Novel coronavirus vaccines and methods for designing and obtaining viral vaccines

Abstract

The present invention relates to a mutated receptor binding domain (mRBD) of coronavirus (mRBD-CORONA) or a fragment thereof, and a mutated spike protein of coronavirus (CORONA-mSpike) or a fragment thereof comprising CORONA-mRBD or a fragment thereof. Moreover, the present invention relates to a polypeptide or protein comprising mRBD-CORONA or a fragment thereof or CORONA-mSpike or a fragment thereof, and a nucleic acid comprising a nucleotide sequence encoding mRBD-CORONA or a fragment thereof or CORONA-mSpike or a fragment thereof. Moreover, the present invention relates to a vaccine composition comprising one or more CORONA-mRBD or a fragment thereof, one or more CORONA-mSpike, one or more polypeptides or proteins, and / or one or more nucleic acids according to the present invention. Moreover, the present invention relates to one or more CORONA-mRBD or a fragment thereof, one or more CORONA-mSpike, one or more polypeptides or proteins, one or more nucleic acids, and / or a vaccine composition according to the present invention for use in the prevention and / or treatment of a disease caused by coronavirus in a subject. Moreover, the present invention relates to a method for designing and / or obtaining an active ingredient for a vaccine composition according to the present invention, as well as a VIRUS-mRBD or a fragment thereof designed and / or obtained by the method for obtaining VIRUS-mRBD.

Description

[Technical field]

[0001] The present invention relates to a mutated receptor binding domain (mRBD) of a coronavirus (mRBD-CORONA) or a fragment thereof, and a mutated spike protein of a coronavirus (CORONA-mSpike) or a fragment thereof comprising CORONA-mRBD or a fragment thereof.

[0002] Moreover, the present invention relates to a polypeptide or protein comprising mRBD-CORONA or a fragment thereof or CORONA-mSpike or a fragment thereof, and to a nucleic acid comprising a nucleotide sequence encoding mRBD-CORONA or a fragment thereof or CORONA-mSpike or a fragment thereof.

[0003] Furthermore, the present invention relates to a vaccine composition comprising one or more CORONA-mRBDs or fragments thereof, one or more CORONA-mSpikes, one or more polypeptides or proteins, and / or one or more nucleic acids according to the present invention.

[0004] Moreover, the present invention relates to one or more CORONA-mRBDs or fragments thereof, one or more CORONA-mSpikes, one or more polypeptides or proteins, one or more nucleic acids, and / or vaccine compositions according to the present invention for use in the prevention and / or treatment of a disease caused by a coronavirus in a subject.

[0005] Moreover, the present invention relates to a method for designing and / or obtaining an active ingredient for a vaccine composition according to the present invention, as well as a VIRUS-mRBD or a fragment thereof designed and / or obtained by the method for obtaining VIRUS-mRBD. [Background technology]

[0006] Angiotensin-converting enzyme 2 (ACE2) acts as a receptor that mediates the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into cells. The membrane-bound ACE2 receptor is cleaved by proteases, such as ADAM17 (tumor necrosis factor alpha convertase) and TMPRSS2 (transmembrane serine protease subtype), which promotes the release of soluble ACE2 (sACE2) into the extracellular space. Soluble ACE2 exists in an enzymatically active form (active ACE2) and an enzymatically inactive form (inactive ACE2).

[0007] Importantly, the ability of sera to neutralize the virus depends on neutralizing antibodies (nABs). Induction of antibodies that interfere with the interaction of ACE2 with spike is important to generate a neutralizing immune response and confer protective immunity. However, activation of B cells and affinity maturation of potent antibodies are highly dependent on the availability of the antigen, i.e., the spike protein or the receptor binding domain (RBD) of the spike protein. Summary of the Invention

[0008] The present invention is based on our discovery that binding of soluble receptors to immunogens interferes with humoral immunity by masking antigens (Figure 1). For SARS-CoV-2, we could show that high concentrations of sACE2 mask the SARS-CoV-2 spike during infection and immunization, which consequently interferes with the maturation of antibodies to the SARS-CoV-2 receptor binding motif (RBM), the receptor binding interface of the RBD. We could show that high concentrations of soluble receptors and / or high binding affinity enhance epitope masking. Importantly, we could show that the introduction of mutations that block ACE2 binding abrogates masking by soluble ACE2, thereby favoring the production of RBM-binding antibodies in the presence of soluble receptors. This discovery is particularly important for the design of vaccine antigens, since the immune response to a vaccine can be improved by blocking binding to the host receptor.

[0009] Similar to ACE2, other viral receptors can be shed, such as DPP4, the receptor for the MERS-CoV coronavirus, which is present in high concentrations in the blood of healthy human subjects. Similarly, other viruses use receptors for viral entry, which are also present in soluble versions at concentrations that can interfere with the immune response.

[0010] On the other hand, it has been found that certain vaccinations / immunizations / natural infections do not confer sufficient immunity against SARS-CoV-2 in 5–40% of individuals. Moreover, antibodies produced against the RBD fade more rapidly than those against other parts of the viral spike (https: / / www.cell.com / action / showPdf?pii=S0092-8674%2821%2900706-6). Moreover, antibodies produced by memory B cells induced after vaccination have been shown to be less potent compared to those induced by natural infection (https: / / www.biorxiv.org / content / 10.1101 / 2021.07.29.454333v1.full.pdf).

[0011] Moreover, it has been found that newly emerged virus variants may have improved affinity to these receptors (Figure 11). Therefore, we conclude that future vaccines based on these high affinity variants may be more susceptible to masking by sACE2. Higher affinity antibodies are required to achieve the same level of protection against new variants. Therefore, vaccines that avoid masking of soluble receptors will become even more important if the evolution of current viruses causes the emergence of virus strains with higher affinity.

[0012] In view of the above, there is an urgent need for improved vaccines that overcome the above mentioned problems.It is a primary object of the present invention to provide a method for obtaining such a vaccine.

[0013] The present invention is based on the surprising discovery that all of the above mentioned objectives can be achieved by using for immunization a coronavirus receptor binding domain that has reduced avidity for the receptor.

[0014] The present invention is further based on the surprising discovery that all of the above mentioned objectives can be achieved by the use of a mutated coronavirus receptor binding domain having one or more amino acid substitutions.

[0015] Moreover, the present invention is further based on the surprising discovery that all of the above mentioned objectives can be achieved by use of a combination of mutated coronavirus receptor binding domains, each having a different amino acid substitution.

[0016] The present invention is further based on the surprising finding that all of the above mentioned objectives can be achieved by using a method for designing and / or obtaining a viral receptor binding domain by screening for receptor binding domains with reduced binding affinity to their receptor.

[0017] Thus, the present invention provides a mutated coronavirus receptor binding domain (mRBD) (CORONA-mRBD) or a fragment thereof, which has reduced binding avidity to a coronavirus RBD receptor (CORONA-RBD receptor) compared to a wild-type coronavirus receptor binding domain (CORONA-wtRBD).

[0018] The inventors were able to show that sACE2 levels are increased in some individuals and developed an assay to properly evaluate this blood concentration. As a result, people with increased sACE2 are at risk of developing a less protective immune response. Individuals with increased release of sACE2 could be identified and benefit from the design of an improved vaccine as described above. In addition, the inventors were able to show that in severely ill patients, sACE2 levels increase immediately after infection. Thus, an improved vaccine may be particularly useful when administered not only prior to infection but also immediately after infection to promote the development of protective antibodies.

[0019] It is therefore an object of the present invention to provide mutated coronavirus receptor binding domains (mRBDs) (CORONAmRBDs) that allow for the induction of highly efficient immune responses in subjects against coronaviruses even in the presence of high levels of soluble receptors and / or against coronavirus mutants with increased masking activity. A further object of the present invention is to provide a method for timely and efficient access to such mutated receptor binding domains (mRBDs) to block the spread of viruses, in particular newly emerged viral variants, and to protect as many individuals as possible, including those who are not sufficiently immunized by conventional vaccination / immunization and / or natural infection.

[0020] In summary, we found that in both humans and mice, soluble viral receptors, and in particular soluble coronavirus receptors, mediate the masking of important epitopes through binding to these antigens, thereby impairing protective antibody responses. This mechanism of epitope masking has previously only been described for antibodies, but not for viral receptors.

[0021] The inventors were able to show that increased levels of sACE2 in the blood are associated with severe COVID-19 disease. Moreover, patients with high levels of sACE2 showed a significant reduction in antibody titers specific for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Thus, these patients also showed a significant reduction in the ability to block the binding of the SARS-CoV-2 spike protein to ACE2.

[0022] In particular, the inventors are the first to show that a soluble receptor interferes with a protective immune response and provide mechanistic evidence.

[0023] Moreover, we were able to show in mice that sACE2 significantly reduces the generation of B cells producing RBD-specific antibodies that block the binding of the virus to the ACE2 receptor (Figure 1). Analysis of mouse sera confirmed that the antibody response to immunization was less efficient.

[0024] Furthermore, by in silico calculations, the inventors were able to show that mutations such as those in the SARS-CoV-2 variants B.1.1.7 and 501.V2 that emerged in the UK and South Africa, respectively, improve the masking effect (Figure 8).

[0025] The present invention provides a mutated coronavirus spike protein (CORONA-mSpike) or a fragment thereof comprising CORONA-mRBD or a fragment thereof.

[0026] Moreover, the present invention provides a polypeptide or protein comprising a CORONA-mRBD or a fragment thereof according to the present invention or a CORONA-mSpike or a fragment thereof according to the present invention.

[0027] Moreover, the present invention provides - a CORONA-mRBD according to the invention or a fragment thereof, - CORONA-mSpike or a fragment thereof according to the invention, or - a polypeptide or protein according to the invention A nucleic acid comprising a nucleotide sequence encoding the

[0028] Moreover, the present invention provides, as an active ingredient, - one or more CORONA-mRBDs or fragments thereof according to the invention, and / or - one or more CORONA-mSpikes or fragments thereof according to the invention, and / or - one or more polypeptides or proteins according to the invention, and / or - one or more nucleic acids according to the invention The present invention provides a vaccine composition comprising:

[0029] Moreover, the present invention relates to a method for the prevention and / or treatment of a disease caused by a coronavirus in a subject, comprising: one or more CORONA-mRBDs or fragments thereof according to the invention, and / or one or more CORONA-mSpikes or fragments thereof according to the invention, and / or one or more polypeptides or proteins according to the invention, and / or one or more nucleic acids according to the invention, and / or Vaccine composition according to the present invention to provide.

[0030] Moreover, the present invention relates to a method for designing and / or obtaining an active ingredient for a vaccine composition, comprising the steps of: (i) providing a mutated viral receptor binding domain (VIRUS-mRBD) or a fragment thereof, comprising one or more mutations in a wild-type viral receptor binding domain (VIRUS-wtRBD); (ii) VIRUS-mRBD or a fragment thereof a) Reduced binding of the viral receptor-binding domain to the receptor (RBD receptor) (VIRUS-RBD receptor) compared to VIRUS-wtRBD determining whether (iii) selecting VIRUS-mRBD or a fragment thereof as an active ingredient when a) is satisfied; The present invention relates to a method comprising the steps of:

[0031] Moreover, the present invention provides VIRUS-mRBD or a fragment thereof obtained as an active ingredient by the method for designing and / or obtaining an active ingredient for a vaccine composition according to the present invention.

[0032] Wild type (wt) is the typical phenotype of a naturally occurring species. Thus, in the present invention, wtRBD is any RBD of a naturally occurring pathogen, i.e., a pathogen that has been observed to spread in a population, which has been isolated from an infected individual (human or animal) and has had its genome defined by sequencing.

[0033] In particular, VIRUS-wtRBD belongs to naturally occurring viruses, specifically those defined by the WHO as official virus variants, including variants of interest (VOI), variants of concern (VOC), and viruses of special concern. Such naturally occurring virus variants may also be referred to as mutants, since they include mutations compared to the first-appearing Wuhan variant. However, in the present invention, naturally occurring viruses, specifically virus variants, have wtRBD.

[0034] Thus, VIRUS-wtRBD includes viral sequences available on the Centers for Disease Control platform as well as on common repositories, such as https: / / www.ecdc.europa.eu / en, https: / / www.cdc.gov, https: / / www.gisaid.org, https: / / nextstrain.org, https: / / cov-lineages.org or https: / / microbiologysociety.org.

[0035] Mutation is a new genetic trait resulting from or arising from the expression of mutation, generally a change in the DNA sequence of the genome or chromosome of an organism.Therefore, in the present invention, VIRUS-mRBD is any VIRUS-RBD that is genetically modified compared to this VIRUS-wtRBD defined above, and that has a reduced binding affinity to this receptor compared to VIRUS-wtRBD.Therefore, in particular, VIRUS-mRBD does not belong to naturally occurring pathogens, in particular viruses as defined above.Therefore, VIRUS-mRBD is different from VIRUS-wtRBD.Virus mutants that grow in populations cannot have VIRUS-mRBD as defined herein.

[0036] Coronavirus Mutated Receptor Binding Domain (CORONA-mRBD) The present invention provides a mutated coronavirus receptor binding domain (mRBD) (CORONA-mRBD) or a fragment thereof.

[0037] In particular, the present invention provides CORONA-mRBD or a fragment thereof, which has reduced binding affinity to a coronavirus RBD receptor (CORONA-RBD receptor) compared to the wild-type coronavirus receptor binding domain (CORONA-wtRBD).

[0038] Preferably, the CORONA-mRBD or a fragment thereof is a mutated receptor binding domain (mRBD) of the coronavirus spike protein (CORONA-mRBD) or a fragment thereof.

[0039] Preferably, the CORONA-mRBD or a fragment thereof is a peptide or protein, preferably a protein.

[0040] Preferably, CORONA-mRBD or a fragment thereof is obtained as an active ingredient by the method according to the invention for designing and / or obtaining an active ingredient for a vaccine composition, as described below.

[0041] Preferably, the CORONA-RBD receptor is a soluble and / or membrane-bound CORONA-RBD receptor, preferably a soluble CORONA-RBD receptor (soluble CORONA-RBD receptor). The soluble CORONA-RBD receptor may include active and inactive soluble CORONA-RBD receptors.

[0042] The binding strength of CORONA-mRBD or its fragment and CORONA-wtRBD to CORONA-RBD receptor can be measured by any method known to those skilled in the art.For example, the binding strength can be determined by, for example, staining cells expressing CORONA-mRBD or CORONA-wtRBD on the cell surface with CORONA-RBD receptor directly or indirectly labeled with fluorophore, or conversely, staining cells expressing CORONA-RBD receptor on the cell surface with soluble CORONA-mRBD or soluble CORONA-wtRBD directly or indirectly labeled with fluorophore, performing flow cytometry analysis, and counting positively stained cells, or by determining the Kd (dissociation constant) or EC50 (effective concentration at 50% of maximum binding) of CORONA-wtRBD or CORONA-mRBD to each CORONA-RBD receptor by using ELISA. The reduction in binding affinity is then obtained by comparing the binding affinity of CORONA-wtRBD to the CORONA-RBD receptor with the binding affinity of CORONA-mRBD to the CORONA-RBD receptor.

[0043] Preferably, the avidity is measured by determining the EC50 or Kd, where a higher EC50 or Kd indicates a lower avidity. Alternatively, the avidity is measured by flow cytometry analysis, where a lower number of positively stained cells indicates a lower avidity. Thus, the reduced avidity is preferably determined by comparing the EC50, Kd, ​​and / or the number of positively stained cells resulting from the binding of CORONA-wtRBD to the CORONA-RBD receptor with the EC50, Kd, ​​and / or the number of positively stained cells resulting from the binding of CORONA-mRBD to the CORONA-RBD receptor, respectively. An example for determining the EC50 based on ACE2 as the CORONA-RBD receptor, SARS-CoV-2 as the coronavirus, and the corresponding wild-type and mutant RBDs of SARS-CoV-2 (SARS-wtRBD and SARS-mRBD) is provided in Example 4. Another example for determining the EC50 based on DPP4 as the CORONA-RBD receptor, MERS-CoV as the coronavirus, and the wild-type and mutated RBD of MERS-CoV (MERS-wtRBD and MERS-mRBD) is provided in Example 7.

[0044] When the reduction in binding ability is determined by determining EC50 or Kd, the reduction in the binding ability of CORONA-mRBD to CORONA-RBD receptor is preferably indicated by an increase in EC50 and / or Kd of 1.5 times or more, more preferably 2 times or more, even more preferably 10 times or more, even more preferably 100 times or more, even more preferably 500 times or more, and most preferably 1000 times or more. In order to obtain the best effect of vaccination, when CORONA-mRBD is used as a vaccine, the binding ability to CORONA-RBD receptor must be reduced as much as possible. Therefore, preferably, there is no upper limit for the reduction in binding ability to CORONA-RBD receptor. Therefore, preferably, there is no upper limit for the increase in EC50 or Kd. However, the upper limit for the increase in EC50 or Kd can be 500.000 times or less, more preferably 100.000 times or less, even more preferably 10.000 times or less. An example for determining the EC50 based on ACE2 as the CORONA-RBD receptor, SARS-CoV-2 as the coronavirus, and wild-type and mutant RBDs of SARS-CoV-2 (SARS-wtRBD and SARS-mRBD) is provided in Example 4. Another example for determining the EC50 based on DPP4 as the CORONA-RBD receptor, MERS-CoV as the coronavirus, and wild-type and mutant RBDs of MERS-CoV (MERS-wtRBD and MERS-mRBD) is provided in Example 7.

[0045] When the reduction in binding ability is determined by flow cytometry analysis, the reduction in the binding ability of CORONA-mRBD to the CORONA-RBD receptor is preferably indicated by a reduction in the number of positively stained cells of 50% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more. In order to obtain the best effect of vaccination, when CORONA-mRBD is used as a vaccine, the binding ability to the CORONA-RBD receptor must be reduced as much as possible. Therefore, preferably, there is no upper limit for the reduction in the binding ability to the CORONA-RBD receptor. Therefore, preferably, there is no upper limit for the reduction in the number of positively stained cells, and this reduction can even be 100%. However, the upper limit for the reduction in the number of positively stained cells can be 100% or less, more preferably 99% or less.

[0046] Moreover, CORONA-mRBD or a fragment thereof preferably exhibits binding to anti-CORONA-wtRBD neutralizing antibodies (anti-CORONA-wtRBD-nAB), i.e., the binding avidity to anti-CORONA-wtRBD neutralizing antibodies (anti-CORONA-wtRBD-nAB) is preferably maintained, i.e., ranging from a slight to moderately decreased binding avidity compared to CORONA-wtRBD, to an increased binding avidity.

[0047] Anti-CORONA-wtRBD-nABs may belong to different antibody classes, such as class I, class II, class III, and / or class IV, and / or may be specific for different epitopes of CORONA-wtRBD. Anti-CORONA-wtRBD-nABs may be monoclonal and / or polyclonal antibodies reactive against CORONA-wtRBD. Monoclonal anti-CORONA-wtRBD-nABs may be recombinantly obtained. Polyclonal anti-CORONA-wtRBD-nABs may be obtained from plasma or serum derived from an immunized subject who has experienced a coronavirus infection, e.g., a convalescent subject and / or an immunized subject who has been immunized, e.g., by vaccination, where such immunized subjects are preferably immunologically reactive to CORONA-wtRBD. The subject is preferably a vertebrate, more preferably an immunized mammal, more preferably an ape, monkey, lemur, canine, such as dog, wolf or fox, feline, such as large or small cat, rodent, such as mouse, rat, guinea pig, hamster or rabbit, bovine, such as buffalo, antelope, sheep, goat or cow, deer, horse, donkey, bear, marten, bat, and / or human. Even more preferably, the immunized subject is a mouse, rat, rabbit, hamster, goat, donkey, horse, dog, cat, sheep or human, most preferably a human.

[0048] The binding strength of CORONA-mRBD or its fragments and CORONA-wtRBD to anti-CORONA-wtRBD-nAB can be measured by any method known to those skilled in the art. For example, the binding strength can be determined by staining cells expressing CORONA-mRBD or CORONA-wtRBD on the cell surface with monoclonal anti-CORONA-wtRBD-nAB directly or indirectly labeled with a fluorophore, or conversely, staining cells expressing monoclonal anti-CORONA-wtRBD-nAB on the cell surface with soluble CORONA-mRBD or soluble CORONA-wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, and counting positively stained cells, or by staining cells expressing monoclonal anti-CORONA-wtRBD-nAB on the cell surface with soluble CORONA-mRBD or soluble CORONA-wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, and counting positively stained cells, or by staining cells expressing monoclonal anti-CORONA-wtRBD-nAB on the cell surface with soluble CORONA-wt ... It can be determined by using ELISA to determine the Kd (dissociation constant) or EC50 (effective concentration at 50% of maximum binding) of CORONA-wtRBD and CORONA-mRBD for CORONA-wtRBD-nAB, or by determining the ED50 (effective dilution at which 50% of serum antibodies bind) of polyclonal antibodies in plasma or serum from immunized subjects who have experienced a coronavirus infection, e.g., convalescent subjects and / or immunized subjects who have been immunized, e.g., by vaccination, where such immunized subjects are preferably immunologically reactive with CORONA-wtRBD. An immunized subject is as defined above. Examples for determining EC50, Kd, ​​and ED50 based on anti-SARS-CoV-2-wtRBD neutralizing antibodies (anti-SARS-wtRBD-nABs) as anti-CORONA-wtRBD-nABs, SARS-CoV-2 as coronavirus, and wild-type and mutant RBDs of SARS-CoV-2 (SARS-wtRBD and SARS-mRBD) are provided in Example 4. Examples for determining EC50 based on anti-SARS-CoV-2-wtRBD neutralizing antibodies (anti-SARS-wtRBD-nABs) as anti-CORONA-wtRBD-nABs, SARS-CoV-2 as coronavirus, and wild-type and mutant RBDs of SARS-CoV-2 (SARS-wtRBD and SARS-mRBD) are provided in Example 4.Another example for determining EC50 based on anti-MERS-CoV-wtRBD neutralizing antibodies (anti-MERS-wtRBD-nAB) as anti-CORONA-wtRBD-nAB, MERS-CoV as coronavirus, and wild-type and mutated RBD of MERS-CoV (MERS-wtRBD and MERS-mRBD) is provided in Example 7.

