The present invention relates to a codon-pair deoptimized polynucleotide encoding a respiratory syndrome coronavirus 2 (SARS-CoV-2) protein, to a live attenuated SARS-CoV-2 comprising such polynucleotide, to a pharmaceutical composition comprising such a live attenuated SARS-CoV-2, to a vaccination method for administering this pharmaceutical composition, to a vector comprising such polynucleotide, to a host cell comprising such polynucleotide, as well as to method of producing a virus.

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). It has spread rapidly around the world within a matter of weeks and the world is still battling with the ongoing COVID-19 pandemic.

SARS-CoV-2 primarily replicates in the upper respiratory tract (Zou et al., 2020). The infection with SARS-CoV-2 can cause a wide spectrum of clinical manifestations, ranging from asymptomatic to life-threatening disease conditions (Chen et al., 2020; Zhou et al., 2020a). Especially the elderly and patients with pre-existing conditions are at greater risk of developing more severe disease such as pneumonia, acute respiratory distress syndrome and multiple organ failure (Chen et al., 2020; Garg et al., 2020; Zhou et al., 2020a). The ongoing pandemic imposes an enormous health, psychological, economic, and social burden. To date (December 2021 ) more than 270 million people have been infected with the virus, of whom more than 5.3 million have died as a result of the infection (https://coronavirus.jhu.edu/map.html) (Dong et al., 2020).

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

SARS-CoV-2 is rapidly evolving (Tegally et al., 2021 ; Faria et al., 2021 ; Davies et al., 2021 ). Benefiting from its global presence, the virus continues to adapt to its new host and to infection- or vaccine- induced immunity. During the course of the pandemic, a number of genetic variants have emerged (Tegally et al., 2021 ; Faria et al., 2021 ; Davies et al., 2021 ). Variants that exhibit increased infectivity, cause greater morbidity and mortality, or have the ability to evade infection- or vaccine-induced immunity pose an increased threat to public health. The World Health Organization (WHO) and other national health agencies have independently established classification systems that categorize emerging variants as variants of interest (VOIs), variants under investigation (VUIs), or variants of concern (VOCs) based on their risk to public health (cf. Table 1 of Trimpert et al. “Live attenuated virus vaccine protects against SARS-CoV-2 variants of concern B.1.1.7 (Alpha) and B.1.351 (Beta)", Science Advances, Vol. 7, No. 49 (2021 )). In addition, to simplify communication with the public, the WHO recommends that VOIs and VOCs should also be labeled using the letters of the Greek alphabet. As of 12 August 2021 , viruses belonging to lineages B.1.1.7 (Alpha), B.1.351 (Beta), B.1.1.28.1 (Gamma), B.1.617.2 (Delta), and, most recently, B.1 .159.1 (Omicron) are classified by several health agencies as VOCs. In countries where they emerged, these variants rapidly supplanted the preexisting variants and started to spread globally.

The B.1.1.7 variant, first detected in the United Kingdom in December 2020, is 50 to 100% more transmissible and possibly also more lethal than earlier variants but shows no tendency to evade immunity induced by infection or vaccination (Davies et al., 2021 ; Volz et al., 2021 ; Abu-Raddad et al., 2021 ). The B.1.1.7 variant has been detected in 132 countries and rapidly became the dominant variant in Europe and the United States. The B.1.351 variant, first detected in South Africa in May 2020, is not only more transmissible but also capable of reinfecting individuals and of breaking through vaccine protection (Madhi et al., 2021 ; Johnson & Johnson; Naveca et al., 2021 ). The B.1 .1 .28.1 variant is similar to B.1 .351 in that both share some important mutations in the spike glycoprotein (E484K, K417N/T, and N501 Y). B.1 .1 .28.1 emerged in late 2020 in Manaus, Brazil (Faria et al., 2021 ). Similar to the B.1.351 variant, it can cause reinfection because it can bypass immunity developed after infection with other virus variants (Faria et al., 2021 ; Naveca et al., 2021 ). It is estimated that B.1.1.28.1 is 40 to 140% more transmissible, more pathogenic, and 10 to 80% more lethal than other variants (Faria et al., 2021 ). On 7 May 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 largely outcompeted B.1.1.7 and became the predominant variant in Europe and the United States. According to the WHO, B.1.617.2 is the most dangerous strain worldwide, and it has attracted considerable attention for its ability to evade infection- and vaccine-mediated protection (Dyer et al., 2021 ). Variant B.1 .1 .529 of SARS-CoV-2 was first detected in Botswana in November 2021 . It has become the predominant variant in circulation around the world. After the original BA.1 variant, several subvariants of Omicron have evolved: BA.2, BA.3, BA.4, and BA.5, with BA.5 dominating the world as of August 2022.

WO 2021/154828 A1 and WO 2023/283106 A1 describe modified SARS-CoV-2 coronaviruses. These viruses have been recoded, for example, codon deoptimized or codon pair bias deoptimized and are useful for reducing the likelihood or severity of a SARS-CoV-2 coronavirus infection, preventing a SARS-CoV-2 coronavirus infection, eliciting and immune response, or treating a SARS-CoV-2 coronavirus infection.

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

CN 1 12175913 A describes a SARS-CoV-2 attenuated strain and its application in the prevention and/or treatment of a novel coronavirus pneumonia.

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

This object is achieved with a specific polynucleotide encoding a) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein; and optionally b) at least one non- structural SARS-CoV-2 protein selected from the group consisting of non-structural protein 7, non-structural protein 8, non-structural protein 9, non-structural protein 10, non-structural protein 1 1 , non-structural protein 12, an endoribonuclease (also referred to as non-structural protein 15), and a 2'-O-methyltransferase (also referred to as non-structural protein 16). In this context, the polynucleotide comprises or consists of at least one sequence part comprising codon-pair deoptimizations in comparison to the corresponding SARS-CoV-2 genome part. Furthermore, the polynucleotide comprises a furin cleavage site modification resulting in a functional loss of a furin cleavage site being naturally present in the SARS-CoV-2 genome. Thus, the polynucleotide is lacking some genetic information being present in naturally occurring SARS-CoV-2. The term “furin cleavage site modification”, as used herein, refers to a site in the nucleotide sequence of the polynucleotide that corresponds to a site in the SARS-CoV-2 genome encoding for a furin cleavage site but having a modification such that it results in a substantial reduction or loss of (furin) cleavage susceptibility of the encoded cleavage site. Examples of such a modification are demonstrated herein below, e.g., in the examples. The furin cleavage site modification described herein can be any modification that alters the cleavage susceptibility, for example a (partial or full) deletion, an insertion, or a replacement of nucleotides or sequence parts in comparison to the SARS-CoV-2 genome. In some embodiments, the furin cleavage site modification described herein embodies not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9, not more than 10, not more than 11 , not more than 12, not more than 13, not more than 14, not more than 15, not more than 16, not more than 17, not more than 18, not more than 19, not more than 20, not more than 21 , not more than 22, not more than 23, or not more than 24 nucleotide modifications compared to the sequence part of the SARS-CoV-2 encoding the furin cleavage site.

The term “polynucleotide”, as used herein, refers to a molecule containing multiple nucleotides (e.g. mRNA, RNA, cRNA, cDNA or DNA). The term typically refers to oligonucleotides 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. The polynucleotide of the present invention either essentially consists of the nucleic acid sequences described herein or comprises the aforementioned nucleic acid sequences. Thus, it may contain further nucleic acid sequences as well. The term polynucleotide encompasses single stranded as well as double stranded polynucleotides. Moreover, encompassed herein are also modified polynucleotides including chemically modified polynucleotides, artificial modified polynucleotides, or naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides.

The term “SARS-CoV-2” or “severe acute respiratory syndrome coronavirus 2”, as used herein, refers to any variant that is classified as SARS-CoV-2. In some embodiments, the SARS-CoV- 2 described herein is at least one SARS-CoV-2 variant selected from the group consisting of Alpha, Beta, Gamma, Delta or Omicron variant. In some embodiments, the SARS-CoV-2 Omicron variant is at least one SARS-CoV-2 Omicron sub-lineage such as BA.1 , BA.2, BA.3, BA.4, or BA.5. In some embodiments, the SARS-CoV-2 refers to a SARS-CoV-2 Spike variant comprising at least one mutation selected from the group consisting of del 69-70, RSYLTPGD246-253N, N440K, G446V, L452R, Y453F, S477G/N, E484Q, E484K, F490S, N501Y, N501 S, D614G, Q677P/H, P681 H, P681 R, and A701 V. In some embodiments, the SARS-CoV-2 refers to a SARS-CoV-2 variant comprising at least one mutation selected from the group consisting of G142D, G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681 H, N764K, D796Y, Q954H, N969K. In some embodiments, the SARS-CoV-2 refers to a SARS-CoV-2 variant comprising at least one mutation selected from the group consisting of L452R, F486V, and R493Q. Any combination of these mutations is possible and herewith disclosed. Specifically, all mutations listed in either of the precedingly mentioned groups of mutations can be present at the same time within a SARS-CoV-2 variant. Therefore, “SARS-CoV-2 protein” and “SARS- CoV-2 genome” may also be understood as the protein and genome of a SARS-CoV-2 variant, respectively.

The term “codon”, as used herein, refers to any group of three consecutive nucleotides in a coding part of a polynucleotide such as a messenger RNA molecule, or coding strand of DNA that specifies a particular amino acid, a starting or stopping signal for translation. Typically, codons are specific for one amino acid, however cases of a codon sharing at least one nucleotide with another codon are known for SARS-CoV-2.

The term “codon pair”, as used herein refers to two consecutive codons.

The term “codon-pair deoptimization” (CPD), as used herein, refers to a “reformulation” of codons or an exchange of codons by other codons encoding the same amino acid such that the encoded protein is the same, but suboptimal codon pairs and/or CpG dinucleotides emerge. Methods for codon-pair deoptimizations 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 comprises increasing the number of underrepresented or suboptimal codon pairs and CpG dinucleotides in recoded genomes. In some embodiments, the codon-pair deoptimization(s) described herein result(s) in increased mRNA decay and/or reduced translation efficiency. In some embodiments, the codon-pair deoptimization(s) described herein result(s) in less protein, less virus, reduced virulence, and/or a live-attenuated virus.

The polynucleotide of the invention may be used in virus production or in the context of a vaccine. As such, the polynucleotide of the invention emits a decreased risk of uncontrolled replication during production, transport, storage, processing and/or administration compared to the wild type sequence. Typically, this polynucleotide forms part of a live attenuated SARS-CoV-2. In comparison to a wild type virus, a live attenuated virus provokes less and/or less severe or even no symptoms in a host organism after the host organism has been confronted (infected) with the attenuated virus. At the same time, the live attenuated virus induces an immune response of the host to the attenuated virus that is at least partially protective against a wild type virus infection and/or at least one symptom thereof.

In contrast to most of the vaccines under development, the inventors generated attenuated but replicating SARS-CoV-2 vaccine candidates by genetic modification of the SARS-CoV-2 genome via codon pair deoptimization (Coleman et al., 2008). CPD is a virus attenuation strategy which 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., 2021 a; Trimpert et al., 2021 b). CPD rearranges the positions of existing synonymous codons in one or more viral genes, without changing the codon bias or amino acid composition of the encoded protein (Eschke et al., 2018; Groenke et al., 2020; Khedkar et al., 2018; Kunec and Osterrieder, 2016; Osterrieder and Kunec, 2018; Trimpert et al., 2021 a; Trimpert et al., 2021 b). Naturally underrepresented codons can become overrepresented by CPD. Since the effect of CPD is highly dependent on the genome sequence to be deoptimized, no exact instructions can be given on how the position of codons in the recoded sequence should be changed during deoptimization. However, the person skilled in the art is aware how to identify or estimate codon pairs that can be replaced by naturally underrepresented codon pairs at the target site (e.g. in a target species or a target tissue) at which the polynucleotide is intended to be translated. The inventors provide herein examples which codon pairs to recode to achieve overrepresentation of naturally underrepresented codon pairs and codon-pair deoptimization of the polynucleotide. The skilled person could therefore - at least from the codon pairs at the target site and the means and methods provided herein - arrive at other polynucleotides according to the invention.

A polynucleotide is to be considered as codon-pair deoptimized if at least one codon pair is deoptimized with respect to the corresponding natural sequence. Recoded viruses typically do not produce proteins from the recoded genes as efficiently as their parents, and can show defects in reproductive fitness, which enables the host to control wild-type virus infection by 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., 2021 b; Wimmer et al., 2009). The conserved antigenic identity and replicative potential enable recoded attenuated viruses to fully engage the immune system of the host and provoke strong immune responses.

Thus, by the codon-pair deoptimization, the resulting proteins are not altered. Rather, even though the genomic sequence of SARS-CoV-2 is altered, the resulting proteins remain the same. However, typically the efficiency of translation is reduced so that the virus replication is also reduced. This leads to an immune response when the live attenuated SARS-CoV-2 is used as vaccine without the risk of a pathologic virus replication in a patient having received the vaccine. Another possible effect of codon-pair deoptimization is a CpG mediated immune response leading to virus attenuation. The present disclosure is not limited to a specific one of these effects.

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

It is a very surprising finding that the vaccine efficacy is indeed not compromised by the modification, in particular deletion, of the FCS and the combined attenuating, but independent attenuating mechanisms of codon-pair deoptimization and modification, in particular deletion, of the FCS. In many cases, one can observe “over-attenuating” by combining different attenuating effects, leading to insufficient virus growth or insufficient immune response. Interestingly, such negative over-attenuating effects were not observed for the presently claimed subject matter. In contrast, the modification, in particular deletion, of the FCS results in an increased virus growth in vitro and in an increased genetic stability of the life attenuated SARS-CoV-2. The latter is particularly important with respect to regulatory requirements, since the higher the stability of the life attenuated SARS-CoV-2, the easier it is to obtain a marketing authorization for a vaccine comprising the life attenuated SARS-CoV-2 and the higher is the medical safety of such vaccine.

Unintentional spread of vaccine viruses from vaccinated to unvaccinated individuals is a factor that can complicate the use of transmissible live attenuated vaccines (LAV) (Bull et al., 2018; Layman et al., 2021 ; Nuismer et al., 2018; Pons-Salort et al., 2016). While self-dissemination is desirable in some scenarios, specifically when herd immunity is sought in wildlife (Smithson et al., 2019), uncontrolled circulation of vaccine viruses potentially increases the likelihood for reversion to virulence (Bull et al., 2018; Layman et al., 2021 ; Nuismer et al., 2018). Recombination between different vaccine viruses or vaccine and field viruses is especially problematic because it can give rise to recombinants with increased virulence, transmissibility or immune evasion capabilities (Burns et al., 2014; Combelas et al., 201 1 ; Lee et al., 2012; Ming et al., 2020). The rapid evolution of SARS-CoV-2 urges extra caution in the use of LAVs with respect to their potentially irrevocable circulation. Moreover, transmission of attenuated viruses to immunocompromised individuals is a danger that accompanies the use of transmissible virus vaccines (Kamboj and Sepkowitz, 2007).

