Live attenuated SARS-CoV-2 and vaccines using same

Abstract

The present invention relates to a) a polynucleotide encoding the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) spike protein, and / or b) at least one nonstructural SARS-CoV-2 protein selected from the group consisting of nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, nonstructural protein 11, nonstructural protein 12, endoribonuclease, and 2'-O-methyltransferase. The polynucleotide comprises or consists of at least one sequence portion that comprises deoptimization of codon pairs compared to the SARS-CoV-2 genome. The present invention further relates to an attenuated SARS-CoV-2 comprising this polynucleotide, a vaccine comprising this attenuated SARS-CoV-2, and related methods.

Description

[Technical field]

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

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

[0003] SARS-CoV-2 replicates primarily in the upper respiratory tract (Zou et al., 2020). SARS-CoV-2 infection can cause a wide range of clinical manifestations, from asymptomatic to life-threatening conditions (Chen et al., 2020; Zhou et al., 2020a). Patients, especially older adults and those with pre-existing conditions, are at higher risk of developing more severe disease, including pneumonia, acute respiratory distress syndrome, and multiple organ failure (Chen et al. 2020; Zhou et al., 2020a). The ongoing pandemic has imposed an enormous health, psychological, economic, and social burden. To date (December 2021), more than 270 million people have been infected with the virus, of which more than 5.3 million have died from the infection (https: / / coronavirus.jhu.edu / map.html). (Dong et al.).

[0004] The unprecedented scale and severity of the COVID-19 pandemic has prompted rapid development of novel diagnostic tests, therapeutics, and vaccines that can be used to stop the spread of the virus and contain the pandemic. Globally, more than 90 vaccines are being tested in clinical trials, although only a few have reached the final stage of trials (Zimmer et al., 2021). Almost all vaccines that have been or are being evaluated in clinical trials are based on inactivated or subunit virus preparations (Ella et al., 2021; Gao et al., 2020; Wang et al., 2020; Zhang et al., 2021), replication-deficient viral vectors (Emary et al., 2021; Logunov et al., 2021; Solforosi et al., 2021; Voysey et al., 2021b; 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., 2021). al., 2020; Mulligan et al., 2020; Polack et al., 2020; Sahin et al., 2020; Walsh et al., 2020).

[0005] SARS-CoV-2 is at the other end of the earlier arms race and 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 hosts and to infection- or vaccine-induced immunity. Over the course of the pandemic, numerous genetic variants have emerged (Tegally et al., 2021; Faria et al., 2021; Davies et al., 2021). Variants with enhanced infectivity, higher morbidity and mortality, or the ability to evade infection or vaccine-induced immunity pose an increased threat to public health. The World Health Organization (WHO) and national health authorities have independently established classification systems to categorize emerging variants into variants of interest (VOI), variants under investigation (VUI) or variants of concern (VOC) based on their risk to public health (see Table 1 in Trimpert et al., “Live attenuated virus vaccine protects against SARS-CoV-2 variants of concern B.1.1.7 (Alpha) and B.1.351 (Beta)”, Science Advances, Vol.7, No.49 (2021)). In addition, to facilitate communication with the public, WHO recommends that VOIs and VOCs also be indicated using Greek alphabet letters. As of August 12, 2021, viruses belonging to the B.1.1.7 (alpha), B.1.351 (beta), B.1.1.28.1 (gamma), B.1.617.2 (delta), and most recently, B.1.159.1 (omicron) lineages have been classified as VOCs by several health authorities. In countries where these variants have emerged, they have rapidly displaced pre-existing variants and begun to spread globally.

[0006] The B.1.1.7 variant, first detected in the UK in December 2020, is 50–100% more infectious and potentially 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 has rapidly become the dominant variant in the West. The B.1.351 variant, first detected in South Africa in May 2020, is not only more infectious but also has the ability to reinfect individuals and break through vaccine defenses (Madhi et al., 2021; Johnson & Johnson; Naveca et al., 2021). The B.1.1.28.1 variant, better known as P.1, is similar to B.1.351 in that it shares several key mutations in the spike glycoprotein (E484K, K417N / T, and N501Y). B.1.1.28.1 emerged in Manaus, Brazil in late 2020 (Faria et al., 2021). Like the B.1.351 variant, B.1.1.28.1 can bypass the immunity developed after infection with other viral variants, so B.1.1.28.1 can cause reinfection (Faria et al., 2021; Naveca et al., 2021). B.1.1.28.1 is estimated to be 40–140% more infectious, more pathogenic, and 10–80% more lethal than other variants (Faria et al., 2021). More recently, on May 7, 2021, the WHO reclassified the B.1.617.2 variant, first detected in India, as a VOC due to its high transmissibility (WHO). As of August 2021, B.1.617.2 has nearly surpassed B.1.1.7 as the predominant variant in Europe and the United States. According to the WHO, B.1.617.2 is the most dangerous strain globally, and B.1.617.2 has attracted considerable attention for its ability to evade infection and vaccine protection (Dyer et al., 2021). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Johnson & Johnson COVID-19 Vaccine Authorized by US FDA For Emergency Use - First Single-Shot Vaccine in Fight Against Global Pandemic https: / / www.jnj.com / johnson-johnson-covid-19-vaccine-authorized-by-us-fda-for-emergency-usefirst-single-shot-vaccine-in-fight-against-global-pandemic#_edn2 (Johnson & Johnson). [Non-Patent Document 2] The World Health Organization. Draft landscape of COVID-19 candidate vaccines. https: / / www.who.int / publications / m / item / draft-landscape-of-covid-19-candidate-vaccines (WHO). [Non-Patent Document 3] Abu-Raddad, LJ, Chemaitelly, H., Butt, AA, and National Study Group for, C.-V. (2021). Effectiveness of the BNT162b2 Covid-19 Vaccine against the B.1.1.7 and B.1.351 Variants. N Engl J Med. [Non-Patent Document 4] Almazan , F. , Gonzalez , JM , Penzes , Z. , Izeta , A. , Calvo , E. , Plana-Duran , J. , and Enjuanes , L. (2000). Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proceedings of the National Academy of Sciences of the United States of America 97, 5516–5521.

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[0008] The object of the present invention is to provide a novel SARS-CoV-2 vaccine candidate. [Means for solving the problem]

[0009] This objective is achieved using a) a specific polynucleotide encoding the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) spike protein and / or b) at least one nonstructural SARS-CoV-2 protein selected from the group consisting of nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, nonstructural protein 11, nonstructural protein 12, endoribonuclease (also referred to as nonstructural protein 15), and 2'-O-methyltransferase (also referred to as nonstructural protein 16). In this context, the polynucleotide comprises at least one sequence portion that comprises deoptimization of codon pairs compared to the corresponding SARS-CoV-2 genome portion or consists of at least one sequence portion that comprises deoptimization of codon pairs.

[0010] The term "polynucleotide" as used herein refers to a molecule that comprises a plurality of nucleotides (e.g., mRNA, RNA, cRNA, cDNA or DNA). This term typically refers to an oligonucleotide of length greater than 200, preferably greater than 300, preferably greater than 400, preferably greater than 500, preferably greater than 600, preferably greater than 700, preferably greater than 800, preferably greater than 900, preferably greater than 998 nucleotide residues. The polynucleotide of the present invention essentially consists of or comprises the nucleic acid sequence described herein. Thus, the polynucleotide of the present invention may also comprise additional nucleic acid sequences. The term polynucleotide encompasses single-stranded and double-stranded polynucleotides. In addition, modified polynucleotides, including chemically modified polynucleotides, artificially modified polynucleotides, or naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides, are also encompassed herein.

[0011] The term "SARS-CoV-2" or "severe acute respiratory syndrome coronavirus 2" as used herein refers to any variant classified as SARS-CoV-2. In some embodiments, the SARS-CoV-2 described herein is at least one SARS-CoV-2 variant selected from the group consisting of alpha, beta, gamma, delta, or omicron variants. In some embodiments, SARS-CoV-2 refers to a SARS-CoV-2 variant that includes a mutation selected from the group consisting of del 69-70, RSYLTPGD246-253N, N440K, G446V, L452R, Y453F, S477G / N, E484Q, E484K, F490S, N501Y, N501S, D614G, Q677P / H, P681H, P681R, and A701V. Therefore, "SARS-CoV-2 protein" and "SARS-CoV-2 genome" can also be understood as the protein and genome, respectively, of a SARS-CoV-2 variant.

[0012] The term "codon," as used herein, refers to a group of three consecutive nucleotides in a coding portion of a polynucleotide, such as a messenger RNA molecule, or a coding strand of DNA that specifies a particular amino acid, a translation start or stop signal, etc. Typically, a codon is specific for one amino acid, although there are known cases in SARS-CoV-2 where a codon shares at least one nucleotide with another codon.

[0013] The term "codon pair," as used herein, refers to two consecutive codons.

[0014] The term "codon pair deoptimization" as used herein refers to recoding codons such that the encoded protein is the same, but non-optimal codon pairs and / or CpG dinucleotides occur. Methods for codon pair deoptimization are known in the art (see, e.g., Coleman et al., 2008; Mueller et al., 2010). In some embodiments, the codon pair deoptimization described herein includes increasing the number of non-optimal codon pairs and CpG dinucleotides in the recoded genome. In some embodiments, the codon pair deoptimization described herein results in increased mRNA decay and / or reduced translation efficiency. In some embodiments, the codon pair deoptimization described herein results in less protein, less virus, reduced pathogenicity, and / or attenuated virus.

[0015] The polynucleotides of the present invention can be used in virus production or in the context of a vaccine. Thus, the polynucleotides of the present invention have a reduced risk of uncontrolled replication during production, transportation, storage, processing and / or administration compared to wild-type sequences.

[0016] Typically, this polynucleotide forms part of a live attenuated SARS-CoV-2. Compared to the wild-type virus, the live attenuated virus causes milder and / or less severe or no symptoms in the host organism after the host organism is confronted (infected) with the attenuated virus. At the same time, the live attenuated virus induces an immune response in the host against the attenuated virus that is at least partially protective against wild-type virus infection and / or at least one symptom thereof.

[0017] In contrast to most vaccines in development, we have generated an attenuated but replication-competent SARS-CoV-2 vaccine candidate by genetically modifying the SARS-CoV-2 genome via codon pair deoptimization (CPD) (Coleman et al., 2008.) CPD is a virus attenuation strategy that can rapidly and efficiently attenuate a variety of viruses (Broadbent et al., 2016; Coleman et al., 2008; Eschke et al., 2018; Groenke et al., 2020; Khedkar et al., 2018; Kunec and Osterrieder, 2016; Le Nouen et al., 2014; Mueller et al., 2010; Shen et al., 2015; Trimpert et al., 2021a; Trimpert et al., 2021b). CPD rearranges the positions of existing synonymous codons in one or more viral genes without changing the codon bias or amino acid composition of the encoded protein (Eschke et al., 2018; Groenke et al., 2020; Khedkar et al., 2018; Kunec and Osterrieder, 2016; Osterrieder and Kunec, 2018; Trimpert et al., 2021a; Trimpert and, 2021b). Codons that are naturally under-represented can be over-represented by CPD. The effect of CPD strongly depends on the genomic sequence to be deoptimized, so a general indication of codons to be deoptimized cannot be given. However, those skilled in the art are aware of methods to identify or estimate codon pairs that can be replaced by non-naturally occurring codon pairs in the target site (e.g., target species or target tissue) where the polynucleotide is intended to be translated. The inventors herein provide examples of which codon pairs are recoded to achieve overexpression of non-naturally occurring codon pairs and deoptimization of codon pairs of polynucleotides. Thus, those skilled in the art can arrive at other polynucleotides according to the invention - at least from the codon pairs of the target site and the means and methods provided herein.

[0018] A polynucleotide is considered non-optimized if at least one codon pair is not optimized relative to the corresponding native sequence. Recoded viruses usually do not produce proteins from recoded genes as efficiently as their parents and exhibit defects in reproductive fitness, allowing 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., 2021b; Wimmer et al., 2009). With preserved antigenic identity and replicative capacity, recoded attenuated viruses are able to fully engage the host immune system and elicit a strong immune response.

[0019] Thus, the deoptimization of codon pairs does not change the resulting protein. Rather, the change in the genome sequence of SARS-CoV-2 does not change the resulting protein. However, translation is usually less efficient, and therefore the replication of the virus is also reduced. Therefore, when live attenuated SARS-CoV-2 is used as a vaccine, an immune response is obtained without the risk of pathological viral replication in vaccinated patients. Another possible effect of deoptimization of codon pairs is a CpG-mediated immune response leading to viral attenuation. The present disclosure is not limited to any particular one of these effects.

[0020] In one embodiment, the polynucleotide encodes nonstructural protein 7. In one embodiment, the polynucleotide encodes nonstructural protein 8. In one embodiment, the polynucleotide encodes nonstructural protein 9. In one embodiment, the polynucleotide encodes nonstructural protein 10. In one embodiment, the polynucleotide encodes nonstructural protein 11. In one embodiment, the polynucleotide encodes nonstructural protein 12. In one embodiment, the polynucleotide encodes an endonuclease. In one embodiment, the polynucleotide encodes a 2'-O-methyltransferase. In one embodiment, the polynucleotide encodes a spike protein (sometimes referred to as a spike glycoprotein).

[0021] In one embodiment, the polynucleotide encodes at least two nonstructural proteins. For example, the polynucleotide encodes an endoribonuclease and a 2'-O-methyltransferase. For another example, the polynucleotide encodes nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, and nonstructural protein 11.

[0022] In one embodiment, the SARS-CoV-2 genome is the genome portion extending from position 11,000 to position 27,000 of the genome of SARS-CoV-2. For the position numbering and definition of the terms "genome of SARS-CoV-2" and "wild type SARS-CoV-2", see GenBank accession number MT108784.1 (freely accessible via the website https: / / www.ncbi.nlm.nih.gov / genbank / ), which contains 29,891 bases or nucleotides. The first (5' end) of these bases or nucleotides is located at position 1. The last (3' end) of these bases or nucleotides is located at position 29,891. The skilled person will know how to adjust the sequence numbering referenced for the embodiment in which the SARS-CoV-2 genome is understood as a sequence from different SARS-CoV-2 variants. In some embodiments, the polynucleotide of the invention is a codon pair deoptimized sequence of a sequence comprised in the SARS CoV-2 genome section from position 11,000 to 24,000. In one embodiment, the genome section extends from position 11,500 to 26,000, in particular from position 11,900 to 25,500, in particular from position 11,950 to 25,350, in particular from position 12,000 to 24,000. In one embodiment, the genome section extends from position 11,950 to 14,400. In one embodiment, the genome section extends from position 11,900 to 13,500. In one embodiment, the genome section extends from position 13,900 to 14,400. In one embodiment, the genome section extends from position 20,300 to 21,600. In one embodiment the genome section extends from position 24,300 to position 25,400. These embodiments can be combined in any desired way.

[0023] In one embodiment, at least one sequence portion comprising deoptimized codon pairs has a length in the range of 750 nucleotides to 2500 nucleotides, in particular in the range of 800 nucleotides to 2400 nucleotides, in particular in the range of 900 nucleotides to 2300 nucleotides, in particular in the range of 999 nucleotides to 2200 nucleotides, in particular in the range of 1000 nucleotides to 2100 nucleotides, in particular in the range of 1100 nucleotides to 2000 nucleotides, in particular in the range of 1146 nucleotides to 1900 nucleotides, in particular in the range of 1200 nucleotides to 1836 nucleotides, in particular in the range of 1300 nucleotides to 1800 nucleotides, in particular in the range of 1400 nucleotides to 1700 nucleotides, in particular in the range of 1500 nucleotides to 1600 nucleotides.

[0024] In one embodiment, 15% to 40%, in particular 20% to 35%, in particular 25% to 30% of the nucleotides of at least one sequence portion containing non-optimized codon pairs differ from the nucleotides of the corresponding (wild-type) SARS-CoV-2 genome. Such a wild-type SARS-CoV-2 genome is a genomic sequence of a virus variant or lineage that has not been artificially modified, 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). A wild-type SARS-CoV-2 genome may also be referred to as an authentic SARS-CoV-2 genome or an authentic SARS-CoV-2 genome sequence.

