (upper limit of detection = 1:1,024; lower limit of detection is indicated by the dotted line). Results are shown as mean ± SEM, and symbols represent individual values. Figure 9B shows SARS-specific IgG levels against B.1 spike, BA.5 spike, nucleocapsid, and ORF3a in serum collected from vaccinated contacts at 6 days post-contact (dpc). Figure 9C shows SARS-specific IgA levels against B.1 spike and BA.5 spike in nasal washes collected from vaccinated contacts at day 6. The findings shown in Figures 9B and 9C are displayed as optical density (OD) read at 450 nm. Box plots show the 25th to 75th percentiles, with the center line representing the median and the whiskers representing the minimum to maximum values. Symbols represent individual values.

[0213] The results in Figures 9A to 9C were statistically evaluated by the Kruskal-Wallis test and Dunn's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001, *** p<0.0001. Results in Figure 9D were obtained by semiquantitative scoring of SARS-CoV-2 N protein immunohistochemistry (IHC) signals in the nasal epithelium of vaccinated contacts. Scoring of inflammatory changes in the nasal epithelium, including lymphocyte and neutrophil influx, olfactory and respiratory epithelial necrosis, apoptosis, loss of cilia, and flattened epithelial cells, is presented as median and range. Symbols represent individual values. Ordinary one-way ANOVA was performed with Tukey's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001, *** p<0.0001.

[0214] As expected, vaccination with sCPD9-ΔFCS elicited robust production of neutralizing antibodies, with similar levels across groups exposed to different challenge viruses (Figure 9A). This finding, combined with the virological results, suggests that mucosal immunity induced by sCPD9-ΔFCS vaccination prevented replication of naturally occurring viruses. However, transient exposure to viral antigens resulted in a slight increase in neutralizing activity. In contrast, mock-vaccinated animals showed increased serum neutralizing activity against specific challenge viruses. A similar trend was observed in mRNA-vaccinated animals, although the difference was less pronounced (Figure 9A). As expected, specific serum neutralizing activity was similar across all shedding animals and was determined by the challenge virus.

[0215] ELISA revealed that levels of anti-B.1 spike and anti-BA.5 spike IgG antibodies were comparable in sCPD9-ΔFCS- and mRNA-vaccinated hamsters (Figure 9B). As expected, nucleocapsid- and ORF3a-specific antibodies were present only in sCPD9-ΔFCS-vaccinated animals, highlighting the broad immunity induced by LAV (Figure 9B). Not surprisingly, IgG levels in shedding animals were relatively uniform and strongly influenced by the challenge virus. Mucosal immunity was examined by measuring SARS-CoV-2-specific IgA levels in nasal washes obtained 6 days after vaccination (Figure 9C). Regardless of the challenge virus, sCPD9-ΔFCS-vaccinated animals displayed comparable levels of anti-B.1 spike and anti-BA.5 spike IgA antibodies (Figure 9C). In contrast, only mRNA-vaccinated hamsters exposed to B.1-shedding bacteria produced appreciable IgA levels. mRNA-vaccinated animals exposed to BA.5 shedding bacteria and both mock-vaccinated groups lacked measurable mucosal IgA responses. The absence of IgA in nasal washes from mRNA-vaccinated hamsters exposed to BA.5 shedding confirmed that mRNA vaccination confers only limited mucosal immunity prior to virus challenge. However, exposure to the homologous B.1 mutant resulted in significant induction of mucosal IgA antibodies (Figure 9C). Shedding animals showed low IgA levels, which corresponded to the virus used for infection.

[0216] LAV vaccination prevents mucosal infection with SARS-CoV-2B.1 and BA.5. To further assess vaccination-induced upper respiratory tract protection, nasal epithelia at 6 days post-vaccination were evaluated for the presence of SARS-CoV-2 nucleocapsids and histological signs of infection and inflammation. SARS-CoV-2-positive cells were absent in the nasal respiratory and olfactory epithelia of sCPD9-ΔFCS-vaccinated contacts, whereas antigen was abundantly detected in mRNA-vaccinated and mock-vaccinated animals (Figure 9D).