[0049] When determining the binding strength of anti-CORONA-wtRBD-nABs by flow cytometry analysis, EC50, and / or Kd, the binding strength is preferably the average binding strength of several different monoclonal anti-CORONA-wtRBD-nABs, preferably 4 or more different arbitrarily selected monoclonal anti-CORONA-wtRBD-nABs. Illustrating again with respect to Example 4 for SARS-CoV-2 and anti-SARS-wtRBD-nABs, the average EC50 of anti-SARS-wtRBD-nABs CC12.1, P2C-1F11, REGN10933, and S309 is determined taking into account SARS-wtRBD or SARS-mRBD. Illustrating again with respect to Example 4 for MERS-CoV and anti-MERS-wtRBD-nABs, the average EC50 of anti-MERS-wtRBD-nABs 4C2h, D12, LCA60, and MERS4V2 is determined.

[0050] The sustained avidity, i.e., the range of avidity from slight to moderate decreases in avidity to increases in avidity, is then obtained by comparing the avidity or average avidity of CORONA-wtRBD to anti-CORONA-wtRBD-nAB with the avidity or average avidity of CORONA-mRBD to anti-CORONA-wtRBD-nAB, respectively.

[0051] Preferably, the avidity is measured by determining EC50 or Kd, where a higher EC50 or Kd indicates a lower avidity. Alternatively, the avidity is measured by flow cytometry analysis or by determining ED50, where a lower number of positively stained cells or a lower ED50 indicates a lower avidity, respectively. Thus, whether the avidity is maintained, i.e., in the range of a slight to moderate decrease in avidity and an increase in avidity, is preferably determined by comparing the EC50, Kd, ​​number of positively stained cells, and / or ED50 resulting from the binding of CORONA-wtRBD to anti-CORONA-wtRBD-nAB with the EC50, Kd, ​​number of positively stained cells, and / or ED50 resulting from the binding of CORONA-mRBD to anti-CORONA-wtRBD-nAB, respectively.

[0052] When determining avidity by EC50 or Kd determination, a slight to moderate decrease in avidity of CORONA-mRBD to anti-CORONA-wtRBD-nAB is preferably indicated by an increase in EC50 and / or Kd of 3-fold or less, even more preferably 2-fold or less, even more preferably 1.5-fold or less, and even more preferably 1.2-fold or less. When determining avidity by flow cytometric analysis or ED50 determination, a slight to moderate decrease in avidity of CORONA-mRBD to RBD-anti-CORONA-wtRBD-nAB is preferably indicated by a decrease in positively stained cell count and / or ED50 of 50% or more, more preferably 80% or more, and even more preferably 90% or more.

[0053] As already mentioned above, when determining avidity by EC50, Kd or flow cytometry analysis, the avidity is preferably the average avidity as defined above. Thus, a slight to moderate decrease in avidity as defined above is preferably a slight to moderate decrease in average avidity as defined above.

[0054] In order to obtain the best effect of vaccination, when CORONA-mRBD is used as a vaccine, the binding ability to anti-CORONA-wtRBD-nAB should be as high as possible.Therefore, preferably, the decrease in the binding ability to anti-CORONA-wtRBD-nAB should be as moderate as possible, and preferably, the binding ability of anti-CORONA-wtRBD-nAB to CORONA-mRBD is preferably not decreased or even increased.That is, EC50 and Kd are not increased, and / or the number of positively stained cells and ED50 are not decreased.

[0055] Therefore, when determining the binding ability by determining EC50 or Kd, the increase in the binding ability of CORONA-mRBD to anti-CORONA-wtRBD-nAB is preferably indicated by a decrease of 10% or more, more preferably 25% or more, even more preferably 50% or more, even more preferably 80% or more, and most preferably 90% or more of EC50 and / or Kd. As mentioned above, in order to obtain the best effect by vaccination, when CORONA-mRBD is used as a vaccine, the binding ability to anti-CORONA-wtRBD-nAB should be as high as possible. Therefore, preferably, there is no upper limit for the increase in the binding ability to anti-CORONA-wtRBD-nAB. Thus, preferably, there is no upper limit for the decrease in EC50 or Kd, and this decrease can even be 100%. However, the upper limit for the decrease in EC50 and / or Kd can be 100% or less, more preferably 99% or less.

[0056] When determining the avidity by flow cytometry analysis or ED50, the increase in the avidity of CORONA-mRBD to anti-CORONA-wtRBD-nAB is preferably indicated by an increase of 1.1 times or more, more preferably 1.2 times or more, even more preferably 1.5 times or more, and even more preferably 2 times or more in the number of positively stained cells and / or ED50, respectively. As mentioned above, in order to obtain the best effect of vaccination, when CORONA-mRBD is used as a vaccine, the avidity to anti-CORONA-wtRBD-nAB should be as high as possible. Therefore, preferably, there is no upper limit for the increase in avidity to anti-CORONA-wtRBD-nAB. Therefore, preferably, there is no upper limit for the increase in positively stained cells and / or ED50. However, the upper limit for the increase in positively stained cells and / or ED50 may be 10.000 times or less, more preferably 1.000 times or less, and even more preferably 100 times or less.

[0057] As already mentioned above, when determining avidity by EC50, Kd or flow cytometric analysis, the avidity is preferably the average avidity as defined above. Thus, an increase in avidity as defined above is preferably an increase in average avidity as defined above.

[0058] Moreover, CORONA-mRBD or a fragment thereof reduces cell-cell fusion.

[0059] The reduction in cell-cell fusion can be measured by any method known to those skilled in the art. For example, cell-cell fusion can be determined by cell-cell fusion assay combined with microscopic cell imaging. In particular, CORONA-wtRBD or CORONA-mRBD transfected and fluorophore-labeled cells are imaged, and syncytia and nuclei are counted or cell diameters are compared. Then, the cell-cell fusion of CORONA-wtRBD is compared with the cell-cell fusion of CORONA-mRBD, i.e., the reduction in cell-cell fusion is obtained by comparing the number of syncytia and nuclei or cell diameters.

[0060] In particular, cell-cell fusion can be measured by means of a luciferase activity-based cell fusion reporting system as described in "A robust reporting system for measurement of SARS-CoV-2 spike fusion efficiency" by Huang et al., Signal Transduction and Targeted Therapy, (2022) 7:179 (https: / / www.nature.com / articles / s41392-022-01037-4) or by flow cytometric analysis of GFP-expressing fusion reporter cells and quantification by fluorescence intensity distribution as described in "Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity" by Bo Mend et al., Nature, Vol 603, 24 March 2022 (https: / / www.nature.com / articles / s41586-022-04474-x). Cell diameter and granularity may be determined by forward or side scatter, by light microscopy, fluorescence microscopy or FACS analysis.

[0061] An example for determining the reduction in cell-cell fusion is provided in Example 12.

[0062] A reduction in cell-cell fusion is preferably indicated by a reduction in the number of syncytia and / or nuclei per cell, or a reduction in cell diameter.

[0063] When the reduction in cell-cell fusion is determined by a reduction in the number of nuclei per cell, the reduction in cell-cell fusion is preferably indicated by a reduction in the number of 10 nuclei per cell to 1 nucleus per cell. More preferably, the reduction in cell-cell fusion is indicated by less than 10 nuclei per cell, preferably less than 5 nuclei per cell, more preferably less than 2 nuclei per cell. Most preferably, all cells have only one nucleus per cell and do not exhibit cell-cell fusion.

[0064] When the reduction in cell-cell fusion is determined by the reduction in the diameter of the cells, the average diameter of the cells is compared to the average diameter of untreated cells. The reduction in cell-cell fusion is preferably indicated by a 10-fold increase in the average diameter compared to untreated cells. More preferably, the reduction in cell-cell fusion is indicated by a less than 10-fold increase in the average diameter compared to untreated cells, even more preferably a less than 5-fold increase in the average diameter compared to untreated cells, even more preferably a less than 2-fold increase in the average diameter compared to untreated cells. Most preferably, all cells have the same average diameter as untreated cells. Cell-cell fusion must be reduced as much as possible, since surface expression of fusion-competent antigens can cause side effects through the formation of syncytia, which are responsible for tissue damage by COVID-19. Thus, there is no limit to this reduction, and the reduction can even be 100%. That is, preferably, cell-cell fusion is completely prevented.

[0065] Moreover, CORONA-mRBD or a fragment thereof reduces cellular antigen uptake and / or receptor internalization.

[0066] The cellular antigen uptake and the reduction of receptor internalization can be measured by any method known to those skilled in the art.For example, the cellular antigen uptake can be determined by live cell imaging or live cell single molecule microscopy.In particular, cells are transfected with CORONA-wtRBD or CORONA-mRBD full spike, the cells and spike are labeled with fluorophores, and the cells are imaged to evaluate the internalization of antigen, i.e., spike, over time, for example, by live cell confocal microscopy, FACS analysis, or single molecule microscopy.

[0067] In particular, cellular antigen uptake as well as receptor internalization can be measured by means of single-molecule microscopy in living cells as described in "Quantification of GPCR internalization by single-molecule microscopy in living cells" by Arnauld Serge et al., Integr Biol (Camb), 2011 Jun;3(6):675-83 (https: / / pubmed.ncbi.nlm.nih.gov / 21541374 / ).

[0068] An example for determining the reduction in cellular antigen uptake as well as receptor internalization is provided in Example 12.

[0069] A decrease in cellular antigen uptake is preferably indicated by a decrease in the amount of fluorescently labeled spike internalized following binding to a cell.

[0070] When determining the reduction in cellular antigen uptake and receptor internalization by cell imaging, the "mean fluorescence intensity" (MFI) is determined for the extreme test conditions after internalization. This MFI is compared to wtRBD / wt-Spike as a positive control, and to no RBD treatment or treatment with a fluorescently labeled irrelevant protein as a negative control.

[0071] The MFI for the extreme test conditions (incubation time and antigen amount) after internalization of wtRBD / wt-Spike (positive control) is expressed as 100%. The MFI with no RBD treatment or treatment with a fluorescently labeled irrelevant protein (negative control) is expressed as 0%. The MFI percentage for the test conditions is calculated according to the positive and negative controls.

[0072] Preferably, cellular antigen uptake and receptor internalization is reduced by 50%, more preferably by 80%, and even more preferably by 90%.

[0073] Cellular antigen uptake and receptor internalization should be reduced as much as possible.Therefore, there is no limit to this reduction, and the reduction can even be 100%.That is, preferably, cellular antigen uptake is completely prevented.However, the upper limit for the reduction of cellular antigen uptake and receptor internalization can be 100% or less, more preferably 99% or less.

[0074] Preferably, the coronavirus is of the family coronaviridae, more preferably of the genus betacoronavirus.

[0075] Even more preferably, (A) The coronavirus is SARS-CoV-2 (severe acute respiratory syndrome coronavirus type 2), CORONA-mRBD is the mutated receptor binding domain of SARS-CoV-2 (SARS-mRBD) or a fragment thereof; The wild-type receptor binding domain is SARS-wtRBD (wild-type receptor binding domain of SARS-CoV-2), The CORONA-RBD receptor is ACE2 (angiotensin-converting enzyme 2), Preferably, the anti-SARS-wtRBD neutralizing antibodies (anti-CORONA-wtRBD-nABs) are anti-SARS-wtRBD-nABs, and / or the plasma or serum is derived from an immune subject, e.g., a convalescent subject, as defined above, where such an immune subject is preferably immunologically reactive to SARS-wtRBD, or (B) The coronavirus is MERS-CoV (Middle East Respiratory Syndrome coronavirus), CORONA-mRBD is the mutated receptor-binding domain of MERS-CoV (MERS-mRBD) or a fragment thereof; The wild-type receptor-binding domain is MERS-wtRBD (the wild-type receptor-binding domain of MERS-CoV), The CORONA-RBD receptor is DPP4 (dipeptidyl peptidase 4), Preferably, the anti-MERS-wtRBD neutralising antibodies (anti-CORONA-wtRBD-nAB) are anti-MERS-wtRBD-nAB and / or the plasma or serum is derived from an immunised subject as defined above, e.g. a convalescent subject, where such an immunised subject is preferably immunologically reactive to MERS-wtRBD.

[0076] Thus, preferably, the present invention relates to (A) a SARS-mRBD or a fragment thereof having reduced binding ability to ACE2 compared to SARS-wtRBD, and / or the present invention relates to (B) a MERS-mRBD or a fragment thereof having reduced binding ability to DDP4 compared to MERS-wtRBD.

[0077] (A) Mutated receptor binding domain of SARS-CoV-2 (SARS-mRBD) As mentioned above, the present invention preferably relates to (A) SARS-mRBD or a fragment thereof, which has reduced binding affinity to ACE2 compared to SARS-wtRBD.

[0078] Preferably, the SARS-mRBD (mutated receptor binding domain of SARS-CoV-2) is a mutated receptor binding domain (mRBD) of the SARS-CoV-2 spike protein (SARS-mRBD) or a fragment thereof.

[0079] Preferably, the SARS-mRBD or fragment thereof is a peptide or protein, preferably a protein.

[0080] Preferably, SARS-mRBD or a fragment thereof is obtained as an active ingredient by the method according to the invention for designing and / or obtaining an active ingredient for a vaccine composition, as described below.

[0081] Preferably, the ACE2 is a soluble and / or membrane-bound ACE2, preferably a soluble ACE2 (sACE2). The sACE2 may include active and inactive sACE2.

[0082] The binding ability of SARS-mRBD or its fragment and SARS-wtRBD to ACE2 can be measured by any method known to those skilled in the art. For example, the binding ability can be determined by, for example, staining cells expressing SARS-mRBD or SARS-wtRBD on the cell surface with ACE2 directly or indirectly labeled with a fluorophore, or conversely, staining cells expressing ACE2 on the cell surface with soluble SARS-mRBD or soluble SARS-wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, and counting positively stained cells, or by determining the Kd (dissociation constant) or EC50 (effective concentration at 50% of maximum binding) of SARS-wtRBD or SARS-mRBD to ACE2, respectively, by using ELISA. The binding ability of SARS-wtRBD to ACE2 is then compared with the binding ability of SARS-mRBD to ACE2 to obtain a decrease in binding ability.

[0083] Preferably, the avidity is measured by determining the EC50 or Kd, where a higher EC50 or Kd indicates a lower avidity. Alternatively, the avidity is measured by flow cytometry analysis, where a lower number of positively stained cells indicates a lower avidity. Thus, the reduced avidity is preferably determined by comparing the EC50, Kd, ​​and / or number of positively stained cells resulting from the binding of SARS-wtRBD to ACE2 with the EC50, Kd, ​​and / or number of positively stained cells resulting from the binding of SARS-mRBD to ACE2, respectively. Preferably, the EC50 and Kd of SARS-wtRBD or SARS-mRBD to ACE2 are determined as described in Example 4.

[0084] When the reduction in binding is determined by determining EC50 or Kd, the reduction in the binding of SARS-mRBD to ACE2 is preferably indicated by an increase in EC50 and / or Kd of 1.5 times or more, more preferably 2 times or more, even more preferably 10 times or more, even more preferably 100 times or more, even more preferably 500 times or more, and most preferably 1000 times or more. In order to obtain the best effect of vaccination, when SARS-mRBD is used as a vaccine, the binding to ACE2 must be reduced as much as possible. Therefore, preferably, there is no upper limit for the reduction in binding to ACE2. Therefore, preferably, there is no upper limit for the increase in EC50 or Kd. However, the upper limit for the increase in EC50 or Kd may be 500.000 times or less, more preferably 100.000 times or less, even more preferably 10.000 times or less. Preferably, the EC50 and Kd of SARS-wtRBD or SARS-mRBD to ACE2 are determined as described in Example 4.

[0085] When the reduction in binding ability is determined by flow cytometry analysis, the reduction in the binding ability of SARS-mRBD to ACE2 is preferably indicated by a reduction in the number of positively stained cells of 50% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more.To obtain the best effect of vaccination, when SARS-mRBD is used as a vaccine, the binding ability to ACE2 must be reduced as much as possible.Therefore, preferably, there is no upper limit for the reduction in the binding ability to ACE2.Therefore, preferably, there is no upper limit for the reduction in the number of positively stained cells, and this reduction can even be 100%.However, the upper limit for the reduction in the number of positively stained cells can be 100% or less, more preferably 99% or less.

[0086] Moreover, SARS-mRBD or a fragment thereof preferably exhibits binding to anti-SARS-wtRBD neutralizing antibodies (anti-SARS-wtRBD-nABs), i.e., the avidity for anti-SARS-wtRBD neutralizing antibodies (anti-SARS-wtRBD-nABs) is preferably maintained, i.e., ranging from a slight to moderately reduced avidity to an increased avidity, as compared to SARS-wtRBD.

[0087] The anti-SARS-wtRBD-nABs may belong to different antibody classes, such as class I, class II, class III, and / or class IV, and / or may be specific for different epitopes of SARS-wtRBD. The anti-SARS-wtRBD-nABs may be monoclonal and / or polyclonal antibodies reactive against SARS-wtRBD. The monoclonal anti-SARS-wtRBD-nABs may be recombinantly obtained. The polyclonal anti-SARS-wtRBD-nABs may be obtained from plasma or serum derived from an immunized subject who has experienced a coronavirus infection, e.g., a convalescent subject and / or an immunized subject who has been immunized, e.g., by vaccination, where such immunized subjects are preferably immunologically reactive to SARS-wtRBD. The subject is preferably a vertebrate, more preferably an immunized mammal, more preferably an ape, monkey, lemur, canine, such as dog, wolf or fox, feline, such as large or small cat, rodent, such as mouse, rat, guinea pig, hamster or rabbit, bovine, such as buffalo, antelope, sheep, goat or cow, deer, horse, donkey, bear, marten, bat, and / or human. Even more preferably, the immunized subject is a mouse, rat, rabbit, hamster, goat, donkey, horse, dog, cat, sheep or human, most preferably a human.

[0088] The binding strength of SARS-mRBD or fragments thereof and SARS-wtRBD to anti-SARS-wtRBD-nAB can be measured by any method known to those skilled in the art. For example, the binding strength can be determined by staining cells expressing SARS-mRBD or SARS-wtRBD on the cell surface with monoclonal anti-SARS-wtRBD-nAB directly or indirectly labeled with a fluorophore, or conversely, staining cells expressing monoclonal anti-SARS-wtRBD-nAB on the cell surface with soluble SARS-mRBD or soluble SARS-wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, and counting positively stained cells, or by ...wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, and counting positively stained cells, or by staining cells expressing monoclonal anti-SARS-wtRBD-nAB on the cell surface with soluble SARS-wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, and counting positively stained cells, or by staining cells expressing monoclonal anti-SARS-wtRBD-nAB on the cell surface with soluble SARS-wtRBD directly or indirect The Kd (dissociation constant) or EC50 (effective concentration at 50% of maximum binding) of SARS-wtRBD and SARS-mRBD against RS-wtRBD-nAB can be determined by using ELISA, or by determining the ED50 (effective dilution at which 50% of serum antibodies bind) of polyclonal antibodies in plasma or serum from immunized subjects who have experienced coronavirus infection, e.g., convalescent subjects and / or immunized subjects who have been immunized, e.g., by vaccination, where such immunized subjects are preferably immunologically reactive to SARS-wtRBD. The immunized subjects are as defined above. Preferably, the EC50, Kd, ​​and ED50 of SARS-wtRBD or SARS-mRBD against anti-SARS-wtRBD-nAB are determined as described in Example 4.

[0089] When the avidity of anti-SARS-wtRBD-nABs is determined by flow cytometry analysis, EC50, and / or Kd, the avidity is preferably the average avidity of several different monoclonal anti-SARS-wtRBD-nABs, preferably four or more different arbitrarily selected monoclonal anti-SARS-wtRBD-nABs. Preferably, the four different monoclonal anti-SARS-wtRBD-nABs are CC12.1, P2C-1F11, REGN10933, and S309 as described in Example 4, and the average avidity is the average EC50 or average Kd of CC12.1, P2C-1F11, REGN10933, and S309, respectively.

[0090] The sustained avidity, i.e., the range of avidity from slight to moderate decreased to increased avidity, is then obtained by comparing the avidity or average avidity of SARS-wtRBD to anti-SARS-wtRBD-nAB with the avidity or average avidity of SARS-mRBD to anti-SARS-wtRBD-nAB, respectively.

[0091] Preferably, avidity is measured by determining EC50 or Kd, where a higher EC50 or Kd indicates lower avidity. Alternatively, avidity is measured by flow cytometry analysis or by determining ED50, where a decrease in positively stained cell count or a decrease in ED50, respectively, indicates lower avidity. Thus, whether avidity is maintained, i.e., in the range of slight to moderately decreased avidity and increased avidity, is preferably determined by comparing the EC50, Kd, ​​positively stained cell count, and / or ED50 resulting from the binding of SARS-wtRBD to anti-SARS-wtRBD-nAB with the EC50, Kd, ​​positively stained cell count, and / or ED50 resulting from the binding of SARS-mRBD to anti-SARS-wtRBD-nAB, respectively.

[0092] When avidity is determined by EC50 or Kd determination, a slight to moderate decrease in avidity of SARS-mRBD to anti-SARS-wtRBD-nAB is preferably indicated by an increase in EC50 and / or Kd of 3-fold or less, even more preferably 2-fold or less, even more preferably 1.5-fold or less, and even more preferably 1.2-fold or less. When avidity is determined by flow cytometric analysis or ED50 determination, a slight to moderate decrease in avidity of SARS-mRBD to RBD anti-SARS-wtRBD-nAB is preferably indicated by a decrease in positively stained cell count and / or ED50 of 50% or more, more preferably 80% or more, and even more preferably 90% or more.

[0093] As already mentioned above, when determining avidity by EC50, Kd or flow cytometry analysis, the avidity is preferably the average avidity as defined above. Thus, a slight to moderate decrease in avidity as defined above is preferably a slight to moderate decrease in average avidity as defined above.

[0094] In order to obtain the best effect of vaccination, when SARS-mRBD is used as a vaccine, the binding ability to anti-SARS-wtRBD-nAB should be as high as possible.Therefore, preferably, the decrease in the binding ability to anti-SARS-wtRBD-nAB should be as moderate as possible, and preferably, the binding ability of anti-SARS-wtRBD-nAB to SARS-mRBD is preferably not decreased or even increased.That is, EC50 and Kd are not increased, and / or the number of positively stained cells and ED50 are not decreased.

[0095] Therefore, when determining the avidity by determining EC50 or Kd, the increase in the avidity of SARS-mRBD to anti-SARS-wtRBD-nAB is preferably indicated by a decrease in EC50 and / or Kd of 10% or more, more preferably 25% or more, even more preferably 50% or more, even more preferably 80% or more, and most preferably 90% or more. As mentioned above, in order to obtain the best effect of vaccination, when SARS-mRBD is used as a vaccine, the avidity to anti-SARS-wtRBD-nAB should be as high as possible. Therefore, preferably, there is no upper limit for the increase in avidity to anti-SARS-wtRBD-nAB. Thus, preferably, there is no upper limit for the decrease in EC50 or Kd, and this decrease can even be 100%. However, the upper limit for the decrease in EC50 and / or Kd can be 100% or less, more preferably 99% or less.