The inventors already found that sCPD9 confers robust immunity against several SARS-CoV- 2 variants in COVID-19 hamster models (Trimpert et al., 2021 a; Trimpert et al., 2021 b). Most importantly, sCPD9 outperformed intra-muscularly administered adenovirus vector and mRNA vaccines in its ability to induce systemic and mucosal immunity (Nouailles et al., 2022). It was a very surprising finding that this robust immunity against several SARS-CoV-2 variants is not compromised by additionally introducing the modification, in particular deletion, of the FCS into the polynucleotide encoding at least some proteins of the live attenuated SARS-CoV-2. Rather, the immune protection conferred by a vaccination with a live attenuated SARS-CoV-2 comprising the novel polynucleotide construct was equally good as the immune protection conferred by a vaccination with the live attenuated SARS-CoV-2 previously described (Trimpert et al., 2021 a; Trimpert et al., 2021 b), even though the biologic safety of the novel construct is significantly higher.

The entry of SARS-CoV-2 into host cells is mediated by its major surface protein, the spike protein. The spike protein initiates infection by binding to its cellular receptor, angiotensinconverting enzyme 2 (ACE2), and enables the actual cell entry through fusion between the viral envelope with host cell membranes. To enable infection, the spike protein must be activated by cellular proteases. Activation involves proteolytic cleavage of the spike protein at approximately the midpoint of the protein at the S1/S2 site, resulting in two subunits S1 and S2 that are held together by non-covalent interactions. Cleavage of the spike protein induces conformational changes that enables the S1 subunit to bind to ACE2 via its receptor binding domain and triggers the fusogenic activity of the membrane-anchored S2 subunit.

Unlike other closely related viruses, SARS-CoV-2 contains a unique polybasic cleavage motif (PRRAj.) at the S1/S2 site, termed furin cleavage site (FCS). While several enzymes can cleave the FCS, it is most efficiently cleaved by the cellular transmembrane protease serine 2 (TMPRSS2), a cell surface trypsin-like protease (Hoffmann and Pohlmann, 2021 ). The TMPRSS2 protease determines the entry pathway of the virus (Koch et al., 2021 ). When the host cell expresses TMPRSS2, the virus is activated at the cell surface and rapidly enters the cells via cell fusion in a pH-independent manner. In contrast, if TMPRSS2 is absent, the virus is endocytosed and the virus entry is mediated by cathepsin L, an endosomal/lysosomal cysteine protease.

To prevent transmission of the vaccine virus sCPD9, the inventors deleted the FCS from its spike protein. It has been shown in preclinical studies that removal of the FCS renders mutant viruses non-transmissible and strongly attenuated (Johnson et al., 2021 ; Lau et al., 2020; Peacock et al., 2021 ; Sasaki et al., 2021 a). However, since removal of the FCS can enhance viral attenuation, combining it with a different attenuation mutation may result in an overly attenuated virus with limited ability to induce strong immunity. Therefore, it is important to compare transmissibility and protective efficacy of LAV candidates that lack the FCS with those that have the intact FCS. On the other hand, if removal of the FCS does not compromise the protective effect of the LAV candidates, then removal of the FCS is desirable because it increases the safety of the LAV candidates by introducing a second and independent attenuating mutation into the viral genome.

In addition, aside from eliminating transmission and increasing vaccine safety, removal of the FCS has a potentially important practical advantage for the production of SARS-CoV-2 LAVs. During propagation in cells that do not express TMPRSS2 such as Vero cells, which are commonly used by vaccine manufacturers, SARS-CoV-2 variants lacking a functional FCS become rapidly dominant because they outcompete variants 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., 2021 b; Wong et al., 2021 ). Consistent with these reports, the inventors found that sCPD9 also rapidly loses its FCS when propagated on cells that do not express TMPRSS2. In contrast, removal of the FCS increases the genetic stability of vaccine viruses during production, and also increases viral titers on TMPRSS2-deficient cell lines.

In an embodiment, the polynucleotide encodes the non-structural protein 7. In an embodiment, the polynucleotide encodes the non-structural protein 8. In an embodiment, the polynucleotide encodes the non-structural protein 9. In an embodiment, the polynucleotide encodes the non- structural protein 10. In an embodiment, the polynucleotide encodes the non-structural protein 11. In an embodiment, the polynucleotide encodes the non-structural protein 12. In an embodiment, the polynucleotide encodes the nsp15, the endonuclease. In an embodiment, the polynucleotide encodes the nsp16, the 2’-O-methyltransferase. In an embodiment, the polynucleotide encodes the spike protein (sometimes also referred to as spike glycoprotein).

In an embodiment, the polynucleotide encodes the spike protein and at least one of the non- structural proteins.

In an embodiment, the polynucleotide encodes at least two of the non-structural proteins. To give an example, the polynucleotide encodes, in an embodiment, the endoribonuclease and the 2’-O-methyltransferase. To give another example, the polynucleotide encodes, in an embodiment, non-structural protein 7, non-structural protein 8, non-structural protein 9, non- structural protein 10, and non-structural protein 1 1.

In an embodiment, the furin cleavage site modification is a partial or full deletion of a furin cleavage site being naturally present in the SARS-CoV-2 genome.

In an embodiment, the furin cleavage site modification is a loss-of-function mutation of a furin cleavage site being naturally present in the SARS-CoV-2 genome.

In an embodiment, the furin cleavage site modification is an at least partial substitution of a furin cleavage site being naturally present in the SARS-CoV-2 genome.

In an embodiment, the SARS-CoV-2 genome is a genome section extending from position 11 ,000 to position 27,000 of the genome of SARS-CoV-2. For position numbering and definition of the terms “genome of SARS-CoV-2” and “wild type SARS-CoV-2 “, reference is made to the gene bank accession number MT108784.1 (freely accessible via the website https://www.ncbi.nlm.nih.gov/genbank/) that comprises 29,891 bases or nucleotides. The first of these bases or nucleotides (at the 5’ terminus) is positioned at position 1 . The last of these bases or nucleotides (at the 3’ terminus) is positioned at position 29,891 . The skilled person is aware of how to adjust the numbering of the referenced sequence to embodiments, wherein the SARS-CoV-2 genome 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 1 1 ,000 to position 24,000. In an embodiment, the genome section extends from position 11 ,500 to position 26,000, in particular from position 1 1 ,900 to position 25,500, in particular from position 1 1 ,950 to position 25,350, in particular from position 12,000 to position 24,000. In an embodiment, the genome section extends from position 11 ,950 to position 14,400. In an embodiment, the genome section extends from position 11 ,900 to position 13,500. In an embodiment, the genome section extends from position 13,900 to position 14,400. In an embodiment, the genome section extends from position 20,300 to position 21 ,600. In an embodiment, the genome section extends from position 24,300 to position 25,400. These embodiments can be combined in any desired way.

In an embodiment, the at least one sequence part comprising codon-pair deoptimizations has a length lying in a range of from 750 nucleotides to 2500 nucleotides, in particular of from 800 nucleotides to 2400 nucleotides, in particular of from 900 nucleotides to 2300 nucleotides, in particular of from 999 nucleotides to 2200 nucleotides, in particular of from 1000 nucleotides to 2100 nucleotides, in particular of from 1100 nucleotides to 2000 nucleotides, in particular of from 1146 nucleotides to 1900 nucleotides, in particular of from 1200 nucleotides to 1836 nucleotides, in particular of from 1300 nucleotides to 1800 nucleotides, in particular of from 1400 nucleotides to 1700 nucleotides, in particular of from 1500 nucleotides to 1600 nucleotides.

In an embodiment, between 15 % and 40 %, in particular between 20 % and 35 %, in particular between 25 % and 30 % of the nucleotides of the at least one sequence part comprising codonpair deoptimizations are different from the nucleotides of a corresponding (wild type) SARS- CoV-2 genome. Such a wild-type SARS-CoV-2 genome is the genomic sequence of a non- artificially modified virus variant or lineage, such as lineages B.1.1.7 (Alpha), B.1.351 (Beta), B.1.1.28.1 (Gamma), B.1.617.2 (Delta), or B.1.159.1 (Omicron), including any sub-variants such as Omicron sub-variants BA.1 , BA.2, BA.3, BA.4, BA.5. It can also be denoted as authentic SARS-CoV-2 genome or authentic SARS-CoV-2 genomic sequence.

In an embodiment, between 200 and 500 nucleotides, in particular between 250 and 450 nucleotides, in particular between 300 and 400 nucleotides of the at least one sequence part comprising codon-pair deoptimizations are different from the (in particular identically positioned) nucleotides of a corresponding SARS-CoV-2 genome.

In an embodiment, between 40 % and 70 %, in particular between 45 % and 65 %, in particular between 50 % and 60 %, in particular between 55 % and 62 % of the codons of the at least one sequence part comprising codon-pair deoptimizations are different from the respective codons of a corresponding SARS-CoV-2 genome.

In an embodiment, between 150 and 400 codons, in particular between 200 and 350 codons, in particular between 250 and 300 codons of the at least one sequence part comprising codon- pair deoptimizations are different from the (in particular identically positioned) codons of a corresponding SARS-CoV-2 genome.

In an embodiment, the at least one sequence part comprising codon-pair deoptimizations comprises a first deoptimized sequence part and a second deoptimized sequence part. Both deoptimized sequence parts are separated from each other by a non-deoptimized sequence section comprising at least 300 nucleotides, e.g., 300 to 1000 nucleotides, in particular 400 to 900 nucleotides, in particular 500 to 800 nucleotides, in particular 600 to 700 nucleotides. By conserving a specific part of the RNA sequence and by deoptimizing flanking parts upstream and downstream of the conserved RNA sequence, a particularly high efficacy in attenuating the SARS-CoV-2 is achieved while maintaining its ability to replicate.

In an embodiment, the first deoptimized sequence part has a length lying in a range of from 1300 nucleotides to 1600 nucleotides, in particular of from 1400 nucleotides to 1500 nucleotides, in particular of from 1450 nucleotides to 1490 nucleotides. At the same time, the second deoptimized sequence part has a length lying in a range of from 100 nucleotides to 400 nucleotides, in particular of from 200 nucleotides to 300 nucleotides, in particular of from 350 nucleotides to 400 nucleotides. The length of the first deoptimized sequence part and of the second deoptimized sequence part is chosen such that other applicable restrictions (such as an overall length of the at least one sequence part comprising codon-pair deoptimizations of not more than 2000 nucleotides) are fulfilled, if desired. If the length of the at least one sequence part comprising codon-pair deoptimizations shall not exceed 2000 nucleotides, 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 parts to fulfil the restriction of the maximum length of the at least one sequence part comprising codonpair deoptimizations, considering that the first deoptimized sequence part and the second deoptimized sequence part are separated by at least 300 nucleotides of the authentic SARS- CoV-2 genome. At the same time, the upper threshold of 1600 nucleotides for the first deoptimized sequence part can be combined with the lower threshold of 100 nucleotides for the second deoptimized sequence part to fulfil a maximum length of 2000 nucleotides, considering the intermediate 300 non-recoded nucleotides.

In an embodiment, the first deoptimized sequence part is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 2. At the same time, the second deoptimized sequence part is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO.

4.

In an embodiment, the polynucleotide is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 6.

In an embodiment, the polynucleotide is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 8.

In an embodiment, the polynucleotide is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 10.

In an embodiment, the polynucleotide is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 15.

In an embodiment, the polynucleotide is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 16.

In an embodiment, the polynucleotide is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 17.

Even though an FCS deletion in the SARS-CoV-2 genome was described in prior art, one could not have assumed that such a deletion could have the effects observed by the present inventors when combining such deletion (or at least an FCS modification) with the specific codon-pair deoptimizations present in the above-mentioned sequences. It is rather surprising to learn that the immune protective properties of these codon-pair deoptimized sequences is not compromised by combining the codon-pair deoptimization with a modification, in particular deletion, of the FCS.

In an embodiment, the furin cleavage site modification comprises a deletion of the nucleotides encoding an amino acid sequence XRRA (i.e., a furin cleavage site), wherein X denotes P, R or H. The following Table 1 lists specific embodiments of the presently claimed subject matter relating to different amino acid sequences missing in the expressed protein due to the deletion of the furin cleavage site. It should be noted that it is less relevant which nucleotides are excised from the SARS-CoV-2 genome by the deletion of the furin cleavage site, as long as the furin cleavage site is no longer present in the resulting protein.

Table 1 : Embodiments of missing amino acids due to the deletion of the furin cleavage site.

In an embodiment, the furin cleavage site modification, in particular deletion, effects that an expression of the polynucleotide results in a protein, in particular a spike protein, in which at least or exactly 5, in particular at least or exactly 6, in particular at least or exactly 7, in particular at least or exactly 8, in particular at least or exactly 9, in particular at least or exactly 10, in particular 5 to 10, in particular 6 to 9, in particular 7 to 8, consecutive amino acids of the naturally expressed protein are replaced by a single amino acid. This can be achieved by an off-reading-frame deletion of nucleotides. By such an off-reading-frame deletion, a single nucleotide of a first triplet is combined with two nucleotides of a second triplet to form a novel triplet that is not present in the natural SARS-CoV-2 genome at this genome position. In an embodiment, the single amino acid that replaces the naturally expressed consecutive amino acids is an isoleucine.

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

In an aspect, the present invention relates to a live attenuated SARS-CoV-2. This live attenuated SARS-CoV-2 comprises a partially recoded genomic RNA sequence, i.e., a partially recoded genomic viral sequence. The partially recoded genomic RNA sequence is a codonpair deoptimized sequence coding for a spike protein and/or a specific non-structural protein (nsp). The non-structural protein is chosen from the group consisting of non-structural protein 7, non-structural protein 8, non-structural protein 9, non-structural protein 10, non-structural protein 11 (a small, only 13 amino acids long protein), non-structural protein 12 (also referred to as RNA-dependent RNA polymerase), non-structural protein 15 (an endoribonuclease), and non-structural protein 16 (a 2’-O-methyltransferase) of the live attenuated SARS-CoV-2. The live attenuated SARS-CoV-2 further comprises a furin cleavage site modification resulting in a functional loss of a furin cleavage site. This furin cleavage site is naturally present in the SARS- CoV-2 genome. As a result, the spike protein of the live attenuated SARS-CoV-2 does not comprise a functional furin cleavage site, whereas non-engineered SARS-CoV-2 does comprise such furin cleavage site.

In an embodiment, the partially recoded genomic RNA sequence codes for the non-structural protein 12. In an embodiment, the partially recoded genomic sequence codes for the spike protein (sometimes also referred to as spike glycoprotein).

In an embodiment, the partially recoded genomic RNA sequence comprises at least two of the non-structural proteins. To give an example, the partially recoded genomic RNA sequence codes, in an embodiment, for the endoribonuclease and the 2’-O-methyltransferase. To give another example, the partially recoded genomic RNA sequence codes, in an embodiment, for non-structural protein 7, non-structural protein 8, non-structural protein 9, non-structural protein 10, and non-structural protein 1 1.