[0025] In one embodiment, 200 to 500 nucleotides, in particular 250 to 450 nucleotides, in particular 300 to 400 nucleotides of at least one sequence portion comprising deoptimized codon pairs differ from the corresponding (in particular identically positioned) nucleotides of the SARS-CoV-2 genome.

[0026] In one embodiment, 40% to 70%, in particular 45% to 65%, in particular 50% to 60%, in particular 55% to 62% of the codons of at least one sequence portion comprising deoptimized codon pairs differ from the respective codons of the corresponding SARS-CoV-2 genome.

[0027] In one embodiment, 150 to 400 codons, particularly 200 to 350 codons, particularly 250 to 300 codons of at least one sequence portion comprising deoptimized codon pairs differ from the corresponding (particularly identically positioned) codons of the SARS-CoV-2 genome.

[0028] In one embodiment, the at least one sequence portion comprising deoptimization of codon pairs comprises a first deoptimized sequence portion and a second deoptimized sequence portion. Both deoptimized sequence portions are separated from each other by a non-optimized sequence portion comprising at least 300 nucleotides, for example 300-1000 nucleotides, in particular 400-900 nucleotides, in particular 500-800 nucleotides, in particular 600-700 nucleotides. By conserving specific portions of the RNA sequence and deoptimizing the flanking portions upstream and downstream of the conserved RNA sequence, a particularly high effect is achieved in attenuating SARS-CoV-2 while maintaining the replication capacity of SARS-CoV-2.

[0029] In one embodiment, the first deoptimized sequence portion has a length ranging from 1300 nucleotides to 1600 nucleotides, particularly from 1400 nucleotides to 1500 nucleotides, particularly from 1450 nucleotides to 1490 nucleotides. At the same time, the second deoptimized sequence portion has a length ranging from 100 nucleotides to 400 nucleotides, particularly from 200 nucleotides to 300 nucleotides, particularly from 350 nucleotides to 400 nucleotides. The lengths of the first and second deoptimized sequence portions are optionally selected such that other applicable restrictions (e.g., the total length of at least one sequence portion including codon pair deoptimization is 2000 nucleotides or less) are met. If the length of at least one sequence portion containing codon pair deoptimization must not exceed 2000 nucleotides, considering that the first and second deoptimized sequence portions are separated by at least 300 nucleotides of the authentic SARS-CoV-2 genome, it is immediately clear that only the lower threshold of 1300 nucleotides can be combined with the upper threshold of 400 nucleotides for the first and second deoptimized sequence portions to meet the maximum length restriction of at least one sequence portion containing codon pair non-optimization. At the same time, the upper threshold of 1600 nucleotides for the first deoptimized sequence portion can be combined with the lower threshold of 100 nucleotides for the second deoptimized sequence portion, meeting the maximum length of 2000 nucleotides when considering the intermediate 300 non-coding nucleotides.

[0030] In one embodiment, the first deoptimized sequence portion is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO: 2. At the same time, the second deoptimized sequence portion is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO: 4.

[0031] In one embodiment the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:6.

[0032] In one embodiment the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:8.

[0033] In one embodiment the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:10.

[0034] In one embodiment the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:15.

[0035] In one embodiment the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:16.

[0036] In one embodiment the polynucleotide is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:17.

[0037] In one embodiment, the present invention relates to an attenuated SARS-CoV-2. The live attenuated SARS-CoV-2 comprises a partially encoded genomic RNA sequence, i.e., a partially encoded genomic viral sequence. The partially encoded genomic RNA sequence is a codon pair optimized sequence that encodes the spike protein and / or a specific nonstructural protein (nsp). The nonstructural protein is selected from the group consisting of nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, nonstructural protein 11 (a small protein only 13 amino acids long), nonstructural protein 12 (also called RNA-dependent RNA polymerase), endoribonuclease, and 2'-O-methyltransferase of the attenuated SARS-CoV-2.

[0038] In one embodiment, the partially encoded genomic RNA sequence encodes nonstructural protein 12. In one embodiment, the partially encoded genomic sequence encodes the spike protein (sometimes referred to as spike glycoprotein).

[0039] In one embodiment, the partially encoded genomic RNA sequence includes at least two of the nonstructural proteins. For example, the partially encoded genomic RNA sequence, in one embodiment, encodes an endoribonuclease and a 2'-O-methyltransferase. For another example, the partially encoded genomic RNA sequence, in one embodiment, encodes nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, and nonstructural protein 11.

[0040] In one embodiment, the partially encoded genomic RNA sequence is present in the genome portion extending from position 11,000 to position 27,000 of the genome of the attenuated SARS-CoV-2. The genome of wild-type SARS-CoV-2, according to GenBank accession number MT108784.1, consists of 29,891 bases or nucleotides. The genomes of the attenuated SARS-CoV-2 are essentially the same length. The only difference is the length of the poly-A tail at the 3' end, which was found by sequencing to be 8 adenine nucleotides longer than the wild-type SARS-CoV-2. It should be noted that there remains uncertainty in determining the length of the poly-A tail by sequencing. Thus, the poly-A tail of the wild-type sequence may be longer or shorter than indicated by the sequence according to GenBank accession number MT108784.1. Similarly, the poly-A tail in the attenuated SARS-CoV-2 sequence may be longer or shorter than currently determined.

[0041] The first base of the 29,891 bases (5' end) of the genome of wild-type SARS-CoV-2 is located at position 1. The last of these bases or nucleotides (3' end) is located at position 29,891. In one embodiment, the genome section extends from position 11,500 to 26,000, in particular from position 11,900 to 25,500, in particular from position 11,950 to 25,350, in particular from position 12,000 to 24,000. In one embodiment, the genome section extends from position 11,950 to 14,400. In one embodiment, the genome section extends from position 11,900 to 13,500. In one embodiment, the genome section extends from position 13,900 to 14,400. In one embodiment, the genome section extends from position 20,300 to 21,600. In one embodiment the genome section extends from position 24,300 to position 25,400. These embodiments can be combined in any desired way.

[0042] In one embodiment, the partially encoded genomic RNA sequence has a length ranging from 750 nucleotides to 2500 nucleotides, particularly from 800 nucleotides to 2400 nucleotides, particularly from 900 nucleotides to 2300 nucleotides, particularly from 999 nucleotides to 2200 nucleotides, particularly from 1000 nucleotides to 2100 nucleotides, particularly from 1100 nucleotides to 2000 nucleotides, particularly from 1146 nucleotides to 1900 nucleotides, particularly from 1200 nucleotides to 1836 nucleotides, particularly from 1300 nucleotides to 1800 nucleotides, particularly from 1400 nucleotides to 1700 nucleotides, particularly from 1500 nucleotides to 1600 nucleotides.

[0043] In one embodiment, 15% to 40%, particularly 20% to 35%, particularly 25% to 30% of the nucleotides of the partially encoded genomic RNA sequence differ from the nucleotides of the corresponding wild-type genomic RNA sequence. Such wild-type genomic RNA sequences are genomic viral sequences of non-artificially modified virus variants or lineages, 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). Such wild-type genomic RNA sequences can also be referred to as authentic SARS-CoV-2 genomic RNA sequences.

[0044] In one embodiment, between 200 and 500 nucleotides, particularly between 250 and 450 nucleotides, particularly between 300 and 400 nucleotides of the partially encoded genomic RNA sequence differ from the nucleotides at the same positions of the corresponding wild-type viral genomic RNA sequence.

[0045] In one embodiment, 40% to 70%, in particular 45% to 65%, in particular 50% to 60%, in particular 55% to 62% of the codons (i.e. the three nucleotides in each case which code for a specific amino acid) of the partially encoded genomic RNA sequence differ from the respective codons of the corresponding wild-type viral genomic RNA sequence.

[0046] In one embodiment, between 150 and 400 codons, particularly between 200 and 350 codons, especially between 250 and 300 codons, of the partially encoded genomic RNA sequence differ from the codons at the same positions of the corresponding wild-type viral genomic RNA sequence.

[0047] In one embodiment, the partially encoded genomic RNA sequence comprises a first encoded portion and a second encoded portion. Both recoded portions are separated from each other by a non-recoded genomic portion consisting of at least 300 nucleotides, for example 300-1000 nucleotides, in particular 400-900 nucleotides, in particular 500-800 nucleotides, in particular 600-700 nucleotides. By conserving certain portions of the RNA sequence and recoding adjacent portions upstream and downstream of the conserved RNA sequence, a particularly high effect of attenuating SARS-CoV-2 is achieved while maintaining the general viability of SARS-CoV-2.

[0048] In one embodiment, the first encoded portion has a length ranging from 1300 to 1600 nucleotides, particularly ranging from 1400 to 1500 nucleotides, particularly ranging from 1450 to 1490 nucleotides. At the same time, the second encoded portion has a length ranging from 100 to 400 nucleotides, particularly ranging from 200 to 300 nucleotides, particularly ranging from 350 to 400 nucleotides. The lengths of the first and second encoded portions are optionally selected to meet other applicable limitations (e.g., total length of the encoded genomic RNA sequence of 2000 nucleotides or less). If the length of the partially coded genomic RNA sequence must not exceed 2000 nucleotides, considering that the first coded portion and the second coded portion are separated by at least 300 nucleotides of the authentic SARS-CoV-2 genome sequence, it is immediately clear that only the lower threshold of 1300 nucleotides can be combined with the upper threshold of 400 nucleotides for the first coded portion and the second coded portion to meet the maximum length limit of the partially coded genomic RNA sequence. At the same time, the upper threshold of 1600 nucleotides for the first coded portion can be combined with the lower threshold of 100 nucleotides for the second coded portion, taking into account the intermediate 300 non-coded nucleotides, to meet the maximum length of 2000 nucleotides.

[0049] In one embodiment, the first coding portion is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO: 2. At the same time, the second coding portion is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO: 4.

[0050] In one embodiment the partially encoded genomic RNA sequence is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:6.

[0051] In one embodiment the partially encoded genomic RNA sequence is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:8.

[0052] In one embodiment the partially encoded genomic RNA sequence is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:10.

[0053] The phrase "% identical" or "having % sequence identity" with respect to a reference sequence is defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to nucleotides or amino acid residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum % sequence identity, and not considering conservative substitutions as part of the sequence identity. Alignment for the purpose of determining % amino acid sequence identity can be achieved in a variety of ways that are within the scope of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including the algorithms required to achieve maximum alignment over the entire length of the sequences to be compared.

[0054] Those skilled in the art recognize that sequences as exemplified herein can be changed to a certain extent without substantially changing or altering biological functions such as attenuation or encoded protein.Therefore, those skilled in the art who use the means and methods described herein can modify codon pairs according to the teachings of the present invention.For example, codon pairs can be replaced with attenuated and / or non-naturally occurring synonymous versions as well as deoptimized codon pairs of sequences described herein.Therefore, by replacing codon pairs in the position ranges described herein, the same degree of deoptimization can be achieved.

[0055] The present invention is based, at least in part, on the discovery that codon optimization of the range of positions described herein is particularly useful for attenuating SARS-CoV-2 virus while substantially maintaining immunogenicity and viral replication capacity.

[0056] The individual recoded sequences and their location in the SARS-CoV-2 genome are summarized in Table 1 below.

[0057] Table 1: Summary of recoded RNA sequences. [Table 1]

[0058] It should be noted that WT6A / CPD6A only encodes 9 nucleotides (3 amino acids) of nsp12 (RNA-dependent RNA polymerase, RdRp), the majority of this protein being encoded by WT6B / CPD6B.

[0059] The central part of fragment WT6 was not encoded in fragment CPD6 because it contains a conserved regulatory RNA sequence essential for viral replication. Fragment WT6 encodes a -1 ribosomal frameshift element, a so-called RNA pseudoknot structure. This structure promotes ribosomal frameshifting, a process in which the translation reading frame changes at the junction of open reading frames (ORFs) 1a and 1b. During this process, one nucleotide in the slippery sequence located downstream of the RNA pseudoknot structure is read twice by the translating ribosome, shifting the reading frame by -1 nucleotide (the ribosome slides back one nucleotide in the slippery sequence). In most cases, translation of ORF1a terminates at the stop codon of ORF1a. However, when a -1 ribosomal frameshift occurs, translation of ORF1a continues directly to ORF1b, producing polyprotein (pp)1ab. Thus, the CPD6A sequence is translated into both polyproteins 1a and 1ab, whereas the CPD6B sequence is translated only into polyprotein 1ab.

[0060] The differences between the CPD sequence and the underlying wild-type sequence are summarized in Table 2 below.

[0061] Table 2: Summary of sequence differences between CPD and wild type. [Table 2]

[0062] It should be noted that the primary structure of the protein encoded by the RNA sequence of the CPD is identical to that of the wild-type (original) RNA sequence. As explained above, the CPD does not change the primary structure of the resulting protein. This is summarized in Table 3.

[0063] Table 3: Summary of protein sequences. [Table 3]

[0064] The sequence portions CPD6A, CPD6B, sCPD9, and sCPD10 were recoded to generate different fragments of the SARS-CoV-2 genome that form part of the attenuated SARS-CoV-2 in the embodiment. These fragments are shown in Table 4 below.

[0065] Table 4: Genomic fragments of SARS-CoV-2. [Table 4]

[0066] In one aspect, the present invention relates to a live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a polynucleotide according to the above description.

[0067] In one embodiment, the attenuated SARS-CoV-2 has a nucleic acid sequence that is at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:18.

[0068] In one embodiment, SARS-CoV-2 has a nucleic acid sequence that is at least 98%, particularly at least 99%, particularly 100% identical to SEQ ID NO:19.

[0069] In one aspect, the present invention relates to a pharmaceutical composition comprising a live attenuated SARS-CoV-2 according to any of the preceding descriptions. Such a pharmaceutical composition may further comprise auxiliary substances, such as adjuvants, for example, to enhance the immune response of the patient. Suitable adjuvants are potassium alum; aluminum hydroxide; aluminum phosphate; calcium hydroxide phosphate; aluminum sulfate hydroxyphosphate; paraffin oil; propolis; killed bacteria of the species Bordetella pertussis or Mycobacterium bovis; plant saponins from Quillaja, soybean, and / or Polygala senega; cytokines IL-1, IL-2, and / or IL-12; and Freund's complete adjuvant.

[0070] In one embodiment, the present invention relates to the further medical use of such a pharmaceutical composition as a vaccine.

[0071] In one aspect, the invention relates to methods for preparing vaccines from such pharmaceutical compositions.

[0072] In one aspect, the present invention relates to a method of vaccinating a human or animal patient in need thereof, the animal patient being in particular a non-human mammal such as a rodent, a dog, a cat or a mustelid, comprising the step of administering to the patient a pharmaceutical composition according to the above description.

[0073] In one embodiment, administration is by parenteral administration, such as intranasal administration, oral administration, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intravenous infusion, or intraperitoneal infusion. In particular, intranasal or oral administration is suitable. These administration routes present the live attenuated SARS-CoV-2 in the body of the vaccinated patient in a manner similar or similar to that of natural exposure to the virus.

[0074] In one embodiment, the vaccination comprises * 10 3 From 1 * 10 8 The focus-forming units (FFUs) between * 10 4 From 1 * 10 7 FFU between, especially 1 * 10 5 From 1 * 10 6 The method is carried out by administering a pharmaceutical composition of live attenuated SARS-CoV-2 at a dose containing an FFU between 100 and 150 mg / kg / day. The dose is selected so that the pharmaceutical composition is well tolerated by the patient but induces an immune response that protects the patient from infection or a severe course of infection with SARS-CoV-2. In one embodiment, the dose is one of the minimum protective dose and the maximum tolerated dose, or is between the minimum protective dose and the maximum tolerated dose.

[0075] A variety of factors can influence the dosage used for a particular application, including frequency of administration, duration of treatment, prophylactic or therapeutic intent, use of multiple therapeutic agents, route of administration, previous treatments, the patient's clinical history, the attending physician's discretion, and the severity of the disease, disorder, and / or condition, which may influence the amount that needs to be administered.

[0076] As with the dosage, various factors can influence the actual frequency of administration used for a particular application, for example, dosage, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the disease, disorder, and / or condition may require more or less frequent administration.

[0077] In some cases, the effective period for administering the pharmaceutical composition of the present invention (and any additional therapeutic agent) can be any period that reduces the severity or occurrence of the symptoms of the disease, disorder and / or condition to be treated without causing significant toxicity to the subject.Several factors can affect the actual effective period used for a particular treatment.For example, the effective period can vary depending on the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of the disease, disorder and / or condition to be treated.