[0096] When determining the avidity by flow cytometry analysis or ED50, the increase in avidity of SARS-mRBD to anti-SARS-wtRBD-nAB is preferably indicated by an increase of 1.1 times or more, more preferably 1.2 times or more, even more preferably 1.5 times or more, and even more preferably 2 times or more in the number of positively stained cells and / or ED50, respectively. As mentioned above, in order to obtain the best effect of vaccination, when SARS-mRBD is used as a vaccine, the avidity to anti-SARS-wtRBD-nAB should be as high as possible. Therefore, preferably, there is no upper limit for the increase in avidity to anti-SARS-wtRBD-nAB. Thus, preferably, there is no upper limit for the increase in positively stained cells and / or ED50. However, the upper limit for the increase in positively stained cells and / or ED50 may be 10.000 times or less, more preferably 1.000 times or less, and even more preferably 100 times or less.

[0097] As already mentioned above, when determining avidity by EC50, Kd or flow cytometric analysis, the avidity is preferably the average avidity as defined above. Thus, an increase in avidity as defined above is preferably an increase in average avidity as defined above.

[0098] Preferably, the present invention relates to a method for producing a pharmaceutical composition comprising the steps of: An amino acid sequence comprising one or more, preferably one to three, more preferably one or two, even more preferably one substitution of amino acid residues at positions selected from G184, Y187, L137, Y171, F138, Q180, F168, Y131 or S55 of SARS-wtRBD of SEQ ID NO:1 or a fragment thereof. and more preferably consisting of, and excluding said substitution, having 85% or greater amino acid sequence identity to SEQ ID NO:1.

[0099] Preferably, the SARS-mRBD or a fragment thereof has, excluding substitutions, greater than 90% amino acid sequence identity to SEQ ID NO:1, more preferably greater than 95%, even more preferably greater than 97%, even more preferably greater than 98%, and most preferably 100%.

[0100] SARS-wtRBD in SEQ ID NO:1 is the Wuhan mutant SARS-wtRBD.

[0101] The amino acid sequence identity of SARS-mRBD to SARS-wtRBD of SEQ ID NO:1 as defined above must be low enough to still encompass the SARS-RBD of SARS-CoV-2 mutants other than SEQ ID NO:1, the Wuhan mutant, thereby making the SARS-mRBD or a fragment thereof adaptable to such mutants or to combinations of mutations, e.g., substitutions, deletions, and insertions, contained in such mutants when compared to SEQ ID NO:1. Examples of such SARS-CoV-2 mutants are: B1.351 (beta), which contains the substitutions K99N, E166K, and N183Y; B.1.1.7 (alpha, 501Y.V1), which contains the N183Y substitution; P.1 (gamma, 501Y.V3), which contains the substitutions K99T, E166K, and N183Y; and / or B.1.617.2 (Delta) containing the substitutions L134R and T160K where the position of each substitution is shown with reference to SARS-wtRBD in SEQ ID NO:1.

[0102] Preferably, the one or more substitutions of amino acid residues are selected from G184, L137, Y171 or Y187, more preferably one to three, preferably one or two, most preferably one substitution of an amino acid residue at a position selected from G184, L137 or Y187 of SARS-wtRBD of SEQ ID NO:1.

[0103] More preferably, the one or more substitutions of amino acid residues are of one or both, preferably one, substitution of an amino acid residue at a position selected from G184 or Y187 or G184 or Y137 of SARS-wtRBD of SEQ ID NO:1.

[0104] Even more preferably, the one or more substitutions of amino acid residues are substitutions of the amino acid residue at position G184 of SARS-wtRBD of SEQ ID NO:1.

[0105] Preferably, the one or more substitutions of amino acid residues are one or more, preferably one to three, more preferably one or two, even more preferably one substitution of amino acid residues at positions selected from G184, Y187, L137, Y171, F138, Q180, F168, Y131 or S55 of SARS-wtRBD of SEQ ID NO: 194, wherein: - the substitution of the amino acid residue at position G184 is selected from G184A, G184C, G184D, G184E, G184F, G184H, G184I, G184K, G184L, G184M, G184N, G184P, G184Q, G184R, G184S, G184T, G184V, G184W or G184Y; - the substitution of the amino acid residue at position Y187 is selected from Y187A, Y187C, Y187D, Y187E, Y187G, Y187I, Y187K, Y187L, Y187M, Y187N, Y187Q, Y187R, Y187S, Y187T or Y187V; - the substitution of the amino acid residue at position L137 is selected from L137D, L137E, L137K, L137R and L137Y; - the substitution of the amino acid residue at position Y171 is selected from Y171A, Y171C, Y171E, Y171I, Y171K, Y171L, Y171M, Y171N, Y171P, Y171Q, Y171R, Y171S, Y171T or Y171V; - the substitution of the amino acid residue at position F138 is selected from F138A, F138C, F138E, F138G, F138I, F138K, F138N, F138Q, F138R, F138S, F138T, F138W or F138Y; - the substitution of the amino acid residue at position Q180 is selected from Q180C, Q180D, Q180I, Q180K, Q180L or Q180V; - the substitution of the amino acid residue at position F168 is selected from F168C, F168D or F168E; - the substitution of the amino acid residue at position Y131 is selected from Y131A, Y131C, Y131D, Y131E, Y131F, Y131G, Y131H, Y131I, Y131L, Y131M, Y131N, Y131P, Y131Q, Y131S, Y131T, Y131V or Y131W, and / or - the substitution of the amino acid residue at position S55 is S55N.

[0106] More preferably, the one or more substitutions of amino acid residues are one to three, preferably one or two, more preferably one substitution of an amino acid residue at a position selected from G184, L137, Y171 or Y187 of SARS-wtRBD having SEQ ID NO:1, more preferably G184, L137, or Y187, wherein - the substitution of the amino acid residue at position G184 is selected from G184R, G184D, G184Y, G184K, G184S, G184P, G184E, G184A, G184V, G184N, G184Q, G184T, G184M, G184H or G184L; - the substitution of the amino acid residue at position L137 is selected from L137R or L137E, - the substitution of the amino acid residue at position Y187 is selected from Y187N or Y187Q, - the substitution of the amino acid residue in position Y171 is selected from Y171S or Y171T.

[0107] Even more preferably, the one or more substitutions of amino acid residues are one or both, even more preferably one, substitution of an amino acid residue at a position selected from G184 or Y187 or G184 or L137, more preferably G184 or L137, of SARS-wtRBD having SEQ ID NO:1, wherein: - the substitution of the amino acid residue at position G184 is selected from G184R, G184D, G184Y, G184K, G184S, G184P, G184E, G184A, G184V, G184N, G184Q, G184T, G184M, G184H or G184L, more preferably G184R, G502D or G184E, most preferably G184R or G184E, - the substitution of the amino acid residue at position L137 is L137R, - the substitution of the amino acid residue at position Y187 is Y187N.

[0108] Even more preferably, the one or more substitutions of amino acid residues are a substitution of one of the amino acid residues at position G184 of SARS-wtRBD having SEQ ID NO:1, where the substitution is G184R, G184D or G184E, more preferably G184R or G184E, and / or the one or more substitutions of amino acid residues are a substitution of one of the amino acid residues at position L137 of SARS-wtRBD having SEQ ID NO:1, where the substitution is L137R, and most preferably the substitution is G184R or G184E.

[0109] Preferably, the SARS-mRBD or a fragment thereof comprises: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, 49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93 or SEQ ID NO:94; More preferably, selected from SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:5, SEQ ID NO:20, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:13, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:11, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:55, SEQ ID NO:53, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:92 or SEQ ID NO:93; Even more preferably, selected from SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:5, SEQ ID NO:20, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:13, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:11, SEQ ID NO:7 or SEQ ID NO:10, SEQ ID NO:55 or SEQ ID NO:30; Even more preferably, selected from SEQ ID NO:15, SEQ ID NO:5, SEQ ID NO:2, SEQ ID NO:55 or SEQ ID NO:30; Even more preferably, selected from SEQ ID NO:15, SEQ ID NO:5, SEQ ID NO:2, or SEQ ID NO:55; Even more preferably, comprising, and preferably consisting of, an amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 2, or SEQ ID NO: 55; Most preferably, it comprises, and preferably consists of, the amino acid sequence of SEQ ID NO:15 or SEQ ID NO:2.

[0110] (B) Mutated receptor-binding domain of MERS-CoV (MERS-mRBD) As described above, the present invention further preferably relates to (B) MERS-mRBD or a fragment thereof, which has a reduced binding ability to DDP4 compared to MERS-wtRBD.

[0111] Preferably, the MERS-mRBD (mutated receptor binding domain of MERS-CoV) is a mutated receptor binding domain (mRBD) of the MERS-CoV spike protein (MERS-mRBD) or a fragment thereof.

[0112] Preferably, the MERS-mRBD or fragment thereof is a peptide or protein, preferably a protein.

[0113] Preferably, MERS-mRBD or a fragment thereof is obtained as an active ingredient by the method according to the invention for designing and / or obtaining an active ingredient for a vaccine composition, as described below.

[0114] Preferably, the DPP4 is a soluble and / or membrane-bound DPP4, preferably a soluble DPP4 (sDPP4).

[0115] The binding ability of MERS-mRBD or its fragment and MERS-wtRBD to DPP4 can be measured by any method known to those skilled in the art.For example, the binding ability can be determined by, for example, staining cells expressing MERS-mRBD or MERS-wtRBD on the cell surface with DPP4 directly or indirectly labeled with a fluorophore, or conversely, staining cells expressing DPP4 on the cell surface with soluble MERS-mRBD or soluble MERS-wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, and counting positively stained cells, or by determining the Kd (dissociation constant) or EC50 (effective concentration at 50% of maximum binding) of MERS-wtRBD or MERS-mRBD to DPP4 by using ELISA.Then, the binding ability of MERS-wtRBD to DPP4 is compared with the binding ability of MERS-mRBD to DPP4 to obtain the decrease in binding ability.

[0116] Preferably, the avidity is measured by determining EC50 or Kd, where a higher EC50 or Kd indicates a lower avidity. Alternatively, the avidity is measured by flow cytometry analysis, where a lower number of positively stained cells indicates a lower avidity. Thus, the reduced avidity is preferably determined by comparing the EC50, Kd, ​​and / or number of positively stained cells resulting from the binding of MERS-wtRBD to DPP4 with the EC50, Kd, ​​and / or number of positively stained cells resulting from the binding of MERS-mRBD to DPP4, respectively. Preferably, the EC50 of MERS-wtRBD or MERS-mRBD to DPP4 is determined as described in Example 7.

[0117] When the reduction in binding ability is determined by determining EC50 or Kd, the reduction in the binding ability of MERS-mRBD to DPP4 is preferably indicated by an increase in EC50 and / or Kd of 1.5 times or more, more preferably 2 times or more, even more preferably 10 times or more, even more preferably 100 times or more, even more preferably 500 times or more, and most preferably 1000 times or more. In order to obtain the best effect of vaccination, when MERS-mRBD is used as a vaccine, the binding ability to DPP4 must be reduced as much as possible. Therefore, preferably, there is no upper limit for the reduction in the binding ability to DPP4. Therefore, preferably, there is no upper limit for the increase in EC50 or Kd. However, the upper limit for the increase in EC50 or Kd may be 500.000 times or less, more preferably 100.000 times or less, even more preferably 10.000 times or less. Preferably, the EC50 of MERS-wtRBD or MERS-mRBD to DPP4 is determined as described in Example 7.

[0118] When the reduction in binding ability is determined by flow cytometry analysis, the reduction in the binding ability of MERS-mRBD to DPP4 is preferably indicated by a reduction in the number of positively stained cells of 50% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more.To obtain the best effect of vaccination, when MERS-mRBD is used as a vaccine, the binding ability to DPP4 must be reduced as much as possible.Therefore, preferably, there is no upper limit for the reduction in the binding ability to DPP4.Therefore, preferably, there is no upper limit for the reduction in the number of positively stained cells, and this reduction can even be 100%.However, the upper limit for the reduction in the number of positively stained cells can be 100% or less, more preferably 99% or less.

[0119] Moreover, MERS-mRBD or a fragment thereof preferably exhibits binding to anti-MERS-wtRBD neutralizing antibodies (anti-MERS-wtRBD-nAB), i.e., the avidity for anti-MERS-wtRBD neutralizing antibodies (anti-MERS-wtRBD-nAB) is preferably maintained, i.e., ranges from a slight to moderately reduced avidity to an increased avidity, as compared to MERS-wtRBD.

[0120] Anti-MERS-wtRBD-nABs may belong to different antibody classes, such as class I, class II, class III, and / or class IV, and / or may be specific for different epitopes of MERS-wtRBD. Anti-MERS-wtRBD-nABs may be monoclonal and / or polyclonal antibodies reactive to MERS-wtRBD. Monoclonal anti-MERS-wtRBD-nABs may be recombinantly obtained. Polyclonal anti-MERS-wtRBD-nABs may be obtained from plasma or serum derived from an immunized subject who has experienced a coronavirus infection, e.g., a convalescent subject and / or an immunized subject who has been immunized, e.g., by vaccination, where such immunized subject is preferably immunologically reactive to MERS-wtRBD. The subject is preferably a vertebrate, more preferably an immunized mammal, more preferably an ape, monkey, lemur, canine, such as dog, wolf or fox, feline, such as large or small cat, rodent, such as mouse, rat, guinea pig, hamster or rabbit, bovine, such as buffalo, antelope, sheep, goat or cow, deer, horse, donkey, bear, marten, bat, and / or human. Even more preferably, the immunized subject is a mouse, rat, rabbit, hamster, goat, donkey, horse, dog, cat, sheep or human, most preferably a human.

[0121] The binding strength of MERS-mRBD or a fragment thereof and MERS-wtRBD to anti-MERS-wtRBD-nAB can be measured by any method known to those skilled in the art. For example, the binding strength can be determined by staining cells expressing MERS-mRBD or MERS-wtRBD on the cell surface with monoclonal anti-MERS-wtRBD-nAB directly or indirectly labeled with a fluorophore, or conversely, staining cells expressing monoclonal anti-MERS-wtRBD-nAB on the cell surface with soluble MERS-mRBD or soluble MERS-wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, and counting positively stained cells, or by ...wtRBD or soluble MERS-wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, and counting positively stained cells, or by staining cells expressing monoclonal anti-MERS-wtRBD-nAB on the cell surface with monoclonal anti-MERS-wtRBD-nAB on the cell surface with monoclonal anti-MERS-wtRBD-nAB on the cell surface with monoclonal anti-MERS-wtRBD-nAB on the cell surface with monoclonal anti-MERS-w It can be determined by using ELISA to determine the Kd (dissociation constant) or EC50 (effective concentration at 50% of maximum binding) of MERS-wtRBD and MERS-mRBD against RS-wtRBD-nAB, or by determining the ED50 (effective dilution at which 50% of serum antibodies bind) of polyclonal antibodies in plasma or serum from immunized subjects who have experienced coronavirus infection, e.g., convalescent subjects and / or immunized subjects who have been immunized, e.g., by vaccination, where such immunized subjects are preferably immunologically reactive to MERS-wtRBD. The immunized subjects are as defined above. Preferably, the EC50 of MERS-wtRBD or MERS-mRBD against anti-MERS-wtRBD-nAB is determined as described in Example 7.

[0122] When the avidity of anti-MERS-wtRBD-nABs is determined by flow cytometry analysis, EC50, and / or Kd, the avidity is preferably the average avidity of several different monoclonal anti-MERS-wtRBD-nABs, preferably four or more different arbitrarily selected monoclonal anti-MERS-wtRBD-nABs. Preferably, the four different monoclonal anti-MERS-wtRBD-nABs are 4C2h, D12, LCA60, and MERS4V2 as described in Example 9, and the average avidity is the average EC50 or average Kd of 4C2h, D12, LCA60, and MERS4V2, respectively.

[0123] The sustained avidity, i.e., the range of avidity from slight to moderately decreased to increased avidity, is then obtained by comparing the avidity or average avidity of MERS-wtRBD to anti-MERS-wtRBD-nAB with the avidity or average avidity of MERS-mRBD to anti-MERS-wtRBD-nAB, respectively.

[0124] Preferably, avidity is measured by determining EC50 or Kd, where a higher EC50 or Kd indicates lower avidity. Alternatively, avidity is measured by flow cytometry analysis or by determining ED50, where a lower number of positively stained cells or a lower ED50 indicates lower avidity, respectively. Thus, whether avidity is maintained, i.e., in the range of a slight to moderate decrease in avidity and an increase in avidity, is preferably determined by comparing the EC50, Kd, ​​positively stained cell count, and / or ED50 resulting from the binding of MERS-wtRBD to anti-MERS-wtRBD-nAB with the EC50, Kd, ​​positively stained cell count, and / or ED50 resulting from the binding of MERS-mRBD to anti-MERS-wtRBD-nAB, respectively.

[0125] When avidity is determined by EC50 or Kd determination, a slight to moderate decrease in avidity of MERS-mRBD to anti-MERS-wtRBD-nAB is preferably indicated by an increase in EC50 and / or Kd of 3-fold or less, even more preferably 2-fold or less, even more preferably 1.5-fold or less, and even more preferably 1.2-fold or less. When avidity is determined by flow cytometric analysis or ED50 determination, a slight to moderate decrease in avidity of MERS-mRBD to RBD-anti-MERS-wtRBD-nAB is preferably indicated by a decrease in positively stained cell count and / or ED50 of 50% or more, more preferably 80% or more, and even more preferably 90% or more.

[0126] As already mentioned above, when determining avidity by EC50, Kd or flow cytometry analysis, the avidity is preferably the average avidity as defined above. Thus, a slight to moderate decrease in avidity as defined above is preferably a slight to moderate decrease in average avidity as defined above.

[0127] In order to obtain the best effect of vaccination, when MERS-mRBD is used as a vaccine, the binding ability to anti-MERS-wtRBD-nAB should be as high as possible.Therefore, preferably, the decrease in the binding ability to anti-MERS-wtRBD-nAB should be as moderate as possible, and preferably, the binding ability of anti-MERS-wtRBD-nAB to MERS-mRBD is preferably not decreased or even increased.That is, EC50 and Kd are not increased, and / or the number of positively stained cells and ED50 are not decreased.

[0128] Therefore, when determining the binding ability by determining EC50 or Kd, the increase in the binding ability of MERS-mRBD to anti-MERS-wtRBD-nAB is preferably indicated by a decrease in EC50 and / or Kd of 10% or more, more preferably 25% or more, even more preferably 50% or more, even more preferably 80% or more, and most preferably 90% or more. As mentioned above, in order to obtain the best effect of vaccination, when MERS-mRBD is used as a vaccine, the binding ability to anti-MERS-wtRBD-nAB should be as high as possible. Therefore, preferably, there is no upper limit for the increase in the binding ability to anti-MERS-wtRBD-nAB. Thus, preferably, there is no upper limit for the decrease in EC50 or Kd, and this decrease can even be 100%. However, the upper limit for the decrease in EC50 and / or Kd can be 100% or less, more preferably 99% or less.

[0129] When determining the avidity by flow cytometry analysis or ED50, the increase in the avidity of MERS-mRBD to anti-MERS-wtRBD-nAB is preferably indicated by an increase of 1.1 times or more, more preferably 1.2 times or more, even more preferably 1.5 times or more, and even more preferably 2 times or more in the number of positively stained cells and / or ED50, respectively. As mentioned above, in order to obtain the best effect of vaccination, when MERS-mRBD is used as a vaccine, the avidity to anti-MERS-wtRBD-nAB should be as high as possible. Therefore, preferably, there is no upper limit for the increase in avidity to anti-MERS-wtRBD-nAB. Therefore, preferably, there is no upper limit for the increase in positively stained cells and / or ED50. However, the upper limit for the increase in positively stained cells and / or ED50 may be 10.000 times or less, more preferably 1.000 times or less, and even more preferably 100 times or less.

[0130] As already mentioned above, when determining avidity by EC50, Kd or flow cytometric analysis, the avidity is preferably the average avidity as defined above. Thus, an increase in avidity as defined above is preferably an increase in average avidity as defined above.

[0131] Preferably, the present invention relates to a method for producing a pharmaceutical composition comprising the steps of: An amino acid sequence comprising one or more, preferably one to three, or two, preferably one or two, more preferably one substitution of amino acid residues at positions selected from L140, D144, E170, D171 or D173 of MERS-wtRBD having SEQ ID NO: 194, or a fragment thereof. and more preferably consisting of, and excluding said substitution, having 85% or greater amino acid sequence identity to SEQ ID NO: 194.

[0132] Preferably, the MERS-mRBD or a fragment thereof has, excluding substitutions, greater than 90% amino acid sequence identity to SEQ ID NO:194, more preferably greater than 95%, even more preferably greater than 98%, even more preferably greater than 99%, and most preferably 100%.

[0133] The MERS-wtRBD of sequence number 194 is, by way of example, the MERS-wtRBD found in the AGN70929.1 variant.

[0134] The amino acid sequence identity of MERS-mRBD to MERS-wtRBD of SEQ ID NO: 194 as defined above must be low enough to still encompass the MERS-RBD of MERS-CoV mutants other than SEQ ID NO: 194, thereby making MERS-mRBD or a fragment thereof accommodative to such mutants or to combinations of mutations, e.g., substitutions, deletions, and insertions, contained in such mutants when compared to SEQ ID NO: 194. Examples of such MERS-CoV mutants and / or mutations contained therein are as follows: - in the receptor binding domain (RBD) of the S1 subunit, a T424I substitution in 2 / 8 isolates and a S459T substitution in 1 / 8 isolates. - In the RBD / RBM overlap region, a W553R substitution was found in the RBD / RBM overlap region in 2 / 8 isolates. - in the fusion peptide, Q1009L in the heptad repeat region 1 (HR1) and C1313S in the transmembrane (TM) region (Supplementary Table 2B, substitutions in the S2 subunit included S950T in the fusion peptide, Q1009L in the heptad repeat region 1 (HR1), and C1313S in the transmembrane (TM) region). - MERS-CoV variants are listed in the following table (retrieved from https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5165220 / ).

[0135] [Table 1]

[0136] It is noted that the mutations in MERS-RBD of MERS-CoV mutants are shown with respect to their position in the MERS-Spike protein. The sequence of MERS-wtRBD in SEQ ID NO: 194 corresponds to the sequence starting at position 367 of MERS-wtSpike in SEQ ID NO: 200. Thus, when 366 amino acids are removed from a particular amino acid position in MERS-wtSpike or MERS-mSpike, the result corresponds to the amino acid position in MERS-wtRBD or MERS-mRBD, respectively. A selection of the corresponding amino acid positions in MERS-wtRBD / MERS-mRBD and MERS-wtSpike / MERS-mSpike is shown in Table B, presented before the description of the figures further below.