In an embodiment, the partially recoded genomic RNA sequence lies in a genome section extending from position 11 ,000 to position 27,000 of the genome of the live attenuated SARS- CoV-2. According to gene bank accession number MT108784.1 , the genome of the wild type SARS-CoV-2 comprises 29,891 bases or nucleotides. The genome of the live attenuated SARS-CoV-2 has essentially a similar length. A difference is the length of a polyA tail at the 3’ terminus that was determined by sequencing to be eight adenine nucleotides longer than in case of the wild type SARS-CoV-2. It should be noted that some uncertainty remains by determining the length of a polyA tail by sequencing. Consequently, it is possible that the polyA tail in the wild type sequence is longer or shorter than indicated in the sequence according to gene bank accession number MT 108784.1 . Likewise, it is possible that the polyA tail in the live attenuated SARS-CoV-2 sequence is longer or shorter than presently determined. A further difference is that the live attenuated SARS-CoV-2 lacks at least 12 nucleotides that encode the furin cleavage site in the wild type SARS-CoV-2.

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

In an embodiment, the partially recoded genomic RNA sequence has a length lying in a range of from 750 nucleotides to 2500 nucleotides, in particular of from 800 nucleotides to 2400 nucleotides, in particular of from 900 nucleotides to 2300 nucleotides, in particular of from 999 nucleotides to 2200 nucleotides, in particular of from 1000 nucleotides to 2100 nucleotides, in particular of from 1 100 nucleotides to 2000 nucleotides, in particular of from 1146 nucleotides to 1900 nucleotides, in particular of from 1200 nucleotides to 1836 nucleotides, in particular of from 1300 nucleotides to 1800 nucleotides, in particular of from 1400 nucleotides to 1700 nucleotides, in particular of from 1500 nucleotides to 1600 nucleotides.

In an embodiment, between 15 % and 40 %, in particular between 20 % and 35 %, in particular between 25 % and 30 % of the nucleotides of the partially recoded genomic RNA sequence are different from the nucleotides of a corresponding wild-type genomic RNA sequence. Such a wild-type genomic RNA sequence is the genomic viral sequence of a non-artif icially modified virus variant or lineage, such as lineages B.1 .1 .7 (Alpha), B.1 .351 (Beta), B.1 .1 .28.1 (Gamma), B.1.617.2 (Delta), or B.1.159.1 (Omicron). It can also be denoted as authentic SARS-CoV-2 genomic RNA sequence.

In an embodiment, between 200 and 500 nucleotides, in particular between 250 and 450 nucleotides, in particular between 300 and 400 nucleotides of the partially recoded genomic RNA sequence are different from the identically positioned nucleotides of a corresponding wildtype virus genomic RNA sequence.

In an embodiment, between 40 % and 70 %, in particular between 45 % and 65 %, in particular between 50 % and 60 %, in particular between 55 % and 62 % of the codons (i.e., three nucleotides in each case that code for a specific amino acid) of the partially recoded genomic RNA sequence are different from the respective codons of a corresponding wild-type virus genomic RNA sequence.

In an embodiment, between 150 and 400 codons, in particular between 200 and 350 codons, in particular between 250 and 300 codons of the partially recoded genomic RNA sequence are different from the identically positioned codons of a corresponding wild-type virus genomic RNA sequence.

In an embodiment, the partially recoded genomic RNA sequence comprises a first recoded part and a second recoded part. Both recoded parts are separated from each other by a nonrecoded genome section comprising at least 300 nucleotides, e.g., 300 to 1000 nucleotides, in particular 400 to 900 nucleotides, in particular 500 to 800 nucleotides, in particular 600 to 700 nucleotides. By conserving a specific part of the RNA sequence and by recoding flanking parts upstream and downstream of the conserved RNA sequence, a particularly high efficacy in attenuating the SARS-CoV-2 while maintaining its general viability is achieved.

In an embodiment, the first recoded part has a length lying in a range of from 1300 nucleotides to 1600 nucleotides, in particular of from 1400 nucleotides to 1500 nucleotides, in particular of from 1450 nucleotides to 1490 nucleotides. At the same time, the second recoded part has a length lying in a range of from 100 nucleotides to 400 nucleotides, in particular of from 200 nucleotides to 300 nucleotides, in particular of from 350 nucleotides to 400 nucleotides. The length of the first recoded part and of the second recoded part is chosen such that other applicable restrictions (such as an overall length of the recoded genomic RNA sequence of not more than 2000 nucleotides) are fulfilled, if desired. If the length of the partially recoded genomic RNA sequence shall not exceed 2000 nucleotides, 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 recoded parts to fulfil the restriction of the maximum length of the partially recoded genomic RNA sequence, considering that the first recoded part and the second recoded part are separated by at least 300 nucleotides of the authentic SARS- CoV-2 genomic sequence. At the same time, the upper threshold of 1600 nucleotides for the first recoded part can be combined with the lower threshold of 100 nucleotides for the second recoded part to fulfil a maximum length of 2000 nucleotides, considering the intermediate 300 non-recoded nucleotides.

In an embodiment, the first recoded part is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 2. At the same time, the second recoded part is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 4.

In an embodiment, the partially recoded genomic RNA sequence is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 6.

In an embodiment, the partially recoded genomic RNA sequence is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 8.

In an embodiment, the partially recoded genomic RNA sequence is at least 95 %, in particular at least 96 %, in particular at least 97 %, in particular at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 10.

The phrase “being % identical” or “having percent (%) 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 with the nucleotides or amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The skilled person is aware that sequences as exemplified herein can be altered to a certain percentage without or without substantially altering biologic functions such as attenuation or the encoded protein. Therefore, the skilled person using means and methods described herein can amend codon pairs according to the teaching of the invention. E.g., codon pairs can be replaced with synonymous versions that are similarly attenuating and/or naturally underrepresented as the deoptimized codon pairs of the sequences described herein. Thus, a similar degree of deoptimization may be achieved by replacing codon pairs in the position ranges described herein.

The invention is based at least in part on the finding that codon deoptimizations in the position ranges described herein are particularly useful for attenuating the SARS-CoV-2 virus while sufficiently maintaining immunogenicity and the ability of the virus to replicate. It is further based on the finding that a furin cleavage site modification, which results in a functional loss of a furin cleavage site being naturally present in the SARS-CoV-2 genome, further attenuates the SARS-CoV-2, while fully maintaining its immunogenicity.

The individual recoded sequences and their position in the SARS-CoV-2 genome are summarized in the following Table 2.

Table 2: Summary of recoded RNA sequences. pp = polyprotein nsp = non-structural protein

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

The middle part of fragment WT6 was not recoded in fragment CPD6, because it contains a conserved regulatory RNA sequence that is essential for virus replication. Fragment WT6 encodes a -1 ribosomal frameshifting element, a so-called RNA pseudoknot structure. This structure promotes a ribosomal frameshifting, a process during which the reading frame of translation is changed at the junction between open reading frames (ORFs) 1 a and 1 b. During this process, a single nucleotide of the slippery sequence that is located downstream from the RNA pseudoknot structure is read twice by the translating ribosome, and the reading frame is shifted by -1 nucleotide (the ribosome slips one nucleotide backwards at the slippery sequence). Often, translation of ORF1 a terminates at the stop codon of ORF1 a. However, when -1 ribosomal frameshifting occurs, translation of ORF1 a continues directly to ORF1 b and polyprotein (pp) 1 ab is produced. Thus, the CPD6A sequence is translated into both polyproteins 1 a and 1 ab; but the CPD6B sequence is translated only into polyprotein 1 ab.

The differences between the CPD sequences and the underlying wild type sequences are summarized in the following Table 3.

Table 3: Summary of difference between CPD and wild type sequences.

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

Table 4: Summary of protein sequences.

By recoding sequence parts CPD6A, CPD6B, sCPD9, and sCPD10, different fragments of the SARS-CoV-2 genome were generated that form part of a live attenuated SARS-CoV-2 in an embodiment. These fragments are listed in the following Table 5.

Table 5: Genome fragments of SARS-CoV-2 comprising recoded sequence parts. In an aspect, the present invention relates to a live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising the polynucleotide according the above explanations.

In an embodiment, the live attenuated SARS-CoV-2 has a nucleic acid sequence being at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 19. Besides a codon-pair deoptimized region, this live attenuated SARS-CoV-2 comprises a 24-nucleotide deletion of a furin cleavage site in the spike protein gene, leading to a replacement of the amino acid sequence NSPRRARSV (SEQ ID NO. 32) comprising a furin cleavage site by the amino acid isoleucine in the spike glycoprotein.

In an embodiment, the SARS-CoV-2 has a nucleic acid sequence being at least 98 %, in particular at least 99 %, in particular 100 % identical to SEQ ID NO. 20. Besides a codon-pair deoptimized region, also this live attenuated SARS-CoV-2 comprises a 24-nucleotide deletion of a furin cleavage site in the spike protein gene, leading to a replacement of the amino acid sequence NSPRRARSV (SEQ ID NO. 32) comprising a furin cleavage site in the spike protein with the amino acid isoleucine.

In an aspect, the present invention relates to a pharmaceutical composition comprising the live attenuated SARS-CoV-2 according to any of the preceding explanations. Such a pharmaceutical composition can further comprise auxiliary substances like adjuvants, e.g., for enhancing an immune response of a patient. Appropriate adjuvants are potassium alum; aluminium hydroxide; aluminium phosphate; calcium phosphate hydroxide; aluminum hydroxyphosphate sulfate; 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; as well as Freund's complete adjuvant.

In an aspect, the present invention relates to the further medical use of such a pharmaceutical composition as a vaccine.

In an aspect, the present invention relates to the further medical use of such a pharmaceutical composition as a vaccine for immunosuppressed individuals, in particular for individuals receiving glucocorticoid treatment such as dexamethasone treatment.

In an aspect, the present invention relates to a method for preparing a vaccine from such a pharmaceutical composition. In an aspect, the present invention relates to a method of vaccinating a human or animal patient in need thereof. The animal patient is in particular a non-human mammal such as a rodents, canines, felines, or mustelids. This method comprises the step of administering a pharmaceutical composition according to the preceding explanations to the patient.

In an embodiment, the human or animal patient is an immunosuppressed patient, in particular a patient receiving glucocorticoid treatment such as dexamethasone treatment.

In an embodiment, the administration is performed by an intranasal administration, an oral administration, a parenteral administration such as a subcutaneous injection, an intramuscular injection, an intravenous injection, an intraperitoneal injection, an intravenous infusion, or an intraperitoneal infusion. An intranasal or oral administration is particularly appropriate. By these routes of administration, the live attenuated SARS-CoV-2 is presented to the body of the patient to be vaccinated in the same or a similar way as in case of a natural exposure to the virus.

In an embodiment, the vaccination is performed by administering the pharmaceutical composition in a dose comprising between 1 *10 ³ and 1 *10 ⁸ focus-forming units (FFLI), in particular between 1 *10 ⁴ and 1 *10 ⁷ FFLI, in particular between 1 *10 ⁵ and 1 *10 ⁶ FFLI, of the live attenuated SARS-CoV-2. The dose is chosen such that the pharmaceutical composition is well tolerated by the patient but evokes an immune response that gives protection to the patient against an infection or a severe course of an infection with SARS-CoV-2. In embodiment, the dose is one of the lowest protective dose and the highest tolerable dose or lies between the lowest protective dose and the highest tolerable dose.

Various factors can influence the dose used for a particular application. For example, the frequency of administration, duration of treatment, preventive or therapeutic purpose, the use of multiple treatment agents, route of administration, previous therapy, the patient's clinical history, the discretion of the attending physician and severity of the disease, disorder and/or condition may influence the required dose to be administered.

As with the dose, various factors can influence the actual frequency of administration used for a particular application. For example, the dose, duration of treatment, use of multiple treatment agents, route of administration, and severity of the disease, disorder and/or condition may require an increase or decrease in administration frequency. In some cases, an effective duration for administering the pharmaceutical composition of the invention (and any additional therapeutic agent) can be any duration that reduces the severity, or occurrence, of symptoms of the disease, disorder and/or condition to be treated without producing significant toxicity to the subject. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the disease, disorder and/or condition being treated.

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

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

In an aspect, the present invention relates to a vector comprising the polynucleotide according to the above explanations.

The term “vector”, as used herein, refers to a nucleic acid molecule, capable of transferring or transporting itself and/or another nucleic acid molecule into a cell. The transferred nucleic acid is generally linked to, i.e., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. In some embodiments, the vector described herein is a vector selected from the group of plasmids (e.g., DNA plasmids or RNA plasmids), shuttle vectors, transposons, cosmids, artificial chromosomes (e.g. bacterial, yeast, human), and viral vectors.

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

In an aspect, the present invention relates to a host cell comprising the polynucleotide according to the above explanations.

The term “host cell”, as used herein, 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”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

In an embodiment, the host cell described herein comprises 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).

In an aspect, the present invention relates to a method for the production of a virus. This method comprises the steps of a) culturing a host cell according to the preceding paragraph; and b) isolating a virus, wherein the virus is a live attenuated SARS-CoV-2.

All embodiments of the polynucleotide can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the live attenuated SARS-CoV- 2, to the pharmaceutical composition, its use, to the method of vaccinating the patient, to the vector, to the host cell, and to the method of producing a virus. All embodiments of the live attenuated SARS-CoV-2 can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the polynucleotide, to the pharmaceutical composition, its use, to the method of vaccinating the patient, to the vector, to the host cell, and to the method of producing a virus. Likewise, all embodiments of the pharmaceutical composition can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the polynucleotide, to the live attenuated SARS-CoV-2, the use of the pharmaceutical composition, to the method of vaccinating a patient, to the vector, to the host cell, and to the method of producing a virus. All embodiments of the use of the pharmaceutical preparation can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the polynucleotide, to the live attenuated SARS- CoV-2, to the pharmaceutical preparation, to the method of vaccinating a patient, to the vector, to the host cell, and to the method of producing a virus. Finally, all embodiments of the method of vaccinating a patient can be combined in any desired way and can be transferred either individually or in any arbitrary combination to the polynucleotide, to the live attenuated SARS- CoV-2, to the pharmaceutical preparation, to the use of the pharmaceutical preparation, to the vector, to the host cell, and to the method of producing a virus.