[0217] Consistent with immunohistochemistry, immune cell influx and inflammatory damage were observed only in the olfactory and respiratory epithelia of mRNA-vaccinated and mock-vaccinated animals, with reduced inflammation in mRNA-vaccinated hamsters. Notably, inflammatory damage was less pronounced in animals exposed to BA.5 shed bacteria (Figure 9D). Consistent with the expected rapid clearance of SARS-CoV-2 (Sia et al., 2020), shed hamsters showed few infected epithelial cells and only signs of mild inflammation at 7 days post-infection (dpi).

[0218] LAV vaccination prevents transmission of SARS-CoV-2 B.1 to naive contacts. The effect of vaccination on suppressing virus transmission from vaccinated and experimentally infected hamsters to naive animals was also examined. Syrian hamsters were administered two doses of sCPD9-ΔFCS or BNT162b2, 21 days apart (days 0 and 21). 14 days later (day 35), the hamsters were infected with either SARS-CoV-2 B.1 or Omicron BA.5. 24 hours after infection, infected animals were housed with uninfected contacts for 6 days and their clinical condition and weight were monitored. Oral swabs were collected daily, and lung samples were taken at 6 days per day.

[0219] The results of these experiments are shown in Figures 10A-10C (SARS-CoV-2 B.1 infection) and Figures 11A-11C (SARS-CoV-2 Omicron BA.5 infection).

[0220] Figure 10A shows the percentage weight loss for vaccinated and B.1-infected groups, as well as for uninfected contact animals. A truncated violin plot shows the weight of uninfected contact animals (n = 6) as group medians and quartiles. Weights of shedding individuals (n = 3) are shown as medians. Figure 10B shows viral gRNA copies in oral swabs from vaccinated hamsters, B.1-infected hamsters, and uninfected contacts. A two-way ordinary ANOVA was performed with Tukey's multiple comparison test. Figure 10C shows viral gRNA copies in oropharyngeal swabs and 2.5 mg homogenized lung tissue collected at termination. Replicated virus in homogenized lung tissue was quantified as focus-forming units (FFU). Results in Figures 10B and 10C for uninfected contact animals (n = 6) are median values ​​with ranges indicated. Symbols represent individual values. Results for vaccinated and superinfected hamsters are displayed as medians. Parametric statistics of log-transformed data are shown in Figures 10B and 10C. The dotted line indicates the limit of detection. In addition, Figure 10C was subjected to a conventional one-way ANOVA with Tukey's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001, *** p<0.0001.

[0221] Figure 11A shows the percent weight loss of vaccinated and BA.5-infected shedders versus uninfected contacts. A truncated violin plot shows the weights of uninfected contacts (n = 6) as group medians and quartiles. Weights for shedders (n = 3) are shown as medians. Figure 11B shows viral gRNA copies in daily oral swabs of vaccinated and challenge-infected BA.5 shedders and uninfected contact hamsters. Ordinary two-way ANOVA with Tukey's multiple comparison test was performed. Figure 11C shows gRNA copy numbers detected in oropharyngeal swabs and 2.5 mg of lung tissue obtained at termination. Replication-competent virus in 50 mg of homogenized lung was quantified as focus-forming units (FFU). Results in Figures 11B and 11C for uninfected contacts (n = 6) are displayed as medians and ranges, with symbols representing individual values. For findings in vaccinated and challenge-infected shedders (n = 3), medians are shown. Parametric statistics were performed on log-transformed data in Figures 11B and 11C. The dotted line indicates the limit of detection. Additionally, Figure 11C was subjected to a conventional one-way ANOVA with Tukey's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001, *** p<0.0001.