[0137] Preferably, the one or more substitutions of amino acid residues are one to three, preferably one or two, preferably one substitution of amino acid residues at a position selected from D173, D144 or D171 of MERS-wtRBD of SEQ ID NO:194.

[0138] More preferably, the one or more substitutions of amino acid residues are one or both, preferably one, substitutions of amino acid residues at positions selected from D144 or D171 of MERS-wtRBD of SEQ ID NO:194.

[0139] Preferably, the one or more substitutions of amino acid residues are one or more, preferably one to three, more preferably one or two, even more preferably one substitution of amino acid residues at positions selected from L140, D144, E170, D171 or D173 of SARS-wtRBD of SEQ ID NO: 194, wherein: - the substitution of the amino acid residue at position L140 is L140A, - the substitution of the amino acid residue at position D144 is D144A, - the substitution of the amino acid residue at position E170 is E170R, - the substitution of the amino acid residue at position D171 is D171K, and / or - the substitution of the amino acid residue at position D173 is D173K.

[0140] More preferably, the one or more substitutions of amino acid residues are one to three, preferably one or two, even more preferably one substitution of amino acid residues at positions selected from D144, D171 or D173 of MERS-wtRBD of SEQ ID NO: 194, wherein: - the substitution of the amino acid residue at position D144 is D144A, - the substitution of the amino acid residue at position D171 is D171K, - the substitution of the amino acid residue at position D173 is D173K.

[0141] Even more preferably, the substitution of one or more amino acid residues is a substitution of one or both, preferably one, of the amino acid residues at positions selected from D144 or D171 or D173 of MERS-wtRBD of SEQ ID NO: 194, wherein: - the substitution of the amino acid residue at position D144 is D144A, - the substitution of the amino acid residue at position D171 is D171K.

[0142] Even more preferably, the MERS-mRBD or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198 or SEQ ID NO:199, more preferably selected from SEQ ID NO:195 or SEQ ID NO:196.

[0143] Mutated coronavirus spike protein (CORONA-mSpike) The present invention further provides a mutated spike protein of a coronavirus (CORONA-mSpike) or a fragment thereof comprising the CORONA-mRBD or a fragment thereof according to the present invention.

[0144] Preferably, the CORONA-mSpike or a fragment thereof comprising the CORONA-mRBD or a fragment thereof is a recombinant polypeptide or protein or a chemically synthesized polypeptide or protein.

[0145] Preferably, the CORONA-mSpike or a fragment thereof comprises, preferably consists of, a CORONA-mSpike or a fragment thereof, wherein the CORONA spike comprises a CORONA-mRBD or a fragment thereof according to the present invention.

[0146] Preferably, CORONA-mSpike or a fragment thereof comprises: (A) a mutated spike protein of SARS-CoV-2 (SARS-mSpike) or a fragment thereof, and the CORONA-mRBD or a fragment thereof is the SARS-mRBD or a fragment thereof according to the invention, or - (B) a mutated spike protein of MERS-CoV (SARS-mSpike) or a fragment thereof, and the CORONA-mRBD or a fragment thereof is the MERS-mRBD or a fragment thereof according to the present invention.

[0147] Thus, preferably, the present invention relates to (A) a SARS-mSpike or a fragment thereof comprising a SARS-mRBD or a fragment thereof according to the present invention, and / or The present invention relates to (B) MERS-mSpike or a fragment thereof comprising the MERS-mRBD or a fragment thereof according to the present invention.

[0148] Moreover, it should be understood that in the present invention, any embodiment disclosing a CORONA-mRBD or a fragment thereof is applicable mutatis mutandis to a CORONA-mRBD or a fragment thereof.

[0149] (A) Mutated spike protein of SARS-CoV-2 (SARS-mSpike) As mentioned above, the present invention preferably relates to SARS-mSpike or a fragment thereof comprising the SARS-mRBD or a fragment thereof according to the present invention.

[0150] Preferably, the SARS-mSpike or a fragment thereof comprising the SARS-mRBD or a fragment thereof is a recombinant or chemically synthesized polypeptide or protein.

[0151] Preferably, the SARS-mSpike or a fragment thereof comprises, preferably consists of, SARS-mSpike or a fragment thereof, wherein the SARS-mSpike comprises the SARS-mRBD or a fragment thereof according to the present invention.

[0152] Preferably, SARS-mSpike or a fragment thereof comprises, preferably consists of, an amino acid sequence comprising the SARS-mRBD or a fragment thereof according to the present invention, and preferably a 2P mutation and / or an abolished Furin cleavage site, more preferably both, wherein SARS-mSpike has 85% or greater amino acid sequence identity to SEQ ID NO: 100, excluding the SARS-mRBD or a fragment thereof, and preferably a 2P mutation and / or an abolished Furin cleavage site.

[0153] The 2P mutation in SARS-wtSpike is a substitution of amino acid residues K986P and V987P in SARS-wtSpike of SEQ ID NO:100.

[0154] The disabled furin cleavage site is the furin cleavage site 682-RRAR-685 of SARS-wtSpike of SEQ ID NO: 100, which is disabled, for example, by mutation through substitution, deletion or insertion of amino acid residues, preferably by substitution with 682-QQAQ-685.

[0155] Preferably, SARS-mSpike or a fragment thereof has 90% or greater, more preferably 95% or greater, even more preferably 98% or greater, still more preferably 99% or greater, and most preferably 100% amino acid sequence identity to SEQ ID NO: 100, excluding the SARS-mRBD or a fragment thereof and preferably the 2P mutation and / or the invalid Furin cleavage site.

[0156] Moreover, it should be understood that in the present invention, any embodiment disclosing SARS-mRBD or a fragment thereof is applicable mutatis mutandis to SARS-mSpike or a fragment thereof.

[0157] Preferably, SARS-mSpike or a fragment thereof comprises: An amino acid sequence or a fragment thereof comprising one or more, preferably one to three, more preferably one or two, even more preferably one substitution of amino acid residues at positions selected from G502, Y505, L455, Y489, F456, Q498, F486, Y449 or S373 of SARS-wtSpike of SEQ ID NO: 100, and preferably a 2P mutation and / or an invalid furin cleavage site, more preferably both. wherein SARS-mSpike or a fragment thereof has 85% or greater amino acid sequence identity to SEQ ID NO: 100, excluding said substitution and preferably 2P mutation and / or abrogated Furin cleavage.

[0158] Preferably, SARS-mSpike or a fragment thereof has, excluding this substitution and preferably the 2P mutation and / or the invalid Furin cleavage site, 90% or more, more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, and most preferably 100% amino acid sequence identity to SEQ ID NO: 100.

[0159] and / or the amino acid sequence identity of SARS-mSpike or a fragment thereof to SARS-wtSpike of SEQ ID NO: 100, excluding the SARS-mRBD or a fragment thereof and preferably 2P mutations and / or invalid Furin cleavage sites; and / or The amino acid sequence identity of SARS-mSpike or a fragment thereof to SARS-wtSpike of SEQ ID NO: 100, excluding substitutions and preferably 2P mutations and / or invalid Furin cleavage sites, is: By still including SARS-Spike of SARS-CoV-2 mutants other than SEQ ID NO: 100, the Wuhan mutant, the mutations, e.g., combinations of substitutions, deletions, and insertions, contained in such mutants, when compared to SEQ ID NO: 100, should be sufficiently low to allow SARS-mSpike or a fragment thereof to be accommodated. Examples of such SARS-CoV-2 mutants are: B1.351 (beta), containing the substitutions D60A, D215G, L242H, K417N, E484K, N501Y, D614G and A701V; B.1.1.7 (alpha, 501Y.V1), containing the N501Y, A570D, D614G, P681H, T716I, S982A and D1118H substitutions as well as the H69 / V70 deletion and the 144 deletion; P.1 (gamma, 501Y.V3), containing the substitutions L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I and V1176F, and / or B.1.617.2 (delta) containing the substitutions T19R, L452R, T478K, D614G, P681R and D950N and the deletion 157-158 where the position of each substitution is shown with reference to SARS-wtSpike in SEQ ID NO:100.

[0160] The underlined amino acid substitutions are located in the RBD of SARS-Spike of the respective SARS-CoV-2 mutants.

[0161] For a better understanding of the invention, it is noted that the sequence of SARS-wtRBD in SEQ ID NO: 1 corresponds to the sequence starting at position 319 of SARS-wtSpike in SEQ ID NO: 100. Thus, when 318 amino acids are removed from a particular amino acid position in SARS-wtSpike or SARS-mSpike, the result corresponds to the amino acid positions in SARS-wtRBD or SARS-mRBD, respectively. A selection of the corresponding amino acid positions in SARS-wtRBD / SARS-mRBD and SARS-wtSpike / SARS-mSpike is shown in Table A, presented before the description of the figures further below in this specification.

[0162] Preferably, the one or more substitutions of amino acid residues are one to three, preferably one or two, most preferably one substitution of amino acid residues at a position selected from G502, L455, Y489 or Y505, more preferably G502, L455 or Y505 of SARS-wtSpike of SEQ ID NO: 100.

[0163] More preferably, the one or more substitutions of amino acid residues are one or both, more preferably one, substitution of the amino acid residues at positions selected from G502 or Y505 or G502 or L455 of SARS-wtSpike of SEQ ID NO:100.

[0164] Even more preferably, the one or more substitutions of amino acid residues are substitutions of the amino acid residue at position G502 of SARS-wtSpike of SEQ ID NO:100.

[0165] Preferably, in SARS-mSpike or a fragment thereof, - the substitution of the amino acid residue at position G502 is selected from G502A, G502C, G502D, G502E, G502F, G502H, G502I, G502K, G502L, G502M, G502N, G502P, G502Q, G502R, G502S, G502T, G502V, G502W or G502Y; - the substitution of the amino acid residue at position Y505 is selected from Y505A, Y505C, Y505D, Y505E, Y505G, Y505I, Y505K, Y505L, Y505M, Y505N, Y505Q, Y505R, Y505S, Y505T or Y505V; - the substitution of the amino acid residue at position L455 is selected from L455D, L455E, L455K, L455R and L455Y; - the substitution of the amino acid residue at position Y489 is selected from Y489A, Y489C, Y489E, Y489I, Y489K, Y489L, Y489M, Y489N, Y489P, Y489Q, Y489R, Y489S, Y489T or Y489V; - the substitution of the amino acid residue at position F456 is selected from F456A, F456C, F456E, F456G, F456I, F456K, F456N, F456Q, F456R, F456S, F456T, F456W or F456Y; - the substitution of the amino acid residue at position Q498 is selected from Q498C, Q498D, Q498I, Q498K, Q498L or Q498V; - the substitution of the amino acid residue at position F486 is selected from F486C, F486D or F486E; - the substitution of the amino acid residue at position Y449 is selected from Y449A, Y449C, Y449D, Y449E, Y449F, Y449G, Y449H, Y449I, Y449L, Y449M, Y449N, Y449P, Y449Q, Y449S, Y449T, Y449V or Y449W, and / or - the substitution of the amino acid residue at position S373 is S373N.

[0166] More preferably, the one or more substitutions of amino acid residues are one to all, preferably one or two, more preferably one substitution of amino acid residues at a position selected from G502, L455, Y489 or Y505, more preferably at a position selected from G502, L455 or Y505 of SARS-wtSpike having SEQ ID NO: 100, wherein - the substitution of the amino acid residue at position G502 is selected from G502R, G502D, G502Y, G502K, G502S, G502P, G502E, G502V, G402A, G502N, G502Q, G502T, G502M, G502H or G502L; - the substitution of the amino acid residue at position L455 is selected from L455R or L455E, - the substitution of the amino acid residue at position Y505 is selected from Y505N and Y505Q, the substitution of the amino acid residue at position Y489 is selected from Y489S and Y489T.

[0167] Even more preferably, the substitution of one or more amino acid residues is a substitution of one or both, preferably one, of an amino acid residue at a position selected from G502 or L505, or G502 or L455, more preferably G502 or L455, of SARS-wtSpike having SEQ ID NO: 100, wherein - the substitution of the amino acid residue at position G502 is selected from G502R, G502D, G502Y, G502K, G502S, G502P, G502E, G502V, G402A, G502N, G502Q, G502T, G502M, G502H or G502L, more preferably G502R, G502D or G502E, most preferably G502R or G502E; - the substitution of the amino acid residue at position L455 is L455R, - the substitution of the amino acid residue at position Y505 is Y505N.

[0168] Even more preferably, the one or more substitutions of amino acid residues are a substitution of one of the amino acid residues at position G502 of SARS-wtSpike having SEQ ID NO: 100, wherein said substitution is G502R, G502D or G502E, more preferably G502R or G502E; and / or the one or more substitutions of amino acid residues are a substitution of one of the amino acid residues at position L455 of SARS-wtRBD having SEQ ID NO: 100, wherein said substitution is L455R, and most preferably said substitution is G502R or G502E.

[0169] Preferably, the SARS-mSpike or a fragment thereof is selected from the group consisting of SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, No. 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193 or SEQ ID NO: 194 is selected from More preferably, selected from SEQ ID NO:114, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:108, SEQ ID NO:115, SEQ ID NO:112, SEQ ID NO:101, SEQ ID NO:117, SEQ ID NO:102, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:110, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:154, SEQ ID NO:152, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:191 or SEQ ID NO:192; Even more preferably, selected from SEQ ID NO:114, SEQ ID NO:104, SEQ ID NO:119, SEQ ID NO:108, SEQ ID NO:115, SEQ ID NO:112, SEQ ID NO:101, SEQ ID NO:117, SEQ ID NO:102, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:110, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:154 or SEQ ID NO:129; Even more preferably, selected from SEQ ID NO:114, SEQ ID NO:104, SEQ ID NO:101, SEQ ID NO:154 or SEQ ID NO:129; Even more preferably, selected from SEQ ID NO:114, SEQ ID NO:104, SEQ ID NO:101 or SEQ ID NO:154; Even more preferably, SEQ ID NO:114, SEQ ID NO:101 or SEQ ID NO:154 comprising, and preferably consisting of, an amino acid sequence selected from Most preferably, it comprises, preferably consists of, the amino acid sequence of SEQ ID NO:114 or SEQ ID NO:101.

[0170] (B) Mutated spike protein of SARS-CoV (MERS-mSpike) As mentioned above, the present invention preferably relates to MERS-mSpike or a fragment thereof comprising the MERS-mRBD or a fragment thereof according to the present invention.

[0171] Preferably, the MERS-mSpike or a fragment thereof comprising the MERS-mRBD or a fragment thereof is a recombinant or chemically synthesized polypeptide or protein.

[0172] Preferably, the MERS-mSpike or fragment thereof comprises, preferably consists of, MERS-mSpike or a fragment thereof, wherein the MERS spike comprises the MERS-mRBD or a fragment thereof according to the present invention.

[0173] Preferably, MERS-mSpike or a fragment thereof comprises, preferably consists of, an amino acid sequence comprising the MERS-mRBD or a fragment thereof according to the present invention, and preferably a 2P mutation and / or an invalid furin cleavage site, more preferably both, wherein MERS-mSpike has 85% or greater amino acid sequence identity to SEQ ID NO: 200, excluding the MERS-mRBD or a fragment thereof, and preferably a 2P mutation and / or an invalid furin cleavage site.

[0174] The 2P mutation in MERS-wtSpike is a substitution of amino acid residues V1060P and L1061P of MERS-wtSpike of SEQ ID NO:200.

[0175] The disabled furin cleavage site is the furin cleavage site 748-RSVR-751 of MERS-wtSpike of sequence number 200, which is disabled, for example, by mutation through substitution, deletion or insertion of amino acid residues, preferably by replacement with 748-ASVG-751.

[0176] Preferably, MERS-mSpike or a fragment thereof has 90% or more, more preferably 95% or more, even more preferably 98% or more, still more preferably 99% or more, and most preferably 100% amino acid sequence identity to SEQ ID NO: 200, excluding MERS-mSpike or a fragment thereof and preferably the 2P mutation and / or the invalid Furin cleavage site.

[0177] Moreover, it should be understood that in the present invention, any embodiment disclosing MERS-mRBD or a fragment thereof is applicable mutatis mutandis to MERS-mSpike or a fragment thereof.

[0178] Preferably, MERS-mSpike or a fragment thereof comprises: An amino acid sequence or a fragment thereof comprising one or more, preferably one to three, or two, preferably one or two, more preferably one substitution of amino acid residues at positions selected from L506, D510, E536, D537, or D539 of MERS-wtSpike of SEQ ID NO: 200. wherein MERS-mSpike or a fragment thereof has 90% or greater amino acid sequence identity to SEQ ID NO: 200, excluding said substitution.

[0179] Preferably, MERS-mSpike or a fragment thereof has, excluding this substitution, greater than 95% amino acid sequence identity to SEQ ID NO:200, more preferably greater than 98%, even more preferably greater than 99%, and most preferably 100%.

[0180] and / or the amino acid sequence identity of MERS-mSpike to MERS-wtSpike of SEQ ID NO: 200, excluding the MERS-mRBD or a fragment thereof and preferably 2P mutations and / or invalid furin cleavage sites; and / or Amino acid sequence identity of MERS-mSpike to MERS-wtSpike of SEQ ID NO: 200, excluding substitutions and preferably 2P mutations and / or invalid furin cleavage sites. must be low enough to still encompass MERS-Spike of MERS-CoV variants other than SEQ ID NO: 200, thereby making MERS-mSpike adaptable to such variants or to combinations of mutations, e.g., substitutions, deletions, and insertions, contained in such variants when compared to SEQ ID NO: 200. Examples of such existing MERS-CoV variants and / or contained mutations are: - in the receptor binding domain (RBD) of the S1 subunit, a T424I substitution in 2 / 8 isolates and a S459T substitution in 1 / 8 isolates. - In the RBD / RBM overlap region, a W553R substitution was found in the RBD / RBM overlap region in 2 / 8 isolates. - In the fusion peptide, Q1009L in the heptad repeat region 1 (HR1) and C1313S in the transmembrane (TM) region (Supplementary Table 2B, substitutions in the S2 subunit included S950T in the fusion peptide, Q1009L in the heptad repeat region 1 (HR1), and C1313S in the transmembrane (TM) region). - MERS-CoV variants are listed in the following table (retrieved from https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5165220 / ).

[0181] [Table 2]

[0182] For a better understanding of the invention, it is noted that the sequence of MERS-wtRBD in SEQ ID NO: 194 corresponds to the sequence starting at position 367 of MERS-wtSpike in SEQ ID NO: 200. Thus, when 366 amino acids are removed from a particular amino acid position in MERS-wtSpike or MERS-mSpike, the result corresponds to the amino acid positions in MERS-wtRBD or MERS-mRBD, respectively. A selection of the corresponding amino acid positions in MERS-wtRBD / MERS-mRBD and MERS-wtSpike / MERS-mSpike is shown in Table B, presented before the description of the figures further below in this specification.

[0183] Preferably, the one or more substitutions of amino acid residues are one to all, preferably one or two, preferably one substitution of amino acid residues at positions selected from D539, D510 or D537 of MERS-wtSpike of SEQ ID NO:200.

[0184] More preferably, the one or more substitutions of amino acid residues are one or both, more preferably one, substitution of an amino acid residue at a position selected from D510 or D537 of MERS-wtSpike of SEQ ID NO:200.

[0185] Preferably, the one or more substitutions of amino acid residues are one or more, preferably one to three, more preferably one or two, even more preferably one substitution of amino acid residues at positions selected from L506, D510, E536, D537 or D539 of MERS-wtSpike of SEQ ID NO: 200, wherein: - the substitution of the amino acid residue at position L506 is L506A, - the substitution of the amino acid residue at position D510 is D510A, - the substitution of the amino acid residue at position E536 is E536R, - the substitution of the amino acid residue at position D537 is D537K, and / or - the substitution of the amino acid residue at position D539 is 539K.

[0186] More preferably, the one or more substitutions of amino acid residues are one to three, preferably one or two, even more preferably one substitution of amino acid residues at positions selected from D510, D537 or D539 of MERS-wtSpike of SEQ ID NO: 200, wherein: - the substitution of the amino acid residue at position D510 is D510A, - the substitution of the amino acid residue at position D537 is D537K, - the substitution of the amino acid residue at position D539 is D539K.

[0187] Even more preferably, the substitution of one or more amino acid residues is a substitution of one or both, more preferably one, of an amino acid residue at a position selected from D510 or D537 of MERS-wtSpike of SEQ ID NO: 200, wherein: - the substitution of the amino acid residue at position D510 is D510A, - the substitution of the amino acid residue at position D537 is D537K.

[0188] Even more preferably, MERS-mSpike or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204 or SEQ ID NO:205, more preferably selected from SEQ ID NO:201 or SEQ ID NO:202.

[0189] Polypeptides and proteins containing CORONA-mRBD or CORONA-mSpike The present invention further relates to a polypeptide or protein comprising a CORONA-mRBD according to the present invention or a fragment thereof or a CORONA-mSpike according to the present invention or a fragment thereof.

[0190] Preferably, the polypeptide and / or protein comprising CORONA-mRBD or a fragment thereof or CORONA-mSpike or a fragment thereof is a recombinant polypeptide or protein, or a chemically synthesized polypeptide or protein.

[0191] The polypeptide or protein may comprise one or more additional amino acid sequences other than the CORONA-mRBD or a fragment thereof or the CORONA-mSpike or a fragment thereof. Such additional amino acid sequences may be domains or fragments of a protein different from the CORONA-mRBD or a fragment thereof and / or the CORONA-spike or a fragment thereof, a localization signal, e.g., a secretion signal, a stability regulating amino acid sequence, e.g., a sequence that promotes or prevents active and / or passive degradation of the polypeptide or protein, a polymerization domain, e.g., a dimerization or trimerization domain, a spacer, a linker, etc. Moreover, the polypeptide or protein may comprise one or more modifications.

[0192] Preferably, the CORONA-mRBD or a fragment thereof is (A) a SARS-mRBD or a fragment thereof according to the present invention or (B) a MERS-mRBD or a fragment thereof according to the present invention.

[0193] Thus, preferably, the present invention relates to (A) a polypeptide or protein comprising a SARS-mRBD or a fragment thereof according to the present invention or a SARS-mSpike or a fragment thereof according to the present invention; and / or The present invention relates to (B) a polypeptide or protein comprising the MERS-mRBD or a fragment thereof according to the present invention, or the MERS-mSpike or a fragment thereof according to the present invention.