Further details of aspects of the present invention will be explained in the following making reference to exemplary embodiments and accompanying Figures. In the Figures:

Figures 1 A to 1 D schematically depict the native structure and exemplary recoding of the SARS-CoV-2 genome;

Figure 2 shows the growth kinetics of sCPD9-AFCS and sCPD9 SARS-CoV-2 in Vero E6 cells;

Figures 3A to 3E illustrate virological and histopathological findings in contact hamsters;

Figures 4A and 4B illustrate clinical parameters of infected Syrian hamsters;

Figures 5A to 5E illustrate clinical, virological, pathological and serological results found in hamsters after challenge-infection with SARS-CoV-2 Delta variant;

Figures 6A to 6I illustrate the setup, the results and the biological background of experiments relating to in vivo and in vitro co-infection with sCPD9- AFCS and BA.5;

Figures 7A to 7J illustrate the setup and the results of experiments on the effect of immunosuppression on safety, efficacy and transmission of sCPD9- AFCS; Figures 8A to 8F illustrate the virological results of sCPD9-AFCS vaccinated contact hamsters with respect to a B.1 and a BA.5 infection, wherein the explanation of the used symbols indicated below Figures 8E and 8F is also valid for Figures 8A to 8D;

Figures 9A to 9D illustrate the systemic and mucosal immunity of vaccinated contact animals, wherein the explanation of the used symbols indicated below Figures 9C and 9D is also valid for Figures 9A and 9B;

Figures 10A to 10C illustrate the clinical and virological results of naive contacts of vaccinated B.1 shedders, wherein the explanation of the used symbols indicated next to Figure 10C is also valid for Figures 10A and 10B;

Figures 1 1 A to 11 C illustrate the clinical and virological results of naive contacts of vaccinated B.5 shedders, wherein the explanation of the used symbols indicated below Figure 11 C is also valid for Figures 11 A and 1 1 B; and

Figures 12A to 12C illustrate the systemic and mucosal immunity of naive contact animals, wherein the explanation of the used symbols indicated below Figure 12C is also valid for Figures 12A and 12B.

EXEMPLARY EMBODIMENTS

The inventors generated a series of recoded SARS-CoV-2 mutants, characterized them in cultured cells and also in vivo using the Syrian and Roborovski hamster models. It was proven that a single-dose intranasal immunization with attenuated live viruses can elicit strong immune responses and offer complete protection against SARS-CoV-2 challenge in a robust small animal model of COVID-19.

Vaccine design

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

In this context, Figures 1 A to 1 D illustrate the structure and recoding of the SARS-CoV-2 genome.

Figure 1 A illustrates that the SARS-CoV-2 genome is a single-stranded, positive-sense RNA molecule of about 30,000 nucleotides (nt), which encodes 1 1 canonical ORFs. “3CL-Pro” denotes 3C-like proteinase; “RdRp” denotes RNA-dependent RNA polymerase; “ExoN” denotes 3’-to-5’ exoribonuclease; “EndoRNAse” denotes endoribonuclease; and “2’-O-MT” denotes 2’-0-ribose methyltransferase.

As illustrated in Figure 1 B, after infection, ORF 1 a/1 ab is directly translated and cleaved into 15 proteins of the replication-transcription complex.

As illustrated in Figure 1 C, recoded SARS-CoV-2 mutants were constructed using a recently established reverse genetics system of SARS-CoV-2, which consists of 12 subgenomic fragments. Fragments 1 , 11 and 12 were not recoded. The dark grey boxes indicate recoded sequences CPD2-10, and the light grey boxes indicate parental, non-recoded sequences in the respective fragments 2-10. The frame-shifting element contained in fragment 6 and the transcription regulatory sequence (TRS) of the spike gene in fragment 9 were excluded from the recoding process (light grey boxes present between two dark grey boxes in CPD6 and CPD9).

As shown in Figure 1 D, the dark grey boxes indicate recoded sequences SCPD3-5 and sCPD8- 10 in different subgenomic fragments.

To preserve the full compatibility with the available reverse genetics system, the inventors recoded only SARS-CoV-2 sequences that were not present in the overlapping parts of the subgenomic fragments (approximately 2,500 bp in each recoded fragment) (Figures 1 A to 1 D). This design enabled the inventors to generate a wide variety of SARS-CoV-2 mutants, carrying a single or more recoded fragments.

The inventors recoded nine fragments of the SARS-CoV-2 reverse genetics system (fragments 2-10). Fragments 1 and 12, which are relatively short, 591 and 1 ,812 bp respectively, and fragment 1 1 , which contains many short ORFs, were excluded from the recoding. To ensure that mutant viruses are replication-competent, two genomic regions containing essential cisacting RNA elements were excluded from the recoding: the frame-shifting element carried by fragment 6 and the transcription regulatory sequence (TRS) of the spike gene within fragment 9. In addition, the first 500 bp of ORF 1 a located in fragment 2 were not recoded either.

Material and Methods

Study design and animal husbandry

This exemplary embodiment aimed to determine the impact of removing the furin cleavage site (FCS) of sCPD9 on vaccine virus spread, on its immunogenicity, protective efficacy and host- to-host transmission. The transmissibility of sCPD9-AFCS (i.e., a SARS-CoV-2 with a sCPD9 sequence and a deletion of the furin cleavage site) was investigated in comparison to WT (B.1 ) SARS-CoV-2 and sCPD9 SARS-CoV-2. Additionally, potential recombination between SARS- CoV-2 variant Omicron BA.5 and the vaccine candidate sCPD9-AFCS virus was assessed in a co-infection experiment. Furthermore, the safety and transmissibility of the sCPD9-AFCS vaccine candidate were investigated in immunosuppressed hamsters.

Syrian hamsters (Mesocricetus auratus', breed RjHamAURA) were purchased from Janvier Labs and housed in pairs in individually ventilated cages. Food and water were provided ad libitum, and cages were enriched with nesting material. The room temperature was maintained at a constant range of 22 to 24 °C with a relative humidity of 40 to 55 %. Prior to the begin of the experiment, the animals were allowed to acclimate to the housing conditions for seven days.

The transmission of B.1 , sCPD9 and sCPD9-AFCS viruses from infected to naive animals was evaluated using 36 male and female Syrian hamsters that were 10 weeks old.

Half of the respective animals received either 1 *10 ⁵ FFLI WT (B.1 ), sCPD9 or sCPD9-AFCS by intranasal instillation. Immediately after WT infection or sCPD9/sCPD9-AFCS vaccination, the hamsters were kept in individual cages and reunited with their non-infected/non-vaccinated conspecifics one day post infection/vaccination. Body weights were recorded daily for all hamsters and clinical conditions were checked two times per day. From 1 - 6 dpc (days post contact) mouth swabs were taken daily from contact hamsters. Contact animals were euthanized 6 dpc to assess viral loads in upper and lower respiratory tract, signs of pneumonia and seroconversion.

WT-infected and sCPD9/sCPD9-AFCS-vaccinated animals were challenged-infected with 1 *10 ⁵ PFU SARS-CoV-2 Delta variant 21 days following infection/vaccination. Moreover, a group of 6 non-infected/non-vaccinated hamster was challenged with the Delta variant for comparative purposes. Subsequently, the hamsters were euthanized 2 and 5 dpch (days post challenge) to collect blood, trachea and lungs for virolog ical, serological and histopathological analysis.

To investigate the potential for recombination between SARS-CoV-2 variant Omicron BA.5 and sCPD9-AFCS, a co-infection experiment was performed using twelve female Syrian hamsters that were 4 weeks old. After acclimation, six hamsters were co-infected with 1 x10 ⁴ FFLI of sCPD9-AFCS and 1 *10 ⁴ FFLI of SARS-CoV-2 variant Omicron BA.5 by intranasal instillation. The infection was conducted under general anesthesia, which was induced as described below. Subsequently, the infected animals were quarantined in separate cages to prevent accidental transmission of the virus inoculum to naive animals. After one day, they were again cohoused with their naive partners. Body weights and oral swab were collected daily from all animals to screen for virus transmission. Clinical conditions were monitored twice daily. After 6 days of cohousing, animals were euthanized to collect blood, trachea and lungs for virological analyses.

To assess vaccine safety and transmissibility in immunosuppressed animals, 12 female Syrian hamsters that were 4 weeks old were immunocompromised by daily subcutaneous injection of dexamethasone (2 mg/kg). On the third day of immunosuppression, six hamsters were vaccinated with 1 x10 ⁴ FFLI of sCPD9-AFCS by intranasal application performed under general anesthesia as described below. The vaccinated animals were housed in individual cages for 24 h and subsequently reunited with their unvaccinated and immunosuppressed conspecifics. During the cohousing period, oral swabs were taken daily from all hamsters to detect potential vaccine virus transmission. The contact animals were euthanized 6 days post contact (dpc), whilst vaccinated hamsters were sacrificed 21 days post vaccination (dpv). Blood, trachea and lungs were collected for serological, virological and histopathological analyses.

Cells

Minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 lll/ml penicillin G, and 100 pg/ml streptomycin was used to cultivate Vero E6 (ATCC CRL-1586) and WHO Vero RCB 10-87 cells. For Vero E6-TMPRSS2 cells (NIBSC 100978) the medium contained additionally 1000 pg/ml geneticin (G418) to select for cells expressing TMPRSS2.

The cells were kept at 37 °C and 5% CO2.

Viruses

The SARS-CoV-2 variants B.1 (B.1 , BetaCoV/Munich/ChVir984/2020, hCoV-19/Germany/BY- ChVir-929/2020, EPI_ISL_406862), Delta (B.1.617.2, Human, 2021 , Germany ex India, 20A/452R, EVAg: 009V-04187), and the SARS-CoV-2 mutants B.1-AFCS, sCPD9 and sCPD9-AFCS were cultured on Vero E6-TMPRSS2 cells. The 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, EPIJSLJ 6221625) were propagated on CaLu-3 cells. The BAC-derived SARS-CoV-2 variant B.1 (GenBank: MT 108784) was grown on Vero E6 cells and used for growth kinetics and plaque size assays. Titers of virus stocks were determined by conducting plaque assays on Vero E6 cells, and the viruses were stored at -80 °C before experimental infection.

Ethic statement

In vivo and in vitro experiments were carried out in the biosafety level three (BSL-3) laboratory at the Institut fur Virologie, Freie Universitat Berlin, Germany. Animal works were conducted according to institutional, national, and international guidelines for care and humane use of animal and authorized by the Landesamt fur Gesundheit und Soziales, Berlin (permit number 0086/20).

Infection/Vaccination

Hamsters were infected or vaccinated under general anesthesia (0.15 mg/kg medetomidine, 2.0 mg/kg midazolam and butorphanol 2.5 mg/kg). 1 *10 ⁵ FFU B.1 (Wildtype), sCPD9 or sCPD9-AFCS were diluted in 60 pl MEM and applied intranasally. Mock vaccinated individuals received 60 pl plain MEM without virus. 21 days after vaccination or initial infection, challengeinfection with 1 *10 ⁵ FFU SARS-CoV-2 Delta variant was conducted as described for initial infection.

RNA extraction and reverse transcription quantitative PCR (RT-qPCR)

Genomic copies were quantified in oropharyngeal swabs and 2.5 mg lung tissue homogenized in a bead mill (Analytic Jena). For RNA extraction the innuPREP Virus DNA/RNA Kit (Analytic Jena, Jena, Germany) was used in accordance with the manufacturer’s instructions. To conduct the RT-qPCR, NEB Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) was utilized. Cycling conditions of 10 min at 55 °C for reverse transcription, 3 min at 94 °C for activation of the enzyme and 40 cycles of 15 s at 94 °C and 30 s at 58 °C were applied on a qTower G3 cycler (Analytic Jena) using Primers and Probes as reported by Corman et al. (Corman et al., 2020).

Plaque assay

To quantify replication-competent virus, 10-fold serial dilutions of 50 mg homogenized lung tissue were prepared and plated on Vero E6 cells grown in 12-well plates. After incubating the cells for 2.5 hours at 37 °C and 5% CO2, cells were overlaid with 1 .5 % carboxymethylcellulose sodium (Sigma Aldrich) diluted in complete growth medium. To fix the cells 72 hours after infection, PBS-buffered 4 % formaldehyde solution (pH 6.5) was used. Plaque-forming units were counted per well after staining with 0.75 % methylene blue (aqueous solution).

Neutralization test

Serum samples from all hamsters were tested for neutralizing capacity against SARS-CoV-2 (B.1 ). Additionally, sera from challenge-infected animals (0, 2, 5 dpch) were tested for neutralizing activity against SARS-CoV-2 Delta variant (B.1 .617). To this end, sera were inactivated for 30 minutes at 56 °C. Subsequently, 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) were added to all wells. After an incubation time of 1 hour at 37 °C, the dilutions were plated on subconfluent Vero E6 cells in 96-well cell culture plates and incubated for another 72 hours. At last, plates were fixed with 4 % formaldehyde solution and stained with 0.75 % methylene blue (aqueous solution). Wells without virus-induced cytopathic effect were considered neutralized and reported as titers for the respective serum. Positive and negative controls were included in all plates.

Histopathology

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

Growth kinetics

T25 flasks of confluent WHO Vero RCB 10-87 cells were infected with either B.1 - BetaCoV/Munich/ChVir984/2020, B.1 , EPI_ISL_406862, B.1-AFCS, sCPD9 or sCPD9-AFCS at a MOI of 0.01 . The virus was diluted in a final volume of 5 ml complete cell culture medium and added to each flask. After the infection, supernatant was collected at 24, 48, 72 and 96 hours post infection and subjected to one freeze-thaw cycle. Ten-fold dilutions were prepared and plated on 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 solution after 48 hours. For plaque visualization, immunofluorescence staining was conducted as described (Trimpert et al., 2021 b).

Co-culture of sCPD9-AFCS and Omicron BA.5

CaLu-3 cells were seeded in T25 flasks and grown to a density of 90 %. Prior to co-infection with 100 FFLI of SARS-CoV-2 variant Omicron BA.5 and 1 ,000 FFLI of sCPD9-AFCS, cell culture medium was changed to 5 ml DMEM/F12 1 :1 containing 10% FBS, 100 lll/ml penicillin G, 100 pg/ml streptomycin and 1 % non-essential amino acids. After 72 h, the supernatant was harvested and cleared by centrifugation at 5,000 rpm for 15 min. Subsequently, 1 % of supernatant was transferred to previously uninfected CaLu-3 cells. The assay was performed in triplicates and continued for a total of 10 passages.

Sequencing

Following RNA extraction from cell culture supernatant as described above, libraries were prepared and sequenced using Illumina technology (Illumina). For library preparation the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (New England Biolabs) was used. This approach relies on standard library preparation steps for Illumina sequencing, such as end repair, adaptor ligation and PCR enrichment. Quantification of enriched sequencing libraries was performed using the NEBNext® Library Quant Kit for Illumina® (New England Biolabs). Libraries were then pooled and sequenced on an Illumina Miseq System (Illumina).