[0078] In one embodiment, the pharmaceutical formulation is administered to the patient at least twice, where the second administration is separated from the first administration by a first period of time. In this context, the first period of time is in the range of 2 weeks to 36 months, particularly in the range of 3 weeks to 30 months, particularly in the range of 4 weeks to 24 months, particularly in the range of 5 weeks to 21 months, particularly in the range of 6 weeks to 18 months, particularly in the range of 7 weeks to 15 months, particularly in the range of 8 weeks to 12 months, particularly in the range of 9 weeks to 10 months, particularly in the range of 10 weeks to 8 months, particularly in the range of 12 weeks to 6 months, particularly in the range of 13 weeks to 4 months.

[0079] In one embodiment, the pharmaceutical formulation is administered to the patient after or before administering a different vaccine (e.g., a vector-based vaccine, an mRNA-based vaccine, a protein-based vaccine, etc.) to the patient, i.e., vaccinating the patient with a different vaccine, offset in time. In this context, the administration of the pharmaceutical composition is offset by a second period of time with respect to the administration of the different vaccine. In this context, the second period of time is in the range of 2 weeks to 36 months, particularly in the range of 3 weeks to 30 months, particularly in the range of 4 weeks to 24 months, particularly in the range of 5 weeks to 21 months, particularly in the range of 6 weeks to 18 months, particularly in the range of 7 weeks to 15 months, particularly in the range of 8 weeks to 12 months, particularly in the range of 9 weeks to 10 months, particularly in the range of 10 weeks to 8 months, particularly in the range of 12 weeks to 6 months, particularly in the range of 13 weeks to 4 months.

[0080] In one aspect, the present invention relates to a vector comprising a polynucleotide according to the above description.

[0081] The term "vector" as used herein refers to a nucleic acid molecule that can transfer or transport itself and / or another nucleic acid molecule into a cell. The transferred nucleic acid is generally linked, i.e., inserted, into the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in the cell, or may contain sequences sufficient to allow integration into the host cell DNA. In some embodiments, the vector described herein is a vector selected from the group of plasmids (e.g., DNA or RNA plasmids), shuttle vectors, transposons, cosmids, artificial chromosomes (e.g., bacterial, yeast, human), and viral vectors.

[0082] In some embodiments, the vectors described herein are used in combination with at least one transfection enhancer, e.g., a transfection enhancer selected from the group of oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides.

[0083] In one aspect, the present invention relates to a host cell comprising a polynucleotide according to the above description.

[0084] As used herein, "host cells" refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. As used herein, mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included.

[0085] In one embodiment, the host cells described herein comprise at least one cell type selected from the group of Chinese Hamster Ovary (CHO), Vero, Vero E6, Vero TMPRSS, MRC 5, Per.C6, PMK, and WI-38.

[0086] In one embodiment, the present invention relates to a method for producing a virus, the method comprising the steps of a) culturing a host cell according to the preceding paragraph; and b) isolating the virus, wherein the virus is a live attenuated SARS-CoV-2.

[0087] All embodiments of the polynucleotides can be combined in any desired way and can be transferred individually or in any combination to the live attenuated SARS-CoV-2, the pharmaceutical composition, its use, the method of vaccinating a patient, the vector, the host cell, and the method of producing the virus. All embodiments of the attenuated SARS-CoV-2 can be combined in any desired way and can be transferred individually or in any combination to the polynucleotide, the pharmaceutical composition, its use, the method of vaccinating a patient, the vector, the host cell, and the method of producing the virus. Similarly, all embodiments of the pharmaceutical composition can be combined in any desired way and can be transferred individually or in any combination to the polynucleotide, the attenuated SARS-CoV-2, the use of the pharmaceutical composition, the method of vaccinating a patient, the vector, the host cell, and the method of producing the virus. All embodiments of the use of the pharmaceutical preparation can be combined in any desired way and can be transferred individually or in any combination to the polynucleotide, to the 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 the virus. Finally, all embodiments of the method of vaccinating a patient can be combined in any desired way and can be transferred individually or in any combination to the polynucleotide, to the 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 the virus. [Brief description of the drawings]

[0088] Further details of aspects of the invention will now be described with reference to exemplary embodiments and the accompanying drawings. [Figure 1A-1D] 1A-1D are schematic diagrams showing the native structure and exemplary recoding of the SARS-CoV-2 genome. [Diagram 2] 2A-B show the areas of infected cell foci and multistep growth kinetics of parental and encoded viruses in Vero E6 cells. [Figure 3A-3B] Figures 3A-B show the decay of encoded SARS-CoV-2 mutants in Syrian hamsters. [Figure 3C-3E] Figures 3C-E show the decay of encoded SARS-CoV-2 mutants in Syrian hamsters. [Fig. 3F-3G] Figures 3F-G show the decay of encoded SARS-CoV-2 mutants in Syrian hamsters. [Fig. 3H-3J] Figures 3H-J show the decay of encoded SARS-CoV-2 mutants in Syrian hamsters. [Fig. 3K-3L] Figures 3K-L show the decay of encoded SARS-CoV-2 mutants in Syrian hamsters. [Figure 3M-3N] Figures 3M-N show the decay of encoded SARS-CoV-2 mutants in Syrian hamsters. [Fig. 3O-3P] Figures 3O-P show the decay of encoded SARS-CoV-2 mutants in Syrian hamsters. [Figure 4A-4N] 4A-4N show lung histology images of infected Syrian hamsters 3 days after vaccination. [Figure 5A] FIG. 5A shows lung histopathology of vaccinated Syrian hamsters from days 2 to 14 post-challenge. [Figure 5B-5C] 5B-5C show lung histopathology images of vaccinated Syrian hamsters from days 2 to 14 after challenge. [Fig. 5D-5E] Figures 5D-5E show lung histopathology of vaccinated Syrian hamsters from days 2 to 14 post-challenge. [Figure 5F] FIG. 5F shows lung histopathology of vaccinated Syrian hamsters from days 2 to 14 post-challenge. [Figure 5G-5V] Figures 5G-5V show lung histopathology images of vaccinated Syrian hamsters from days 2 to 14 post-challenge. [Figure 6]Shows SARS-CoV-2 neutralizing antibody titers in vaccinated Syrian hamsters. [Figure 7A] Figure 7A shows that the encoded SARS-CoV-2 mutant sCPD9s is strongly attenuated in Roborovski dwarf hamsters. [Figure 7B-7C] Figures 7B-C show that the encoded SARS-CoV-2 mutant sCPD9s is strongly attenuated in Roborovski dwarf hamsters. [Figures 7D-7O] Figures 7D-O show that the encoded SARS-CoV-2 mutant sCPD9s is strongly attenuated in Roborovski dwarf hamsters. [Figure 8] Representative images of viral foci formed by the parent virus SARS-CoV-2 (WT) and the recoded virus. [Figure 9] 9A-9B are Western blot analyses showing that sCPD10 virus produces less spike protein in infected Vero E6 cells than the parental or sCPD9 viruses. [Figure 10B-10C] Figures 10B-C show that the live-attenuated virus vaccine candidate sCPD9s is strongly attenuated and protects Roborovski dwarf hamsters against challenge with B.1, B.1.1.7, and B.1.351 viruses (Figure 10A intentionally omitted). [Fig. 10D-10E] Figures 10D-E show that the live-attenuated virus vaccine candidate sCPD9s is strongly attenuated and protects Roborovski dwarf hamsters from challenge with B.1, B.1.1.7, and B.1.351 viruses (Figure 10A intentionally omitted). [Fig. 10F-10G] Figures 10F-G show that the live-attenuated virus vaccine candidate sCPD9s is strongly attenuated and protects Roborovski dwarf hamsters from challenge with B.1, B.1.1.7, and B.1.351 viruses (Figure 10A intentionally omitted). [Fig. 11A-11T]Figures 11A-11T show lung histopathology at different time points in mock / SCPD9 vaccinated and challenged Roborovski dwarf hamsters. [Figure 12A-12B] Figures 12A-B show the susceptibility of SARS-CoV-2 variants B.1, B.1.1.7 (alpha), B.1.351 (beta), B.1.128.1 (gamma), and B.1.617.2 (delta) to neutralization by antibodies in the serum of sCPD9-vaccinated Roborovski dwarf hamsters. [Figure 12C] Figure 12C shows the susceptibility of SARS-CoV-2 variants B.1, B.1.1.7 (alpha), B.1.351 (beta), B.1.128.1 (gamma), and B.1.617.2 (delta) to neutralization by antibodies in the serum of sCPD9-vaccinated Roborovski dwarf hamsters. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0089] (First Exemplary Embodiment)

[0090] We generated a series of encoded SARS-CoV-2 mutants and characterized them in cell culture and in vivo in Syrian and Roborovski hamster models. We demonstrated that a single intranasal immunization with live attenuated virus elicited a robust immune response and provided complete protection against SARS-CoV-2 challenge in a robust small animal model of COVID-19.

[0091] (Vaccine Design) Our goal was to generate an attenuated SARS-CoV-2 vaccine candidate through large-scale recoding of the SARS-CoV-2 genome with CPD (Eschke et al., 2018; Groenke et al., 2020; Khedkar et al., 2018; Kunec and Osterrieder, 2016). To achieve viral attenuation in humans, we recoded the genome of SARS-CoV-2 with the least expressed codon pairs in human genes (Groenke et al., 2020). The engineered SARS-CoV-2 mutants were then transformed into a novel SARS-CoV-2 vaccine using a recently established reverse genetics system for SARS-CoV-2 (Thi Nhu Thao et al. (Thi Nhu Thao et al.) Figures 1A to 1D). The system relies on 12 subgenomic fragments of the SARS-CoV-2 genome, which are assembled into a single yeast / bacterial artificial chromosome (YAC / BAC) by transformation-associated recombination (TAR) cloning in Saccharomyces cerevisiae (Noskov et al. (Noskov et al., 2002)). The subgenomic fragments are approximately 3,000 bp in length, and adjacent fragments overlap by approximately 300 bp, allowing the assembly of a SARS-CoV-2 infectious clone by homologous recombination.

[0092] In this regard, Figures 1A to 1D show the structure and recoding of the SARS-CoV-2 genome.

[0093] Figure 1A shows that the SARS-CoV-2 genome is a single-stranded, positive-sense RNA molecule of approximately 30,000 nucleotides (nt) encoding 11 canonical ORFs. "3CL-Pro" 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'O-ribose methyltransferase.

[0094] As shown in Fig. 1B, after infection, ORF 1a / 1ab is directly translated and cleaved into 15 proteins of the replication-transcription complex.

[0095] As shown in Figure 1C, a SARS-CoV-2 mutant consisting of 12 subgenomic fragments was constructed using a recently established SARS-CoV-2 reverse genetics system. Fragments 1, 11, and 12 were not recoded. The dark grey boxes indicate the recoded sequences CPD2-10, and the light grey boxes indicate the parental non-recoded sequences in fragments 2-10, respectively. The frameshifting element contained in fragment 6 and the transcriptional regulatory sequence (TRS) of the spike gene contained in fragment 9 were excluded from the recoding process (light grey box present between the two dark grey boxes of CPD6 and CPD9).

[0096] As shown in Figure 1D, the dark grey boxes indicate the recoded sequences of sCPD3-5 and sCPD8-10 in different subgenomic fragments.

[0097] To maintain full compatibility with available reverse genetics systems, we encoded only SARS-CoV-2 sequences not present in the overlapping subgenomic fragments (~2,500 bp in each coding fragment) (Figures 1A to 1D). This design allowed us to generate a wide variety of SARS-CoV-2 mutants with single or multiple coding fragments.

[0098] We recoded nine fragments (fragments 2–10) of the SARS-CoV-2 reverse genetics system. Fragments 1 and 12 are relatively short, 591 and 1,812 bp, respectively, and fragment 11, which contains many short ORFs, was excluded from recoding. To ensure that the mutant virus was replication-competent, two genomic regions containing essential cis-acting RNA elements were excluded from recoding: a frameshifting element harbored by fragment 6 and the transcriptional regulatory sequence (TRS) of the spike gene in fragment 9. In addition, the first 500 bp of ORF 1a located in fragment 2 was also not recoded.

[0099] Recovery of encoded viral mutants We attempted to rescue infectious progeny from recoded SARS-CoV-2 constructs in two different ways: directly from DNA in Vero E6 cells and from viral RNA in BHK 21 cells. To enable recovery from DNA, the genome of SARS-CoV-2 was placed under the control of the immediate early promoter of human cytomegalovirus. We recovered infectious viruses with recoded fragments 2 and 6 (hereafter denoted CPD2 and CPD6). It is known that extensive recoding by CPDs can result in a lethal phenotype of recoded mutants (Eschke et al., 2018). We suspected that the degree of recoding in constructs that did not produce infectious viruses was too high, and further constructed mutants with shorter non-optimized sequences (Figure 1A-D). In total, we generated six SARS-CoV-2 mutant constructs with shorter, approximately 1,000 bp long recoded sequences (sCPD3, sCPD4, sCPD5, sCPD8, sCPD9, and sCPD10). As expected, decreasing the degree of deoptimization rescued several more SARS-CoV-2 viruses. Specifically, of the six constructs, we rescued four mutant viruses by DNA transfection: sCPD3, sCPD4, sCPD9, and sCPD10.

[0100] (result) Characterization of encoded SARS-CoV-2 mutants in cell culture The morphological changes induced by infection of Vero E6 cells differed greatly depending on the mutant viruses. CPD6, sCPD3, and sCPD4 viruses produced easily visible plaques on Vero E6 cells within 24 hours post-infection (hpi), whereas sCPD9 and sCPD10 viruses did not produce visible plaques or discernible cytopathic effects (CPE) on Vero E6 cells in the first 72 hours (hpi). Therefore, we determined the viral titer and spread of the viral mutants in cell culture monolayers by what we call an assay (focus-forming assay) that evaluates virus-induced foci and relied on the identification of infected cells by immunofluorescence under semi-solid medium (Avicel). Recoded viruses CPD6, sCPD3, sCPD9, and sCPD10 formed significantly smaller foci of infected cells than the parental viruses (Figure 2A, Figure 8). The foci formed by sCPD3 and sCPD4 viruses were slightly smaller than those formed by the parental viruses. On average, they were approximately 65% ​​and 80% the size of those formed by the parental viruses. In contrast, the foci formed by CPD6, sCPD9, and sCPD10 viruses were significantly smaller, on average approximately 50%, 20%, and 45% the size of the parental viruses, respectively.

[0101] In this regard, Figures 2A and 2B show the areas of infected cell foci and multistep growth kinetics of parental and encoded viruses in Vero E6 cells.

[0102] Figure 2A shows the relative area of ​​infected cell foci at 48 hpi. Focus area was normalized to the average focus size from the parent virus. Bars indicate geometric mean and SD. P values ​​were calculated using the Kruskal-Wallis test with Dunn's post-hoc test, *P=0.0135, ****P<0.0001. See also Figure 8.

[0103] Figure 2B shows the multistep growth kinetics of parental and coded viruses. Vero E6 cells were infected with parental or coded modified viruses at a multiplicity of infection (MOI) of 0.01. Cell cultures were harvested after 6, 12, 24, 48, and 72 h, and virus titers were measured by focus-forming assay. Data are presented as the mean ± SD of three independent biological replicates. Comparison of growth curves by Friedman test and Dunn's post-hoc test showed that sCPD9 and sCPD10 viruses grew significantly worse than the parental viruses, *P=0.0264 and **P=0.0049, respectively.

[0104] Figure 8 shows representative images of viral foci formed by parental SARS-CoV-2 (WT) and recoded viruses. Bars indicate 1 mm.

[0105] These multi-step growth kinetics in Vero E6 cells indicated that the encoded viruses replicated with varying efficiency in Vero E6 cells (Figure 2B). Replication kinetics of CPD6, sCPD3, and sCPD4 viruses were comparable to the parental viruses. In contrast, sCPD9 and sCPD10 viruses replicated at significantly reduced levels at all evaluation time points. The mean titers of sCPD9 and sCPD10 viruses were 100- to 1,000-fold lower than the parental viruses at 24 h and 10- to 100-fold lower at 48 h.