[0222] Both vaccines effectively prevented weight loss, but mock-vaccinated animals showed weight loss upon B.1 infection (Figure 10A). Virological results confirmed the strong protective effect of the sCPD9-ΔFCS vaccine. Naive contacts housed with the sCPD9-ΔFCS-vaccinated and B.1-infected animals maintained stable body weights. In contrast, contacts of mock-vaccinated or mRNA-vaccinated SARS-CoV-2-infected animals lost weight from 2 and 4 days after vaccination, respectively (Figure 10A), and their oral swabs showed high levels of SARS-CoV-2 RNA. However, contacts with virus-shedding mRNA-vaccinated animals showed a slightly delayed viral replication rate compared with contacts with mock-vaccinated animals (Figure 10B). The presence of high RNA levels and detection of replication-competent virus in the lungs further confirmed that all naive animals in contact with mRNA-vaccinated or mock-vaccinated hamsters and B.1-infected hamsters were infected (Figure 10C). In contrast, sCPD9-ΔFCS-vaccinated animals and contacts of B.1-infected animals tested negative for SARS-CoV-2 RNA in the upper and lower respiratory tracts, and no replicating virus was detected in the lower respiratory tract. These findings strongly suggest that vaccination with sCPD9-ΔFCS confers highly effective protection against transmission of B.1 virus to uninfected contacts (Figures 10B and 10C).

[0223] Consistent with these observations, hamsters infected with B.1 from vaccinated and challenged animals exhibited typical signs of COVID-19 pneumonia, including necrotizing pustular bronchitis and bronchitis, proliferation of alveolar type II epithelium, vascular endotheliitis, diffuse alveolar damage, and perivascular and alveolar edema. However, immune cell influx and edema were reduced in hamsters housed with mRNA-vaccinated animals compared with mock-vaccinated animals. Importantly, neither hamsters in contact with sCPD9-ΔFCS-vaccinated nor B.1-infected animals showed signs of pneumonia.

[0224] LAV provides excellent protection against Omicron BA.5 infection and its spread. Hamsters infected with Omicron BA.5 showed no significant weight loss, likely due to its attenuation in Syrian hamsters (Figure 11A). Animals vaccinated with sCPD9-ΔFCS and superinfected with Omicron BA.5 cleared infection within 48 hours, but mRNA vaccination only slightly reduced viral loads in the upper and lower respiratory tract compared with mock vaccination. Importantly, SARS-CoV-2 RNA was detected in oral swabs from mRNA-vaccinated hamsters up to 5 days after vaccination (Figures 11B and 11C). Histopathological examination of vaccinated and B.5-infected hamsters confirmed the efficacy of both vaccines. However, the histopathological changes observed in BA.5-infected animals were generally subtle compared to those observed in B.1-infected animals, suggesting that the pathological changes of this variant are generally milder than those observed in the original virus.

[0225] No weight loss was observed in uninfected animals that had contact with vaccinated or BA.5-infected hamsters (Figure 11A). However, mRNA vaccination did not prevent backward transmission of the BA.5 virus. From 3 days after vaccination, all animals in contact with mRNA- or mock-vaccinated shedding individuals had similar SARS-CoV-2 RNA loads (Figure 11B). Furthermore, high SARS-CoV-2 RNA levels were also observed in swab and lung samples collected from these animals at termination (Figure 11C). In contrast, sCPD9-ΔFCS vaccination significantly reduced transmission to uninfected contacts, with only one animal becoming infected around 2 days after vaccination. A second naive animal in the same cage tested positive at 6 days after vaccination, indicating a secondary infection (Figure 11B). Both hamsters tested positive for SARS-CoV-2 RNA in oral swabs and lungs at the end of infection, but only one animal had replicating virus at the end of infection. Meanwhile, replicating virus was observed in the hamsters. It was present in three contacts of both mRNA- and mock-vaccinated shedding individuals (Fig. 11C).

[0226] Histopathological findings were less pronounced in contacts of 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 collapse. Naive contacts of sCPD9-ΔFCS-vaccinated hamsters showed no signs or mild pneumonia, reflecting the infection status.