The generated Illumina sequencing data were processed with Trimmomatic v.0.39 (Bolger et al., 2014) and mapped against BA.5 (NCBI accession number: ON249995) and sCPD9-AFCS genome references (GenBank: MZ064545.1 ) (Trimpert et al., 2021 b), respectively, using the Burrows-Wheeler aligner v.0.7.17 (Li and Durbin, 2009). Mapping statistics were generated using Samtools v1 .10 (Danecek et al., 2021 ) and alignments were visualized using IGV v2.9.4 for Linux (Robinson et al., 2011 ). For detection of single-nucleotide polymorphisms (SNPs), Freebayes, a Bayesian genetic variant detector (arXiv:1207.3907 [q-bio.GN] 2012) was used. All SNPs with a minimum mapping quality of 5, minimum count of 3 and minimum fraction of 0.1 were initially considered. SNPs detected between the starting BA.5 isolate used in these studies and the BA.5 reference were removed from further analysis as they were present before these experiments. A table containing the removed SNPs is provided. Consensus sequences for each sample were obtained using BCFtools (Danecek et al., 2021 ). Direct sequence comparison to sCPD9-AFCS is not efficient given the slight differences in genomic structure between BA.5 and sCPD9-AFCS and high entropy between the samples and the sCPD9-AFCS reference. To facilitate this process, a sample-wide consensus sequence containing all detected SNPs was created on a backbone of the BA.5 reference (the most similar to all samples) using SNP-sites (Page et al., 2016) and BCFtools. This samplewide consensus was aligned to both BA.5 and sCPD9-AFCS references using EMBOSS Stretcher (Myers and Miller, 1988) and new SNP tables were created using SNP-sites, to verify location and identity of all detected SNPs against both references. All SNPs occurring between the BA.5 starting isolate and sCPD9-AFCS references, and thus not arising during the coinfection experiments, were removed from analysis.

Results

Deletion of FCS moderately increases viral titer on TMPRSS-2-negative cells

To increase the genetic stability and reduce transmissibility of sCPD9, the inventors generated an sCPD9 derivative designated sCPD9-AFCS that lacked the FCS in the spike protein. The sCPD9-AFCS mutant was engineered to contain the same deletion in the spike protein, termed ‘Bristol deletion’, that occurred naturally during serial passage on cultured Vero E6 cells (Davidson et al., 2020). The introduced deletion is 24 nucleotides long. It removes 9 amino acids ‘NSPRRARSV’ from the spike protein, including the entire FCS, and introduces isoleucine as novel amino acid at the same position instead (Davidson et al., 2020). Thus, a total of 8 amino acids is removed from the spike protein.

It has been widely reported that SARS-CoV-2 virus variants lacking FCS have a distinct growth advantage over viruses with intact FCS on different Vero cell lines. In accordance with these results, it was found that removal of the FCS slightly increased titers of sCPD9-AFCS viruses after propagation on Vero cells. Since WHO RCB 10-87 Vero cells are widely used for vaccine production, the ability to generate slightly higher peak virus titers faster on these particular cells is of great practical importance as it reduces the cost of vaccine production (Figure 2). In this context, Figure 2 shows growth kinetics of sCPD9-AFCS and sCPD9 conducted on WHO Vero RCB 10-87 cells in mean ± standard deviation (SD). The time points on which the supernatant was harvested are shown in hours post infection (hpi).

Deletion of FCS prevents transmission of sCPD9-AFCS to unvaccinated contacts

To investigate the ability of sCPD9 and sCPD9-AFCS to transmit to unvaccinated contact animals, Syrian hamsters were infected with sCPD9, sCPD9-AFCS or parental SARS-CoV-2 (WT) on day 0. Twenty-four hours after infection, each infected hamster was placed in an individually ventilated cage with an unvaccinated hamster. Already on the first day of this cohabitation, swabs from the mouths of all naive animals that were in contact with WT-infected animals were strongly positive for SARS-CoV-2 RNA. All naive animals that were in contact with sCPD9-vaccinated subjects contracted the virus on days 1 -3 of cohabitation and displayed a similar, although delayed, course of virus replication in the upper airways when compared with WT-infected animals. In contrast, naive animals in contact with sCPD9-AFCS vaccinated subjects did not become positive for SARS-CoV-2 RNA during the first 7 days of cohabitation which was the duration of the observation period (Figure 3A). In this context, Figure 3A shows genomic RNA (gRNA) copies found in mouth swabs taken daily (1 -6 dpc) from contact hamsters in mean ± SD. Two-way ANOVA and Tukey’s multiple comparisons test were performed (* p < 0.05, ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 ). In this and in all Figures, non-f illed circles or squares (first bar from the left) denote sCPD9-AFCS, light grey circles or squares (second bar from the left) denote sCPD9, and dark grey circles or squares (third bar from the left) denote B.1 (WT SARS-CoV-2).

Natural transmission of WT 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). In agreement with previous findings, the inflammatory changes in the lungs were greatly attenuated in animals that contracted sCPD9. In addition, the lungs of animals cohabitating with the sCPD9-AFCS vaccinated animals showed virtually no signs of inflammation. sCPD9-AFCS is highly attenuated in Syrian hamsters

All hamsters remained clinically healthy after infection with sCPD9 or sCPD9-AFCS, whereas WT-infected animals showed the expected mild to moderate signs of disease, such as forced breathing and considerable weight loss during the first week after infection (Figure 4A). In this context, Figure 4A shows body weight loss in percentage after initial vaccination/infection shown per group and up to challenge-infection time point 21 dpi/dpv (dpi = days post infection, dpv = days post vaccination).

However, in the absence of other visible signs of disease, sCPD9-infected animals showed a trend towards slightly decreasing body weights while sCPD9-AFCS-vaccinated animals presented with relatively stable weights in the week following vaccination.

Although all contact animals that were exposed to sCPD9- or WT-infected animals contracted the respective virus, clinical signs of disease and body weight loss occurred only in animals that contracted the WT virus (Figure 4B, illustrating weight development of contact animals in percent per group during the co-housing period). Both Figure 4A and Figure 4B show violin plots (truncated) with quartiles and median. Similarly, severe histological signs of lung inflammation on day 7 post contact, were present only in contact animals that contracted the WT virus (Figure 3B, illustrating the number of gRNA copies recovered from oropharyngeal swabs and homogenized lungs and replication- competent virus detected in lung tissue; the limit of detection is marked by dotted lines). The data depicted in Figures 3B to 3D was statistically evaluated by conducting Kruskal-Wallis test and Dunn’s multiple comparisons test (* p < 0.05, ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 ).

Figure 3C shows the replication-competent virus detected in lung tissue. The limit of detection is again marked by dotted lines.

Figure 3D shows the histopathological scoring including consolidated lung area in percentage, lung inflammation score with severity of pneumonia, influx of neutrophils, lymphocytes and macrophages, bronchial epithelial necrosis, bronchitis, alveolar epithelial necrosis, perivascular lymphocyte cuffs and pneumocyte type II hyperplasia and edema score accounting for perivascular edema and alveolar edema.

Figure 3E shows the neutralizing activity against SARS-CoV-2 B.1 WT of sera taken from contact hamsters at day 6 after contact.

Contact animals that became infected with sCPD9 showed only mild inflammatory changes in the lungs, these further reduced or absent in the lungs of the sCPD9-AFCS-infected contact animals which had not contracted the virus.

Deletion of FCS does not reduce vaccine efficacy

All infected animals were challenged with the pathogenic SARS-CoV-2 Delta variant on day 21 after infection. None of the challenged animals developed clinical signs of disease or exhibited marked body weight loss which was observed in the unvaccinated control animals (Figure 5A, illustrating body weight changes that were controlled until analysis time points 2 + 5 dpch and are shown in percent per group). In this and all other Figures, medium grey squares (fourth bar from the left) denote mock-vaccinated animals.

As expected, protection against replication of the challenge virus was comparable in all three infected groups. On day 2 after challenge, all groups showed high viral RNA loads in the upper respiratory tract (Figure 5B, illustrating quantification of gRNA in oropharyngeal swabs and lung and infectious virus particles detected in homogenized lung tissue). In Figures 5B to 5E, dotted lines mark the limit of detection. In addition, the data presented in Figures 5B to 5E was statistically analyzed by two-way ANOVA and Tukey’s multiple comparisons test (* p < 0.05, ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 ). The observed high viral RNA loads, however, were reduced to levels near the detection limit by day 5 after challenge in all studied groups. Protection in the lower respiratory tract was more pronounced at day 2 after challenge with significantly lower viral RNA loads and minimal levels of replication-competent virus in the lung of infected animals (Figure 5B). By day 5 after challenge, RNA loads were near or below the detection limit, and no replicating virus was present in the lungs of any of the challenged subjects.

Overall, virological parameters confirmed high protective efficacy of sCPD9 and sCPD9-AFCS vaccine candidates and were comparable to those of the WT virus.

Protective efficacy of sCPD9 and sCPD9-AFCS vaccine viruses was determined by examining the lung histopathology of uninfected and sCPD9-, sCPD9-AFCS- and WT-infected hamsters on days 2 and 5 after challenge infection (Figure 5C, illustrating infectious virus particles detected in homogenized lung tissue, and Figure 5D, illustrating the percentage of lung area consolidated by SARS-CoV-2 infection; lung inflammation score accounts for severity of pneumonia, influx of neutrophils, lymphocytes and macrophages, bronchial epithelial necrosis, bronchitis, alveolar epithelial necrosis, perivascular lymphocyte cuffs and pneumocyte type II hyperplasia; pulmonary edema is scored based on the extent of perivascular and alveolar edema). Here, it became evident that WT-infected animals were more prone to develop inflammation and showed a tendency towards increased influx of immune cells compared with animals that were infected with sCPD9 or sCPD9-AFCS viruses or unvaccinated animals. This situation was ameliorated by day 5 after challenge, suggesting that immune system of the WT- infected animals transiently overreacted shortly after challenge, causing pulmonary damage that was observed on day 2 after challenge. Importantly, all animals showed excellent and comparable protection against COVID-19-like pneumonia at all examined time points after challenge.

Finally, the humoral immune responses were compared against SARS-CoV-2 WT and the Delta variant in infected animals before challenge and on days 2 or 5 after challenge (Figure 5E, illustrating neutralizing activity against B.1 and Delta variant of hamster sera taken on day 0 (prior challenge), 2 and 5 after SARS-CoV-2 Delta challenge; the range of detection reached from dilution 1 :8 to 1 :1024). The results showed that vaccination/infection with sCPD9, sCPD9- AFCS or WT viruses induced comparable levels of neutralizing antibodies at all time points tested. sCPD9-AFCS is slightly more attenuated than sCPD9, but equally protective

The results presented here indicate that removal of the FCS from sCPD9 results in a non- transmissible and highly attenuated virus in the Syrian hamster model. Despite the high level of attenuation, the sCPD9-AFCS virus showed equal protection against SARS-CoV-2 variant Delta as the parental sCPD9 virus that has an intact FCS in the spike protein. In terms of virological, serological and histological parameters, both sCPD9 and sCPD9-AFCS vaccine viruses provided excellent and comparable protection against virus replication and disease. Overall, protection conferred by either vaccine virus was comparable to that of the induced by infection with WT virus. Remarkably, WT-infected animals showed stronger signs of inflammation in the lungs during first days after challenge.

Thorough histological examination revealed another remarkable advantage of a vaccination with sCPD9-AFCS or sCPD9 over vaccination with WT virus with respect to subsequent virus challenge infection. A previous WT virus infection and subsequent challenge with SARS-CoV- 2 led to a significant alveolar bronchiolysis in tested animals. In contrast, no alveolar bronchiolysis was observed in animals that were challenged with SARS-CoV-2 after prior vaccination with sCPD9-AFCS or sCPD9.

Benefits of FCS removal for mass production of LAV SARS-CoV-2 vaccines

Many reports have shown that FCS impairs replication of SARS-CoV-2 in TMPRSS2-deficient cells such as Vero or Vero-E6 cells, because SARS-CoV-2 variants lacking a functional FCS become rapidly dominant upon passage of 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., 2021 b; Wong et al., 2021 ). These results suggest that mutant viruses lacking the functional FCS have a strong selection advantage for replication in cells that do not express TMPRSS2. The results presented here have confirmed these observations, as the sCPD9-AFCS mutant replicated to higher titers than the original sCPD9 virus in Vero cells.

Thus, removal of the FCS from SARS-CoV-2-LAV has two advantages for LAV vaccine production. Because most vaccine manufacturers use Vero cells to produce LAV vaccines, increasing viral titers also helps reduce the production costs, but more importantly, it facilitates purification of sufficient numbers of infectious virus particles that are required for a single vaccination.

The second major advantage of removing FCS is that it greatly improves the genetic stability of the LAV vaccine candidate. One of the prerequisites for the use of LAV vaccines in humans is that the vaccine virus population has a high degree of genetic homogeneity. Since SARS- CoV-2 rapidly loses its FCS at the S1/S2 boundary after passage into Vero cells, removal of the FCS eliminates the problem of genetic instability, and the vaccine virus can be produced with high genetic homogeneity.

Co-infection in vivo does not give rise to viral recombinants

To examine potential recombination between vaccine and circulating field virus in vivo, Syrian hamsters were infected with equal quantities of sCPD9-AFCS and the Omicron variant BA.5 (1 x10 ⁴ FFU/animal). After 24 h, infected animals were co-housed with naive contact hamsters to assess host-to-host transmission. Oral swabs were collected daily; all animals were euthanized after 6 days of cohousing. Figure 6A shows a schematic sketch of the experimental design.

Figure 6B shows a change in body weight of animals after co-infection or contact to co-infected animals. Compared to contacts, infected animals showed a wider distribution of body weights with mild, transient body weight loss in some individuals.

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

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 (upper or left panels) and contact hamsters (lower or right panels), using assays targeting the SARS-CoV-2 E gene (envelope) (results represented in Figures 6D to 6G by circles), which is present in both viruses, or the sequences that are uniquely present in two different viruses - the spike gene of Omicron BA.5 virus (results represented in Figures 6D to 6G by squares), or the recoded sCPD9 region of sCPD9-AFCS virus (results represented in Figures 6D to 6G by triangles). Dotted lines in Figures 6C to 6G show the limits of detection.

Low levels of replication-competent virus were detected in lung tissue of 3 contact hamsters on day 6 of cohabitation, as illustrated in Figure 6D.

RT-qPCRs performed on daily oral swab samples of infected hamsters showed that sCPD9- AFCS was only detectable within the first 3 days after infection, while BA.5 was detected during the entire course of the experiment (Figure 6D). In contrast, minor amounts of sCPD9 specific gRNA copies were found in more sensitive oropharyngeal swabs (Figure 6E). No sCPD9 specific gRNA was found in swab and lung samples collected from contact animals, whilst BA.5-specific RNA was detected abundantly in daily swabs from day 2 post contact on as well as in oropharyngeal swabs and lungs (Figures 6D-F). This indicates transmission of BA.5 while sCPD9-AFCS remained non-transmissible under co-infection conditions. A recombination event that would restore the FCS in the vaccine virus, thereby enabling transmission of vaccine virus, is thus not observed in the chosen experimental setup.