[0106] To evaluate the genetic stability of the encoded viruses, we serially passaged the two most attenuated viruses, sCPD9 and sCPD10, 10 times on Vero E6 cells at MOI 0.1. After 10 passages, the viruses did not appear to have any significant phenotypic changes, as they only formed small foci on Vero E6 cells and did not produce readily observable CPE within 72 hours. We also analyzed the genomes of the passaged viruses by RT-PCR and Sanger sequencing, and we did not detect any mutations in the recoded regions after serial passages.

[0107] Recoding by codon pair optimization does not affect the amino acid sequence of the encoded protein, but the underlying genomic sequence is changed extensively. Suboptimal codon pairs reduce the stability and translation efficiency of the mRNA of the recoded gene, whereas the optimized codon pairs (Groenke et al., 2020; Mueller et al., 2010). As a result, protein production is reduced, leading to virus attenuation. To determine whether reduced protein production from the recoded gene could be the reason for the attenuation of the mutant virus, we examined the production of spike and nucleocapsid proteins in Vero E6 cells infected with wild-type (WT), sCPD9, or sCPD10 viruses. Since the sCPD10 virus has a codon pair-optimized spike gene (Figures 1A to 1D), it is expected to produce relatively less spike protein than either the WT or sCPD9 viruses. Indeed, spike protein production was significantly reduced in cells infected with sCPD10 virus compared with cells infected with WT or sCPD9 viruses (Figures 9A and 9B), indicating that CPD reduced protein production of the target genes.

[0108] In this context, Figure 9 shows that sCPD10 virus produces less spike protein in infected Vero E6 cells than parental or sCPD9 viruses. Figure 9A is a Western blot analysis of viral protein production in infected cells by SARS-CoV-2 (WT), sCPD9 and sCPD10 viruses. Vero E6 cells were infected with WT, sCPD9 or sCPD10 viruses. After 48 h, infected cells were lysed, cell lysates were resolved by SDS-PAGE under reducing conditions and proteins were transferred to a PVDF membrane. The membrane was cut into two parts. The upper membrane contains proteins with higher molecular weight and was incubated with mouse monoclonal anti-S antibody. Note that this antibody binds to the S2 subunit of the spike protein and thus can recognize both the unmodified spike protein (MW ~ 180,000) and its S2 subunit (MW ~ 95,000). The lower membrane section, containing low molecular weight proteins, was incubated with a mouse monoclonal antibody that recognizes the N protein (MW ∼90,000).

[0109] Figure 9B shows an image of the upper membrane containing intact spike protein and its subunits after longer exposure. Data are representative of three independent experiments.

[0110] Vaccination and challenge infection setting for Syrian hamsters The degree of attenuation of SARS-CoV-2 mutants was evaluated in a Syrian hamster model with a single intranasal challenge (Figures 3A to 3P). Hamsters were housed in individual ventilated cages and randomly assigned to four groups of 15 each. On day 0, they were either mock-vaccinated or vaccinated with 1 × 10 4 or 1×10 5 Animals were vaccinated with 1 × 10 5Mice were intranasally infected with FFU of WT SARS-CoV-2, variant B.1 (Wolfel et al.). Body weight and clinical signs were recorded daily during the vaccination experiment (35 days). Three mice from each group were euthanized on days 3, 23, 24, 26, and 35 post-vaccination (corresponding to days 2, 3, 5, and 14 post-challenge) to measure the extent of viral replication in different organs and to evaluate pathological changes in the lungs.

[0111] Figures 3A to 3P show the attenuation of encoded SARS-CoV-2 mutants in Syrian hamsters. The attenuation level of the encoded viral vaccine candidate was evaluated in two consecutive studies. In the first study, Syrian hamsters were mock vaccinated or inoculated with CPD6, sCPD3, or sCPD4 viruses (Figures 3A to 3E and 3K to 3M). In the second study, Syrian hamsters were mock vaccinated or vaccinated with sCPD9 or sCPD10 viruses (Figures 3F to 3J and 3N to 3P). 21 days after vaccination, all animals were infected with WT SARS-CoV-2.

[0112] Figures 3A, 3B, 3F and 3G show changes in body weight of Syrian hamsters after vaccination (n=15; Figures 3A and 3F) and challenge (n=12; Figures 3B and 3G). Data are expressed as mean ± SD.

[0113] Figures 3C, 3D, 3H and 3I show the viral load in the upper (Figures 3C and 3H) and lower (Figures 3D and 3H) respiratory tracts on day 3 post-vaccination.

[0114] Figures 3E and 3J show the number of infectious viral particles detected in 50 mg of lung tissue 3 days post-vaccination.

[0115] Figures 3K, 3L, 3N and 3O show the viral load in the upper (Figures 3K and 3N) and lower (Figures 3L and 3O) respiratory tracts of animals on days 2, 3, 5 and 14 post-challenge.

[0116] Figures 3M and 3P show the infectious viral load detected in 50 mg of lung tissue on days 2, 3, and 5 after challenge. The Kruskal-Wallis test was used to determine whether the differences in viral load between groups were significant (Figures 3N-3P), *P<0.05.

[0117] Due to space limitations, we investigated the attenuation of the five coding viruses in two animal studies. In the first study, hamsters were administered 1 × 10 5 In the second study, hamsters were vaccinated with 1 × 10 4 We vaccinated mice with FFU of the viruses encoding sCPD9 or sCPD10. We used lower doses because we were initially unable to grow the sCPD9 and sCPD10 mutants to sufficiently high levels, as the peak titers of these mutants in Vero E6 cells were 10- to 100-fold lower than the parental viruses (Figure 2B).

[0118] Attenuation of the coding viruses CPD6, CPD9 and CPD10 in Syrian hamsters At day 3 post-vaccination, all viruses tested replicated efficiently in the upper and lower respiratory tract of vaccinated hamsters, as determined by viral loads in oropharyngeal swabs (Figures 3C and 3H) and lung tissues (Figures 3D and 3I). Unexpectedly, despite weak replication in Vero E6 cells, the encoded viruses sCPD9 and sCPD10 replicated efficiently in the upper respiratory tract at day 3 post-vaccination (Figure 3H). Indeed, the amount of RNA copies detected in oropharyngeal swabs (~10 6The viral loads (RNA copies / swab) were comparable for all encoded viruses (Figures 3C and 3H) and were also comparable to the viral loads of WT virus in mock-infected animals 3 days post-challenge (Figures 3K and 3N). These results indicate that the viruses exhibited significantly different replication capabilities in cultured Vero E6 cells and that all recoded viruses replicated with comparable efficiency in the upper respiratory tract of infected animals, despite the animals being vaccinated with different doses of recoded viruses.

[0119] We successfully reisolated replicating virus from the lungs of vaccinated hamsters 3 days after vaccination, confirming that all recoded viruses were also replicating in the lungs of vaccinated animals (Figures 3E and 3J). Viral titers were lower in the lungs of hamsters vaccinated with CPD6 virus than in those vaccinated with sCPD3 or sCPD4 viruses (Figure 3E), indicating that CPD6 was the most attenuated virus of the three viruses examined. We found that less infectious virus was detected in the lungs of hamsters vaccinated with sCPD9 virus than in those vaccinated with sCPD10 virus (Figure 3J), indicating that sCPD9 is more attenuated than sCPD10 virus in vivo. In general, lung viral titers correlated well with the size of viral foci (Figure 2A) and also with lung histopathology 3 days after vaccination (Figures 4A-4N).

[0120] In this context, Figures 4A to 4N show the lung histopathology of infected Syrian hamsters 3 days after vaccination.

[0121] Figures 4A to 4N show representative whole-section scans of the left lung lobe (top row, Figures 4A to 4G) and photomicrographs of the bronchial epithelium (bottom row, Figures 4H to 4N) of formalin-fixed, paraffin-embedded, hematoxylin-eosin-stained tissue. Hamsters were mock vaccinated (Mock), SARS-CoV-2 infected (WT), or vaccinated with viruses CPD6, sCPD3, sCPD4, sCPD9, or sCPD10. Bars: 1 mm (Figures 4A to 4G) or 100 μm (Figures 4H to 4N).

[0122] Hamsters inoculated with sCPD3 and sCPD4 viruses lost weight significantly starting on day 2 after inoculation (Figure 3A), and lung histological examination on day 3 revealed significant necrotizing bronchointerstitial pneumonia (Figures 4A-4N). Four to five days after vaccination, the clinical symptoms of the hamsters peaked, and they gradually began to recover and gain weight. Because the clinical and histological signs were reminiscent of those caused by infection with pathogenic WT SARS-CoV-2, we discontinued the vaccination trial with sCPD3 and sCPD4 viruses on day 10 after vaccination.

[0123] CPD6 virus was moderately attenuated in vivo. Individual hamsters lost up to 6% of their body weight during the first 3 days after vaccination. Vaccinated animals lost an average of 0.4% body weight on day 1 and 2.8% on day 2 after vaccination (Figure 3A). Body weight regain began on day 3. In contrast, mock-vaccinated hamsters had a significantly greater average daily weight loss after challenge with WT virus in both vaccination studies (Figures 3B and 3G). The average body weight of the hamsters was reduced by 1.1%, 5.0%, 7.1%, 7.6%, and 9.6% during the first 5 days after challenge compared to the day of challenge. Furthermore, individual hamsters lost up to 14% of their body weight during this period (Figures 3B and 3G). In both studies, hamsters began to gain weight starting on day 5 after challenge.

[0124] Because Syrian hamsters were inoculated with a 10-fold lower virus dose in the second animal study than in the first study, the degree of attenuation of sCPD9 and sCPD10 viruses cannot be directly compared with that of CPD6, sCPD3, and sCPD4 viruses, or with that of the WT virus after challenge in mock-vaccinated hamsters. Nevertheless, the results obtained were consistent with those obtained with a 1 × 10 4 These results indicate how pathogenic the sCPD9 and sCPD10 viruses are to Syrian hamsters when vaccinated at FFU / animal, and whether this dose can protect vaccinated Syrian hamsters from challenge with WT virus.

[0125] Syrian hamsters vaccinated with sCPD9 and sCPD10 viruses did not develop any side effects or signs consistent with SARS-CoV-2 infection. Hamsters lost weight for 2 days after vaccination, but the average daily weight loss was minor. Animals vaccinated with sCPD9 virus lost weight by an average of 0.3% on day 1 and 1.9% on day 2 after vaccination (Figure 3F). Weight loss was more pronounced but still moderate in hamsters vaccinated with sCPD10 virus. Animals lost weight by an average of 1.3% on day 1 and 3.8% on day 2 after vaccination compared to baseline weight. We recorded weight gain in both groups on day 3 after vaccination, and when the animals had fully recovered, the average daily weight gain was comparable to the mock-vaccinated group.

[0126] Vaccination of Syrian hamsters confers complete protection against SARS-CoV-2 challenge Vaccination with CPD6 virus induced strong protective immunity against challenge with WT SARS-CoV-2. CPD6-vaccinated animals did not lose weight (Figure 3B) and did not show signs of disease after challenge. CPD6-vaccinated animals had lower viral loads in the upper and lower respiratory tracts on days 2, 3, 5, and 14 after challenge than mock-vaccinated animals (Figures 3K and 3L). To better understand the efficacy of vaccination, we also performed virus isolation from lung tissue (Figure 3M). We were able to detect high titers of replicating virus in lung tissue from mock-vaccinated animals, but not from CPD6-vaccinated animals, suggesting that vaccination with live attenuated CPD6 virus induced immunity that efficiently blocked replication of the challenge virus in the lungs.

[0127] Similarly, vaccination with sCPD9 and sCPD10 viruses induced strong protective immunity against challenge infection with WT SARSCoV2. Vaccinated animals did not lose weight and did not show clinical symptoms of disease after infection. RT-qPCR analysis showed active virus replication in the upper respiratory tract of infected animals (Figure 3N). The viral load 5 days after challenge was approximately 1,000-fold lower compared to the mock-vaccinated group. Similarly, the viral load in the lower respiratory tract was significantly lower in vaccinated animals compared to mock-vaccinated animals (Figure 3O). To evaluate the efficacy of the vaccine, we also attempted to isolate virus from lung tissue of challenged animals. As expected, we were able to easily isolate virus from tissue samples of mock-vaccinated animals, but no infectious virus was isolated from lung samples of sCPD9- and sCPD10-vaccinated animals (Figure 3P). However, these animals showed persistent viral load in the pharynx, indicating active viral replication (Figure 3N).

[0128] (Vaccination prevents major tissue damage) Histopathological examination of the lungs 3 days after vaccination confirmed striking differences in the intensity and distribution of bronchial and pulmonary lesions between animals infected with some of the encoded viruses and those infected with the WT virus (Figures 4A to 4N). However, animals within each group developed fairly similar lesions, indicating that the lungs of animals were effectively infected and that the disease outcome was homogeneous within each group. sCPD3 and sCPD4 viruses caused a relatively similar degree of bronchitis and pneumonia as the WT virus, whereas sCPD10 virus caused less severe pathology, and infection with CPD6 and sCPD9 viruses caused the weakest pathology and inflammation in both bronchial and pulmonary tissues (Figures 4A to 4N). More detailed histopathological examination was performed to characterize the protective capacity of the attenuated viruses. To this end, hamsters vaccinated with CPD6, sCPD9 or sCPD10 viruses and challenged 21 days later with WT SARS-CoV-2 were examined according to standard criteria for evaluation of the COVID-19 Syrian hamster model (Gruber et al.). Results showed that all three attenuated viruses caused overall smaller lung area and less severe bronchitis, pneumonia and other associated lesions (Figures 5A-5F, 5G-5V).

[0129] Figures 5A through 5V show lung histopathology in vaccinated Syrian hamsters from days 2 through 14 post-challenge.

[0130] Figures 5A to 5F show the histopathological assessment and scoring of lung pathology. Parameters evaluated: estimated percentage of affected lung area (Figure 5A), degree of bronchitis (Figure 5B), pulmonary inflammation (Figure 5C), endotheliitis (Figure 5D), edema (Figure 5E), and epithelial hyperplasia (Figure 5F). Scores and parameters in Figures 5B to 5F were categorized as none (0), minimal (1), mild (2), moderate (3), or severe (4).

[0131] Figures 5G to 5V are representative photomicrographs of formalin-fixed, paraffin-embedded, hematoxylin-eosin-stained lung tissues: bronchial epithelium (Figures 5G to 5J), air spaces (Figures 5K to 5V). Insets in Figures 5G to 5J show the distribution of SARS-CoV-2 RNA in Syrian hamster lungs 2 days post-challenge visualized by in situ hybridization. Red: viral RNA, blue: hemalaun counterstain. Insets in Figures 5O to 5R show blood vessels affected by endotheliitis 5 days post-challenge. Pathological changes are shown from representative animals that were mock vaccinated (Mock) or vaccinated with CPD6, sCPD9, or sCPD10. Bars: 50 μm (Figures 5G to 5V), 1 mm (inserts in Figures 5G to 5J), 100 μm (inserts in Figures 5O to 5R).

[0132] Of particular importance in COVID-19 pneumonia, we further scored the intensity of endotheliitis and perivascular and alveolar edema, which were also significantly reduced in vaccinated hamsters (Figures 5D and 5E). Furthermore, we compared bronchial and alveolar epithelial cell hyperplasia as evidence of tissue regeneration after parenchymal injury. Again, signs of the need for tissue repair were largely absent in hamsters vaccinated with one of the three recoded viruses (Figures 5F and 5O-5R). These observations further support the notion that all three attenuated viruses protect hamsters from extensive tissue damage upon subsequent challenge with WT virus.

[0133] In situ hybridization was used to examine the protective effect of vaccination 2 days after challenge, when the viral load in the lung tissue of hamsters peaks (Osterrieder et al.). The results showed that no signal for SARS-CoV-2 RNA was detected in the lungs of vaccinated hamsters, whereas a strong positive signal was detected in the lungs of mock-vaccinated and challenged hamsters (Figure 5G to 5J insets). This is consistent with the viral load in the lungs of vaccinated hamsters determined by RT-qPCR and virus titration assays (Figure 3K to 3P).

[0134] (Vaccination induces high levels of neutralizing antibodies) SARS-CoV-2 neutralizing antibodies were quantified in the sera of vaccinated hamsters by serum virus neutralization assay (Figure 6).