[0227] LAV-induced humoral and mucosal immunity reduced SARS-CoV-2 transmission. The results of the infection experiment are shown in Figures 12A-12C. Figure 12A shows the concentrations of neutralizing antibodies against SARS-CoV-2 variants B.1, BA.5, Delta, and BA.1 in the serum of naive hamsters in contact with vaccine recipients and shedding individuals infected with B.1 or BA.5. The lower limit of detection is indicated by a dotted line, and the upper limit of detection is indicated by 1:1,024. Results are presented as mean ± SEM. Figure 12B shows SARS-specific IgG levels against B.1 spike, BA.5 spike, nucleocapsid, and ORF3a in the serum of naive contacts collected at 6 dpc. Figure 12C shows SARS-specific IgA levels against B.1 spike and BA.5 spike in nasal washes collected at termination. The findings shown in Figures 12B and 12C are displayed as optical density (OD) read at 450 nm. Boxplots show the 25th-75th percentiles with a center line indicating the median, whiskers ranging from the minimum to the maximum, and individual values ​​indicated by symbols. A Kruskal-Wallis test with Dunn's multiple comparison test was performed on the data in Figures 12A-12C. * p<0.05, ** p<0.01, *** p<0.001, *** p<0.0001.

[0228] Uninfected animals vaccinated with sCPD9-ΔFCS and exposed to infected shepherds did not seroconvert, whereas animals vaccinated with mRNA or mock and exposed to superinfected shepherds seroconverted depending on the challenge virus (Figures 12A and 12B). As expected, sCPD9-ΔFCS-vaccinated animals displayed broad and robust humoral immune responses prior to infection, whereas mRNA-vaccinated hamsters directed humoral responses exclusively against the B.1 spike protein. Neutralizing antibody titers against BA.5 were detected only in animals vaccinated with sCPD9-ΔFCS. Superinfection enhanced antibody responses in all groups.

[0229] Consistent with the virological results, only one sCPD9-ΔFCS-vaccinated contact animal showed seroconversion. Antibodies to SARS-CoV-2 B.1, Delta, and BA.1 were not detected in any of the serum samples obtained from contacts of the BA.5 shedding individual. Furthermore, antibodies targeting the B.1 S, N, and ORF3a proteins were not detectable by ELISA (Figure 12B). Overall, only sCPD9-ΔFCS vaccination induced broad humoral immunity and effectively reduced BA.5 infection in naive contact animals.

[0230] Nasal washes from sCPD9-ΔFCS-vaccinated and infected hamsters showed high IgA levels against B.1 and OmicronBA.5 spikes, regardless of the challenge virus. In contrast, mRNA-vaccinated, mock-vaccinated, and superinfected hamsters had low or no IgA levels. No IgA antibodies were detected in uninfected contacts at 6 days post-transplant (dpc), but a slight trend toward IgA expression was observed in contacts of mRNA-vaccinated, mock-vaccinated, and infected animals, consistent with the virological and serological findings (Figure 12C).

[0231] Uninfected contacts of mRNA-vaccinated hamsters were infected with both SARS-CoV-2 subtypes and had abundant nucleocapsid expression in the nasal epithelium. Contacts of mock-vaccinated and B.1-vaccinated hamsters had lower numbers of SARS-CoV-2-positive cells in the nasal epithelium compared with contacts of mRNA-vaccinated hamsters, which was consistent with viral RNA levels in oral swabs at 6 days of age.

[0232] Consistent with previous observations, SARS-CoV-2 nucleoprotein was not detected in the nasal epithelium of naive contacts from sCPD9-ΔFCS-vaccinated and B.1-infected hamsters, except for one BA.5-infected hamster. Similar to the immunohistochemical results, inflammation and immune cell recruitment were detected to varying degrees in all hamsters infected with either of the two variants, confirming the effectiveness of sCPD9-ΔFCS in preventing viral infection. Histological findings confirmed complete eradication of infection in all vaccinated and infected groups.