In vitro co-culture does not suggest important recombination events

Figure 6G shows the replication of Omicron BA.5 and sCPD9-AFCS in CaLu-3 cells. CaLu-3 cells were infected with 1 ,000 FFLI of sCPD9-AFCS and 100 FFU of BA.5 and after 72 hours of incubation, 1% of the supernatant was used as an inoculum for next virus passage (n=3). The resulting virus population was passaged 10 times serially on CaLu-3 cells to assess recombination events between the vaccine and field virus. RNA was extracted from cell culture supernatant of each passage. A qPCR assay targeting a conserved region within the SARS- CoV-2 E gene was used to assess total SARS-CoV-2 gRNA content, while assays targeting the FCS region in the spike gene of the BA.5 virus and the genetically recoded sCPD9 region were employed to discriminate between the vaccine and field viruses. Consistent with in vivo data, qPCR results showed that the sCPD9-AFCS was outgrown by the BA.5 virus within one passage. The sCPD9-specific RNA levels dropped to levels around the limit of detection by passage 1 , while BA.5 maintained replication, resulting in high levels of gRNA over the entire range of the experiment (Figure 6G). This suggests that sCPD9-AFCS has a considerable growth disadvantage in cell culture which limits the possibility of recombination between the vaccine and field virus in the same replication compartments. Consequently, the risk of recombination between sCPD9-AFCS and wild type viruses is reduced.

To ascertain the absence of high-fitness recombinants, total RNA sequencing of cell culture supernatants from different passages and replicates of co-cultured viruses was performed. The sequencing analysis showed that, from passage one onwards, all sequences above the detection threshold were derived only from the BA.5 virus. No evidence was found for the presence of sCPD9-AFCS derived sequences in any of the performed analyses. While some de-novo mutations appear to have been selected, likely indicating adaptation to cell culture, the emergence of sCPD9-AFCS/BA.5 recombinants that would have a selective advantage over the BA.5 virus in cultured human cells can be excluded in the performed experimental setup (Figure 6H, I). Figure 6H illustrates SNPs identified in passages 1 , 2, 3, 6, 7, 10 of the co-infection experiments and their respective location within the BA.5 reference genome. The panel shows the SNPs identified in the three replicates that contained the most SNPs in each passage, irrespective of the passaging condition. Only SNPs that were identified in >10% sequence reads are depicted. (Figure 6I shows all unique SNPs that emerged during the co-infection experiments (with >10% read support) in comparison with both BA.5 and sCPD9-AFCS genome reference sequences. sCPD9-AFCS is safe and immunogenic also for immunosuppressed hamsters

Figure 7A gives a schematic overview of the experimental setup. Syrian hamsters were immunosuppressed by daily subcutaneous injections of dexamethasone (Dex) at a dose of 2mg/kg, starting 3 days prior to vaccination or contact. After 3 days of treatment, the hamsters were vaccinated with sCPD9-AFCS allowing to determine the effects of immunosuppression on vaccine safety and the humoral immune response to vaccination. Subsequently, the vaccinated hamsters were cohoused with naive and immunosuppressed contact animals 24 h after vaccination. Oral swabs were collected daily from all hamsters. Contacts were euthanized 6 days post contact (dpc), whilst vaccinated hamsters were euthanized 21 days post vaccination (dpv).

Figure 7B illustrates the change in body weight of immunosuppressed animals after vaccination or contact with vaccinated hamsters. Upon vaccination, immunosuppressed hamsters presented with stable body weights and absence of clinical illness (Figure 7B). In Figures 7B to 7J, triangles denote immunosuppressed contact hamsters, wherein squares denote immunosuppressed hamsters vaccinated with sCPD9-AFCS.

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

Figure 7C shows the neutralizing activity of sera collected from sCPD9 -A FCS- vaccinated immunocompetent animals (IC; illustrated by circles) and sCPD9-AFCS-vaccinated immunosuppressed animals 21 days post vaccination (dpv) against SARS-CoV-2 variant B.1 (upper detection limit = 1 :1 ,024). The significance was tested with Mann-Whitney test (p < 0.05). These serum neutralization assays made it possible to determine humoral immune response in immunosuppressed animals 21 days after receiving a single dose of sCPD9- AFCS. By trend, serum neutralization titers are lower in immunosuppressed animals compared to immunocompetent animals that received the same vaccination. Nevertheless, despite immunosuppression, all hamsters developed a substantial humoral response to vaccination (Figure 7F). Moreover, lung histopathology was assessed and revealed no evidence of pneumonia in immunosuppressed hamsters after vaccination with sCPD9-AFCS (Figure 7G- J). Overall, sCPD9-AFCS remains safe and immunogenic in animals receiving high-dose glucocorticoid treatment.

The dotted lines in Figures 7C to 7F show the limits of detection. sCPD9-AFCS is not transmissible between immunosuppressed hamsters

Next, it was determined whether the sCPD9-AFCS virus was transmissible between immunosuppressed animals. To test this, six immunosuppressed and sCPD9-AFCS- vaccinated animals described above were brought into contact with six dexamethasone- treated and immunologically naive hamsters and cohabited for six consecutive days (Figure 7A). Throughout the entire cohabitation period, no clinical signs of disease or significant body weight loss were observed (Figure 7B). Although vaccine virus RNA was detected in oral swabs from vaccinated individuals (Figure 7C), no transmission of vaccine virus was observed, as evidenced by the absence of detectable virus RNA in both upper and lower respiratory tracts of the immunosuppressed contact animals (Figure 7D, E). sCPD9-AFCS is safe in immunosuppressed animals and shows no recombination with a circulating SARS-CoV-2 variant

The infection experiments in dexamethasone-treated animals indicate that sCPD9-AFCS is also safe for individuals undergoing glucocorticoid treatment. Glucocorticoids are well known for their immunosuppressive effects and are frequently used to treat airway inflammation, making this a specifically relevant immunosuppressive treatment in the context of COVID-19. It was previously demonstrated that dexamethasone treatment exerts strong immunosuppressive effects and increases SARS-CoV-2 replication in Syrian hamsters (Wyler, 2022). Despite this, it was found that replication of vaccine virus sCPD9-AFCS remained moderate under dexamethasone treatment, with RNA levels decreasing towards the limit of detection within a week following vaccination. Dexamethasone treated hamsters neither show clinical signs of disease nor any apparent pathology. Moreover, the vaccine virus remained non-transmissible and induced a considerable humoral immune response in dexamethasone- treated animals. These results present an indication of safety and efficacy of sCPD9-AFCS in immunosuppressed patients, in particular in individuals receiving glucocorticoid treatment. According to preliminary evaluation of initial data, such effects of humoral immune response, safety and efficacy in immunosuppressed patients cannot be observed to the same extent upon vaccination with sCPD9 still comprising the FCS.

Both the in vitro and in vivo co-infection experiments demonstrated that LAV virus sCPD9- AFCS is rapidly and consistently outcompeted by the Omicron BA.5 field isolate, indicating a strong selective disadvantage of the attenuated vaccine virus. This limits the replication of vaccine and field viruses in the same host or host compartment, which in turn, limits the potential for recombination events between the two viruses. While recombination events between the two viruses cannot be formally exclude in the chosen experimental setup, the sequence analysis made it possible to confirm that no recombinant has gained a selective advantage over the BA.5 variant in the co-infection experiments. Furthermore, the in vivo coinfection experiments indicate that co-infection with vaccine and field virus does not result in increased virulence, nor does it yield a transmissible vaccine virus.

Additional experiments illustrating the superiority of sCPD9-AFCS

In the following, additional experiments illustrating the superiority of sCPD9-AFCS will be explained in more detail.

Material and Methods

Study design

The aim of this study was to compare the protection against SARS-CoV-2 transmission provided by two different types of vaccines. The extent of SARS-CoV-2 transmission from infected shedder hamsters to vaccinated animals and from vaccinated and subsequently infected shedders to naive contact hamsters was investigated in two different trials. For this purpose, hamsters were randomly assigned into groups of 12 animals. The experimental design of both studies included three groups following a prime-boost schedule in which the hamsters were vaccinated on day 0 and day 21 respectively. The three groups received either two doses of sCPD9-AFCS (10 ⁴ FFU) intranasally, 5 pg of the mRNA vaccine BNT162b2 (Comirnaty®, Pfizer-BioNTech) intramuscularly or two doses of mock vaccination.

In the first trial, vaccinated hamsters were co-housed with infected shedder animals two weeks after receiving the booster vaccination. Twenty-four hours prior to co-habitation, the shedder hamsters were infected with 10 ⁵ FFU of either SARS-CoV-2 variant B.1 or the Omicron subvariant BA.5 by intranasal instillation. For the second experiment, previously prime-boost vaccinated animals were infected with either SARS-CoV-2 variant B.1 or Omicron subvariant BA.5 two weeks after the second vaccination. Subsequently, latter were co-housed with naive contact hamsters 24 h after infection. In both trials, hamsters were co-habituated for 6 days. In this period, oral mucosal swabs were taken daily to determine viral loads in the upper airways of shedder and contact hamsters. Additionally, body weights and clinical conditions were assessed. On day 6 after contact, all hamsters were euthanized to collect blood, nasal washes, lungs and skulls for virolog ical, serological and histopathological examinations.

Cells

Vero E6 (ATCC CRL-1586) and Vero E6-TMPRSS2 (NIBSC 100978) cells were cultivated in minimal essential medium (MEM) containing 10% fetal bovine serum (PAN Biotech), 100 lll/ml penicillin G and 100 mg/ml streptomycin (Carl Roth). To ensure selection of TMPRSS2- expressing cells, the medium for Vero E6-TMPRSS2 cells contained additional 1000 pg/ml geneticin (G418). CaLu-3 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) with 20% fetal bovine serum (PAN Biotech), 1% non-essential amino acids, 100 lll/ml penicillin G and 100 mg/ml streptomycin (Carl Roth). All cells were cultivated at 37 °C and 5% CO ₂ .

Viruses

The ancestral SARS-CoV-2 variant B.1 (B.1 , hCoV-19/Germany/BY-ChVir-929/2020, EPI ISL 406862) propagated on Vero E6 cells and the Omicron subvariant BA.5 (BE.1.1 , hCoV-19/Germany/SH-ChVir29057_V34/2022, EPIJSL 6221625) grown on CaLu-3 cells, were used to infect the shedder hamsters. Additionally, Delta variant B.1.617.2 (B.1.617.2, Human, 2021 , Germany ex India, 20A/452R, EVAg: 009V-04187) propagated on Vero E6- TMPRSS2 cells and Omicron subvariant BA.1 (BA.1.18, hCoV-19/Germany/BE- ChVir26335/2021 , EPI ISL 7019047) grown on CaLu-3 cells were used to conduct serum neutralization assays. Plaque assays were performed on Vero E6 cells to determine the titer of all virus stocks prior to infection experiment. Vials were stored at -80°C.

Ethics statement

Animal works were performed in compliance with all applicable national and international regulations and approved by the regulatory state authority, Landesamt fur Gesundheit und Soziales in Berlin, Germany (permit number 0086/20). All in vitro and animal experiments were conducted in the certified BSL-3 laboratory at the Institut fur Virologie, Freie Universitat Berlin, Berlin, Germany.

Animal husbandry Syrian hamsters (Mesocricetus auratus; breed RjHamAURA) were purchased from Janvier Labs at 5 - 7 weeks of age. They were kept in groups of 2 to 3 animals in individually ventilated cages (IVCs; Tecniplast) equipped with nesting material. Prior to vaccination, the animals were allowed to habituate to the housing conditions for seven days. The hamsters had free access to water and food at all times. During all experiments, cage temperature and relative humidity were monitored and ranged between 22 to 24 °C and 40 and 55%.

Vaccine preparation and Vaccination

The live-attenuated vaccine candidate sCPD9-AFCS was propagated on Vero E6-TMPRSS2 cells. Titers were determined by plaque assays conducted on Vero E6 cells. Prior to vaccination, the stock was adjusted to a final titer of 2 x 10 ⁵ FFU/ml. Intranasal vaccination with 10 ⁴ FFU per animal was performed under general anesthesia (0.15 mg/kg medetomidine, 2.0 mg/kg midazolam and 2.5 mg/kg butorphanol).

BNT162b2 (Comirnaty®) was prepared according to the manufacturer’s instructions. The final concentration of mRNA was diluted to 50 pg/ml instead of 100 pg/ml as recommended for use in humans. The dilution was prepared with 0.9% NaCI sterile water immediately prior to vaccination and applied intramuscularly at a dose of 5 pg per hamster.

Animals assigned to the mock group received minimal essential medium (MEM) intranasally under general anesthesia.

Nasal washes

To obtain nasal washes of all animals, the skulls paramedian of the nasal septum were punctured with a cannula. Subsequently, a pipette tip was inserted and 200 pl of PBS were applied. The wash fluid was picked up on the nostrils and the washing procedure was repeated twice. Per animal approximately 150 pl of sample were collected.

RNA isolation and RT-qPCR

In preparation for RNA extraction, 25 mg lung tissue were 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) as recommended by the manufacturer. To detect SARS-CoV-2 RNA, reverse transcription quantitative PCR (RT-qPCR) was performed on a qTower G3 cycler (Analytik Jena) using the NEB Luna Universal Probe One-Step RT- qPCR Kit (New England Biolabs) under the following cycling conditions: 10 min at 55 °C for reverse transcription, 3 min at 94 °C for activation of the enzyme and 40 cycles of 15 s at 94 °C and 30 s at 58 °C (Corman et al., 2020). Plaque assay and indirect immunofluorescence staining

Replicating virus was determined in 50mg lung tissue. For quantification, lung samples were homogenized in a bead mill (Analytik Jena), serially diluted in MEM and plated on 12-well plates containing confluent Vero E6 cells. After 2.5 hours at 37 °C and 5% CO2, the inoculum was removed, and cells were overlaid with 2X Eagle’s Minimum Essential Medium (EMEM; Lonza™ BioWhittaker™) medium containing 1.5% microcrystalline cellulose and carboxymethyl cellulose sodium (Vivapur 61 1 p; JRS Pharma). Seventy-two hours after infection the plates were fixed with 4% PBS-buffered formaldehyde.

To conduct indirect immunofluorescence staining, the cells were permeabilized with 0.1% Triton X-100 and blocked for 30 minutes with 3% BSA diluted in PBS. After washing the plates with PBS, the primary polyclonal anti-SARS Coronavirus nucleocapsid antibody (Invitrogen) was added for 1 h followed by the goat anti-rabbit IgG-AlexaFluor 488 secondary antibody (Invitrogen) for 45 min. To determine the titers, the plaques were counted using an inverted fluorescence microscope (Axiovert S100, Zeiss).