[0135] In this context, Figure 6 shows the titers of SARS-CoV-2 neutralizing antibodies in vaccinated Syrian hamsters. SARS-CoV-2 neutralizing antibodies were quantified in sera from mock-vaccinated hamsters and hamsters vaccinated with CPD6, sCPD9, and sCPD10 after challenge with WT virus. Serum from naive hamsters was used as a negative control. The dashed line indicates the detection limit of the assay.

[0136] Analysis showed that mock-vaccinated hamsters had moderate to high titers of neutralizing antibodies as early as day 5 after challenge with WT virus (Figure 6). This indicates that humoral immunity mounts a robust response very quickly after virus infection. Furthermore, in hamsters previously vaccinated with CPD6, sCPD9, or sCP10 viruses, neutralizing titers after challenge were very high and surprisingly stable (Figure 6). Because the highest dilution we tested was 1:512, we estimate that individual animals had even higher neutralizing antibody titers.

[0137] Attenuation of sCPD9 virus in Roborovski dwarf hamsters Because the mutant viruses sCPD9 and sCPD10 replicated with delayed kinetics in Vero E6 cells compared to the parent viruses and did not produce visible plaques, the inventors were initially unable to generate virus stocks of the two virus mutants at sufficiently high titers. This drawback led the inventors to use a high virus dose (1 × 10 5 We were unable to compare the pathogenicity of these two viruses to that of the parental viruses in Syrian hamsters infected with 10000000000000000 focus forming units [FFU] / animal. To better evaluate the degree of attenuation of our lead attenuated vaccine candidate, sCPD9, we decided to study its pathogenicity in Roborovskii dwarf hamsters (Phodopus roborovskii), a rodent species highly susceptible to severe COVID-19-like disease (Trimpert et al., 2020). When infected with WT SARS-CoV-2, Roborovskii dwarf hamsters developed fulminant clinical symptoms 2–4 days after infection, exhibiting rapid weight loss, significant hypothermia, signs of respiratory distress, and death (Trimpert et al.).

[0138] Roborovski dwarf hamsters were randomly assigned to two groups: 12 hamsters were mock infected and 30 hamsters were infected with 1 × 10 5 Hamsters were infected with FFU of mutant sCPD9 virus. Body weight and body temperature were recorded daily during the experimental period (21 days). On average, the body weight of hamsters infected with sCPD9 was barely reduced (3.4%) on the first day, but had already started to increase by the second day and continued to increase steadily until the end of the experiment (Figure 7A).

[0139] Figures 7A-O show that the SARS-CoV-2 mutant sCPD9s is strongly attenuated in Roborovski dwarf hamsters. Figure 7A shows the weight change in mock-infected (n=12) and sCPD9-infected (n=30) hamsters. Data are presented as mean ± SD.

[0140] Figure 7B shows daily body temperatures in mock-infected (n=12) and sCPD9-infected (n=30) hamsters. Data are presented as mean ± SD.

[0141] FIG. 7C shows the viral load in the upper respiratory tract (oropharyngeal swabs) and lower respiratory tract (lungs) 3 days after infection, and infectious viral particles detected in 50 mg of lung tissue (lung titer).

[0142] Figures 7D-7N show histopathological evaluation of infection with sCPD9 (Figures 7D-7H) or SARS-CoV-2 mutant (WT; Figures 7I-7N) in lung tissues 3 days after infection. Mild inflammatory lesions were observed in the left lung lobe (Figure 7D). The bronchial lobe had mostly normal columnar epithelium (Figure 7E) with only occasional mild bronchitis (Figure 7F); neutrophils (black arrows); alveolar septa showed a mild increase in macrophages and few neutrophils (Figure 7G), along with small areas of obvious pneumonia with macrophages (white arrows), neutrophils and alveolar epithelial cell necrosis (Figure 7H); normal pulmonary blood vessels (Figure 7I). Typical lung of a hamster infected with WT virus (Figure 7J); necrotizing pustular bronchiolitis with early hyperplasia of bronchiolar epithelial cells (Figure 7K); intrabronchiolar cellular debris (hash); infiltrating neutrophils (black arrows). Bronchointerstitial pneumonia (Figure 7L) with necrosis of alveolar epithelial cells, infiltration of macrophages and neutrophils (Figure 7M, black arrows) and alveolar edema (Figure 7M, asterisks). Endotheliitis with mild to moderate edema around blood vessels (arrowheads) (Figures 7N and 7O, asterisks). Scale bars: 1 mm (Figures 7D and 7J); 50 µm (Figures 7E, 7G, 7K and 7L); 100 µm (Figures 7I and 7N); 20 µm (inserts 7F, 7H, 7M and 7O).

[0143] Individual hamsters showed a maximum weight loss of 8.0% or a maximum weight gain of 3.6% on day 1 postinfection. Mock-infected hamsters did not gain weight during the first 3 days after mock infection, but gained weight rapidly on day 4 and steadily thereafter. Daily weight gain was comparable between the two groups, and both groups showed similar weight distribution at the end of the experiment on day 21 (Figure 7A). For mock-infected hamsters, the average daily weight shows a greater variability, likely due to the smaller group size. More importantly, no reduction in body temperature (Figure 7B) or other clinical signs indicative of severe disease, such as forced breathing or lethargy, were observed in any of the hamsters vaccinated with the sCPD9 virus (Trimpert et al.). Three animals from each group were euthanized on day 3 postinfection, and RT-qPCR analysis confirmed that the sCPD9 virus was replicating in the upper and lower respiratory tracts of infected animals (Figure 7C). Virus reisolation from lung samples was successful in 2 / 3 of infected sCPD9 animals (Fig. 7B).

[0144] (Strong attenuation of sCPD9 virus in Roborovski dwarf hamsters confirmed by lung histopathology) To evaluate the lung pathology induced by sCPD9 infection, three infected Roborovski dwarf hamsters were euthanized on day 3 postinfection. In these animals and in previous studies, 1 × 10 5Lungs from Roborovski dwarf hamsters euthanized 3 days after infection with FFU WT SARS-CoV-2 variant B.1 were used (Trimpert et al.). Detailed histopathological examination was performed after infection with WT SARS-CoV-2 variant B.1 (Figures 7D-7O). Virological parameters confirmed productive viral lower respiratory tract infection in approximately two-thirds of the animals (Figure 7C), but the histology of these animals was much milder compared to WT SARS-CoV-2 infection (Figures 7D-7O). In Roborovski dwarf hamsters infected with sCPD9 virus, mild inflammatory lesions were only observed in small areas of the lungs (Figure 7D). Of note, pulmonary vessels did not show endotheliitis. In contrast, lungs from Roborovski dwarf hamsters infected with WT SARS-CoV-2 showed widespread inflammation involving 70% of the lung tissue (Figure 7J). Detailed analysis revealed that bronchitis, pneumonia, and endotheliitis were significantly reduced in sCPD9-infected animals (Figs. 7E to 7I) compared with WT-infected animals (Figs. 7K to 7O).

[0145] (discussion) Several vaccines have demonstrated efficacy in large-scale Phase 3 clinical trials and have been approved for human use. (Baden et al., 2021; Dagan et al., 2021; Ella et al., 2021; Emary et al., 2021; Gao et al., 2020; Logunov et al., 2021; Solforosi et al., 2021; Voysey et al., 2021b; Wang et al., 2020; Zhang et al., 2021; Zhu et al., 2020; Zimmer et al., 2021). Despite considerable progress, the majority of the world's population remains unvaccinated. As of December 12, 2021, more than 4.4 billion vaccine doses have been administered worldwide. However, there are significant disparities in vaccination progress between high-income and low-income countries. (Zimmer et al., 2021). As a result, the COVID-19 pandemic continues to rage, destroying people's lives in different parts of the world. To mitigate the social and economic burden of the pandemic, safe, effective and affordable vaccines need to be developed, manufactured and administered in billions.

[0146] The most widely administered vaccines include the mRNA vaccines developed by Pfizer-BioNTech (BNT162b2, Comirnaty) and Moderna (mRNA-1273), the Gamaleya Institute (Sputnik V), the University of Oxford-AstraZeneca (AZD1222, Vaxzevria or Covishield) and Janssen-Johnson & Johnson (Ad26.COV2.S), as well as inactivated virus vaccines developed by The Beijing Institute of Biological Products-Sinopharm (BBIBP-CorV), Sinovac Biotech (CoronaVac, or PiCoVacc), Wuhan Institute of Biological Products-Sinopharm (unnamed), and the Indian Council of Medical Research-Bharat Biotech (Covaxin) (Zimmer et al., 2021).

[0147] Although mRNA-based vaccines have not been approved for human use so far, they are the most effective and, to date, the safest of the approved COVID-19 vaccines. They have shown approximately 95% efficacy in preventing symptomatic disease, including severe disease, against the original Wuhan virus, with few severe side effects in various clinical trials and real-world settings (Baden et al. 2021; Haas et al., 2021; Hall et al., 2021). The drawbacks of mRNA vaccines are their high cost and the requirement for cold storage (Comirnaty), which makes them unaffordable and logistically impractical for many low-income countries.

[0148] Vaxzebria is the most widely administered adenovirus-based vaccine (Zimmer et al., 2021). The vaccine's efficacy in preventing symptomatic COVID-19 is considerably lower than that of mRNA vaccines, at around 75%, but its efficacy against severe disease and hospitalization is 100% (Voysey et al. 2021a). The main advantages of this vaccine are its low cost and its long-term stability at standard refrigeration temperatures (2-8 °C). However, in rare cases, the vaccine can cause the formation of diffuse thrombi with a dramatic reduction in platelet counts (Greinacher et al., 2021). As a result, adenovirus-based vaccination has been suspended, restricted, or completely stopped in many countries, as the adenovirus vector is suspected of triggering rare but severe side effects.

[0149] Preliminary reports from various clinical trials have shown that inactivated virus vaccines are 50-80% effective in preventing COVID-19 caused by the WT virus (Mallapaty, 2021). The vaccine also appears to be safe, as no serious side effects have been observed to date. Inactivated virus vaccines are being widely administered in several countries, including China. The World Health Organization (WHO) has approved BBIBP-CorV and CoronaVac for emergency use. (Mallapaty, 2021).

[0150] With the exception of inactivated virus vaccines, all currently licensed vaccines are based solely on the viral spike glycoprotein. SARS-CoV-2 is rapidly evolving. During the course of the pandemic, many viral variants have accumulated identical or similar mutations in the spike protein through convergent evolution. Recent evidence suggests that some of the acquired mutations may compromise the efficacy of licensed vaccines. Viruses of lineages B.1.1.7 (UK), B.1.1.28.1 (Brazil), B.1.1.28.3 (Philippines), B.1.525 (West Africa), and B.1.526 (USA), but especially viruses of lineages B.1.351 and B.1.1.529 (South Africa), have been associated with reinfection and vaccine breakthrough. (Abu-Raddad et al., 2021; Madhi et al., 2021). Widespread vaccination could also increase selection pressure and lead to the emergence of SARS-CoV-2 escape variants that cannot be efficiently controlled by vaccination. The emergence of such variants would reduce the efficacy of spike-based vaccines and require vaccine revision. Indeed, three of the largest manufacturers of licensed vaccines, Pfizer, AstraZeneca, and Moderna, are developing updated vaccines targeting the B.1.351 variant (ClinicalTrials.gov Identifier: NCT04785144; Zimmer et al., 2021).

[0151] An important advantage of live attenuated virus vaccines is that they replicate within the vaccinated individual and therefore stimulate immune responses against the entire ensemble of viral antigens, not just the major surface glycoproteins. Thus, improved live attenuated virus vaccines should, in theory, be better able to protect against a wide variety of recently emerged viral variants such as B.1.1.7, B.1.351, B.1.1.28.1, B.1.617.2, and B.1.1.529. Preliminary data suggest that a single vaccination with sCPD9 variants indeed induces robust protection. Furthermore, live attenuated virus vaccines can be administered intranasally, thus inducing mucosal immune responses directly at the site of viral entry. This may better protect target tissues from infection and further limit disease severity and viral shedding. For similar reasons, the University of Oxford and CanSino Biologics have initiated clinical trials to investigate whether intranasal administration of the Vaxzevria and Ad5-nCoV vaccines, respectively, could enhance protection and limit viral transmission (ClinicalTrials.gov Identifiers: NCT04816019, NCT04840992).

[0152] Live attenuated virus vaccines also have many practical advantages that could enable them to play an important role in pandemic response in countries where price, distribution and administration are key factors.An important advantage of an improved live virus vaccine against SARS-CoV-2 is the relative ease of producing, storing, distributing and administering such a vaccine.

[0153] The virus stocks of sCPD9 and sCPD10 viruses prepared by the present inventors initially had relatively low titers (2 × 10 5 FFU / ml, but the inventors were able to significantly increase the virus yield (1 × 10 7They found that it was possible to produce large amounts of vaccine in a short time following standard procedures, since SARS-CoV-2 replicates rapidly in Vero E6 cells to relatively high titers. Moreover, it was possible to produce large amounts of vaccine in a short time using a relatively low viral load (2 × 10 4 Experiments have shown that vaccination with 1000 mg of 10000 FFU / animal can fully protect Syrian hamsters against WT challenge. The optimal vaccine dose is likely to be a relatively low dose for humans. This means that a large number of vaccine doses can be produced very quickly and cheaply from a small number of cells. Vero cells are easy to culture and have been approved by many regulatory agencies for the production of various human vaccines, including the recent inactivated SARS-CoV-2 vaccine (Ella et al., 2021; Zhang et al., 2021). In addition, the biosafety level for highly attenuated viruses has been reduced from the current level 3 to level 2, making the production and transportation of such vaccines even easier. Frozen or lyophilized viruses are stable and only moderately reduce viral titers upon thawing or reconstitution. A single-dose vaccine against SARS-CoV-2 would offer distribution advantages over a two-dose vaccine in mass vaccination campaigns against the COVID-19 pandemic.

[0154] The main drawback of vaccines with live attenuated viruses is the theoretical risk of reversion to virulence. For example, the oral poliovirus vaccine contains 51 mutations, only five of which are attenuating. As a result, this vaccine is prone to reversion and can, in rare cases, revert to hypervirulence and cause paralytic poliomyelitis in vaccinees (Kew et al.). In contrast, recoded viruses contain hundreds of nucleotide mutations, but many of these mutations are attenuating. Attenuation of CPD viruses is thought to occur through the addition of small defects that each "bad" codon pair has on gene expression of the recoded genes. Due to the large number of mutations, CPD viruses are generally genetically stable and unlikely to revert to virulence. However, it cannot be denied that vaccination of a large number of individuals increases the chance of reversion. Another theoretical possibility is that recoded variants can adapt to humans and eventually become prevalent in human populations.

[0155] None of the current vaccine candidates are modified live or attenuated virus vaccines (WHO). Of the three basic types of viral vaccines, modified live virus vaccines are considered the most effective in healthy individuals and are generally superior to inactivated, vectored, and subunit vaccines. (Bazin, 2003; Kusters and Almond, 2008; Lauring et al.). Traditionally, modified live virus vaccines have been prepared empirically by an iterative process of passaging of pathogenic viruses in cell cultures and / or experimental animals (Kusters and Almond, 2008; Lauring et al. (Kusters and Almond, 2008; Lauring et al.)). Attenuation by CPD is an alternative to approaches that rely on serendipity as the main attenuation principle: attenuating mutations are purposefully introduced into the viral genome, based on rational design (Eschke et al. (Eschke et al.,2018; Groenke et al.,2020;Osterrieder and Kunec,2018; Trimpert et al. In CPD, viral genes are recoded with statistically insufficient codon pairs, which results in perturbation of gene expression and attenuation of the recoded virus (Coleman et al., 2008; Groenke et al., 2020). Codon pair bias is a species-specific feature, but phylogenetically closely related species have similar codon pair bias. For example, all mammals have approximately the same codon pair bias (Kunec and Osterrieder, 2016). Therefore, a mutant virus with a codon pair deoptimized genome sequence based on human codon pair bias should be attenuated not only in human cells but also in all permissive cells derived from different mammalian species (Groenke et al., 2020). Results are consistent with this assumption, as the recoded SARS-CoV-2 mutant was attenuated not only in cells derived from African green monkeys but also in cells derived from two different rodents.