[0194] Moreover, in the present invention, it should be understood that any embodiment disclosing CORONA-mRBD or a fragment thereof and / or CORONA-mSpike or a fragment thereof is applicable mutatis mutandis to a polypeptide or protein comprising CORONA-mRBD or a fragment thereof or CORONA-mSpike or a fragment thereof.

[0195] (A) Polypeptides and proteins containing SARS-mRBD or SARS-mSpike As mentioned above, the present invention preferably relates to a polypeptide or protein comprising a SARS-mRBD or a fragment thereof according to the present invention or a SARS-mSpike or a fragment thereof according to the present invention.

[0196] Preferably, the polypeptides and / or proteins comprising SARS-mRBD or a fragment thereof or SARS-mSpike or a fragment thereof are recombinant or chemically synthesized polypeptides or proteins.

[0197] The polypeptides and proteins may contain one or more additional amino acid sequences other than SARS-mRBD or a fragment thereof or SARS-mSpike or a fragment thereof. Such additional amino acid sequences may be domains or fragments of proteins different from SARS-mRBD or a fragment thereof and / or SARS spike or a fragment thereof, localization signals, such as cytosol localization signals or secretion signals, stability regulating amino acid sequences, such as sequences that promote or prevent active and / or passive degradation of the polypeptide or protein, polymerization domains, such as dimerization or trimerization domains, spacers, linkers, tags, such as FLAG tags or His tags, etc. Moreover, the polypeptides or proteins may contain one or more modifications, such as glycosylation.

[0198] Moreover, it should be understood that in the present invention, any embodiment disclosing SARS-mRBD or a fragment thereof and / or SARS-mSpike or a fragment thereof is applicable mutatis mutandis to a polypeptide or protein comprising SARS-mRBD or a fragment thereof or SARS-mSpike or a fragment thereof.

[0199] (B) Polypeptides and proteins containing MERS-mRBD or MERS-mSpike As mentioned above, the present invention preferably relates to a polypeptide or protein comprising the MERS-mRBD or a fragment thereof according to the present invention or the MERS-mSpike or a fragment thereof according to the present invention.

[0200] Preferably, the polypeptide and / or protein comprising MERS-mRBD or a fragment thereof or MERS-mSpike or a fragment thereof is a recombinant polypeptide or protein or a chemically synthesized polypeptide or protein.

[0201] The polypeptide and protein may comprise one or more additional amino acid sequences other than MERS-mRBD or a fragment thereof or MERS-mSpike or a fragment thereof. Such additional amino acid sequences may be domains or fragments of proteins different from MERS-mRBD or a fragment thereof and / or MERS-Spike or a fragment thereof, localization signals, such as cytosol localization signals or secretion signals, stability regulating amino acid sequences, such as sequences that promote or prevent active and / or passive degradation of the polypeptide or protein, polymerization domains, such as dimerization or trimerization domains, spacers, linkers, tags, such as FLAG tags or His tags, etc. Moreover, the polypeptide or protein may comprise one or more modifications, such as glycosylation.

[0202] Moreover, it should be understood that in the present invention, any embodiment disclosing MERS-mRBD or a fragment thereof and / or MERS-mSpike or a fragment thereof is applicable mutatis mutandis to a polypeptide or protein comprising MERS-mRBD or a fragment thereof or MERS-mSpike or a fragment thereof.

[0203] nucleic acid Moreover, the present invention provides - a CORONA-mRBD according to the invention or a fragment thereof, - CORONA-mSpike or a fragment thereof according to the invention, or - a polypeptide or protein according to the invention comprising a CORONA-mRBD according to the invention or a fragment thereof or a CORONA-mSpike according to the invention or a fragment thereof The present invention relates to a nucleic acid comprising a nucleotide sequence encoding

[0204] Preferably, the nucleic acid is DNA or RNA, such as mRNA.

[0205] Preferably, the nucleic acid is a recombinant nucleic acid or a chemically synthesized nucleic acid.

[0206] The nucleic acid may include one or more additional nucleotide sequences other than the nucleotide sequence encoding.

[0207] Moreover, the nucleic acid may comprise one or more additional sequences other than the nucleotide sequence encoding the CORONA-mRBD or a fragment thereof, the CORONA-mSpike or a fragment thereof, or a polypeptide or protein according to the present invention. Such additional nucleotide sequences may be transcriptional regulatory sequences, such as promoters, enhancers, terminators, transcription factor binding motifs or DNA polymerase or reverse transcriptase binding sites, translational regulatory sequences, such as ribosome binding motifs, exons, introns, 5'-cap, polyA tails, localization signals, such as core or cytosolic localization signals, hybridization stretches and / or stability regulatory sequences, such as nucleotide sequences that promote or prevent active and / or passive degradation of the nucleic acid, spacers, linkers, tags, such as sequences encoding FLAG tags or His tags, etc. Moreover, the nucleic acid may comprise modifications, such as methylation.

[0208] More preferably, the nucleic acid is preferably - a CORONA-mRBD according to the invention or a fragment thereof, - CORONA-mSpike or a fragment thereof according to the invention, or - a polypeptide or protein according to the invention comprising a CORONA-mRBD according to the invention or a fragment thereof or a CORONA-mSpike according to the invention or a fragment thereof It consists of a nucleic acid encoding

[0209] Preferably, the CORONA-mRBD or a fragment thereof is (A) a SARS-mRBD according to the invention and / or (B) a MERS-mRBD according to the invention, and / or Preferably, CORONA-mSpike or a fragment thereof is (A) SARS-mSpike according to the present invention and / or (B) MERS-mSpike according to the present invention.

[0210] Therefore, preferably, the present invention provides a method for producing a composition comprising: - SARS-mRBD or a fragment thereof according to the invention, - SARS-mSpike or a fragment thereof according to the invention, or - a polypeptide or protein according to the invention comprising a SARS-mRBD or a fragment thereof according to the invention or a SARS-mSpike or a fragment thereof according to the invention and / or The present invention relates to (B) - MERS-mRBD or a fragment thereof according to the invention, - MERS-mSpike or a fragment thereof according to the invention, or - a polypeptide or protein according to the invention comprising a MERS-mRBD or a fragment thereof according to the invention or a MERS-mSpike or a fragment thereof according to the invention The present invention relates to a nucleic acid comprising a nucleotide sequence encoding

[0211] Moreover, in the present invention, CORONA-mRBD or a fragment thereof, and / or CORONA-mSpike or a fragment thereof, and / or A polypeptide or protein comprising CORONA-mRBD or a fragment thereof, or CORONA-mSpike or a fragment thereof It should be understood that any embodiment disclosed for is applicable mutatis mutandis to the nucleic acids according to the present invention.

[0212] (A) Nucleic acid Therefore, the present invention preferably comprises: (A) - SARS-mRBD or a fragment thereof according to the invention, - SARS-mSpike or a fragment thereof according to the invention, or - a polypeptide or protein according to the invention comprising a SARS-mRBD or a fragment thereof according to the invention or a SARS-mSpike or a fragment thereof according to the invention The present invention relates to a nucleic acid comprising, preferably consisting of, a nucleotide sequence encoding

[0213] Preferably, the nucleic acid is DNA or RNA, such as mRNA.

[0214] Preferably, the nucleic acid is a recombinant nucleic acid or a chemically synthesized nucleic acid.

[0215] The nucleic acid may include one or more additional nucleotide sequences other than the nucleotide sequence encoding.

[0216] Moreover, the nucleic acid may comprise one or more additional sequences other than the nucleotide sequence encoding the SARS-mRBD or a fragment thereof, the SARS-mSpike or a fragment thereof, or the polypeptide or protein according to the present invention. Such additional nucleotide sequences may be transcriptional regulatory sequences, such as promoters, enhancers, terminators, transcription factor binding motifs or DNA polymerase or reverse transcriptase binding sites, translational regulatory sequences, such as ribosome binding motifs, exons, introns, 5'-cap, polyA tails, localization signals, such as core or cytosolic localization signals, hybridization stretches and / or stability regulatory sequences, such as nucleotide sequences that promote or prevent active and / or passive degradation of the nucleic acid, spacers, linkers, tags, such as sequences encoding FLAG tags or His tags, etc. Moreover, the nucleic acid may comprise modifications, such as methylation.

[0217] Preferably, the nucleotide sequence encoding the SARS-mRBD is SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:266.

[0218] Preferably, the nucleotide sequence encoding SARS-mSpike is SEQ ID NO:255, SEQ ID NO:256 or SEQ ID NO:265.

[0219] Moreover, in the present invention, SARS-mRBD or a fragment thereof, and / or SARS-mSpike or a fragment thereof, and / or A polypeptide or protein comprising SARS-mRBD or a fragment thereof, or SARS-mSpike or a fragment thereof It should be understood that any embodiment disclosed for is applicable mutatis mutandis to the nucleic acids according to the present invention.

[0220] (B) Nucleic acid Moreover, the present invention preferably comprises: (B) - MERS-mRBD or a fragment thereof according to the invention, - MERS-mSpike or a fragment thereof according to the invention, or - a polypeptide or protein according to the invention comprising a MERS-mRBD or a fragment thereof according to the invention or a MERS-mSpike or a fragment thereof according to the invention The present invention relates to a nucleic acid comprising, preferably consisting of, a nucleotide sequence encoding

[0221] Preferably, the nucleic acid is DNA or RNA, such as mRNA.

[0222] Preferably, the nucleic acid is a recombinant nucleic acid or a chemically synthesized nucleic acid.

[0223] The nucleic acid may include one or more additional nucleotide sequences other than the nucleotide sequence encoding.

[0224] Moreover, the nucleic acid may comprise one or more additional sequences other than the nucleotide sequence encoding the MERS-mRBD or a fragment thereof, the MERS-mSpike or a fragment thereof, or a polypeptide or protein according to the present invention. Such additional nucleotide sequences may be transcriptional regulatory sequences, such as promoters, enhancers, terminators, transcription factor binding motifs or DNA polymerase or reverse transcriptase binding sites, translational regulatory sequences, such as ribosome binding motifs, exons, introns, 5'-cap, polyA tails, localization signals, such as core or cytosolic localization signals, hybridization stretches and / or stability regulatory sequences, such as nucleotide sequences that promote or prevent active and / or passive degradation of the nucleic acid, spacers, linkers, tags, such as sequences encoding FLAG tags or His tags, etc. Moreover, the nucleic acid may comprise modifications, such as methylation.

[0225] Moreover, in the present invention, MERS-mRBD or a fragment thereof, and / or MERS-mSpike or a fragment thereof, and / or A polypeptide or protein comprising MERS-mRBD or a fragment thereof, or MERS-mSpike or a fragment thereof It should be understood that any embodiment disclosed for is applicable mutatis mutandis to the nucleic acids according to the present invention.

[0226] Vaccine Compositions and Medical Uses The present invention relates to a compound comprising the following active ingredient: - one or more CORONA-mRBDs or fragments thereof according to the invention, - one or more CORONA-mSpikes or fragments thereof according to the invention, - one or more polypeptides or proteins according to the invention, comprising a CORONA-mRBD or a fragment thereof according to the invention or a CORONA-mSpike or a fragment thereof according to the invention, and / or - one or more nucleic acids according to the invention, comprising a nucleotide sequence encoding one or more CORONA-mRBDs or fragments thereof, one or more CORONA-mSpikes or fragments thereof, or one or more polypeptides or proteins; The present invention further relates to a vaccine composition comprising, and preferably consisting of,

[0227] Preferably, the vaccine composition further comprises a pharma- ceutically acceptable adjuvant, such as Matrix-M1 adjuvant, aluminum-containing adjuvant, MF59, AS01, AS02, AS03, AS04, virosomes, ISA51, CpG ODN, carriers, diluents, lipid nanoparticles, and / or excipients.

[0228] Preferably, the one or more nucleic acids according to the present invention are mRNA and the vaccine comprises the mRNA packaged in a lipid nanoparticle.

[0229] Preferably, the vaccine uses a viral vector to deliver the nucleic acid, eg, DNA.

[0230] Preferably, the vaccine composition comprises an effective amount of the active ingredient.

[0231] Preferably, the vaccine composition is capable of inducing an immune response in a subject against a coronavirus. Preferably, the coronavirus is SARS-CoV-2 and / or the coronavirus is MERS-CoV.

[0232] Preferably, the vaccine composition is a vaccine for the prevention and / or treatment of a disease caused by a coronavirus in a subject. Preferably, the disease caused by a coronavirus is COVID-19 caused by SARS-CoV-2, and / or the disease caused by a coronavirus is MERS caused by MERS-CoV.

[0233] Moreover, the present invention relates to a method for the prevention and / or treatment of a disease caused by a coronavirus in a subject. One or more CORONA-mRBDs or fragments thereof according to the present invention, One or more CORONA-mSpikes or fragments thereof according to the present invention, one or more polypeptides or proteins according to the invention, comprising a CORONA-mRBD or a fragment thereof according to the invention or a CORONA-mSpike or a fragment thereof according to the invention, one or more nucleic acids according to the invention, comprising a nucleotide sequence encoding one or more CORONA-mRBDs or fragments thereof, one or more CORONA-mSpikes or fragments thereof, or one or more polypeptides or proteins; and / or The above vaccine composition takes into account CORONA-mRBD and CORONA-mSpike.

[0234] Preferably, in vaccine compositions and / or uses in prevention and / or treatment, one or more is one to three, more preferably one or two, and even more preferably two.Preferably, for example, when two, three or more CORONA-mRBDs are present, they differ from each other in their sequences, for example, due to the presence of various substitutions.The same applies to the presence of two, three or more CORONA-mSpikes, polypeptides or proteins, and / or nucleic acids.

[0235] Preferably, the term subject as used for the vaccine composition and / or for use in prophylaxis and / or treatment, the subject is a vertebrate, more preferably a mammal, even more preferably an ape, monkey, lemur, canine, such as dog, wolf or fox, feline, such as big or small cat, rodent, such as mouse, rat, guinea pig, hamster or rabbit, bovine, such as buffalo, antelope, sheep, goat or cow, deer, horse, donkey, bear, marten, bat, and / or human, and even more preferably the subject is a human.

[0236] Preferably, in the vaccine composition and / or in the use in prevention and / or treatment, the subject is not infected with coronavirus and / or is infected with coronavirus. The subject not infected with coronavirus may or may not have previously experienced coronavirus infection. The subject infected with coronavirus may currently have coronavirus infection. The determination of whether a subject is not infected with coronavirus or is infected with coronavirus may use any method known to those skilled in the art. An example of a suitable method is PCR test.

[0237] Preferably, in the vaccine composition and / or in the prophylactic and / or therapeutic use, the disease caused by a coronavirus is COVID-19 caused by SARS-CoV-2 and / or the disease caused by a coronavirus is MERS caused by MERS-CoV.

[0238] Preferably, SARS-CoV-2 as used in the present invention includes any variant thereof, such as Wuhan, B.1.1.7 (alpha, UK), B.1.617.2 (delta, India), B.1.351 (beta, South Africa), P.1 (gamma, Brasilia), and / or B.1.1.28., preferably the Wuhan variant of SARS-CoV-2.

[0239] Preferably, the MERS-CoV as used in the present invention includes any of its variants, such as AF88936.1, AFY13307.1, AGG22542.1, AGV08379.1, AGV08584.1, AHI48528.1, AHI48733.1, AHC74088.1, AGV08438.1, AID55090.1, AID55095.1, AID55087.1, AKL59401.1, ALB08322.1, ALB08289.1, AHY22545.1, AHL18090.1, AHX00711.1, AHX0072.1, and AHY22555.1.

[0240] Preferably, the vaccine composition and / or prophylactic and / or therapeutic use comprises the CORONA-mRBD or a fragment thereof is (A) a SARS-mRBD or a fragment thereof according to the invention, or (B) a MERS-mRBD or a fragment thereof according to the invention, and / or CORONA-mSpike or a fragment thereof is (A) SARS-mSpike or a fragment thereof according to the present invention or (B) MERS-mSpike or a fragment thereof according to the present invention.

[0241] Moreover, it should be understood that in the present invention, any embodiment disclosing CORONA-mRBD or a fragment thereof and / or CORONA-mSpike or a fragment thereof, a polypeptide or protein comprising CORONA-mRBD or a fragment thereof or CORONA-mSpike, and / or a nucleic acid encoding the same, is applicable mutatis mutandis to a vaccine composition.

[0242] (A) Vaccine Compositions and Medical Uses Preferably, the present invention relates to a composition comprising, as an active ingredient (A): - one or more SARS-mRBDs or fragments thereof according to the invention, - one or more SARS-mSpike or fragments thereof according to the invention, - one or more polypeptides or proteins according to the invention, comprising a SARS-mRBD or a fragment thereof according to the invention or a SARS-mSpike or a fragment thereof according to the invention, and / or - one or more nucleic acids according to the invention comprising a nucleotide sequence encoding one or more SARS-mRBDs or fragments thereof, one or more SARS-mSpikes or fragments thereof, or one or more polypeptides or proteins; The present invention relates to a vaccine composition comprising, and preferably consisting of,

[0243] Preferably, the vaccine composition (A) further comprises a pharma- ceutically acceptable adjuvant, carrier, diluent and / or excipient.

[0244] Preferably, the vaccine composition (A) comprises an effective amount of the active ingredient.

[0245] Preferably, the vaccine composition (A) is capable of inducing an immune response against SARS-CoV-2.

[0246] Preferably, the vaccine composition (A) is a vaccine for the prevention and / or treatment of COVID-19 caused by SARS-CoV-2.

[0247] Moreover, the present invention preferably relates to a method for the prevention and / or treatment of COVID-19 caused by SARS-CoV-2 in a subject (A). - one or more SARS-mRBDs or fragments thereof according to the invention, - one or more SARS-mSpikes or fragments thereof according to the invention, - one or more polypeptides or proteins according to the invention, comprising a SARS-mRBD or a fragment thereof according to the invention or a SARS-mSpike or a fragment thereof according to the invention, and / or - one or more nucleic acids according to the invention comprising a nucleotide sequence encoding one or more SARS-mRBDs or fragments thereof, one or more SARS-mSpikes or fragments thereof, or one or more polypeptides or proteins, and / or - regarding a vaccine composition as described above taking into account SARS-mRBD and SARS-mSpike.

[0248] Preferably, in the vaccine composition (A) and / or in the prophylactic and / or therapeutic use (A), one or more is one to three, more preferably one or two, and even more preferably two.Preferably, for example, when two, three or more SARS-mRBDs are present, they differ from each other in their sequences, for example, due to the presence of various substitutions.The same applies to the presence of two, three or more SARS-mSpikes, polypeptides or proteins, and / or nucleic acids.

[0249] Preferably, the term subject as used for the vaccine composition (A) and / or the prophylactic and / or therapeutic use (A) is a vertebrate, more preferably a mammal, even more preferably an ape, monkey, lemur, canine, such as dog, wolf or fox, feline, such as big or small cat, rodent, such as mouse, rat, guinea pig, hamster or rabbit, bovine, such as buffalo, antelope, sheep, goat or cow, deer, horse, donkey, bear, marten, bat and / or human, even more preferably the subject is a human.

[0250] Preferably, in the vaccine composition (A) and / or in the prophylactic and / or therapeutic use (A), the subject is not infected with SARS-CoV-2 and / or is infected with SARS-CoV-2. A subject not infected with SARS-CoV-2 may or may not have previously experienced a SARS-CoV-2 infection. A subject infected with SARS-CoV-2 may currently have a SARS-CoV-2 infection. Any method known to those skilled in the art may be used to determine whether a subject is not infected with SARS-CoV-2 or is infected with SARS-CoV-2. An example of a suitable method is a PCR test.

[0251] Preferably, SARS-CoV-2 as used in the present invention includes any variant thereof, such as Wuhan, B.1.1.7 (alpha, UK), B.1.617.2 (delta, India), B.1.351 (beta, South Africa), P.1 (gamma, Brasilia), and / or B.1.1.28., preferably the Wuhan variant of SARS-CoV-2.

[0252] Preferably, one or more is two or more, more preferably one to five, even more preferably two to four, and even more preferably two. Preferably, for example, when two or more SARS-mRBDs are present, these substitutions are different. The same applies to the presence of two, three or more SARS-mSpikes, polypeptides or proteins, and / or nucleic acids.

[0253] More preferably, the one or more SARS-mRBDs or fragments thereof is any one or any combination of SARS-mRBDs according to the present invention.

[0254] Even more preferably, the one or more SARS-mRBDs or fragments thereof is a SARS-mRBD or fragments thereof comprising an amino acid sequence or fragments thereof comprising one substitution of an amino acid residue at a position selected from G184, L137, Y187 or Y171 of SARS-wtRBD of SEQ ID NO:1, preferably G184, L137 or Y187, even more preferably G184 or L137.

[0255] Even more preferably, the one or more SARS-mRBDs or fragments thereof is a combination of two or more SARS-mRBDs or fragments thereof each comprising an amino acid sequence or fragments thereof that includes a substitution of an amino acid residue at a position selected from G184, L137, Y187 or Y171 of SARS-wtRBD of SEQ ID NO:1, preferably G184, L137 or Y187, even more preferably G184 or L137.

[0256] Preferably, - the substitution of the amino acid residue at position G184 is selected from G184R, G184D, G184Y, G184K, G184S, G184P, G184E, G184A, G184V, G184N, G184Q, G184T, G184M, G184H or G184L, more preferably G184R, G184D or G184E, even more preferably G184R or G184E, still more preferably the SARS-mRBD or a fragment thereof is selected from SEQ ID NO: 1 5, SEQ ID NO:17, SEQ ID NO:5, SEQ ID NO:20, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:13, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:11, SEQ ID NO:7 or SEQ ID NO:10, more preferably comprising, preferably consisting of, SEQ ID NO:15, SEQ ID NO:5 or SEQ ID NO:2, most preferably comprising, preferably consisting of, SEQ ID NO:15 or SEQ ID NO:2, - the substitution of the amino acid residue at position L137 is selected from L137R or L137E, more preferably L137R, even more preferably the SARS-mRBD or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 55 or SEQ ID NO: 53, most preferably comprises, preferably consists of SEQ ID NO: 55; - the substitution of the amino acid residue at position Y187 is selected from Y187N or Y187Q, more preferably Y187N, even more preferably the SARS-mRBD or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 30 or SEQ ID NO: 31, most preferably comprises, preferably consists of SEQ ID NO: 30; - the substitution of the amino acid residue at position Y171 is selected from Y171S or Y171T, even more preferably, the SARS-mRBD or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 92 or SEQ ID NO: 93.

[0257] Preferably, two or more SARS-mRBDs or fragments thereof differ in these substitutions.