Serum neutralization assay

Neutralizing activity against the SARS-CoV-2 variant B.1 and the Omicron subvariant BA.5 was determined in all hamster sera (0 days post challenge, 6 dpc. In addition, neutralizing capacity against the Delta variant and the Omicron subvariant BA.1 was tested in day 6 serum samples. Twofold 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 FFLI of SARS-CoV-2 diluted in MEM were applied per well and incubated for 1 h at 37 °C. Subsequently, the dilutions were plated on Vero E6 cells cultivated in 96-well plates and incubated for 72 h (B.1 , Delta) or 96 h (Omicron BA.1 , BA.5) at 37 °C. Thereafter, cells were fixed with PBS-buffered formaldehyde (4%, pH 6.5) and stained with methylene blue (0.75% aqueous solution). Neutralization was considered effective in wells that showed no cytopathic effect. The last neutralized well was reported as the titer. Positive and negative controls were included in all plates. To plot the results, samples without neutralizing activity were set to a titer of 1 :4.

Enzyme-linked immunosorbent assay (ELISA)

SARS-CoV-2-specific IgG levels against the spike protein of the B.1 and BA.5 variants, as well as the nucleocapsid and ORF3a proteins were measured in hamster sera using an in-house ELISA. Clear 96-well plates with flat bottom (MEDISORP, Thermo Fisher Scientific, catalog number: MW96F) were coated with 5 pl of purified, recombinant, His-tagged SARS-CoV-2 antigens: the spike protein (D614G) of the B.1 variant (Aero Biosystems, catalog number: SPN- C52H3), spike protein of the BA.5.5 variant (GenBank accession: QHD43416, Aero Biosystems, catalog number: SPN-C522p), nucleocapsid protein (GenBank accession: QHD43423, Ray Biotech, catalog number: 230-01104), and ORF3a protein (Thermo Fisher Scientific, catalog number: RP-87667). The antigens were diluted in 1 x PBS to a final concentration of 20 pg/ml. Additionally, each well was supplemented with 45 pl of coating buffer (50 mM Na2COs, 50 mM NaHCOs, pH 9.6). After incubating the plates for 12 h at 4 TD, the plates were washed four times with a washing buffer (0.05% Tween 20 in 1 x PBS) and blocked with a blocking buffer (1 x PBS, 1% BSA, 10% FCS) for 1 h. The serum samples were diluted 1 :100 in a dilution buffer (1 x PBS, 2% BSA, 0.1 % Tween 20) and plated in duplicates of 50 pl per well. The plates were then incubated for 2 h at RT, followed by another washing step. Next, 50 pl of a secondary, horseradish peroxidase (HRP)-conjugated polyclonal goat anti-hamster IgG (H+L) antibody (Thermo Fisher Scientific, catalog number: 10537453), which was diluted 1 :1000 in 1 x PBS, was added to each well. After a 1 -h incubation at RT, the plates were washed again, and 50 pl of the chromogenic substrate, 3,3’,5,5’-Tetramethylbenzidine (TMB; TCI chemicals, catalog number: T3854) was added to each well. The reaction was stopped with 1 M H2SO4 after 15 min. The optical density was measured at 450 nm and 570 nm using a SpectraMax Plus 384 plate reader (Molecular Devices).

Moreover, SARS-CoV-2-specific IgA levels against the spike protein of the B.1 and BA.5 variants were measured in nasal washes following the protocol described above with minor adaptions. Nasal washes were diluted 1 :50 in a dilution buffer and plated in duplicates. For IgA detection, a polyclonal HRP-conjugated rabbit anti-hamster IgA (Brookwood Biomedical, catalog number: sab3003a) was diluted 1 :750 and used as a secondary antibody. After 1 h of incubation at RT, plates were washed, and 50 pl of 1-Step™ Ultra TMB ELISA substrate solution (Thermo Fisher Scientific, catalog number: 34028) was added into each well. The reaction was stopped after 20 minutes of incubation at room temperature.

Histopathology and immunohistochemistry

For histopathological analysis, left lung lobes and skinned skulls were fixed in PBS-buffered formaldehyde solution (4%) for 48 h. Skulls were rinsed under tap water for 30 min and decalcified in buffered EDTA solution (pH=7.0) for three days at 65 °C. Skulls were trimmed to obtain rostral sections at the tip of the first triangular ruga of the hard palate and sections from further caudal at the level of the first molar teeth. Sections of 2 pm thickness were cut from routinely formalin-fixed, paraffin-embedded samples, stained with hematoxylin and eosin or prepared for immunohistochemistry. Histopathological analyses of lung sections were carried out as described (Osterrieder et al., 2020). Nose sections were scored for the presence of lymphocytes, granulocytes, necrosis, epithelial flattening, and loss of cilia as 0 = less than 5% of the epithelium affected, 1 = 5 to 40% of the epithelium affected, 2 = 41 to 80% of the epithelium affected, or 3 = more than 80% of the epithelium affected. Additionally, the airway exudate was characterized. For immunohistochemical analyses, nasal sections were dewaxed in xylene and rehydrated in descending grades of ethanol. Endogenous peroxidase was blocked using H2O2. Antigen retrieval was achieved by microwaving sections at 600 W in 750 ml buffered citric acid with 1 % Triton X 100 (Roth) for 12 min. The primary monoclonal mouse anti SARS-CoV-2 nucleocapsid protein antibody (Sino Biological, dilution: 1 :500) was incubated overnight at 4 °C. For universal negative controls, sections were incubated with irrelevant purified mouse IgG (BioGenex) instead of anti-SARS-CoV-2 antibody. Nonspecific binding was blocked with 20% goat serum for 30 min. After washing with PBS/Triton buffer, the secondary antibody, goat-anti-mouse IgG (Vector Laboratories, diluted at 1 :200), was applied and incubated for 30 min. The signal was developed with diaminobenzidine tetrahydrochloride (Merck) following 8 min of signal enhancement with Vectastain Elite ABC Kit (Vector Laboratories). Hematoxylin was used as counterstain. For histopathological evaluation, an Olympus BX41 microscope with a DP80 Microscope Digital Camera (Olympus) and cellSensTM Imaging Software, Version 1.18 (Olympus Soft Imaging Solutions) was used. Automatic digitization was facilitated using an Aperio CS2 slide scanner (Leica Biosystems). Microphotographs were generated with image Scope Software (Leica Biosystems). Adobe Photoshop or GIMP Software was used to generate figure panels.

LAV vaccine prevent the development of clinical symptoms following natural transmission of SARS-CoV-2 B.1 and BA.5

Syrian hamsters received two doses of sCPD9-AFCS, BNT162b2 or a mock vaccine at an interval of 3 weeks (on day 0 and day 21 ). After another fourteen days (on day 35 after vaccination), the hamsters were brought in contact with shedder animals, which had been infected with either the ancestral virus variant B.1 , or the Omicron BA.5 variant one day prior. The animals were cohoused for 6 days and closely monitored for disease symptoms and virus loads in the upper airways.

Figures 8A and 8B show the body weight loss in percentage of B.1 shedder and contact animals (Figure 8A), as well as BA.5 shedder and contacts animals (Figure 8B). Violin plots (truncated) show weights of vaccinated contacts (n=6), group medians and quartiles. Weights of shedders (n=3) are displayed as median. All three groups of shedder animals infected with the ancestral SARS-CoV-2 variant B.1 showed moderate weight loss and clinical symptoms typical of COVID-19-like pneumonia (Figure 8A). As expected, shedder animals infected with the BA.5 variant (Wolter et al. 2022, Uraki et al. 2022) showed less pronounced weight loss, with some variability among the infected groups (Figure 8B). The sCPD9-AFCS and mRNA vaccines effectively prevented body weight loss in vaccinated contact animals exposed to both B.1 or BA.5 shedders (Figures 8A and 8B). As anticipated, mock-vaccinated animals exposed to B.1 shedders exhibited a progressive decline in body weight starting from day 3 after contact (Figure 8A). Meanwhile, mock-vaccinated animals that were exposed to BA.5 shedders did not experience weight loss, likely due to the lower pathogenicity of the BA.5 variant and the consequently milder disease course observed in infected animals (Figure 8B).

Only LAV prevents transmission of SARS-CoV-2 B.1 and BA.5

To monitor virus replication and transmission, oral swabs were collected daily during cohousing of infected and contact animals. Furthermore, oral swabs and lungs were obtained on day 6 post contact (dpc) to quantify viral RNA levels and replicating virus. Viral gRNA copies in oropharyngeal swabs and lung tissue, and replicating virus quantified as focus forming units (FFU) of B.1 infected (Figure 8E) and BA.5 infected (Figure 8F) shedders and their respective vaccinated contacts. In Figures 8C to 8F, the results of vaccinated contact animals (n=6) are displayed as median with range with symbols indicating individual values. For shedder hamsters (n=3), medians are shown. Parametric statistics on log transformed data was done. In Figures 8C and 8D, ordinary two-way ANOVA with Tukey's multiple comparisons test was performed. In Figures 8E and 8F, ordinary one-way ANOVA with T ukey's multiple comparisons test was conducted. * p < 0.05, ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 .

Infected shedder animals exhibited high levels of SARS-CoV-2 RNA with a gradual decrease towards day 6. In general, virus loads were higher in B.1 shedders compared to BA.5 shedders (Figures 8C and 8D).

Consistent with the observed body weight loss, oral swabs of mock-vaccinated contacts exposed to B.1 shedders had high levels of SARS-CoV-2 RNA from 2 dpc onward (Figure 8C). Although mRNA vaccination prevented body weight loss, it did not protect against B.1 infection, as evidenced by persistently high SARS-CoV-2 RNA levels in oral swabs after day 2. Nevertheless, mRNA vaccination resulted in lower viral RNA levels compared to mock vaccination. In contrast, sCPD9-AFCS-vaccinated contacts displayed minimal SARS-CoV-2 RNA levels, oscillating around the detection limit, suggesting repeated exposure to the virus without signs of productive infection in the upper airways (Figure 8C).

Similar results were obtained in vaccinated hamsters exposed to BA.5-infected shedders.

SARS-CoV-2 RNA levels in oral swabs of mRNA- and mock-vaccinated animals peaked on dpc 4 to 5 (Figure 8D), while virus RNA levels in sCPD9-AFCS-vaccinated hamsters were near the detection limit, suggesting effective prevention of BA.5 infection (Figure 8D).

Consistent with these findings, both mRNA- and mock-vaccinated hamsters exposed to B.1 or BA.5 shedders showed high SARS-CoV-2 RNA levels in oral swabs and lungs on 6 dpc (Figures 8E and 8F). Additionally, replication-competent virus was detected in lung tissue of mock-vaccinated animals exposed to B.1 shedders, while most shedders had cleared the infection by that time (Figure 8E). Despite slightly lower viral RNA levels, replicating virus was found in the lungs of two mRNA-vaccinated and three mock-vaccinated hamsters exposed to BA.5 shedders, reflecting the delayed peaking of virus replication compared to B.1 infection (Figure 8F).

Pulmonary lesions were largely absent in sCPD9-AFCS-vaccinated animals and less reduced in mRNA-vaccinated animals exposed to B.1 shedders. Hamsters experimentally infected with B.1 virus or animals that contracted B.1 after mock vaccination developed lesions typical of COVID-19-like pneumonia. Specifically, the infected animals had pronounced patchy bronchointerstitial pneumonia with necrosuppurative bronchitis and bronchiolitis, proliferation of alveolar type II epithelia, vascular endotheliitis, diffuse alveolar damage, as well as perivascular and alveolar edema. Histopathological analysis confirmed significant reduction of tissue alteration, immune cell infiltration, and edema in sCPD9-AFCS- and mRNA-vaccinated animals. Inflammatory damage was milder in all animals infected with Omicron BA.5, and most pronounced in experimentally infected shedder hamsters. However, mock-vaccinated contacts still developed mild pneumonia. Protection was slightly less effective in mRNA vaccinated hamsters. In contrast, sCPD9-AFCS-vaccinated animals exposed to B.1 or BA.5 shedders failed to develop substantial evidence of pneumonia, confirming the highly effective protection provided by this vaccine.

Strong humoral immune response accompanies the protective efficacy of the LAV

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

The results are shown in Figures 9A to 9D. In this context, Figure 9A shows the neutralizing capacity against SARS-CoV-2 variants B.1 , BA.5, Delta and BA.1 of hamster sera taken from vaccinated animals in contact with either B.1 or BA.5 shedders (upper limit of detection = 1 :1 ,024, lower limit of detection is indicated by dotted lines). The results are shown in mean ± SEM with symbol representing individual values. Figure 9B illustrates the SARS-specific IgG levels against B.1 spike, BA.5 spike, nucleocapsid and ORF3a in serum collected from vaccinated contacts on day 6 after contact (dpc). Figure 9C shows SARS-specific IgA levels against B.1 and BA.5 spike in nasal washes obtained from vaccinated contacts 6 dpc. The finding illustrated in Figures 9B and 9C are displayed as optical density (OD) read at 450 nm. Box plots show 25th to 75th percentiles with center lines indicating medians and whiskers from minimum to maximum. Symbols represent individual values.

The results of Figures 9A to 9C were statistically evaluated by a Kruskal-Wallis test with Dunn's multiple comparisons test. * p < 0.05, ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 . The results of Figure 9D ware obtained by a semi-quantitative scoring of SARS-CoV-2 N protein immunohistochemistry (IHC) signal in nasal epithelium of vaccinated contacts. Scoring of inflammatory changes in nasal epithelium including influx of lymphocytes, neutrophils, olfactory and respiratory epithelial necrosis, apoptosis, loss of cilia and flattened epithelial cells displayed as median with range. The symbols indicate individual values. Ordinary one-way ANOVA with Tukey's multiple comparisons test was performed. * p < 0.05, ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 .

As expected, vaccination with sCPD9-AFCS elicited robust production of neutralizing antibodies, with comparable levels in groups exposed to different challenge viruses (Figure 9A). This finding, in conjunction with the virological results, suggests that mucosal immunity induced by sCPD9-AFCS vaccination prevented replication of naturally transmitted virus. However, transient exposure to virus antigens resulted in a minor increase in neutralizing activity. In contrast, mock-vaccinated animals displayed increased serum neutralization capacity against the specific challenge virus. A similar trend was observed in mRNA- vaccinated animals, although the differences were less pronounced (Figure 9A). As expected, specific serum-neutralization capacity was similar in all shedder animals and was determined by the challenge virus.

ELISAs revealed comparable levels of anti-B.1 spike and anti-BA.5 spike IgG antibodies in sCPD9-AFCS- and mRNA-vaccinated hamsters (Figure 9B). As anticipated, nucleocapsid- and ORF3a-specific antibodies were solely present in sCPD9-AFCS-vaccinated animals, highlighting the broad immunity provided by LAVs (Figure 9B). Not surprisingly, IgG levels in shedder animals were relatively uniform and strongly influenced by the challenge virus.

Mucosal immunity was investigated by measuring SARS-CoV-2-specific IgA levels in nasal washes obtained on 6 dpc (Figure 9C). Irrespective of the challenge virus, sCPD9-AFCS- vaccinated animals showed similar levels of anti-B.1 spike and anti-BA.5 spike IgA antibodies (Figure 9C). In contrast, only mRNA-vaccinated hamsters that were exposed to B.1 shedders produced appreciable IgA levels. mRNA-vaccinated animals exposed to BA.5 shedders and both mock-vaccinated groups lacked measurable mucosal IgA response. The absence of IgA in nasal washes of mRNA-vaccinated hamsters that were in contact with BA.5 shedders confirms that mRNA vaccination confers only limited mucosal immunity before virus exposure. However, exposure to the homologous B.1 variant caused significant induction of mucosal IgA antibodies (Figure 9C). Shedder animals exhibited low IgA levels, which corresponded to the virus used for infection.