[0156] Because CPDs significantly alter genome sequences, further phenomena may be involved in the attenuation of recoded viruses. For example, recoding usually increases the number of CpG and UpA dinucleotides in the recoded sequence, which results in the activation of the host's natural defense mechanisms (Odon et al., 2019). In addition, recoding may disrupt unknown cis-regulatory regions, thereby suppressing viral replication (Song et al.). Alternative attenuation strategies based on large-scale recoding of viral genomes take advantage of this knowledge and intentionally increase the number of CpG or UpA dinucleotides, rare codons, or "near-stop" codons in the viral genome (Lauring et al., 2012;Osterrieder and Kunec, 2018).

[0157] The data presented here show that a single vaccination with CPD6, sCPD9 or sCPD10 viruses protects vaccinated hamsters against disease development after challenge infection with pathogenic SARS-CoV-2 (Figures 3A-7O). This was evidenced by histological evaluation of lung pathology after vaccination with CPD6, sCPD9 and sCPD10, which showed virtually no correlation of damage induced by challenge virus replication. Pathological lesions were only detected in a small portion of lung tissue, generally showing only mild bronchitis, pneumonia and edema (Figures 5A-5C). Similarly, epithelial hyperplasia was very limited in vaccinated animals at late times after challenge. This lack of cellular repair and regeneration in the lungs supports the conclusion that all three vaccine candidates protected hamsters from the associated tissue damage due to challenge virus infection (Figure 5F).

[0158] The amount of viral RNA detected in the lungs of challenged animals was very low on days 2, 3, and 5 post-challenge, and more importantly, we were unable to isolate replicating virus from the lungs of animals following challenge infection (Figures 3K to 3P). This data indicates that the recoded virus was unable to protect the upper respiratory tract from reinfection with pathogenic WT virus, but did very well to protect the lower respiratory tract from infection.

[0159] We speculate that a single vaccination with a live attenuated modified virus vaccine should provide protective immunity against infection with pathogenic SARS-CoV-2, since we could not detect any infectious viral particles in the lungs of any of the vaccinated animals. Animals vaccinated with CPD6-, sCPD9- and sCPD10-vaccines did not lose weight after challenge, and histopathological evaluation of the lungs indicates a clear protective effect of all three vaccine candidates tested. The same data suggested that a two-dose regimen may also be feasible if development of more durable immunity is desired.

[0160] Because sCPD9 was the most attenuated mutant virus tested, yet still induced strong protective immunity against disease induced by virulent viruses, we decided to test the safety of this virus in a recently identified non-transgenic rodent model of COVID-19, the Roborovski dwarf hamster (Trimpert et al.). In this experiment, the sCPD9 virus proved to be highly attenuated, as no signs of illness were observed in any of the 30 Roborovski dwarf hamsters and only minimal pathological changes occurred in the lungs of infected animals.

[0161] The durability of protective immunity in vaccinated animals requires further study. A known limitation of this study is that we did not evaluate the extent to which live attenuated virus is transmitted from infected animals to sentinels. Although Syrian hamsters readily transmit pathogenic SARS-CoV-2 to cohabiting uninfected contact animals during the first few days after infection (Sia et al.), we observed no clinical signs of disease and limited histological signs in naturally infected contact animals. In our opinion, the Syrian hamster model is of limited utility for studying the transmission of attenuated viruses and disease outcomes of natural infection. Therefore, for the purpose of this study, we focused on characterizing the attenuation of lead vaccine candidate sCPD9 by experimental mass infection of a species highly susceptible to severe COVID-19-like disease. Furthermore, we are preparing experiments in ferrets, a well-known model for studying respiratory virus transmission (Kutter et al.) to answer questions regarding the spread of the recoded virus as well as its effectiveness against recent SARS-CoV-2 variants of concern in the near future.

[0162] In summary, among all the engineered live attenuated viruses tested, sCPD9 provided the best balance of attenuation, safety, immunogenicity, and protective efficacy.We believe that the safety, immunogenicity, and vaccine efficacy of this candidate virus should be investigated in a variety of animal and primate species, and ultimately in humans.

[0163] (Details of the experimental model and subjects) (Cells and viruses) Vero E6 (ATCC CRL-1586) and BHK-21 (ATCC CCL-10) cells were cultured at 37 °C and 5% CO2 in minimum essential medium (MEM) containing 10% fetal bovine serum (PAN Biotech), 100 IU / ml penicillin G, and 100 μg / ml streptomycin (Carl Roth). Parental SARS-CoV-2 and mutant SARS-CoV-2 were propagated in Vero E6 cells. The ancestral SARS-CoV-2 mutant B.1 (SARS-CoV-2 / Munchen-1.1 / 2020 / 929) (Wolfel et al.) was used as the challenge virus. Virus stocks were stored at -80 °C prior to experimental infection.

[0164] (Ethics statement) In vitro and animal experiments were performed under biosafety conditions in the BSL-3 facility of the Institut fur Virologie, Free University Berlin, Germany. All animal experiments were approved by the Landesamt fur Gesundheit und Soziales, Berlin, Germany (permit number 0086 / 20) and were performed in accordance with the relevant national and international guidelines for the care and humane use of animals.

[0165] (Livestock) Animal experiments were performed in a BSL-3 certified facility. Six-week-old Syrian hamsters (Mesocricetus auratus; breed RjHan:AURA) were purchased from Janvier Labs and kept in individually ventilated cages (IVC; Tecniplast) with extensive enrichment (Carfil). Five- to seven-week-old Roborovskii dwarf hamsters (Phodopus roborovskii) were purchased from a single breeding facility through the German pet trade. They were housed in groups of six in the IVC. The animals had free access to food and water and were allowed to acclimate to this environment for 7 days before infection. In both experiments, cage temperatures were 22–24 °C and relative humidity was 40–55%.

[0166] (Infection experiment) To examine attenuation of Recording virus variants and vaccine protection in Syrian hamsters, male and female hamsters were randomly assigned to groups of 15 with seven or eight male and female animals in each group. In the first study, hamsters were mock vaccinated or vaccinated with CPD6, sCPD3, or sCPD4 viruses. Hamsters were either mock vaccinated with 60 μl of medium from uninfected Vero E6 cells or vaccinated with 1 × 10 60 μl of ... 5 In a second study, hamsters were mock vaccinated or vaccinated with sCPD9 or sCPD10 mutant viruses. Vaccination was performed as above, but vaccinated hamsters received 1 × 10 of the mutant viruses. 4 Twenty-one days after vaccination, hamsters were challenged with 1 × 10 5 FFU, 60 μl, was administered intranasally. During the experiment, all hamsters were monitored twice daily for clinical signs of illness, and body temperature and body weight were recorded. Hamsters that lost more than 10% of their body weight during the 72 h period were euthanized according to the animal use protocol. Three hamsters per group were euthanized on day 3, 23, 24, 26, and 35 post-infection (days 2, 3, 5, and 14 post-challenge) (Nakamura et al., 2017). Blood, oropharyngeal swabs, and lungs (left and right) were collected for virus titration, RT-qPCR, and / or histopathology. All organs were preserved in 4% formalin for subsequent detailed histopathology examination.

[0167] To evaluate the pathogenicity of the encoded mutant virus sCPD9 in Roborovski dwarf hamsters, animals were randomly divided into two groups, with 60% of each group being female. Twelve hamsters were mock infected with cell culture medium and 30 hamsters were infected with 1 × 10 5Hamsters were infected with FFU of mutant virus sCPD9. Anesthetized hamsters were infected as described above, except that the inoculum was 20 μL. Infected hamsters were monitored twice daily for clinical signs of infection. Body weights and temperatures were recorded daily. Three hamsters were sacrificed on day 3 postinfection, and virological and histological parameters of infection were determined on day 3 postvaccination. Infected hamsters were observed twice daily for clinical signs of infection, and body weights and temperatures were recorded once daily. Three hamsters from each group were euthanized on day 3 postinfection, and virological and histological parameters of infection were determined by molecular and virological assays as described above.

[0168] (method details) (Decoding the SARS-CoV-2 genome) Since we planned to generate SARS-CoV-2 mutants via TAR cloning, we designed the recoded SARS-CoV-2 fragment to be fully compatible with the available reverse genetics system of SARS-CoV-2 (Thi Nhu Thao et al.). All indicated sequence positions in the SARS-CoV-2 genome correspond to the SARS-CoV-2 reference sequence NC_045512.2.

[0169] The coding sequence of SARS-CoV-2 was recoded by CPD (Coleman et al.). CPD creates codon pair combinations that are not statistically present in the protein-coding sequence of the viral host by rearranging the positions of synonymous codons in the recoded viral sequence. Because the goal was to create SARS-CoV-2 mutants that should be attenuated for humans, we recoded the SARS-CoV-2 sequence using the codon pair scores (CPS) calculated for human protein-coding genes (Groenke et al.).

[0170] Subgenomic fragments were individually recoded using only existing codons present in each fragment. Creation of novel transcriptional regulatory sequences (TRS; ACGAAC) and EagI restriction sites (CGGCCG) in the recoded sequences was prohibited. Creation of novel TRS sites was prohibited to avoid causing transcriptional aberrations, and the EagI restriction site was reserved for release of the subgenomic fragments from the plasmid.

[0171] Two genomic sequences containing essential cis-acting RNA elements were excluded from recoding. The first sequence excluded from recoding was a 503 bp sequence (NC_045512.2; nucleotides 13,451-13,953) containing a frameshift stimulatory element (FSE). In the SARS-CoV-2 genome, the FSE resides between the overlapping genes ORF1a and ORF1b and is essential for -1 programmed ribosomal frameshifting. A 106 bp sequence (NC_045512.2; nucleotides 21,505-21,610) containing the transcriptional regulatory sequence (TRS) of the spike gene was also omitted from recoding. The TRS of the spike gene is located at the end of ORF1b of the SARS-CoV-2 genome. The non-recoded sequences are carried by fragments 6 and 9, respectively, of the yeast-based SARS-CoV-2 reverse genetics system (Figure 1A to 1D). The sequences of the recoded DNA fragments have been deposited in the NCBI GenBank database (MZ064531-MZ064546).

[0172] (Recovery of mutant SARS-CoV-2) Infectious virus was recovered directly from BAC / YAC SARS-CoV-2 DNA in Vero E6 cells or from in vitro transcribed viral RNA in BHK-21 cells. To rescue viral progeny from DNA, the cytomegalovirus immediate early promoter was inserted upstream of the SARS-CoV-2 genome in a BAC / YAC SARS-CoV-2 clone (Almazan et al., 2000; Noskov et al., 2002). To facilitate infectious virus recovery and monitor transfection efficiency, we constructed a dual expression plasmid, pVITRO2-EGFP-N, expressing EGFP and SARS-CoV-2 nucleocapsid protein from two different eukaryotic promoters. To recover infectious virus, Vero E6 cells were grown to 95% confluence in T25 cell culture flasks and transfected with 4 μg of BAC / YAC DNA and 1 μg of pVITRO2-EGFP-N plasmid using Xfect Single Shot Transfection Reagent (Takara Bio Inc.).

[0173] To recover infectious virus in BHK-21 cells, viral RNA was produced by in vitro transcription using the mMESSAGE mMACHINETM T7 Transcription Kit (Thermo Fisher Scientific). 10 μg of in vitro transcribed viral RNA and 1 μg of pVitro2-EGFP-N plasmid were mixed with 1 × 10 , suspended in 100 μl of OptiPro medium (Thermo Fisher Scientific). 6 The virus was mixed with BHK-21 cells and electroporated in a 2 mm gap cuvette by applying a single 140 V pulse for 25 ms with a Gene Pulser Xcell (Bio-Rad Laboratories). Electroporated BHK-21 cells were co-cultured with susceptible Vero E6. Recovered progeny virus was harvested from the cell culture medium and propagated on Vero E6 cells.

[0174] (Multi-stage growth kinetics) To assess the kinetics of viral growth, Vero E6 cells grown in 6-well plates were infected with parental or encoded viruses at a multiplicity of infection (MOI) of 0.01. After 2 h of incubation, the viral inoculum was removed and replaced with growth medium. Cell culture medium was harvested 6, 12, 24, 48, and 72 h postinfection, and viral titers were measured by focus-forming assay.

[0175] (Virus titration, indirect immunofluorescence, focusing assay) To measure virus titers as focus forming units (FFU) and foci size, Vero E6 cells grown in 12-well plates were infected with 100 μl of serial 10-fold dilutions of virus. To measure virus titers in the lungs, 50 mg of lung tissue was first homogenized in a bead mill (Analytic Jena) and the homogenate was serially diluted. After 2 h of incubation, the virus inoculum was removed and the cells were overlaid with MEM containing 0.6% microcrystalline cellulose Avicel (FMC BioPolymer). 48 h after infection, cells were fixed with 4% formalin, permeabilized with 0.1% Triton-X 100, and blocked with 3% BSA in PBS. Cells were then incubated with monoclonal mouse anti-SARS-CoV-2 nucleocapsid antibody (kindly provided by Sven Reiche, Friedrich Loeffler Institute, Riems, Germany) for 1 h and goat anti-mouse IgG-Alexa Fluor 568 secondary antibody (Invitrogen) for 45 min. To examine the cell-to-cell spread of mutant viruses within cells, images of foci from 30 randomly selected infected cells were taken at 50x magnification using an inverted fluorescence microscope (Axiovert S100, Zeiss). Foci areas were then calculated from their diameters using ImageJ software (Schneider et al.).

[0176] (Histology and in situ hybridization) For histopathology and in situ hybridization (ISH) to localize viral RNA, the left lung lobe was carefully removed and immersion fixed in buffered 4% formalin (pH 7.0) for 48 h. Sections were stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) reactions, followed by blinded microscopic evaluation by a board-certified veterinary pathologist (KD, ADG) (Gruber et al., 2020). For ISH, ViewRNA was used. TM The ISH Tissue Assay Kit (Invitrogen by Thermo Fisher Scientific, Darmstadt, Germany) was used according to the manufacturer's instructions with minor adjustments. A probe for detecting SARS-CoV-2 N gene RNA (NCBI database NC_045512.2, nucleotides 28,274 to 29,533, assay ID: VPNKRHM) was used. Lung sections 2 μm thick mounted on adhesive slides were defatted with xylol and dehydrated in graded ethanol. The tissue was incubated at 95 °C for 10 min, followed by protease digestion for 20 min. The sections were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and hybridized with the probe. Hybridization of the amplifier and label probes was performed according to the manufacturer's instructions using Fast Red as the chromogen. Sections were counterstained with hematoxylin for 45 s, washed in tap water for 5 min, and then transferred to Rotif. (R)-Mount Fluor-Care DAPI (4,6-diaminidino-2-phenylindole; Carl Roth). An irrelevant probe for the detection of streptococcal pneumolysin was used as a control for sequence-specific binding. HE-, PAS-stained and ISH slides were analyzed and photographed using an Olympus BX41 microscope equipped with a DP80 Microscope Digital Camera and cellSensTM Imaging Software, Version 1.18 (Olympus Corporation, Munster, Germany). For low-magnification overviews, slides were automatically digitized using an Aperio CS2 slide scanner (Leica Biosystems Imaging Inc., Vista, CA, USA) and image files were generated using IMAGE Scope software (Leica Biosystems Imaging Inc.).

[0177] (RNA isolation and RT-qPCR) RNA was extracted from 25 mg of lung homogenate and oropharyngeal swabs using the innuPREP Virus RNA kit (Analytik Jena AG). SARS-CoV-2 RNA copies were quantified using the NEB Luna Universal Probe One-Step RT-qPCR kit (New England Biolabs) on a StepOnePlus RealTime PCR System (Thermo Fisher Scientific) as previously described (Corman et al., 2020).

[0178] (Serum virus neutralization assay) Neutralization assays were performed on subconfluent monolayers of Vero E6 cells in 96-well plates using two-fold serial dilutions (1:4 to 1:512) of complement-inactivated (56°C, 2 h) hamster sera. 50 FFU of SARS-CoV-2 was added to each well and incubated at 37°C for 72 h, followed by fixation in 4% formalin for 24 h and staining with crystal violet (0.75% in water). Serum neutralization was considered effective in wells that did not show a cytotoxic effect, and the last effective dilution was counted.