[0258] Even more preferably, the one or more SARS-mRBDs or fragments thereof is a combination of two SARS-mRBDs or fragments thereof, where one comprises an amino acid sequence or fragment thereof that includes the G184E, G184D or G184R, more preferably the G184R or G184E substitution of SARS-wtRBD of SEQ ID NO:1, and the other comprises an amino acid sequence or fragment thereof that includes the L137R substitution.

[0259] Even more preferably, the one or more SARS-mRBDs or fragments thereof is a combination of two SARS-mRBDs or fragments thereof, where one consists of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:15, more preferably SEQ ID NO:15 or SEQ ID NO:2, and the other consists of SEQ ID NO:55.

[0260] More preferably, the one or more SARS-mSpike or fragments thereof is any one or any combination of SARS-mSpike according to the present invention.

[0261] Even more preferably, the one or more SARS-mSpikes or fragments thereof are one SARS-mSpike or fragments thereof comprising an amino acid sequence or fragments thereof comprising one substitution of an amino acid residue at a position selected from G502, L455, Y505 or Y489 of SARS-wtSpike of SEQ ID NO: 100, preferably G502, L455 or Y505, even more preferably G502 or L455.

[0262] Even more preferably, the one or more SARS-mSpikes or fragments thereof are a combination of two or more SARS-mSpikes or fragments thereof each comprising an amino acid sequence or fragments thereof comprising one substitution of an amino acid residue at a position selected from G502, L455, Y505 or Y489 of SARS-wtSpike of SEQ ID NO: 100, preferably G502, L455 or Y505, even more preferably G502 or L455, wherein preferably the substitution of an amino acid residue at a position selected from G502, L455, Y505 or Y489 of SARS-wtSpike of SEQ ID NO: 100.

[0263] Preferably, - the substitution of the amino acid residue at position G502 is selected from G502R, G502D, G502Y, G502K, G502S, G502P, G502E, G502A, G502V, G502N, G502Q, G502T, G502M, G502H or G502L, more preferably G502R, G502D or G502E, more preferably G502R or G502E, even more preferably the SARS-mSpike or a fragment thereof is selected from SEQ ID NO: 114, SEQ ID NO: 104, SEQ ID NO: 11 9, SEQ ID NO:108, SEQ ID NO:115, SEQ ID NO:112, SEQ ID NO:101, SEQ ID NO:117, SEQ ID NO:102, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:110, SEQ ID NO:106 or SEQ ID NO:109, more preferably comprising, preferably consisting of, SEQ ID NO:114, SEQ ID NO:104 or SEQ ID NO:101, most preferably comprising, preferably consisting of, SEQ ID NO:114 or SEQ ID NO:101, - the substitution of the amino acid residue at position L455 is selected from L455R or L455E, more preferably L455R, even more preferably the SARS-mSpike or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 154 or SEQ ID NO: 152, most preferably comprises, preferably consists of SEQ ID NO: 154; - the substitution of the amino acid residue at position Y505 is selected from Y505N or Y505Q, more preferably Y505N, even more preferably the SARS-mSpike or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 129 or SEQ ID NO: 130, more preferably comprises, preferably consists of SEQ ID NO: 129; - the substitution of the amino acid residue at position Y489 is selected from Y489S or Y489T, even more preferably, the SARS-mSpike or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 191 or SEQ ID NO: 192.

[0264] Preferably, two or more SARS-mSpike or fragments thereof differ in these substitutions.

[0265] Even more preferably, the one or more SARS-mSPIKE or fragments thereof is a combination of two SARS-mSPIKE or fragments thereof, where one comprises an amino acid sequence or fragment thereof that includes the G502R, G502D or G502E, more preferably the G502R or G502E substitution of SARS-wtSPIKE of SEQ ID NO:1, and the other comprises an amino acid sequence or fragment thereof that includes the L455R substitution.

[0266] Even more preferably, the one or more SARS-mSPIKE or fragments thereof is a combination of two SARS-mSPIKE or fragments thereof, where one consists of SEQ ID NO:114, SEQ ID NO:104 or SEQ ID NO:101, more preferably SEQ ID NO:114, more preferably SEQ ID NO:104 or SEQ ID NO:114, and the other consists of SEQ ID NO:154.

[0267] More preferably, the one or more nucleic acids according to the present invention comprising a nucleotide sequence encoding one or more SARS-mRBDs or fragments thereof, one or more SARS-mSpikes or fragments thereof or one or more polypeptides or proteins is any one or any combination of the nucleic acids according to the present invention.

[0268] Even more preferably, the nucleic acid or nucleic acids according to the present invention are selected from SEQ ID NO: 255, SEQ ID NO: 256 or SEQ ID NO: 266, even more preferably a combination of SEQ ID NO: 255 and SEQ ID NO: 256 or a combination of SEQ ID NO: 256 and SEQ ID NO: 266.

[0269] Moreover, in the present invention, SARS-mRBD or a fragment thereof, and / or SARS-mSpike or a fragment thereof, and / or A polypeptide or protein comprising SARS-mRBD or a fragment thereof or SARS-mSpike, and / or Nucleic acids encoding these It should be understood that any embodiment disclosed is applicable mutatis mutandis to a vaccine composition (A) and / or to a prophylactic and / or therapeutic use (A).

[0270] (B) Vaccine Compositions and Medical Uses Preferably, the present invention additionally or alternatively relates to (B). Preferably, the present invention relates to (B) an active ingredient, - one or more MERS-mRBDs or fragments thereof according to the invention, - one or more MERS-mSpike or fragments thereof according to the invention, - one or more polypeptides or proteins according to the invention, comprising a MERS-mRBD or a fragment thereof according to the invention or a MERS-mSpike or a fragment thereof according to the invention, and / or - one or more nucleic acids according to the invention comprising a nucleotide sequence encoding one or more MERS-mRBDs or fragments thereof, one or more MERS-mSpikes or fragments thereof, or one or more polypeptides or proteins; The present invention relates to a vaccine composition comprising, and preferably consisting of,

[0271] Preferably, the vaccine composition (B) further comprises a pharma- ceutically acceptable adjuvant, carrier, diluent and / or excipient.

[0272] Preferably, the vaccine composition (B) comprises an effective amount of the active ingredient.

[0273] Preferably, the vaccine composition (B) is capable of inducing an immune response against MERS-CoV.

[0274] Preferably, the vaccine composition (B) is a vaccine for the prevention and / or treatment of MERS caused by MERS-CoV.

[0275] Moreover, the present invention preferably relates to a method for the prevention and / or treatment of MERS caused by MERS-CoV in a subject (B). - one or more MERS-mRBDs or fragments thereof according to the invention, - one or more MERS-mSpikes or fragments thereof according to the invention, - one or more polypeptides or proteins according to the invention, comprising a MERS-mRBD or a fragment thereof according to the invention or a MERS-mSpike or a fragment thereof according to the invention, and / or - one or more nucleic acids according to the invention comprising a nucleotide sequence encoding one or more MERS-mRBDs or fragments thereof, one or more MERS-mSpikes or fragments thereof, or one or more polypeptides or proteins, and / or - regarding a vaccine composition as described above taking into account MERS-mRBD and MERS-mSpike.

[0276] Preferably, in the vaccine composition (B) and / or in the use in prophylaxis and / or treatment (B), one or more is two or more, more preferably one to five, even more preferably two to four, and even more preferably two. Preferably, for example, when two or more MERS-mRBDs are present, they differ from each other in their sequences, for example, due to the presence of various substitutions. The same applies to the presence of two, three or more MERS-mSpikes, polypeptides or proteins, and / or nucleic acids.

[0277] Preferably, the term subject as used for the vaccine composition (B) and / or for the prophylactic and / or therapeutic uses (B) is a vertebrate, more preferably a mammal, more preferably an ape, monkey, lemur, canine, such as dog, wolf or fox, feline, such as big or small cat, rodent, such as mouse, rat, guinea pig, hamster or rabbit, bovine, such as buffalo, antelope, sheep, goat or cow, deer, horse, donkey, bear, marten, bat and / or human, even more preferably the subject is a human.

[0278] Preferably, in the vaccine composition (B) and / or in the prophylactic and / or therapeutic use (B), the subject is not infected with MERS-CoV and / or is infected with MERS-CoV. The subject not infected with MERS-CoV may or may not have previously experienced MERS-CoV infection. The subject infected with MERS-CoV may currently have MERS-CoV infection. The determination of whether a subject is not infected with MERS-CoV or is infected with MERS-CoV may use any method known to those skilled in the art. An example of a suitable method is PCR testing.

[0279] Preferably, the MERS-CoV as used in the present invention includes any of its variants, such as AF88936.1, AFY13307.1, AGG22542.1, AGV08379.1, AGV08584.1, AHI48528.1, AHI48733.1, AHC74088.1, AGV08438.1, AID55090.1, AID55095.1, AID55087.1, AKL59401.1, ALB08322.1, ALB08289.1, AHY22545.1, AHL18090.1, AHX00711.1, AHX0072.1 and AHY22555.1.

[0280] Preferably, one or more is two or more, more preferably 1-5, even more preferably 2-4, and even more preferably 2. Preferably, for example, when two or more MERS-mRBDs are present, they differ from each other in their sequences, for example, due to the presence of various substitutions. The same applies to the presence of two, three or more MERS-mSpikes, polypeptides or proteins, and / or nucleic acids.

[0281] More preferably, the one or more MERS-mRBDs or fragments thereof is any one or any combination of MERS-mRBDs according to the present invention.

[0282] Even more preferably, the one or more MERS-mRBDs or fragments thereof is a MERS-mRBD or fragments thereof comprising an amino acid sequence or fragment thereof comprising one substitution of an amino acid residue at a position selected from D144, D171 or D173 of MERS-wtRBD of SEQ ID NO: 194, preferably D144 or D171.

[0283] Even more preferably, the one or more MERS-mRBDs or fragments thereof is a combination of two or more MERS-mRBDs or fragments thereof each comprising an amino acid sequence or fragments thereof that includes a substitution of one amino acid residue at a position selected from D144, D171 or D173 of MERS-wtRBD of SEQ ID NO: 194, preferably D144 or D171.

[0284] Preferably, - the substitution of the amino acid residue at position D144 is D144A, more preferably the MERS-mRBD or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 195; - the substitution of the amino acid residue at position D171 is D171K, more preferably the MERS-mRBD or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 196, - the substitution of the amino acid residue at position D173 is D173K, more preferably the MERS-mRBD or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 197.

[0285] Preferably, two or more MERS-mRBDs or fragments thereof differ in these substitutions.

[0286] More preferably, the one or more MERS-mSpike or fragments thereof is any one or any combination of MERS-mSpike according to the present invention.

[0287] Even more preferably, the one or more MERS-mSpikes or fragments thereof is one SARS-mSpike or fragments thereof comprising an amino acid sequence or fragment thereof comprising one substitution of an amino acid residue at a position selected from D510, D537 or D539 of MERS-wtRBD of SEQ ID NO: 200, preferably D510 or D537.

[0288] Even more preferably, the one or more MERS-mSpikes or fragments thereof are a combination of two or more MERS-mSpikes or fragments thereof each comprising an amino acid sequence or fragments thereof comprising one substitution of an amino acid residue at a position selected from D510, D537 or D539 of MERS-wtRBD of SEQ ID NO: 200, preferably D510 or D537.

[0289] Preferably, - the substitution of the amino acid residue at position D510 is D510A, more preferably, the MERS-mSpike or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 201, - the substitution of the amino acid residue at position D537 is D537K, more preferably, the MERS-mSpike or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 202, - the substitution of the amino acid residue at position D539 is D539K, more preferably, MERS-mSpike or a fragment thereof comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 203.

[0290] Preferably, two or more MERS-mSpike or fragments thereof differ in these substitutions.

[0291] More preferably, the one or more nucleic acids according to the present invention comprising a nucleotide sequence encoding one or more MERS-mRBDs or fragments thereof, one or more MERS-mSpikes or fragments thereof or one or more polypeptides or proteins is any one or any combination of the nucleic acids according to the present invention.

[0292] Moreover, in the present invention, MERS-mRBD or a fragment thereof, and / or MERS-mSpike or a fragment thereof, and / or A polypeptide or protein comprising MERS-mRBD or a fragment thereof or MERS-mSpike, and / or Nucleic acids encoding these It should be understood that any embodiment disclosed for is applicable mutatis mutandis to a vaccine composition (B) and / or to a prophylactic and / or therapeutic use (B).

[0293] Methods for designing and / or obtaining VIRUS-mRBD or fragments thereof The present invention relates to a method for designing and / or obtaining an active ingredient for a vaccine composition, comprising the steps of: (i) providing a mutated viral receptor binding domain (VIRUS-mRBD) or a fragment thereof, comprising one or more mutations in a wild-type viral receptor binding domain (VIRUS-wtRBD); (ii) VIRUS-mRBD or a fragment thereof a) Reduced binding of the viral receptor-binding domain to the receptor (RBD receptor) (VIRUS-RBD receptor) compared to VIRUS-wtRBD determining whether (iii) selecting VIRUS-mRBD or a fragment thereof as an active ingredient when a) is satisfied; The present invention further relates to a method comprising the steps of:

[0294] Preferably, the method comprises: In step (ii), the VIRUS-mRBD or a fragment thereof obtained in step (i) is b) binding to anti-VIRUS-wtRBD neutralizing antibodies (VIRUS-wtRBD-nAB); and c) Stability of proteins and / or peptides, as appropriate determining whether to indicate In step (iii), selecting VIRUS-mRBD or a fragment thereof as an active ingredient when a) and b) are satisfied simultaneously, and optionally a), b), and c) are satisfied simultaneously. Further includes:

[0295] Preferably, steps (i), (ii) and (iii) are carried out in that order.

[0296] Preferably, the VIRUS-mRBD or fragment thereof is a mutated receptor binding domain (mRBD) of the viral spike protein (VIRUS-mRBD) or fragment thereof.

[0297] Preferably, the VIRUS-mRBD or fragment thereof is a peptide or protein, preferably a protein.

[0298] Preferably, the VIRUS-RBD receptor or a fragment thereof is a soluble and / or membrane-bound VIRUS-RBD receptor, preferably a soluble VIRUS-RBD receptor.

[0299] Preferably, the mutation is a substitution of an amino acid residue.

[0300] Preferably, the selection in step (iii) is carried out by sequentially carrying out a) or b) in any order starting from either one of a) or b). More preferably, the VIRUS-mRBD or a fragment thereof that satisfies the one that starts the selection among a) and b) is subjected to any one of the remaining ones of a) and b). Then, the VIRUS-mRBD or a fragment thereof that satisfies the remaining one of a) or b) is selected as one that simultaneously satisfies a) and b). More preferably, the selection in step (iii) is carried out by sequentially carrying out a), b), and c) in any order starting from either one of a), b), or c). Even more preferably, the VIRUS-mRBD or a fragment thereof that satisfies the one that starts the selection among a), b), or c) is subjected to any one of the remaining two of a), b), or c). Then, the VIRUS-mRBD or a fragment thereof that satisfies one of the remaining two of a), b) or c) is subjected to the last one of a), b) or c).The VIRUS-mRBD or a fragment thereof that satisfies the last one of a), b) or c) is then selected as one that simultaneously satisfies a), b), and c).Preferably, a), b), and optionally c) are performed consecutively in the order of a), b), and optionally c), or b), a), and optionally c), more preferably in the order of a), b), and optionally c).

[0301] Preferably, step (ii)c) is carried out in vitro. In vitro determination of whether the stability of the protein and / or peptide is indicated can be carried out by any method known to the skilled artisan. For example, the stability of the protein and / or peptide is measured by ELISA using a specific antibody capable of detecting the amount of protein using a suitable standard and the conservation of protein folding. For example, sufficient protein yield can be obtained at a concentration of more than 0.5 μg / ml, preferably higher, by producing and secreting the protein produced by transfection of HEK293 cells as described in Example 4 into the culture supernatant. Similarly, protein stability can be measured by FACS analysis after expression of the protein on the cell surface and detection by a specific fluorescently labeled antibody. In the present invention, deep mutation scanning experiments can be carried out on a platform that couples genotype to phenotype (https: / / www.nature.com / articles / nmeth.3027.pdf).

[0302] Preferably, Step (i) comprises, and is preferably carried out by, providing a library of VIRUS-mRBDs or fragments thereof each comprising one or more mutations in VIRUS-wtRBD, wherein the mutations in the VIRUS-mRBDs or fragments thereof contained in the library are at least partially distinct; The step (ii) a) Reduced binding to the VIRUS-RBD receptor compared to VIRUS-wtRBD The method is carried out by screening the library for VIRUS-mRBD or a fragment thereof, which exhibits Step (iii) involves selecting, from the library, one or more VIRUS-mRBDs or fragments thereof that satisfy a) as active ingredients.

[0303] More preferably, The step (ii) is a) reduced binding to the VIRUS-RBD receptor compared to VIRUS-wtRBD; and b) Binding to VIRUS-wtRBD-nAB, and c) Stability of proteins and / or peptides, as appropriate by screening the library for VIRUS-mRBD or a fragment thereof, which exhibits Step (iii) involves selecting from the library one or more VIRUS-mRBDs or fragments thereof that simultaneously satisfy a) and b), and optionally a), b), and c), as active ingredients.

[0304] Preferably, the selection in step (iii) is carried out by sequentially carrying out a) or b) in any order starting from either one of a) or b). More preferably, one or more VIRUS-mRBDs or fragments thereof that satisfy the one that starts the selection among a) and b) are subjected to any one of the remaining ones of a) and b). Then, one or more VIRUS-mRBDs or fragments thereof that satisfy the remaining one of a) or b) are selected as those that simultaneously satisfy a) and b). More preferably, the selection in step (iii) is carried out by sequentially carrying out a), b), and c) in any order starting from either one of a), b), or c). Even more preferably, one or more VIRUS-mRBDs or fragments thereof that satisfy the one that starts the selection among a), b), or c) are subjected to any one of the remaining two of a), b), or c). Then, one or more VIRUS-mRBDs or fragments thereof that satisfy one of the remaining two of a), b) or c) are subjected to the last one of a), b) or c). Then, one or more VIRUS-mRBDs or fragments thereof that satisfy the last one of a), b) or c) are selected as those that simultaneously satisfy a), b), and c). Preferably, a), b), and optionally c) are performed consecutively in the order of a), b), and optionally c), or b), a), and optionally c), more preferably in the order of a), b), and optionally c).

[0305] Step (ii)c) may be performed in vitro or in silico, more preferably in vitro. Screening of libraries in vitro for VIRUS-mRBD or fragments thereof exhibiting protein and / or peptide stability may be performed by any method known to the skilled artisan. By way of example, stability may be measured by deep mutation scanning experiments. By way of example, protein and / or peptide stability may be measured by ELISA using specific antibodies capable of detecting protein amount using suitable standards as well as preservation of protein folding. By way of example, sufficient protein yields may be obtained by producing and secreting the protein produced by transfection of HEK293 cells as described in Example 4 into the culture supernatant at concentrations above 0.5 μg / ml, preferably higher. Similarly, protein stability may be measured by FACS analysis after expression of the protein on the cell surface and detection by specific fluorescently labeled antibodies. In the present invention, deep mutation scanning experiments can be performed on a platform that couples genotype to phenotype (https: / / www.nature.com / articles / nmeth.3027.pdf).

[0306] Preferably, steps (i), (ii)a) and (ii)b) are performed in vitro and / or in silico, preferably in vitro. More preferably, steps (i), (ii)a) and (ii)b) are performed in silico. Even more preferably, step (i) is performed in vitro and steps (ii)a) and (ii)b) are performed in silico. Even more preferably, steps (i), (ii)a) and (ii)b) are performed in vitro. Details are described below.

[0307] When steps (i), (ii)a) and / or (ii)b) are carried out in vitro VIRUS-mRBD or a fragment thereof comprising one or more mutations in VIRUS-wtRBD can be prepared in vitro by any method known to one of skill in the art. By way of example, one or more point mutations can be introduced into VIRUS-wtRBD by recombinant DNA techniques and VIRUS-mRBD can be expressed by any suitable expression system, preferably the HEK293 expression system as described in Example 4, provided that VIRUS-mRBD is expressed on the cell surface and thus includes a domain encoding a transmembrane portion of the polypeptide chain.

[0308] The binding and reduced avidity of VIRUS-mRBD or a fragment thereof and VIRUS-wtRBD to the VIRUS-RBD receptor can be measured and determined as described for CORONA-mRBD or a fragment thereof and CORONA-wtRBD to the CORONA-RBD receptor. It should therefore be understood that any disclosure or embodiment relating to reduced binding and avidity disclosed for CORONA-mRBD or a fragment thereof and CORONA-wtRBD to the CORONA-RBD receptor is applicable mutatis mutandis to the measurement of the binding and reduced avidity of VIRUS-mRBD or a fragment thereof and VIRUS-wtRBD to the VIRUS-RBD receptor in the method for designing and / or obtaining an active ingredient for a vaccine composition according to the present invention.

[0309] Similarly, the binding of VIRUS-mRBD or a fragment thereof and VIRUS-wtRBD to anti-VIRUS-wtRBD-nAB can be measured and determined as described for CORONA-mRBD or a fragment thereof and CORONA-wtRBD to anti-CORONA-wtRBD-nAB. It should therefore be understood that any disclosure or embodiment relating to binding disclosed for CORONA-mRBD or a fragment thereof and CORONA-wtRBD to anti-CORONA-wtRBD-nAB is applicable mutatis mutandis to the measurement of binding of VIRUS-mRBD or a fragment thereof and VIRUS-wtRBD to anti-VIRUS-wtRBD-nAB in the method for designing and / or obtaining active ingredients for vaccine compositions according to the present invention.

[0310] Preferably, step (i) comprises, and is preferably carried out by, providing a library of VIRUS-mRBDs or fragments thereof, each of which comprises one or more mutations in VIRUS-wtRBD, and wherein the VIRUS-mRBDs or fragments thereof comprised in the library differ at least in part by the mutations. Such a library of VIRUS-mRBDs or fragments thereof may be provided by any method known to the skilled artisan. Preferably, the library is generated using a strategy that generates exactly one codon mutation per gene, for example by overlap extension PCR (https: / / www.sciencedirect.com / science / article / pii / S0022283613004300?via%3Dihub), error-prone PCR (https: / / link.springer.com / protocol / 10.1385 / 1-59259-395-X:3) or chemical mutagenesis (https: / / academic.oup.com / nar / article / 32 / 4 / 1448 / 1038612?login=true) (https: / / journals.plos.org / plosone / article?id=10.1371 / journal.pone.0052031; https: / / www.sciencedirect.com / science / article / pii / S0022283613004300?via%3Dihub). ct.com / science / article / pii / S0003269713005782?casa_token=d5JPaeUuTwYAAAAA:-F2ChRH8nmjiVhQYPsl1BF4JpK-m_E0CoVBibRWGkDKxCPd7D3IVxW7n-f7rRFCxRPGYJc8Q), where the design accommodates mutation of multiple codons per gene to explore the average effect of mutations (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4104320 / ), and may be cloned and expressed by plasmid or virus, or in vitro systems such as T7 or M13 bacteriophage display, ribosome display, E. coli display, or most preferably, mammalian cell display.