Vaccination with LAV prevents mucosal infection with SARS-CoV-2 B.1 and BA.5

To further assess the protection in the upper airways induced by vaccination, nasal epithelium at 6 dpc was evaluated for the presence of SARS-CoV-2 nucleocapsid and histological signs of infection and inflammation. While SARS-CoV-2-positive cells were absent in nasal respiratory and olfactory epithelium of sCPD9-AFCS-vaccinated contacts, antigen was detected in abundance in mRNA- and mock-vaccinated animals (Figure 9D).

In line with the immunohistochemistry results, influx of immune cells and inflammatory damage were observed exclusively in the olfactory and respiratory epithelium of mRNA- and mock- vaccinated animals, with reduced inflammation in mRNA-vaccinated hamsters. Notably, inflammatory damage was less pronounced in animals exposed to BA.5 shedders (Figure 9D). Consistent with the expected rapid clearance of SARS-CoV-2 (Sia et al., 2020), shedder hamsters exhibited only few infected epithelial cells and mild signs of inflammation on 7 days post infection (dpi).

Vaccination with LAV blocks transmission of SARS-CoV-2 B.1 to naive contacts

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

The results of these experiments are shown in Figures 10A to 10C (with respect to an infection with SARS-CoV-2 B.1 ) and in Figures 11 A to 11 C (with respect to an infection with SARS- CoV-2 Omicron BA.5). Figure 10A shows body weight loss in percentage from vaccinated and B.1 -infected shedders and naive contact animals. Violin plots (truncated) represent weights of naive contacts (n=6) in group medians and quartiles. Weights of shedders (n=3) are shown as median. Figure 10B illustrates viral gRNA copies in oral swabs from vaccinated and B.1 -infected shedder hamsters and naive contacts. Ordinary two-way ANOVA with Tukey's multiple comparisons test was performed. Figure 10C shows viral gRNA copies in oropharyngeal swabs and 2.5 mg homogenized lung tissue collected at termination. Replicating virus in homogenized lung tissue was quantified as focus forming units (FFU). The results in Figures 10B and 10C of naive contact animals (n=6) are shown in median with range. Symbols represent individual values. The results of vaccinated and superinfected hamsters are displayed as median. In Figures 10 B and 10C, parametric statistics on log transformed data. Dotted line shows limit of detection. Additionally, an ordinary one-way ANOVA with T ukey's multiple comparisons test was done in Figure 10C. * p < 0.05, ** p < 0.01 , “ p < 0.001 , and **** p < 0.0001 .

Figure 1 1 A shows body weight loss in percent of vaccinated and BA.5-infected shedder animals and naive contacts. Violin plots (truncated) represent weights of naive contacts (n=6) in group medians and quartiles. Weights of shedders (n=3) are shown as median. Figure 11 B illustrates viral gRNA copies in daily oral swabs of vaccinated and challenge-infected BA.5 shedders and naive contact hamsters. Ordinary two-way ANOVA with Tukey's multiple comparisons test was performed. Figure 1 1 C shows gRNA copy numbers detected in oropharyngeal swabs and 2.5 mg lung tissue obtained at termination. Replication-competent virus in 50 mg homogenized lung was quantified as focus forming units (FFU). The results in Figures 1 1 B and 1 1 C of naive contact animals (n=6) are displayed as median with range with symbols representing individual values. For findings in vaccinated and challenge-infected shedders (n=3), the median value is shown. In Figures 11 B and 11 C parametric statistics on log transformed data was done. Dotted line represents limit of detection. Additionally, ordinary one-way ANOVA with Tukey's multiple comparisons test was done in Figure 1 1 C. * p < 0.05,

** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 .

Both vaccines efficiently prevented body weights loss, whereas mock-vaccinated animals exhibited decreasing body weights upon B.1 -infection (Figure 10A). Virological results confirmed the strong protective efficacy of the sCPD9-AFCS vaccine. Naive contacts cohoused with sCPD9-AFCS-vaccinated and B.1 -infected shedders maintained stable body weights. In contrast, contacts of mock- or mRNA-vaccinated and SARS-CoV-2-infected shedders experienced weight loss starting from 2 and 4 dpc, respectively (Figure 10A), and their oral swabs exhibited high levels of SARS-CoV-2 RNA. However, contacts of mRNA- vaccinated shedders showed slightly delayed virus replication kinetics compared to contacts of mock-vaccinated animals (Figure 10B). The presence of high RNA levels in lungs, and the detection of replication-competent virus further confirmed that all naive animals in contact with mRNA- or mock- vaccinated and B.1 -infected hamsters contracted the infection (Figure 10C). In contrast, contacts of sCPD9-AFCS-vaccinated and B.1 -infected animals remained negative for SARS-CoV-2 RNA in the upper and lower airways, with no replicating virus detected in the lower airways. These findings strongly indicate that vaccination with sCPD9-AFCS confers highly effective protection against onward transmission of the B.1 virus to naive contacts. (Figures 10B and 10C).

Consistent with these observations, hamsters that contracted B.1 infection from vaccinated and challenged animals showed typical signs of COVID-19 pneumonia, including necrosuppurative bronchitis and bronchiolitis, proliferation of alveolar type II epithelia, vascular endotheliitis, diffuse alveolar damage as well as perivascular and alveolar edema. However, the influx of immune cells and the edema were reduced in hamsters cohoused with mRNA- vaccinated animals compared to mock-vaccinated counterparts. Importantly, none of the hamsters in contact with sCPD9-AFCS-vaccinated and B.1 -infected animals showed signs of pneumonia.

LAV provides superior protection against Omicron BA.5 infection and onward transmission

Owing to its attenuation for Syrian hamsters, no significant weight loss was observed in hamsters infected with Omicron BA.5 (Figure 1 1 A). Animals vaccinated with sCPD9-AFCS and superinfected with Omicron BA.5 cleared the infection within 48 hours, while mRNA vaccination only marginally reduced the viral load in upper and lower respiratory tract compared to mock vaccination. Importantly, SARS-CoV-2 RNA was detected in oral swabs of mRNA-vaccinated hamsters until 5 dpc (Figures 11 B and 11 C). Histopathological examination of vaccinated- and B.5-infected hamsters confirmed the efficacy of both vaccines, although the histopathological changes observed in BA.5-infected animals were generally more subtle compared to changes observed in B.1 -infected animals, suggesting an overall milder pathology with this variant compared to the primordial virus.

No weight loss was observed in naive groups in contact with vaccinated and BA.5-infected hamsters (Figure 11 A). However, mRNA vaccination failed to prevent onward transmission of the BA.5 virus. Starting from 3 dpc, all animals in contact with mRNA- or mock-vaccinated shedders had comparable SARS-CoV-2 RNA loads (Figure 11 B). Additionally, high SARS- CoV-2 RNA levels were also found in swab and lung samples collected from these animals at termination (Figure 1 1 C). In contrast, sCPD9-AFCS vaccination greatly reduced transmission to naive contacts, with only one animal contracting the infection around 2 dpc. A second naive animal in the same cage tested positive at 6 dpc, indicating secondary transmission (Figure

I I B). Both hamsters tested positive for SARS-CoV-2 RNA in oral swabs and lungs at termination, but only one animal had replicating virus at termination. Meanwhile, replicating virus was present in three contacts of both mRNA- and mock-vaccinated shedders (Figure

I I C).

Histopathological findings were less pronounced in contacts exposed to vaccinated and BA.5- infected hamsters. Contacts of mock- or mRNA-vaccinated hamsters showed mild to moderate pneumonia with an increased influx of immune cells, but no lung consolidation. Naive contacts of sCPD9-AFCS-vaccinated animals showed either no signs or mild lung inflammation, reflecting their infectious status.

Humoral and mucosal immunity induced by LAV reduced onward transmission of SARS-CoV-2

The results of transmission experiments are shown in Figures 12A to 12C. Figure 12A shows the concentration of neutralizing antibodies against SARS-CoV-2 variants B.1 , BA.5, Delta and BA.1 in sera from naive hamsters in contact with vaccinated and B.1 or BA.5 infected shedders. Lower limit of detection is indicated by dotted lines, upper detection limit = 1 :1 ,024. Result are displayed in mean ± SEM. Figure 12 B illustrates SARS-specific IgG levels against B.1 spike, BA.5 spike, nucleocapsid and ORF3a in sera from naive contacts collected at 6 dpc. Figure 12 C shows SARS-specific IgA levels against B.1 spike and BA.5 spike in nasal washes obtained at termination. The findings illustrated in Figures 12B and 12C are displayed as optical density (OD) read at 450 nm. Box plots show 25th to 75th percentiles with centerlines indicating medians, whiskers from minimum to maximum and individual values indicated by symbols. A Kruskal-Wallis test with Dunn's multiple comparisons test was performed on the data illustrated in Figures 12A to 12C. * p < 0.05, ** p < 0.01 , *** p < 0.001 , and ““ p < 0.0001 .

Naive animals in contact with sCPD9-AFCS-vaccinated and infected shedders showed no seroconversion, while contacts of mRNA- or mock-vaccinated and superinfected shedders exhibited seroconversion dependent on the challenge virus (Figures 12A and 12B). As expected, sCPD9-AFCS-vaccinated animals exhibited broad and strong humoral immune responses prior to infection, while mRNA-vaccinated hamsters directed their humoral response solely against the B.1 spike protein. Neutralizing antibody titers against BA.5 were only detected in animals that were vaccinated with sCPD9-AFCS. Superinfection boosted antibody response in all groups. In agreement with virological results, only one sCPD9-AFCS-vaccinated contact showed seroconversion. No antibodies against SARS-CoV-2 B.1 , Delta and BA.1 were detected in any of the serum samples obtained from contact animals of BA.5 shedders. Moreover, antibodies targeted against B.1 S, N, and ORF3a proteins were not detectable by ELISA (Figure 12B). Overall, only sCPD9-AFCS vaccination induced broad humoral immunity and effectively reduced BA.5 transmission to naive contacts.

Nasal washes of sCPD9-AFCS-vaccinated and infected shedders showed high IgA levels against B.1 and Omicron BA.5 spikes, irrespective of the challenge virus. Meanwhile, mRNA- and mock-vaccinated and superinfected hamsters had low or no IgA levels, confirming the superior mucosal immunity provided by intranasal vaccination. Naive contacts had no IgA antibodies on 6 dpc, but there was a minor tendency towards IgA development in contacts of mRNA- and mock-vaccinated and infected animals, aligning with virological and serological findings (Figure 12C).

Naive contacts of mRNA-vaccinated shedders contracted both SARS-CoV-2 variants and showed abundant expression of the nucleocapsid in nasal epithelium. Contacts of mock- vaccinated and B.1 -challenged hamsters had fewer SARS-CoV-2-positive cells in nasal epithelium compared to contacts of mRNA-vaccinated animals, consistent with virus RNA levels in oral swabs on 6 dpc.

In line with previous observations, nasal epithelium of naive contacts of sCPD9-AFCS- vaccinated and B.1 -infected shedders was free of SARS-CoV-2 nucleoprotein, except for the single hamster that contracted BA.5 infection. In accordance with immunohistochemical results, a variable degree of inflammation and immune cell recruitment was detected in all hamsters that contracted the infection with either of the two variants, corroborating the effectiveness of sCPD9-AFCS in preventing virus transmission. Histological findings confirmed complete clearance of the infection in all vaccinated and infected groups.

Summarizing discussion

The presently used LAV presents the virus’s entire antigenic repertoire at the respiratory mucosa, which among other things, triggers formation of tissue-resident memory T cells (TRM cells), a specialized subset of T cells that remain stationary in specific tissues, such as the respiratory mucosa, after an initial infection or vaccination (Schenkel and Masopust, 2014; Lavelle and Ward, 2022). These cells provide a first line of defense against reinfection, by rapidly recognizing and responding to pathogens that re-enter the tissue at body surfaces (Lavelle and Ward, 2022; Nouailles et al., 2022). In the context of SARS-CoV-2, TRM cells in the respiratory mucosa play a critical role in mounting a swift immune response upon viral exposure. When encountering the virus, TRM cells can quickly activate and release antiviral cytokines, recruit other immune cells to the site of infection, and directly eliminate virus-infected cells. Their ability to reside in the respiratory mucosa allows them to survey and respond to local viral threats more efficiently. By establishing this local immune surveillance network, TRM cells contribute to the early control of viral replication and limit virus spread within the respiratory tract. Together with neutralizing (IgA) antibodies, TRM cells contribute to a comprehensive defense against SARS-CoV-2, effectively targeting the virus at its entry point and initial replication site, thereby reducing the likelihood of respiratory tract infection and transmission (Nouailles et al., 2023).

The effectiveness of the attenuated virus sCPD9-AFCS and the mRNA vaccine BNT162b2, both of which encode the original form of the SARS-CoV-2 spike protein, was compared regarding controlling the transmission of SARS-CoV-2. The superior capacity of the attenuated virus to prevent or significantly reduce virus transmission is demonstrated. Importantly, this remains true even for BA.5, an evolved, highly transmissible and strongly immune evasive SARS-CoV-2 variant. The emergence of Omicron variants, which carry numerous amino acid changes in their spike protein (Wolter et al., 2022; Madhi et al., 2022), spurred the development of bivalent mRNA vaccines. These vaccines contain the spike protein of the ancestral B.1 variant, and of the BA.4/BA.5 variant, providing superior protection against Omicron variants compared to monovalent vaccines (Link-Gelles et al., 2022; Wang et al., 2023). However, it becomes increasingly clear that SARS-CoV-2 transmission is not, or not sufficiently controlled by intramuscular spike-based vaccines (Franco-Paredes, 2022).

The present data indicates that, in the case of SARS-CoV-2, mucosal vaccines have the ability to efficiently prevent or reduce virus infection and onward transmission. Additionally, they apparently do not require frequent updates of the viral antigens, as the B.1 -based vaccine provided highly efficient protection against the BA.5 variant that is antigenically far distant from the B.1 variant. It could be proven that administration of two consecutive doses of sCPD9 effectively enhances immunity. This indicates that pre-existing immunity, such as that conferred by previous SARS-CoV-2 infection, does not impede the effectiveness of the mucosal vaccine tested here. On the contrary, periodic boosting of existing immunity through mucosal vaccines could serve an important strategy for the long-term control of SARS-CoV-2.

In conclusion, the findings presented here underscore the significance and benefits of developing mucosal vaccines to enhance control of not only SARS-CoV-2 but potentially also other respiratory viruses. Reducing virus transmission may constrain and slow respiratory RNA virus circulation and evolution.

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