[0179] (Western Blotting) Confluent Vero E6 cells grown in 6-well plates were infected at an MOI of 0.1. After 48 h, infected cells were frozen at -80°C, resuspended in cell culture supernatant, and lysed in 2× Laemmli buffer containing protease inhibitors (Roche). Cell lysates were incubated at 95°C for 30 min, and proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) under reducing conditions. Proteins were transferred to PVDF membranes by semi-dry blotting. Membranes were blocked with 3% whey in PBS containing 0.05% Tween-20 for 1 h at room temperature and incubated with primary antibodies for 16 h at 4°C. Nuclear proteins were detected with mouse monoclonal anti-N antibody (see above) and spike proteins with mouse monoclonal anti-S2 antibody (1A9, GeneTex). Horseradish peroxidase-conjugated anti-mouse IgG (1:2000, Merck) was used as secondary antibody. Antibody binding was visualized using chemiluminescent Amersham ECL prime western blotting detection reagent (Thermo Fisher Scientific).

[0180] (Quantification and statistical analysis) Statistical tests were performed using GraphPad / Prism. Statistical details of the experiments are described in the figure legends. Exact values ​​of n and statistical tests are given in the figure legends. P values ​​are indicated in the figures. Data are presented as mean ± SD.

[0181] Second Exemplary Embodiment

[0182] (result) As described in connection with the first exemplary embodiment, the inventors have constructed a series of attenuated SARS-CoV-2 live vaccine candidates by extensive recoding of the SARS-CoV-2 genome (Trimpert et al., 2021b). The SARS-CoV-2 genome was modified by codon pair optimization (CPD), an approach that has led to rapid and efficient attenuation of various RNA (Coleman et al., 2008; Groenke et al., 2020) and DNA viruses (Eschke et al., 2018; Khedkar et al., 2018). CPD is based on the observation that in protein-coding sequences, the frequency of occurrence of codon pairs is significantly lower or higher than the statistically expected frequency (Gutman et al., 1989). In CPD, viral genes are designed to be recoded to contain codon pairs that are statistically underrepresented (suboptimal) in the host, leading to attenuation of the recoded virus (Coleman et al., 2008; Groenke et al., 2020; Eschke et al., 2018). During the process of recoding by CPD, the positions of synonymous codons in the recoded sequence are exchanged such that the frequency of the codon pairs increases. Because CPD only exchanges the positions of synonymous codons in the recoded sequence, the recoded attenuated live virus contains exactly the same antigens as the pathogenic parent virus. The introduced genetic changes reduce protein production from the encoded genes and also decrease the replicative fitness of the mutant viruses (Groenke et al.). Due to the conserved antigenic identity and remaining replicative capacity, the recoded attenuated viruses are able to fully engage the host immune system and elicit a strong immune response (Coleman et al., 2008; Groenke et al., 2020; Eschke et al., 2018).

[0183] In a first exemplary embodiment and in a previous study, the inventors evaluated attenuated virus candidates and showed that some of them were strongly attenuated, induced a strong immune response, and protected Syrian hamsters from challenge with ancestral wild-type (WT) SARS-CoV-2 (Trimpert et al., 2021b). In that study, the inventors also showed that the lead attenuated virus candidate, sCPD9, was strongly attenuated in Roborovski dwarf hamsters (Phodopus roborovskii), which are highly susceptible to severe COVID-19-like disease (Trimpert et al.). Despite this hamster species' high susceptibility, sCPD9 did not cause clinical disease or significant lung pathology (Trimpert et al., 2021b).

[0184] The sCPD9 virus is also highly attenuated in vitro, growing to titers 100-fold lower than the WT virus under normal conditions on Vero E6 cells (Trimpert et al., 2021b). Nevertheless, high titers (1 × 10 7 It is possible to generate virus stocks of 1000 infectious viral particles / ml. In this second embodiment, we evaluated the immunogenicity and protective efficacy of sCPD9 virus against the ancestral B.1 and two VOCs, B.1.1.7 and B.1.351.

[0185] FIG. 10 shows that the live attenuated virus vaccine candidate sCPD9s is strongly attenuated and protects Roborovski dwarf hamsters against challenge with B.1, B.1.1.7, and B.1.351 viruses.

[0186] Figure 10B shows the weight change of Roborovski dwarf hamsters after vaccination. The thick black line indicates the median, and the thin lines indicate the quartiles. Figure 10C shows the viral load in the upper respiratory tract (oropharyngeal swabs) and lower respiratory tract (lungs) on day 3 post-vaccination, as well as the infectious viral load (lung titer) detected from 50 mg of lung tissue. Figure 10D shows the weight change of Roborovski dwarf hamsters after challenge with pathogenic B.1, B.1.1.7, or B.1.351 viruses. Figures 10E and 10F show the viral load in the upper respiratory tract (Figure 10E) and lower respiratory tract (Figure 10F) of animals on days 2, 3, 5, and 5 post-challenge. Figure 10G shows the infectious viral load detected from 50 mg of lung tissue on days 2, 3, and 5 post-challenge.

[0187] (Vaccination) Hamsters were randomly assigned to two groups, 12 and 30. On day 0, 12 hamsters were mock vaccinated and 30 hamsters were vaccinated with 1 × 10 5 On day 21, hamsters were inoculated intranasally with 1 × 10 focus-forming units (FFUs) of the B.1, B.1.1.7, or B.1.351 subtypes. 5 Plaque-forming units (PFU) were administered. Body weight and clinical signs were recorded daily throughout the experimental period (26 days). Three mice per group were euthanized at day 3 post-vaccination and days 2, 3, and 5 post-challenge to measure the extent of viral replication in various organs and to evaluate lung pathology (Trimpert et al., 2021b).

[0188] On average, sCPD9-vaccinated hamsters gained slightly less weight than mock-vaccinated hamsters 21 days after vaccination, but the difference was not significant (Figure 10B). No adverse reactions or clinical signs such as forced breathing or lethargy that would indicate severe disease were observed in either mock- or sCPD9-vaccinated hamsters. The attenuated sCPD9 virus replicated efficiently in the upper and lower respiratory tracts of sCPD9-vaccinated hamsters 3 days after vaccination (Figure 10C). Furthermore, we detected infectious virus in the lungs of two-thirds of sCPD9-vaccinated animals 3 days after vaccination (Figure 10C). Histopathological examination of the lungs of sCPD9-vaccinated hamsters 3 days after vaccination showed that sCPD9 was strongly attenuated, as only a few mild inflammatory lesions were detected in the lungs of vaccinated animals (Trimpert et al., 2021b).

[0189] (Challenge infection) Vaccination with sCPD9 virus induced robust protective immunity against challenge with all three SARS-CoV-2 variants. None of the vaccinated hamsters showed clinical symptoms of disease, and none of the groups lost weight after challenge (Figure S10D). In contrast, mock-vaccinated hamsters showed typical signs of severe COVID-19-like disease and began to lose weight 2 days after challenge, which continued until the end of the study (day 5 after challenge) (Figure S10D).

[0190] Virological evaluation of the challenge infections confirmed viral replication in the upper respiratory tract of infected animals on days 2, 3, and 5 after challenge (Figure 10E). Viral RNA was detected in the upper respiratory tract of many vaccinated animals, suggesting that the virus was still able to establish local infection regardless of the variant used in the challenge. Nevertheless, vaccination dramatically reduced the amount of viral genomic RNA in the lower respiratory tract of challenged hamsters (Figure 10F). Vaccinated hamsters had clearly lower viral loads than control animals on all days examined. Viral loads in the control group were 1-6-fold higher on day 2 and 4-5-fold higher on days 3 and 5 after challenge (Figure 10F). As expected, the variants replicated with unequal efficiency in the lungs of challenged hamsters. Hamsters infected with the ancestral SARS-CoV-2 variant B.1, which is antigenically identical to the sCPD9 virus, had the lowest viral loads, whereas hamsters infected with the B.1.351 virus had the highest viral loads.

[0191] To further evaluate the immunity established by vaccination, we attempted to isolate the challenge virus from the lungs of the animals in cultured Vero E6 cells. This evaluation showed sterile immunity in the lower respiratory tract of all subjects except one. While we were unable to isolate B.1 and B.1.1.7 viruses, we detected infectious and replication-competent virus in one hamster (one of three) in the B.1.351 group, but only immediately after challenge infection, on the second day after challenge (Figure 10G).

[0192] (Post-challenge histopathology) To determine the protective potential of vaccination with sCPD9 virus against COVID-19-like disease caused by the three different SARS-CoV-2 variants, lungs of challenged hamsters were subjected to detailed histopathological examination (Figures S1A-S1T).

[0193] Figures 11A-T show lung histopathology from mock / sCPD9 vaccinated and challenged Roborovski dwarf hamsters at days 2 (left column), 3 (middle column), and 5 (right column) post-challenge.

[0194] Figures 11A-11T are representative photomicrographs of hematoxylin and eosin (H&E)-stained, formalin-fixed, paraffin-embedded lung tissue from infected hamsters. sCPD9-vaccinated hamsters were challenged with B.1, B.1.1.7, or B.1.351 viruses on day 21 post-vaccination. Lungs from sCPD9-vaccinated hamsters challenged with the three different SARS-CoV-2 variants showed highly similar lung morphology. Bronchi at days 2 and 3 post-challenge had regular columnar bronchial epithelium (Figures 11A, 11C, 11F, 11H, 11K, and 11M). On days 3 (Figures 6B, 6G, and 6L) and 5 (Figures 6D, 6E, 6I, 6J, 6N, and 6O) postchallenge, alveoli showed mild to moderate infiltration by macrophages and a few neutrophils, with scattered mild alveolar edema (asterisks).

[0195] On the other hand, mock-vaccinated hamsters challenged with SARS-CoV-2 variant B.1 showed typical lesions of COVID-19 at all time points tested (Figures 11P-11T). After 2 days, the bronchial epithelium was flattened with necrosis of bronchial epithelial cells and necrotic cellular debris, and degenerated neutrophils and macrophages were present in the bronchial lumen (hash) (P). Over time, moderate to severe bronchointerstitial pneumonia was observed with necrosis of alveolar epithelial cells and massive infiltration of macrophages and neutrophils into the alveolar septa and alveolar spaces (Figure 11Q). Similarly, the bronchoalveolar epithelium was infiltrated with increasing numbers of neutrophils (arrows), and the lumen showed accumulation of cellular debris (hash) (Figure 11R). Five days after challenge, the lungs showed predominantly interstitial pneumonia with residual alveolar edema fluid (asterisks) (Figure 11S), neutrophils (Figure 11T, arrow), and increased macrophages with prominent foamy cytoplasm (Figure 11T, arrow). Scale bars indicate 50 μm (Figure 11A, Figure 11B, Figure 11D, Figure 11F, Figure 11G, Figure 11I, Figure 11K, Figure 11L, Figure 11N, Figure 11P, Figure 11Q, Figure 11S) and 20 μm (Figure 11C, Figure 11E, Figure 11H, Figure 11J, Figure 11M, Figure 11O, Figure 11R, Figure 11T).

[0196] As a result, all hamsters vaccinated with sCPD9 were fully protected from SARS-CoV-2-induced lung inflammatory damage, regardless of the variant used in challenge (Figures 11A-11T). In contrast, mock-vaccinated and B.1-infected hamsters developed severe lung pathology typical of experimental SARS-CoV-2 infection (Trimpert et al.). These observations support the conclusion that vaccination with sCPD9 virus protects hamsters from extensive tissue damage and fully protects them from COVID-19-like disease after challenge with three different SARS-CoV-2 variants.

[0197] (Serum neutralization after challenge infection) We quantified the ability to neutralize SARS-CoV-2 variants B.1, B.1.1.7, B.1.351, B.1.128.1, and B.1.617.2 by microneutralization assays of 17 hamster sera collected on days 2 and 3 post-challenge (Figure 12A).

[0198] Figure 12 shows the susceptibility of SARS-CoV-2 variants B.1, B.1.1.7 (alpha), B.1.351 (beta), B.1.128.1 (gamma), and B.1.617.2 (delta) to neutralization by antibodies in the serum of Roborovski dwarf hamsters vaccinated with sCPD9 vaccine.

[0199] Figure 12A shows neutralizing antibody titers of hamster sera collected on days 2 and 3 postchallenge. Geometric mean and SD of titers for each group are shown. Kruskal-Wallis test and Dunn's post hoc test showed that hamster sera neutralized virus variants B.1.351, B.1.128.1, and B.1.617.2 significantly less efficiently than variants B.1 or B.1.1.7 (*P=0.0264, **P=0.0012, ***P<0.001, ****P<0.0001).

[0200] Figure 12B shows the titers of neutralizing antibodies plotted as a function of challenge virus. Figure 12C shows the ratio of serum neutralizing titers plotted against different virus variants. This ratio indicates how well each hamster serum neutralizes the different virus variants. The ratio, geometric mean, and SD of the neutralizing titers of the individual sera are shown. The geometric mean is also indicated numerically at the top of the figure.

[0201] Sera from mock-vaccinated hamsters did not show neutralizing activity against all SARS-CoV-2 subtypes tested. Sera from sCPD9-vaccinated hamsters showed strong neutralizing activity against the B.1 and B.1.1.7 variants, with geometric mean titers of 873 and 1129, respectively, but low neutralizing activity against the B.1.351, B.1.128.1, or B.1.617.2 variants, with geometric mean titers of 243, 341, and 182, respectively (Figure 12A). It is expected that the challenge infection will boost the vaccine-induced immune response and produce antibodies that better neutralize the antigens of the respective challenge SARS-CoV-2 variants. Because we collected sera immediately after challenge, we did not expect to observe the induction of mutant-specific humoral responses. Nevertheless, to take into account possible challenge-induced effects, we also compared serum neutralizing activity as a function of challenge virus (Figure 12B). This comparison showed that none of the different challenge viruses had any discernible effect on the neutralizing activity of hamster sera. In other words, hamsters challenged with a certain variant, e.g., B.1.351, did not generate a better humoral response against the same variant within 3 days after challenge. Furthermore, there was no significant difference in the neutralizing activity of sera taken on the second or third day after challenge.

[0202] Vaccination with sCPD9 induced high levels of neutralizing antibodies that were effective not only against the ancestral B.1 variant, which is antigenically identical to the sCPD9 virus, but also against the evolutionarily distant B.1.1.7 variant, which has 17 mutations (8 in the spike protein) that alter or delete amino acids compared to the original virus. Hamster sera also effectively neutralized the B.1.351 variant, albeit with a 6- to 9-fold reduced potency (Figure 12C). This result is in good agreement with previous studies showing that B.1.351 significantly reduced the neutralizing activity of convalescent plasma from individuals infected with earlier SARS-CoV-2 lineages (~9-15-fold reduction) (Cele et al., 2021; Dejnirattisai et al., 2021; Wang et al., 2021) and sera from individuals vaccinated with a vaccine containing the original version of the spike protein (~10-14-fold reduction) (Wang et al., 2021; Planas et al., 2021a).

[0203] Furthermore, sera from vaccinated hamsters taken on days 2 and 3 postchallenge also efficiently neutralized the SARS-CoV-2 variants B.1.128.1 and B.1.617.2 (Figures 12A-12C). These two variants were not used for challenge infection in this study, but were still neutralized with similar efficacy (~5-8 fold reduction compared to B.1) as the B.1.351 variant (~6 fold reduction). This result confirms that vaccination with the vaccine candidate sCPD9 induces broad and potent neutralizing antibody responses against a wide variety of SARS-CoV-2 variants. The data also show that among the viral variants studied here, variant B.1.617.2 is the most resistant to neutralization by antibodies induced by vaccination with a virus containing the ancestral form of the spike protein. Recent studies have shown similar results, showing that sera from vaccinated individuals and convalescent sera from individuals previously infected with variants other than B.1.617.2 neutralize the B.1.617.2 variant by 3-6 fold (Planas et al., 2021; Liu et al., 2021).

[0204] (discussion) This is because it not only reduces the efficacy of vaccines targeting SARS-CoV-2, but also raises concerns that the virus may become resistant to current vaccines (Moore et al., 2021). Mutations in the spike protein have been shown to be the main cause of reduced neutralization of the B.1.351 virus, as they inhibit efficient binding of neutralizing antibodies. Of particular importance is the E484K mutation in the receptor-binding domain of the spike protein, which prevents binding of the most potent neutralizing antibodies found in convalescent and post-vaccination sera (Yuan et al., 2021). This mutation is also present in the B.1.1.28.1 and B.1.525 lineages, and has also been identified in some viruses of the B.1.1.7 and B.1.526 lineages.