[0311] Screening of the library for VIRUS-mRBD or fragments thereof exhibiting a) can be performed by any method known to those skilled in the art. Preferably, the binding strength is determined, for example, by staining cells expressing VIRUS-mRBD or VIRUS-wtRBD on the cell surface with a VIRUS-RBD receptor directly or indirectly labeled with a fluorophore, or conversely, by staining cells expressing VIRUS-RBD receptor on the cell surface with a soluble VIRUS-mRBD or soluble VIRUS-wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, counting stained cells, and performing positive and / or negative selection, where a decrease in the number of positively stained cells indicates a decrease in binding strength. The decrease in binding strength is then obtained by comparing the binding strength of VIRUS-wtRBD to the VIRUS-RBD receptor with the binding strength of VIRUS-mRBD to the VIRUS-RBD receptor. When the binding force is determined by flow cytometry analysis, the reduction in binding force is preferably determined by comparing the number of positively stained cells resulting from the binding of VIRUS-wtRBD to the VIRUS-RBD receptor with the number of positively stained cells resulting from the binding of VIRUS-mRBD to the VIRUS-RBD receptor. Preferably, the reduction in the binding force of VIRUS-mRBD to the VIRUS-RBD receptor is preferably indicated by a reduction in the number of positively stained cells of 50% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more. In order to obtain the best effect of vaccination, when VIRUS-mRBD is used as a vaccine, the binding force to the VIRUS-RBD receptor must be reduced as much as possible. Therefore, preferably, there is no upper limit for the reduction in the binding force to the VIRUS-RBD receptor. Therefore, preferably, there is no upper limit for the reduction in the number of positively stained cells, and this reduction can even be 100%. However, the upper limit for reduction in positively staining cells may be up to 100%, more preferably up to 99%.

[0312] Screening of libraries for VIRUS-mRBD or fragments thereof exhibiting b) can be performed by any method known to the skilled artisan. Exhibiting b) preferably means that the binding avidity to anti-VIRUS-wtRBD-nAB is maintained, i.e. ranging from a slight to moderately reduced to increased binding avidity compared to VIRUS-wtRBD. Anti-VIRUS-wtRBD-nABs may belong to different antibody classes and are defined mutatis mutandis as above for anti-CORONA-wtRBD-nAB, anti-SARS-wtRBD-nAB and / or anti-MERS-wtRBD-nAB.

[0313] The binding strength of VIRUS-mRBD or a fragment thereof and VIRUS-wtRBD to anti-VIRUS-wtRBD-nAB can be measured by any method known to those skilled in the art. Preferably, the binding strength is determined, for example, by staining cells expressing VIRUS-mRBD or VIRUS-wtRBD on the cell surface with anti-VIRUS-wtRBD-nAB directly or indirectly labeled with a fluorophore, or conversely, staining cells expressing anti-VIRUS-wtRBD-nAB on the cell surface with soluble VIRUS-mRBD or soluble VIRUS-wtRBD directly or indirectly labeled with a fluorophore, performing flow cytometry analysis, and counting positively stained cells. When the avidity of the anti-VIRUS-wtRBD-nABs is determined by flow cytometry analysis, the avidity is preferably the average avidity of several different monoclonal anti-VIRUS-wtRBD-nABs, preferably four or more different arbitrarily selected monoclonal anti-VIRUS-wtRBD-nABs.

[0314] The sustained avidity, i.e., the range of avidity from slight to moderate decreases in avidity to increases in avidity, is then obtained by comparing the avidity or average avidity of VIRUS-wtRBD to anti-VIRUS-wtRBD-nAB with the avidity or average avidity of VIRUS-mRBD to anti-VIRUS-wtRBD-nAB, respectively.

[0315] Preferably, avidity is measured by flow cytometry analysis, where a decrease in the number of positively stained cells indicates a lower avidity. Thus, whether avidity is maintained, i.e., in the range of slight to moderately decreased avidity and increased avidity, is preferably determined by comparing the number of positively stained cells resulting from binding of VIRUS-wtRBD to anti-VIRUS-wtRBD-nAB with the number of positively stained cells resulting from binding of VIRUS-mRBD to anti-VIRUS-wtRBD-nAB, respectively.

[0316] When binding is determined by flow cytometric analysis, a slight to moderate decrease in binding of VIRUS-mRBD to RBD-anti-VIRUS-wtRBD-nAB is preferably indicated by a decrease in positively stained cell count and / or ED50 of 50% or more, more preferably 80% or more, and even more preferably 90% or more.

[0317] As already mentioned above, when determining avidity by flow cytometric analysis, the avidity is preferably the average avidity as defined above. Thus, the slight to moderate decrease in avidity as defined above is preferably a slight to moderate decrease in the average avidity as defined above.

[0318] For best vaccination effect, when VIRUSA-mRBD is used as a vaccine, the binding affinity to anti-VIRUS-wtRBD-nAB should be as high as possible. Therefore, preferably, the decrease in binding affinity to anti-VIRUS-wtRBD-nAB should be as moderate as possible, and preferably, the binding affinity of anti-VIRUS-wtRBD-nAB to VIRUS-mRBD is preferably not decreased or even increased. That is, the number of positively stained cells is not decreased.

[0319] When determining the binding strength by flow cytometry analysis, the increase in the binding strength of VIRUS-mRBD to anti-VIRUS-wtRBD-nAB is preferably indicated by an increase of 1.1 times or more, more preferably 1.2 times or more, even more preferably 1.5 times or more, and even more preferably 2 times or more in the number of positively stained cells. As mentioned above, in order to obtain the best effect of vaccination, when VIRUS-mRBD is used as a vaccine, the binding strength to anti-VIRUS-wtRBD-nAB should be as high as possible. Therefore, preferably, there is no upper limit for the increase in the binding strength to anti-VIRUS-wtRBD-nAB. Therefore, preferably, there is no upper limit for the increase in positively stained cells. However, the upper limit for the increase in positively stained cells can be 10.000 times or less, more preferably 1.000 times or less, and even more preferably 100 times or less.

[0320] Preferably, steps (i), (ii)a) and / or (ii)b) are performed by using deep mutation scanning experiments.

[0321] Preferably, when all three steps (i), (ii)a) and / or (ii)b) are carried out in vitro, optionally step (ii)c) is also carried out in vitro as described above.

[0322] When step (i) is performed in vitro or in silico and steps (ii)a) and (ii)b) are performed in silico When all three steps (i), (ii)a) and / or (ii)b) are performed in silico by using a computer, data relating to VIRUS-wtRBD is provided to the computer and processed by bioinformatic analysis, wherein preferably the data provided to the computer is data from a crystal structure of VIRUS-wtRBD or a fragment thereof.

[0323] When providing data of a crystal structure, steps (i) and (ii)a) are combined by providing data of a crystal structure of VIRUS-wtRBD or a fragment thereof in complex with a VIRUS-RBD receptor, and providing data of a library of VIRUS-mRBD or a fragment thereof, or preferably VIRUS-mRBD or a fragment thereof, to a computer, processing the data by bioinformatics analysis to determine whether VIRUS-mRBD or a fragment thereof exhibits a), or more preferably, performing screening of the library for VIRUS-mRBD or a fragment thereof exhibiting a). Preferably, the determination of whether a) is exhibited is made by identifying amino acid residues of VIRUS-wtRBD or a fragment thereof that interfere with the VIRUS-RBD receptor based on the crystal structure of VIRUS-wtRBD or a fragment thereof in complex with a VIRUS-RBD receptor, and selecting a VIRUS-mRBD, preferably a VIRUS-mRBD from the library that contains one or more mutations in the interfering amino acid residues, as exhibiting a).

[0324] Preferably, the crystal structure data relating to a complex between VIRUS-wtRBD and a VIRUS-RBD receptor is preferably one or more experimental structures or computational models of VIRUS-wtRBD complexed with a VIRUS-RBD receptor, or a crystal structure of a relevant domain of the VIRUS-RBD receptor bound to VIRUS-mRBD or a fragment thereof.

[0325] Similarly, when providing crystal structure data, preferably steps (i) and (ii)b) are combined by providing to a computer data of the crystal structure of VIRUS-wtRBD or a fragment thereof complexed with an anti-VIRUS-wtRBD-nAB, and providing data of VIRUS-mRBD or a fragment thereof or preferably a library of VIRUS-mRBD or a fragment thereof, and processing the data by bioinformatic analysis to determine whether VIRUS-mRBD or a fragment thereof exhibits b), or more preferably, performing screening of the library for VIRUS-mRBD or a fragment thereof that exhibits b). Preferably, the determination of whether b) is exhibited is made by identifying amino acid residues in VIRUS-wtRBD or a fragment thereof that do not interfere with anti-VIRUS-wtRBD-nAB based on a crystal structure of VIRUS-wtRBD or a fragment thereof complexed with anti-VIRUS-wtRBD-nAB, and selecting a VIRUS-mRBD, preferably a VIRUS-mRBD from a library containing one or more mutations in the non-interfering amino acid residues, as exhibiting b).

[0326] More preferably, the non-interfering amino acid residues are selected from the interfering amino acid residues identified in determining a).

[0327] Preferably, the crystal structure data for a complex with an anti-VIRUS-wtRBD-nAB is preferably one or more experimental structures or computational models of VIRUS-wtRBD complexed with an anti-VIRUS-wtRBD-nAB, or a crystal structure of a relevant domain of anti-VIRUS-wtRBD-nAB binding to VIRUS-mRBD or a fragment thereof.

[0328] Preferably, all three steps (i), (ii)a) and / or (ii)b) are performed in silico by providing crystal structure data for a complex between VIRUS-wtRBD and a VIRUS-RBD receptor, and, where appropriate, step (ii)c) is performed in vitro as described above.

[0329] Where step (i) is performed in vitro and steps (ii)a) and (ii)b) are performed in silico, optionally step (ii)c) is also performed in silico, wherein preferably step (i) is performed in vitro by using one or more deep mutation scanning experiments. Preferably steps (ii)a) and (ii)b) and optionally step (ii)c) are performed in silico by using a computer, wherein data relating to VIRUS-mRBD and VIRUS-wtRBD are provided to the computer and processed by bioinformatic analysis, wherein the data provided to the computer is preferably data from one or more deep mutation scanning experiments of VIRUS-mRBD or a fragment thereof and VIRUS-wtRBD or a fragment thereof.

[0330] Preferably, step (ii)a) is performed using data from one or more deep mutation scanning experiments and a fitness measure reflecting the binding of VIRUS-mRBD or a fragment thereof (query antigen, s) to the VIRUS-RBD receptor (Rendo(s)), where Rendo is the log-scale difference in the dissociation constant K of query antigen s compared to a reference antigen (e.g. wild type) r, e.g. by titration, where the formula is Log(K(s))-Log(K(r)).

[0331] Similarly, step (ii)b) is performed using data from one or more deep mutation scanning experiments and a measure of fitness of VIRUS-mRBD or fragment(s) thereof that reflects escape (Rnab(s)) from one or more VIRUS-wtRBD-nABs, where Rnab(s) is the escape rate, preferably identifying the number of reduced binding compared to the total number, as quantified by FACS.

[0332] Preferably, step (ii)c) is further performed, which uses data from one or more deep mutation scanning experiments and a fitness measure reflecting the stability of VIRUS-mRBD or fragment(s) thereof (Rstab(s)), where Rstab is the log-scale difference in number c compared to a reference antigen r, e.g., by fluorescence intensity, where the formula is Log(c(s))-Log(c(r)).

[0333] Even more preferably, Step (iii) comprises: a) their ability to reduce binding to the VIRUS-RBD receptor; and b) their ability to bind to VIRUS-wtRBD-nAB, and c) and, where appropriate, their ability to indicate the stability of proteins and / or peptides. and scoring the Based on the scoring, selecting from the library one or more VIRUS-mRBD or fragments thereof having the highest score for a), preferably the highest score for the combination of a) and b), and more preferably the highest score for the combination of a), b), and c) as an active ingredient. Further includes:

[0334] Preferably, a), b), and c) are all weighted equally relative to one another, eg, a):b):c)=1:1:1.

[0335] Preferably, the scoring is performed in silico using a computer. Preferably, scoring one or more VIRUS-mRBD or fragments thereof based on normalized data of Rendo(s), optionally Rnab(s), and further optionally Rstab(s), Stot(s')=Nendo(s'), Where Stot(s') = Nnab(s') - Nendo(s'), Further, where appropriate, Stot(s') = Nstab(s') - Nnab(s') - Nendo(s'); During the ceremony, Stot(s') is the total score of VIRUS-mRBD or a fragment thereof; Nendo(s') is the normalized binding of VIRUS-mRBD or fragment(s) thereof to the VIRUS-RBD receptor (Rendo(s)); Nnab(s') is the normalized escape of VIRUS-mRBD or a fragment thereof from one or more VIRUS-wtRBD-nABs (Rnab(s)); Nstab(s') is the stability of VIRUS-mRBD or a fragment thereof (Rstab(s)); and selecting from the library one or more VIRUS-mRBDs or fragments thereof to be used as active ingredients having the highest score Stot(s'); Includes.

[0336] Preferably, the scoring of one or more VIRUS-mRBDs or fragments thereof is based on Rendo(s), Rnab(s), and Rstab(s) normalization, preferably, Rstab(s) is preselected prior to scoring of the Rendo(s) and Rstab(s) data, and wherein: Rstab(s) of VIRUS-mRBD or a fragment thereof must be at least a factor of exp(-alpha) of VIRUS-wtRBD, the Rend(s) of VIRUS-mRBD or a fragment thereof must be at least a factor of exp(-beta) of VIRUS-wtRBD, Here, preferably, alpha and beta thresholds are set, which allow the degree of destabilization and binding strength of VIRUS-mRBD to be taken into account for scoring.

[0337] Preferably, the process for designing and / or obtaining is a process for obtaining an active ingredient.

[0338] Preferably, the method for designing and / or obtaining an active ingredient is a method for designing and / or obtaining an active ingredient for a vaccine composition against a virus.

[0339] Preferably, as an active ingredient, the VIRUS-mRBD or a fragment thereof or one or more VIRUS-mRBD or fragments thereof - one or more VIRUS-mRBDs or fragments thereof, and / or - one or more mutant spike proteins of the virus (VIRUS-mSpike) or fragments thereof, including the VIRUS-mRBD or fragments thereof, and / or - one or more polypeptides or proteins comprising VIRUS-mRBD or a fragment thereof or VIRUS-mSpike or a fragment thereof, and / or - VIRUS-mRBD or a fragment thereof, VIRUS-mSpike or any fragment thereof, or Polypeptides or proteins One or more nucleic acids encoding It can exist as:

[0340] Preferably, the method for designing and / or obtaining an active ingredient for a vaccine composition is applicable to a virus selected from coronaviruses, more preferably SARS-CoV-2 and / or MERS-CoV, Hepatitis C virus (HCV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Influenza virus, Respiratory Syncytial Virus (RSV), Dengue virus, Human Immunodeficiency Virus (HIV), Yellow Fever Virus, Zika Virus, West Nile Virus (WNV), Japanese Encephalitis Virus, Tick-Borne Encephalitis Virus (TBE), Epstein-Barr Virus (EBV), and / or Cytomegalovirus (CMV).

[0341] Preferably, the method for designing and / or obtaining an active ingredient comprises: - the VIRUS-mRBD is a CORONA-mRBD according to the invention, a Hepatitis C virus mRBD, a Porcine Reproductive and Respiratory Syndrome virus mRBD, an Influenza virus mRBD, a Respiratory Syncytial Virus mRBD, a Dengue virus mRBD, a Human Immunodeficiency Virus mRBD, a Yellow Fever virus mRBD, a Zika virus mRBD, a West Nile virus mRBD, a Japanese Encephalitis virus mRBD, a Tick-borne Encephalitis virus mRBD, an Epstein-Barr virus (EBV) mRBD and / or a Cytomegalovirus mRBD, more preferably the CORONA-mRBD according to the invention is a SARS-mRBD and / or a MERS-mRBD according to the invention, - the VIRUS-wtRBD is a CORONA-wtRBD according to the present invention, Hepatitis C virus wtRBD, Porcine Reproductive and Respiratory Syndrome virus wtRBD, Influenza virus wtRBD, Respiratory Syncytial Virus wtRBD, Dengue virus wtRBD, Human Immunodeficiency Virus wtRBD, Yellow Fever virus wtRBD, Zika virus wtRBD, West Nile virus wtRBD, Japanese Encephalitis Virus wtRBD, Tick-borne Encephalitis Virus wtRBD, Epstein-Barr Virus (EBV) wtRBD and / or Cytomegalovirus wtRBD, more preferably the CORONA-wtRBD according to the present invention is a SARS-wtRBD (SEQ ID NO: 1) and / or a MERS-wtRBD (SEQ ID NO: 194) according to the present invention, - the VIRUS-RBD receptor is CORONA-RBD receptor, more preferably ACE2 and / or DPP4, Hepatitis C virus RBD receptor, more preferably CD81 and / or ApoE, Porcine reproductive and respiratory syndrome virus RBD receptor, more preferably DC163, Influenza virus RBD receptor, more preferably sialic acid, Respiratory syncytial virus RBD receptor, more preferably RSV G protein and / or RSV F protein, even more preferably CX3CR1, heparan sulfate proteoglycan (HSPG), nucleolin, EGFR, IGF1R, ICAM-1 and / or insulin-like growth factor 1 receptor (IGF1R), dengue virus RBD receptor, more preferably mannose receptor, Fc gamma receptor, TIM / TAM, heparin sulfate, DC-SIGN, human immunodeficiency virus RBD receptor, more preferably CD4, yellow fever virus RBD receptor, more preferably heparan sulfate proteoglycan (HSPG), Zika virus RBD receptor, more preferably phosphatidylserine receptor, even more preferably T cell immunoglobulin (TIM) and TYRO3, AXL, and MERTK (TAM), West Nile virus RBD receptor, more preferably phosphatidylserine receptor, even more preferably T more preferably, T-cell immunoglobulin (TIM) and TYRO3, AXL, and MERTK (TAM), Japanese encephalitis virus RBD receptor, more preferably, phosphatidylserine receptor, even more preferably, T-cell immunoglobulin (TIM) and TYRO3, AXL, and MERTK (TAM), tick-borne encephalitis virus RBD receptor, more preferably, phosphatidylserine receptor, even more preferably, T-cell immunoglobulin (TIM) and TYRO3, AXL, and MERTK (TAM), Epstein-Barr virus receptor, more preferably, complement receptor 2 (CD21) and / or cytomegalovirus RBD receptor, more preferably, platelet-derived growth factor (PDGF) receptor alpha (PDGFRα), heparan sulfate proteoglycan (HSPG), epidermal growth factor receptor (EGFR), integrin; - the VIRUS-wtRBD-nAB is a CORONA-wtRBD-nAB according to the invention, a Hepatitis C virus nAB, a Porcine Reproductive and Respiratory Syndrome virus nAB, an Influenza virus nAB, a Respiratory Syncytial Virus nAB, a Dengue virus nAB, a Human Immunodeficiency Virus nAB, a Yellow Fever Virus nAB, a Zika virus nAB, a West Nile virus nAB, a Japanese Encephalitis Virus nAB, a Tick-Borne Encephalitis Virus nAB, an Epstein-Barr Virus nAB and / or a Cytomegalovirus nAB, more preferably the CORONA-wtRBD-nAB according to the invention is a SARS-wtRBD-nAB and / or a MERS-wtRBD-nAB according to the invention, - the VIRUS-mSpike is a CORONA-mSpike according to the present invention, a Hepatitis C virus mSpike, a Porcine Reproductive and Respiratory Syndrome virus mSpike, an Influenza virus mSpike, a Respiratory Syncytial Virus mSpike, a Dengue virus mSpike, a Human Immunodeficiency Virus mSpike, a Yellow Fever Virus mSpike, a Zika virus mSpike, a West Nile virus mSpike, a Japanese Encephalitis Virus mSpike, a Tick-Borne Encephalitis Virus mSpike, an Epstein-Barr Virus mSpike and / or a Cytomegalovirus mSpike, more preferably the CORONA-mSpike according to the present invention is a SARS-mSpike and / or a MERS-mSpike according to the present invention.

[0342] More preferably, the method for designing and / or obtaining an active ingredient comprises - the VIRUS-mRBD is a CORONA-mRBD according to the invention, more preferably a SARS-mRBD and / or a MERS-mRBD according to the invention, - the VIRUS-wtRBD is a CORONA-wtRBD according to the present invention, more preferably a SARS-wtRBD (SEQ ID NO: 1) and / or a MERS-wtRBD (SEQ ID NO: 194) according to the present invention; - the VIRUS-RBD receptor is a CORONA-RBD receptor, more preferably ACE2 and / or DPP4; - the VIRUS-wtRBD-nAB is a CORONA-wtRBD-nAB according to the invention, more preferably a SARS-wtRBD-nAB and / or a MERS-wtRBD-nAB according to the invention, - VIRUS-mSpike is a CORONA-mSpike according to the present invention, more preferably a SARS-mSpike and / or a MERS-mSpike according to the present invention.

[0343] Moreover, in the present invention, CORONA-mRBD, SARS-mRBD, and / or MERS-mRBD or fragments thereof, and / or CORONA-mSpike, SARS-mSpike, and / or MERS-mSpike or fragments thereof; A polypeptide or protein comprising CORONA-mRBD, SARS-mRBD or MERS-mRBD or a fragment thereof, or CORONA-mSpike, SARS-mSpike or MERS-mSpike; Nucleic acids encoding these according to the invention, Vaccine compositions comprising the same according to the invention, and / or Prophylactic and / or therapeutic use It should be understood that any embodiment disclosed is applicable mutatis mutandis to a method for designing and / or obtaining an active ingredient for a vaccine composition.

[0344] Moreover, the present invention relates to the VIRUS-mRBD or a fragment thereof obtained as an active ingredient by the method for designing and / or obtaining an active ingredient for a vaccine composition according to the present invention.