[0233] (Summary of discussion) Currently used LAVs present the entire viral antigen repertoire to the respiratory mucosa and, in particular, induce the formation of tissue-resident memory T cells (TRM cells), a specialized subset of T cells that reside in specific tissues, such as the respiratory mucosa, even after primary infection or vaccination (Schenkel and Masopust, 2014; Lavelle and Ward, 2022). These cells (Lavelle and Ward, 2022; Nouailles et al., 2022) provide a first line of defense against reinfection by rapidly recognizing and responding to pathogens that re-enter tissues from the body surface. In the context of SARS-CoV-2, TRM cells in the respiratory mucosa play a critical role in mounting a rapid immune response upon viral exposure. Upon encountering the virus, TRM cells rapidly activate to release antiviral cytokines, recruit other immune cells to the site of infection, and directly eliminate virus-infected cells. The presence of TRM cells in the respiratory mucosa allows them to more efficiently survey and respond to local viral threats. By establishing such a local immune surveillance network, TRM cells contribute to the early control of viral replication and limit viral spread within the respiratory tract. Together with neutralizing (IgA) antibodies, TRM cells contribute to comprehensive defense against SARS-CoV-2, effectively targeting the viral entry point and initial replication site, thereby reducing the likelihood of respiratory tract infection and transmission (Nouailles et al., 2023).

[0234] We compared the efficacy of the attenuated virus sCPD9-ΔFCS, encoding the prototype SARS-CoV-2 spike protein, with the mRNA vaccine BNT162b2 in inhibiting SARS-CoV-2 transmission. The attenuated virus demonstrated superior ability to prevent or significantly reduce viral infection. Importantly, this was also true for the SARS-CoV-2 variant BA.5. The emergence of omicron variants, which contain multiple amino acid changes in the spike protein (Wolter et al., 2022; Madhi et al., 2022), has spurred the development of bivalent mRNA vaccines. These vaccines contain spike proteins from the ancestral B.1 and BA.4 / BA.5 variants and provide superior protection against omicron variants compared to monovalent vaccines (Link-Gelles et al., 2022; Wang et al., 2023). However, it is becoming increasingly clear that SARS-CoV-2 transmission cannot be controlled, or is not adequately controlled, by intramuscular spike-based vaccines ( Franco-Paredes, 2022 ).

[0235] These data demonstrate that mucosal vaccines can effectively prevent or reduce SARS-CoV-2 infection and subsequent transmission. Furthermore, the B.1-based vaccine provided highly effective protection against the BA.5 variant, which is antigenically distant from the B.1 variant, suggesting that frequent updating of viral antigens is not required. Two consecutive doses of sCPD9 effectively boosted immunity, indicating that pre-existing immunity, such as that conferred by a previous SARS-CoV-2 infection, does not inhibit the efficacy of the mucosal vaccine tested here. On the contrary, periodically boosting pre-existing immunity with mucosal vaccines may be an important strategy for long-term control of SARS-CoV-2.

[0236] In conclusion, the findings presented here highlight the significance and benefits of developing mucosal vaccines to enhance the control of not only SARS-CoV-2 but also other respiratory viruses. By reducing viral transmission, we may be able to suppress and slow the circulation and evolution of respiratory RNA viruses.

Claims

1. A polynucleotide comprising: a) the 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; the polynucleotide comprises at least one sequence portion that comprises a deoptimization of codon pairs compared to the SARS-CoV-2 genome; The polynucleotide further comprises a furin cleavage site variant, wherein the furin cleavage site variant results in the loss of a furin cleavage site naturally present in the SARS-CoV-2 genome.

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

3. 3. The polynucleotide of claim 1, 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 to SEQ ID NO:

6.

4. 3. The polynucleotide of claim 1, 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 to the SEQ ID NO:

8.

5. 3. 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 claims 1 to 5, wherein the modification of the furin cleavage site comprises a deletion of the nucleotides encoding the amino acid sequence XRRA, where X represents P, R or H.

7. 7. The polynucleotide of any one of claims 1 to 6, wherein the modification of the furin cleavage site is such that expression of the polynucleotide results in a protein in which at least 5 consecutive amino acids of the naturally expressed protein are replaced with a single amino acid.

8. 8. The polynucleotide of any one of claims 1 to 7, wherein the modification of the pre-furin cleavage site consists of 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, or consists of 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 the polynucleotide of any one of claims 1 to 8.

10. 10. The live, attenuated SARS-CoV-2 of claim 9, wherein the SARS-CoV-2 has a nucleic acid sequence set forth in SEQ ID NO:19, a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:19, a nucleic acid sequence set forth in SEQ ID NO:20, or a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:

20.

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

12. 12. The pharmaceutical composition of 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 of any one of claims 1 to 8.

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

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