[0205] Recent evidence suggests that some acquired mutations in the spike protein partially or significantly impair the efficacy of licensed vaccines based solely on that protein. For example, the Pfizer-BioNTech vaccine (BNT162b2, Comirnaty) prevents 75% of mild and 90% of severe cases caused by the B.1.351 variant (Abu-Raddad et al., 2021), whereas the Johnson & Johnson vaccine (Ad26.COV2.S) has an efficacy of 57% and 100%, respectively (Johnson&Johnson), and the Novavax vaccine (NVX-CoV2373) has an efficacy of 49% and 100%, respectively (Shinde et al.). The AstraZeneca vaccine (Vaxzevria or Covishield) has the lowest efficacy of all vaccines licensed in Europe, preventing only 10% of infections caused by the B.1.351 variant (Madhi et al., 2021). Similarly, inactivated virus vaccines contain the entire antigenic repertoire of the virus and should, in theory, be more effective against emerging SARS-CoV-2 subtypes; however, in practice, they have the lowest efficacy against early viral subtypes of all licensed vaccines and therefore only offer partial protection against emerging subtypes. For example, in Brazil, where 75% of infections were caused by the B.1.1.28.1 subtype, the Sinovac vaccine (CoronaVac) was only 50% effective in preventing symptomatic infection (Hitchings et al., 2021).

[0206] As SARS-CoV-2 continues to evolve, this study has provided several important insights. We showed that a single intranasal vaccination with sCPD9, a live attenuated virus vaccine candidate, induced complete protection against clinical disease and lung pathology caused by three different SARS-CoV-2 variants in a highly sensitive animal model. Challenged hamsters did not develop signs of disease and demonstrated strong neutralizing antibody responses. We show that the B.1.351 virus was well controlled by vaccination with sCPD9, despite being significantly more resistant to neutralization by serum antibodies than the B.1 and B.1.1.7 variants. The fact that the lungs of hamsters exposed to B.1.351 were completely protected from any damage suggests that vaccination with sCPD9 induced an effective immune response, thereby compensating for the reduced humoral immunity to the B.1.351 variant. Sera from vaccinated and challenged hamsters also showed neutralizing activity against B.1.1.28.1 and B.1.617.2, similar to the results against B.1.351, suggesting that vaccination with sCPD9 provides protection against multiple SARS-CoV-2 subtypes.

[0207] Safe blood collection from dwarf hamsters before euthanasia does not provide sufficient serum to measure serum neutralization titers before challenge infection. Lack of serum does not allow evaluation of vaccine-induced antibody responses in the absence of infection. However, the excellent clinical protection, consistent with the absence of replication-competent virus in the lungs of challenged animals, and the robust antibody responses immediately after challenge demonstrate the strong protective capacity of this vaccine against different SARS-CoV-2 variants. In addition, vaccination with live attenuated virus may provide better protection from (re)infection than a vaccine with only a single antigen.

[0208] While other studies have shown that new variants threaten vaccination success and reignite the pandemic by reducing the efficacy of licensed vaccines, this study shows that a single intranasal vaccination with a lead live attenuated viral vaccine candidate, sCPD9, confers excellent protective immunity against currently circulating VOCs. If fully developed, live attenuated viral vaccines may provide a more practical solution on how to confer protective immunity against newly emerging dangerous variants, instead of developing vaccines specific to each of them. Live attenuated vaccines such as those described here may be particularly effective in boosting and broadening immune responses after primary immunization with other vaccines. It remains to be determined to what extent vaccination with sCPD9 virus can protect against emerging VOCs in non-primate models and in humans.

[0209] Materials and Methods (Cells and viruses) Vero E6 cells (American Type Culture Collection, CRL-1586) were cultured in minimum essential medium (MEM) containing 10% fetal bovine serum (PAN Biotech), penicillin G (100 IU / ml), and streptomycin (100 μg / ml; Carl Roth) at 37°C and 5% CO . SARS-CoV-2 variants B.1 (BetaCoV / Munich / BavPat1 / 2020) (Wolfel et al., 2020), B.1.1.7 (BetaCoV / Germany / ChVir21652 / 2020), B.1.351 (hCoV-19 / Netherlands / NoordHolland_20159 / 2021), B.1..1.28.1 (hCoV-19 / Netherlands / NoordHolland_10915 / 2021), and B.1.617.2 [SARS-CoV-2,Human,2021,Germany ex India,20A / 452R (B.1.617)] were propagated in Vero E6 cells. Viral stocks were stored at -80 °C before experimental infections.

[0210] (Production of high-titer virus stocks of sCPD9) To generate high titer virus stocks, confluent monolayers of Vero E6 cells grown in T-75 flasks were infected with sCPD9 virus at a multiplicity of infection of 1. Infected cells were cultured in 7 ml of cell culture medium and harvested at the onset of cytopathic effects 2–3 days post-infection. Virus stocks prepared in this way typically contained approximately 1 × 10 virus per ml of cell culture medium. 7 contains infectious virus particles.

[0211] (Virus titration) Concentrations of viral stocks and infectious virus in the lungs of infected animals were determined by titration on Vero E6 cells grown in 12-well plates. Stock titers of WT SARS-CoV-2 variants were determined by plaque assay. Because sCPD9 virus does not produce easily visible plaques on Vero E6 cells, titers of sCPD9 virus stocks were determined by focus-forming assay. Briefly, Vero E6 cells were infected with 100 μl of serial 10-fold dilutions of virus. After 2 h of incubation, the viral inoculum was removed and the cells were overlaid with Dulbecco's modified Eagle's medium (DMEM) containing 2.5% microcrystalline cellulose (Avicel, FMC BioPolymer). 72 h after infection, cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS). Cells were then incubated with monoclonal mouse anti-SARS-CoV-2 nucleocapsid antibody (provided by S. Reiche, Friedrich Loeffler Institute, Riems, Germany) for 1 h and goat anti-mouse immunoglobulin G-Alexa Fluor 568 secondary antibody (Thermo Fisher Scientific) for 45 min. To measure lung virus titers, 50 mg of lung tissue was first homogenized in a bead mill (Analytic Jena), the homogenates were serially diluted, and virus titers were measured by focus-forming assay as described above.

[0212] After challenge infection of hamsters, virus titers in the lungs of challenged animals were measured by plating 10-fold serial dilutions of 50 mg of lung homogenate onto Vero E6 cells. After 2 h of incubation at 37°C, homogenized tissue was aspirated, cells were washed with PBS, and overlaid with 2.5% microcrystalline cellulose (Avicel, FMC BioPolymer). 72 h after infection, cells were fixed with 4% formaldehyde, stained with crystal violet (0.75% in water), and plaques were counted.

[0213] (Ethics statement) In vitro and animal experiments were performed under biosafety conditions in a biosafety level-3 (BSL-3) facility at the Institut fur Virologie, Free University Berlin, Germany. All animal experiments were approved by the Landesamt fur Gesundheit und Soziales, Berlin, Germany (permit number 0086 / 20) and were performed in compliance with all relevant institutional, national, and international guidelines for the care and humane use of animal subjects.

[0214] (Livestock) Animal experiments were performed in a BSL-3 certified facility. Five- to seven-week-old Roborovski dwarf hamsters (P. roborovskii) were purchased from a single breeding facility through the German pet trade. They were housed in groups of six in individually ventilated cages. The animals had free access to food and water and were allowed to acclimate to the environment for 7 days before infection. In both experiments, the cage temperature was 22–24 °C and the relative humidity was 40–55%.

[0215] (Vaccination and challenge experiments) To evaluate the vaccine efficacy of the lead attenuated virus vaccine candidate sCPD9 in Roborovski dwarf hamsters, animals were randomly assigned to two groups, with 60% females in each group. Twelve hamsters were mock vaccinated using cell culture medium, and 30 hamsters were vaccinated with the vaccine candidate sCPD9 virus at 1 × 10 in 20 μl.5 FFU were inoculated intranasally under anesthesia (Osterrieder et al., 2020). 21 days after vaccination, 1 × 10 5 Hamsters were challenged intranasally with PFU of challenge virus. Nine mock-vaccinated hamsters were challenged with SARS-CoV-2 variant B.1, and nine sCPD9-vaccinated hamsters were challenged with either SARS-CoV-2 variants B.1, B.1.1.7, or B.1.351. During the experiment, all hamsters were monitored twice daily for clinical signs of disease and body weights were recorded. Three hamsters from each group were euthanized on day 3 post-vaccination and days 2, 3, and 5 post-challenge (experimental days 23, 24, and 26). Blood, tracheal swabs, and lungs were collected to determine virological and histological parameters of vaccination or infection. The left lung was preserved in 4% formalin for subsequent histopathological detailed examination.

[0216] (Histology and in situ hybridization) For histopathological examination, the left lung lobe was carefully removed and immersion fixed in buffered 4% formalin (pH 7.0) for 48 h. Lungs were embedded in paraffin and sectioned at 2 μm thickness. Sections were stained with hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) reactions, followed by blinded microscopic evaluation by a board-certified veterinary pathologist (ADG) (Gruber et al., 2020). H&E- and PAS-stained and in situ hybridization slides were analyzed and photographed using an Olympus BX41 microscope and a DP80 microscope digital camera, cellSens Imaging Software version 1.18 (Olympus Corporation). For low-magnification overview, slides were automatically digitized using an Aperio CS2 slide scanner (Leica Biosystems), and image files were created using Image Scope Software (Leica Biosystems).

[0217] (RNA isolation and reverse transcription quantitative polymerase chain reaction) RNA was extracted from 25 mg of lung homogenate and tracheal swabs using the innuPREP Virus RNA Kit (Analytik Jena). SARS-CoV-2 RNA copies were quantified using the NEB Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) as previously described (Cormanet al., 2020).

[0218] (Serum virus neutralization assay) Microneutralization assays were performed with six sera from mock-vaccinated hamsters and challenged with the B.1 virus variant, and five, six, and six sera from hamsters vaccinated with sCPD9 and challenged with the B.1, B.1.1.7, or B.1.351 virus variants, respectively. Each serum was tested for neutralization of the B.1, B.1.1.7, B.1.351, B.1.1.28.1, or B.1.617.2 variants. Microneutralization assays were performed by plating two-fold serial dilutions (1:16 to 1:2048) of complement-inactivated (56°C, 30 min) hamster sera onto subconfluent monolayers of Vero E6 cells in 96-well plates. Cells in each well were infected with 100 PFU of each SARS-CoV-2 variant, cultured at 37°C for 72 h, fixed in 4% formalin for 24 h, and stained with crystal violet (0.75% in water). Serum neutralization was considered effective in wells that showed no cytotoxicity, and the final effective dilution was counted.

[0219] Numerous modifications and variations of the described examples and embodiments will be apparent to those skilled in the art in light of the above teachings. The disclosed examples and embodiments are presented for illustrative purposes only. Other alternative embodiments may incorporate some or all of the features disclosed herein. Accordingly, it is intended to cover all such modifications and alternative embodiments as may fall within the true scope of the present invention.

Claims

1. A polynucleotide comprising: a) the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, and / or b) encoding at least one nonstructural SARS-CoV-2 protein selected from the group consisting of nonstructural protein 7, nonstructural protein 8, nonstructural protein 9, nonstructural protein 10, nonstructural protein 11, nonstructural protein 12, endoribonuclease, and 2'-O-methyltransferase; The polynucleotide comprises at least one sequence portion that comprises deoptimization of codon pairs compared to the SARS-CoV-2 genome, or consists of at least one sequence portion that comprises deoptimization of codon pairs.

2. The polynucleotide of claim 1 , wherein the polynucleotide encodes at least two of the nonstructural proteins.

3. 3. The polynucleotide of claim 1 or 2, wherein the SARS-CoV-2 genome is a portion of the SARS-CoV-2 genome extending from position 11,000 to position 27,000.

4. 4. The polynucleotide of claim 1, wherein the at least one sequence portion comprising deoptimized codon pairs has a length ranging from 750 nucleotides to 2500 nucleotides.

5. 5. The polynucleotide of any one of claims 1 to 4, wherein 15% to 40% of the nucleotides of at least one sequence portion comprising deoptimized codon pairs differ from the nucleotides of the corresponding SARS-CoV-2 genome.

6. 6. The polynucleotide of any one of claims 1 to 5, wherein the at least one sequence portion comprising deoptimized codon pairs comprises between 200 and 500 nucleotides that differ from the nucleotides of the corresponding SARS-CoV-2 genome.

7. 7. The polynucleotide of any one of claims 1 to 6, wherein 40% to 70% of the codons of the at least one sequence portion comprising deoptimized codon pairs differ from the codons of the corresponding SARS-CoV-2 genome.

8. 8. The polynucleotide of any one of claims 1 to 7, wherein the at least one sequence portion comprising deoptimized codon pairs comprises between 150 and 400 codons that differ from the codons of the corresponding SARS-CoV-2 genome.

9. 9. The polynucleotide of any one of claims 1 to 8, wherein the at least one sequence portion comprising deoptimized codon pairs comprises a first deoptimized sequence portion and a second deoptimized sequence portion, the first deoptimized sequence portion and the second deoptimized sequence portion being separated from each other by a deoptimized sequence portion comprising at least 300 nucleotides.

10. 10. The polynucleotide of claim 9, wherein the first deoptimized sequence portion has a length ranging from 1300 nucleotides to 1600 nucleotides and the second deoptimized sequence portion has a length ranging from 100 nucleotides to 400 nucleotides.

11. 11. The polynucleotide of claim 9 or 10, wherein the first non-optimized sequence portion has at least 95% sequence identity to SEQ ID NO:2 and the second deoptimized sequence portion has at least 95% sequence identity to SEQ ID NO:

4.

12. 11. The polynucleotide of any one of claims 1 to 10, wherein the polynucleotide comprises a nucleic acid sequence defined by SEQ ID NO:6 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:

6.

13. The polynucleotide according to any one of claims 1 to 8, wherein the polynucleotide comprises a nucleic acid sequence defined by SEQ ID NO:8 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:

8.

14. The polynucleotide of any one of claims 1 to 8, wherein the polynucleotide comprises a nucleic acid sequence defined by SEQ ID NO:10 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:

10.

15. The polynucleotide of any one of claims 1 to 8, wherein the polynucleotide comprises a nucleic acid sequence defined by SEQ ID NO:15 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:

15.

16. The polynucleotide of any one of claims 1 to 8, wherein the polynucleotide comprises a nucleic acid sequence defined by SEQ ID NO:16 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:

16.

17. The polynucleotide of any one of claims 1 to 8, wherein the polynucleotide comprises a nucleic acid sequence defined by SEQ ID NO:17 or a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:

17.

18. A live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising the polynucleotide of any one of claims 1 to 17.

19. 19. The live attenuated SARS-CoV-2 of claim 18, wherein the SARS-CoV-2 has a nucleic acid sequence defined by SEQ ID NO:18 or a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:

18.

20. 19. The live attenuated SARS-CoV-2 of claim 18, wherein the SARS-CoV-2 has a nucleic acid sequence defined by SEQ ID NO:19 or a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:

19.

21. A pharmaceutical composition comprising the live attenuated SARS-CoV-2 of any one of claims 18 to 20.

22. 22. The pharmaceutical composition according to claim 21 for use as a vaccine.

23. 22. A method of vaccinating a human or animal patient comprising administering to the patient a pharmaceutical composition according to claim 21.

24. 24. The method of claim 23, wherein the pharmaceutical composition is administered by intranasal, oral, or parenteral administration.

25. The single dose of the pharmaceutical preparation is ＊ 10 ３ From 1 ＊ 10 ８ The method of claim 23 or 24, comprising live attenuated SARS-CoV-2 focus forming units during

26. 26. The method of any one of claims 23 to 25, wherein the pharmaceutical formulation is administered to the patient at least twice, wherein a second administration is separated from a first administration by a first period ranging from 2 weeks to 36 months.

27. A vector comprising the polynucleotide according to any one of claims 1 to 17.

28. A host cell comprising a polynucleotide according to any one of claims 1 to 17.

29. A method for producing a virus, comprising: a) culturing the host cell of claim 28; b) isolating said virus, wherein said virus is a live attenuated SARS-CoV-2.

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