[0345] Preferably, the VIRUS-mRBD is a CORONA-mRBD according to the present invention, Hepatitis C virus mRBD, Porcine Reproductive and Respiratory Syndrome virus mRBD, Influenza virus mRBD, Respiratory Syncytial Virus mRBD, Dengue virus mRBD, Human Immunodeficiency Virus mRBD, Yellow Fever virus mRBD, Zika virus mRBD, West Nile virus mRBD, Japanese encephalitis virus mRBD, Tick-borne encephalitis virus mRBD, Epstein-Barr virus mRBD, and / or Cytomegalovirus mRBD, more preferably, the CORONA-mRBD according to the present invention is a SARS-mRBD and / or MERS-mRBD according to the present invention.

[0346] More preferably, the VIRUS-mRBD is a CORONA-mRBD according to the present invention, more preferably a SARS-mRBD and / or a MERS-mRBD according to the present invention.

[0347] Moreover, the present invention relates to a method for the prevention and / or treatment of a disease caused by a coronavirus, preferably COVID-19 caused by SARS-CoV-2 and / or MERS caused by MERS-CoV, in a subject, comprising: - one or more CORONA-mRBDs according to the invention, preferably SARS-mRBD or MERS-mRBD, or a fragment thereof, - one or more CORONA-mSpikes according to the invention, preferably SARS-mSpike or MERS-mSpike, or a fragment thereof, - one or more polypeptides or proteins according to the invention, - one or more nucleic acids according to the invention, and / or - Vaccine composition according to the invention The present invention relates to a method comprising administering to a subject

[0348] Moreover, the present invention provides a method for inducing an immune response in a subject, preferably against a coronavirus, SARS-CoV-2, MERS-CoV, comprising: - one or more CORONA-mRBDs according to the invention, preferably SARS-mRBD or MERS-mRBD, or a fragment thereof, - one or more CORONA-mSpikes according to the invention, preferably SARS-mSpike or MERS-mSpike, or a fragment thereof, - one or more polypeptides or proteins according to the invention, - one or more nucleic acids according to the invention, and / or - Vaccine composition according to the invention The present invention relates to a method comprising administering to a subject

[0349] Moreover, it should be understood that in the present invention any embodiment disclosing relating to CORONA-mRBD / SARS-mRBD and / or MERS-mRBD or fragments thereof, and / or CORONA-mSpike / SARS-mSpike and / or MERS-mSpike or fragments thereof, CORONA-mRBD / SARS-mRBD or MERS-mRBD or fragments thereof, or polypeptides or proteins comprising CORONA-mRBD / SARS-mRBD or MERS-mRBD or fragments thereof, nucleic acids encoding same, vaccine compositions comprising same, uses in prevention and / or treatment, and / or methods for obtaining same, is applicable mutatis mutandis to methods for the prevention and / or treatment of diseases caused by coronaviruses and / or methods for inducing an immune response.

[0350] [Table 3]

[0351] [Table 4]

[0352] The invention is further illustrated by the following figures and examples. [Brief description of the drawings]

[0353] [Figure 1] Figures 1A-G show in vivo that sACE2 interferes with the generation of B cells secreting cAbs (used synonymously for competing antibodies, "nAbs"). Figure 1A is the structure of modeled mouse ACE2 with humanized residues and the RBD footprint. Figure 1B outlines the immunization schedule and time points for the DropMap analysis. Figure 1C shows the frequency of B cells secreting cAbs. Figure 1D shows ELISA assessment of blockade of ACE2 binding to the RBD. Figure 1E shows a dose-response analysis of the data in Figure 1C. Figure 1F shows a comparative analysis of cAb secretion upon immunization. Figure 1G shows a DropMap analysis of splenocytes after full spike immunization with and without mhACE2. [Diagram 2] Figures 2A-C show in silico identification of conserved binding of SARS-mRBD- and SARS-wtRBD-specific nABs with reduced ACE2 binding using bioinformatic analysis. [Diagram 3] 3A-C show the binding avidity of ACE2 and various monoclonal nABs to SARS-wtRBD and various SARS-mRBDs with single, double, and triple amino acid substitutions, determined in vitro by use of ELISA. [Figure 4] 4A-F show the binding avidity of ACE2 and various monoclonal nABs to SARS-wtRBD and various SARS-mRBD determined in vitro by use of ELISA. [Diagram 5] FIG. 5 lists the EC50 values ​​determined for various monoclonal nABs and ACE2 considering SARS-wtRBD and various SARS-mRBD. [Figure 6] FIG. 6 shows the binding avidity of ACE2 and various monoclonal nABs to SARS-wtRBD and various SARS-mRBD determined in vitro by use of ELISA. [Figure 7]FIG. 7 shows the frequency of B cells following immunization with either SARS-wtRBD or various SARS-mRBDs, as determined in vivo using a mouse model with controlled sACE2 levels. [Figure 8] Figures 8A and B show in silico modeling of epitope masking by sACE2 for the newly emerged SARS-CoV-2 variant containing the N501Y substitution. [Figure 9] FIG. 9 shows the binding avidity of DPP4 and various monoclonal nABs to either MERS-wtRBD or various MERS-mRBDs as determined in vitro by use of ELISA. [Figure 10] FIG. 10 shows the results of pull-down assays of human DPP4 and rabbit DPP4 from serum using either MERS-wtRBD or various MERS-mRBDs. [Figure 11] Figure 11 shows the peptide sequence of the wild-type SARS-CoV-2 complete spike protein with regions highlighted. [Figure 12] FIG. 12 shows the peptide sequence of the wild-type MERS-CoV complete spike protein with regions highlighted. [Figure 13A] FIG. 13 shows the binding avidity of ACE2 and various monoclonal nABs to SARS-wtRBD and various SARS-mRBD determined in vitro by use of ELISA. [Figure 13B] FIG. 13 shows the binding avidity of ACE2 and various monoclonal nABs to SARS-wtRBD and various SARS-mRBD determined in vitro by use of ELISA. [Figure 13C] FIG. 13 shows the binding avidity of ACE2 and various monoclonal nABs to SARS-wtRBD and various SARS-mRBD determined in vitro by use of ELISA. [Figure 13D]FIG. 13 shows the binding avidity of ACE2 and various monoclonal nABs to SARS-wtRBD and various SARS-mRBD determined in vitro by use of ELISA. [Figure 14A] FIG. 14 shows the membrane fusion ability of SARS-mRBD as determined by cell-cell fusion assay. [Figure 14B] FIG. 14 shows the membrane fusion ability of SARS-mRBD as determined by cell-cell fusion assay. [Figure 14C] FIG. 14 shows the membrane fusion ability of SARS-mRBD as determined by cell-cell fusion assay. [Figure 14D] FIG. 14 shows the membrane fusion ability of SARS-mRBD as determined by cell-cell fusion assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0354] Note: In the following examples, in some cases, the positions of amino acid substitutions in the RBD are given with respect to their positions in Spike. In this regard, see Tables A and B above for assigning positions in Spike to respective positions in the RBD. [Example 1]

[0355] Masking of SARS-wtRBD by sACE2 or nAB impairs B cell and antibody responses in an in vivo mouse model - Figure 1A-G Figure 1A-G) Soluble ACE2 interferes with the generation of B cells secreting antibodies (cAbs) that interfere / compete with ACE2-RBD interaction in vivo.

[0356] Figure 1A) Structure of modeled mouse ACE2, with humanized residues in blue and the RBD footprint in black.

[0357] Figure 1B) Overview of immunization schedule and time points for DropMap analysis. Individual splenocytes were analyzed by encapsulation in microdroplets, and binding of secreted antibodies to the RBD and competition with sACE2 was measured by fluorescence relocation.

[0358] FIG. 1C) The frequency of cAb-secreting B cells (as a percentage of IgG-secreting cells) on day 32 is shown for immunizations with RBD (3 μg / immunization, 120 pmol) presented with masking-competent nAb (18 pmol or 86 pmol) or mhACE2 (4 pmol, 37 pmol or 92 pmol).

[0359] FIG1D) Mouse serum was collected on day 32 and blockade of ACE2 binding to the RBD was assessed by ELISA. Optical density and area under the curve (AAC) values ​​versus reciprocal serum dilutions are shown.

[0360] FIG. 1E) Dose-response trend of the data shown in c when normalized to %RBM masking.

[0361] FIG. 1F) Comparative analysis of cAb secretion 4 days (day 32) and 21 days (day 49) after the second immunization.

[0362] Figure 1G) Dropmap analysis of splenocytes on day 49 after full spike immunization (3 μg / immunization, 18 pmol immunization) with or without mhACE2 (0 pmol, 4 pmol, or 92 pmol).

[0363] All experiments shown in Figure 1 used N = 3 mice per condition, and statistical analysis was performed by two-tailed unpaired Student's t-test (***p<0.001, **p<0.01, and *p<0.05). Graphs in c, e-f show mean frequency of occurrence + / - SD.

[0364] This example was carried out as described in Example 5, except that in addition, blocking of ACE2 binding capacity was determined for mouse sera.

[0365] Serial dilutions of sera in PBS with 1% BSA were added to RBD-coated 96-well plates. Recombinant RBD was immobilized on high-binding 96-well ELISA plates (Corning, #CLS3690) at 4 μg / ml in PBS (Sigma Aldrich, MO, USA) overnight at +4°C. Plates were blocked for 1 h at room temperature with 1% BSA in PBS (Thermo Fisher, Gibco, MA, USA). After 1 h of incubation at room temperature, in-house generated biotinylated ACE2-hFcg1 was added to a final effective concentration of 70 (EC70) in PBS with 1% BSA. After another hour of incubation at room temperature, biotinylated ACE2-hFcg1 was detected by incubating the plates with AP-conjugated streptavidin (Southern Biotech, #SBA-7100-04) diluted 1:500 in PBS with 1% BSA. This was not prevented by serum antibodies from binding to the RBD. The 50% maximal ACE2 blocking capacity (BD50) was determined by fitting the sigmoidal curves by nonlinear regression performed with R (stats package). The area under or on the curve was determined with GraphPadPrism software. The upper and lower plateaus of the non-biotinylated ACE2-hFcg1 control served as references. [Example 2]

[0366] In silico identification of the conservation of binding of SARS-mRBD- and SARS-wtRBD-specific nABs with reduced ACE2 binding using bioinformatic analysis - Figures 2A, B, and C Methods for generating mutant protein or peptide antigens capable of inducing stronger immune responses would be particularly useful since the antigens would be prevented from binding to or being masked by membrane-bound or soluble receptors, respectively, and the antigens would be designed such that important epitopes continue to induce neutralizing immune responses specific to the immunogen.

[0367] The generation of "unmasked" antigens involves introducing a minimal number of amino acid exchanges into the antigen to prevent binding to receptors expressed in humans or animals, followed by screening for retention of binding to neutralizing antibodies or convalescent donor sera.

[0368] Select an immunogen and identify the receptor binding motif, for example, by structural testing or antigen mutagenesis. The receptor binding motif is the direct binding interface between the receptor and the antigen. If applicable, identify multiple receptor binding motifs. For example, MERS-CoV recognizes not only DPP4 but also sialic acid with two separate RBMs (https: / / www.pnas.org / content / 114 / 40 / E8508).

[0369] Deep mutational scans of RBMs are performed on platforms that couple genotype to phenotype (https: / / www.nature.com / articles / nmeth.3027.pdf). Such platforms include cell-based assays, where proteins are expressed by plasmids or viruses, or in vitro systems such as T7 or M13 bacteriophage display, ribosome display, E. coli display, or most preferably, mammalian cell display. Gene libraries of mutated variants of antigens are synthesized and cloned into appropriate plasmids. Mutant libraries can be generated by, for example, overlap extension PCR (https: / / www.sciencedirect.com / science / article / pii / S0022283613004300?via%3Dihub), error-prone PCR (https: / / link.springer.com / protocol / 10.1385 / 1-59259-395-X:3) or chemical mutagenesis (https: / / academic.oup.com / nar / article / 32 / 4 / 1448 / 1038612?login=true). Some strategies create mutations at exactly one codon per gene (https: / / journals.plos.org / plosone / article?id=10.1371 / journal.pone.0052031 ; https: / / www.sciencedirect.com / science / article / pii / S0003269713005782?casa_token=d5JPaeUuTwYAAAAA:-F2ChRH8nmjiVhQYPsl1BF4JpK-m_E0CoVBibRWGkDKxCPd7D3IVxW7n-f7rRFCxRPGYJc8Q), and this design can be adapted to mutate multiple codons per gene to explore the average effect of mutations (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4104320 / ).Mutagenesis can be enhanced by designing primers with equal melting temperatures to prevent bias due to certain mutations (https: / / www.sciencedirect.com / science / article / pii / S1931312817301968). For SARS-CoV-2 RBD, deep mutation scanning was applied by expressing an RBD mutant library in yeast (https: / / www.sciencedirect.com / science / article / pii / S0092867420310035). Yeast expression systems are suitable when the antigen does not contain glycosylation in or near the receptor binding motif. When glycosylation affects the binding of the antigen to the host receptor, expression in mammalian cells is preferred. For the production of a vaccine intended for humans, human cell lines are most preferred. Similarly, for the production of a vaccine intended for pigs, porcine cell lines are most preferred.

[0370] Screening of the library can be performed by flow cytometric cell sorting after transfection or transduction of cells with the mutant library. Antigens can be cloned into a backbone that allows membrane expression and tagged with a fluorescent reporter, e.g., GFP, to (i) preselect antigen-expressing cells and (ii) determine the expression level of the antigen. Transfection conditions are preferably such that a single coding variant is obtained per cell (https: / / www.jimmunol.org / content / 200 / 11 / 3825, https: / / www.jbc.org / article / S0021-9258(20)35531-9 / fulltext, https: / / science.sciencemag.org / content / 369 / 6508 / 1261). FACS selection is performed by staining cells with a soluble receptor that binds to RBM expressed on the cell surface. The soluble receptor can be either directly labeled with a fluorophore, or biotinylated and stained with a streptavidin-conjugated fluorophore, or tagged with his-tag, strep-tag, or myc-tag, followed by staining with a secondary agent that detects the tag. Preferably, the protein is tagged with Twin-Strep-Tag and stained with a StrepTactin-conjugated fluorophore. Even more preferably, the protein is directly labeled with a fluorophore. In a first step, cells are stained with the soluble receptor and sorted by a suitable method, for example, FACS sorting. The binding of the soluble receptor is detected by a fluorophore that allows separation from the reporter protein signal fused to the RBM. The cells are positively selected to express the RBM protein / peptide fused to a fluorescent reporter, for example, GFP. Moreover, the cells are selected for which the binding to the soluble receptor is lost.

[0371] Because both soluble receptors and neutralizing antibodies bind to RBM, selection for conservation of the epitope recognized by the neutralizing antibody is preferably performed after selection of RBM mutants with reduced binding to the soluble receptor.

[0372] In a second step, sorted cells with reduced binding to the soluble receptor are stained with an antibody that recognizes the RBM. This positively selects the sub-library of RBM mutants for the preservation of the epitope bound by the neutralizing antibody. The cells of the sub-library are stained with one or, more preferably, multiple recombinant neutralizing antibodies. Most preferably, an equimolar mixture of a set of neutralizing antibodies targeting distinct RBM epitopes is used. Optimally, the neutralizing antibodies are affinity matched or otherwise adjusted to account for different avidities. The sum of the neutralizing antibodies should encompass the entire RBM. The neutralizing antibodies can be individually labeled with either distinct fluorophores or biotin to detect antibody binding en masse. Similarly, antibody binding can be detected with antibody-specific secondary agents. If recombinant neutralizing antibodies are not available, screening can be performed by staining the cells with serum from convalescent donors (human or immunized animals) and detecting bound antibodies with a suitable secondary reagent.

[0373] If a transient expression system is used, the binding of the distinct recombinant neutralizing antibodies or convalescent plasma is examined by a single staining. If a stable expression system is used, the sub-library can be cultured and expanded after selection for loss of receptor binding. The sub-library can be further cultured in bulk or, for example, subcloned as single cells to expand monoclonal cell lines. Cells stably expressing the selected RBM mutants can then be stained with a series of recombinant neutralizing antibodies or convalescent serum. Preferably, staining with distinct recombinant neutralizing antibodies is performed by multiple individual stainings. Mutant antigens expressed by cells that maintain binding of the neutralizing antibodies are positively selected. Most preferably, binding of all selected neutralizing antibodies to the mutant RBM is preserved.

[0374] Cells selected after transient transfection are sorted directly into medium after first staining / before second staining and sorting. After second sorting, cells are sorted in medium or, more preferably, directly into lysis buffer. Selected cells stably expressing mutant RBMs can be expanded and then analyzed. Total gDNA or, more preferably, RNA is purified by a kit such as an RNA extraction kit (Quiagen) or a GeneJET RNA purification kit (Thermo Scientific). The target region of interest is amplified by gene-specific primers spanning the mutated region across the entire mutant library. For RNA extraction, cDNA is reverse transcribed by specific primers using a high fidelity kit such as the Transcriptor High Fidelity cDNA Synthesis Kit (Roche) or high fidelity Accuscript (Agilent). The diversified region of the RBM is amplified by PCR to one or more fragments depending on the size of the mutated region. The PCR amplicon is flanked by adapters for high-throughput sequencing, for example, Illumina sequencing, which include sequencing primers, unique barcodes, and flow cell binding sequences. The amplicon is sequenced by high-throughput sequencing, which is selected to suit the length of the amplified fragment and the expected number of mutants. For example, Illumina's MiniSeq, MiSeq, NextSeq or HiSeq system can be used.

[0375] The data are analyzed by determining the frequency of occurrence of RBM mutant variants in the transcripts and comparing their frequency in the selected library to their frequency in the naive vector library, such that the change in frequency from input to selection for each variant acts as a measure of its ability to abrogate receptor binding and preserve neutralizing antibody binding.

[0376] Screening of mutant libraries serves as a basis for the selection of mutants with minimal binding to host receptors and preservation of binding to neutralizing antibodies. In addition to the data obtained by screening of mutant libraries, these analyses can be combined with structural information of the receptor complexed with antigen. Furthermore, the analyses can be combined with structural information of the neutralizing antibodies complexed with antigen. This allows extended bioinformatic analysis to help identify the most suitable mutations that minimize binding to host receptors while maximizing preservation of neutralizing antibodies.

[0377] Screening data provides associations between single or multiple residues in the RBM sequence with respect to protein expression / stability (E, expression), affinity to receptor (B, binding), and binding to neutralizing antibodies (N, binding). RBM variants are selected such that E is maximized while B is minimized and N is maximized. In addition, the selected residues must significantly differ in the physical interface of the receptor-antigen structure. Moreover, the selected residues must not significantly differ in the physical interface of any structure of the neutralizing antibody complexed with the antigen. Most optimally, the selected residues are not targeted by any known neutralizing antibodies, nor are they targets of serum antibodies from convalescent individuals.

[0378] In the simplest scenario, scanning of mutant libraries reveals the consequences of every single mutation. In addition, libraries can be adapted to test the effects of multiple mutations in combination. To address the latter bioinformatically, evolutionary coupling between sites by sequence covariation analysis can be estimated.

[0379] When RBM mutations are identified by screening mutant libraries and / or bioinformatic screening, mutants can be selected by ELISA binding or any other method to appropriately determine avidity for a receptor or neutralizing antibody.

[0380] For example, the binding strength of RBM to a soluble receptor, measurable by Kd or EC50 (effective concentration at 50% of maximal binding), must be reduced such that the Kd or EC50 exceeds the serum or tissue concentration of the respective soluble receptor.

[0381] Bioinformatics methods The following describes a procedure for prioritizing the selection of stable primary sequence variants of an antigen that preserve binding to neutralizing antibodies while reducing binding to specific binding proteins. The method relies on data from deep mutational scanning experiments that measure the fitness (here binding and stability in a particular selection assay) for a library S of primary sequence variants on a target antigen A.

[0382] procedure Description of experimental data The procedure relies on the following experimental inputs: 1) One or more deep mutational scanning experiments in which the fitness measure reflects antigen stability (Rstab). 2) one or more deep mutational scanning experiments in which the fitness measure reflects escape from one or more neutralizing antibodies (Rnabs). 3) One or more deep mutational scanning experiments in which the fitness measure reflects binding to an endogenous binding partner (Rendo). 4) One or more experimental structures or computational models of the antigen complexed with a neutralizing antibody. 5) An experimental structure or computational model of one or more of the antigens complexed with endogenous protein partners or associated domains that bind to this antigen.

[0383] Note: The sequence libraries in experiments 1-3 may or may not have complete overlap in coverage. Experiments 4 and 5 will be as appropriate.

[0384] Experiments 1-3: These data provide fitness measures for three different properties for each sequence library S. We call the fitness of sequence s' from each library S Rstab(s), Rnab(s), and Rendo(s). The units of fitness are: 1. Rstab - the log scale difference in a number c compared to a reference antigen r, e.g. by fluorescence intensity. Formula: Log(c(s))-Log(c(r)). 2. Rnab-Escape ratio, identifying reduced bound numbers compared to total numbers, quantified by FACS. 3. Rendo - the log scale difference in the dissociation constant K compared to a reference antigen r, e.g. by titration. Formula: Log(K(s))-Log(K(r)).

[0385] Experiments 4-5: These experiments provide information about the spatial structure of the antigen with respect to the neutralizing antibody or endogenous binding partner in the form of a three-dimensional coordinate of the atomic positions. The structural interface, any amino acid residue of the antigen, is defined as having a certain shortest distance to any atom in the endogenous partner of 0.5 nanometers or less. The epitope on the antigen for the neutralizing antibody is also defined in the same way. The primary and secondary epitopes are defined as follows: the primary epitope directly overlaps with the structural interface, the secondary epitope indirectly overlaps, and the antibody binding may interfere with ACE2 binding by excluded volume effects. In Figure 2A, it is observed that the structural information overlaps with other data given and is therefore relevant.

[0386] Pretreatment and Standardization Select data from Experiments 1–3 based on the following two constraints: 1. Rstab of the mutant must be at least one factor of exp(-alpha) of the wild type. 2. The RendO of the mutant should be at most one factor of the wild type exp(-beta).

[0387] The alpha and beta thresholds control the degree of destabilization and binding strength of the variants you want to consider in scoring. Recommended values ​​are provided in Table 1 along with their interpretation.

[0388] [Table 5]

[0389] After selecting mutants that satisfy the constraints outlined above, we calculate normalized fitness scores by first subtracting the sample fitness mean and then dividing by the sample standard deviation. We map all fitness values ​​to floating-point values ​​with zero mean and unit standard deviation. We call the corresponding normalized fitness scores Nstab(s), Nnab(s), and Nendo(s).

[0390] Calculating the score A score is compiled based on the information extracted above to identify and experimentally test possible candidate mutants.

[0391] For all variants, a total score is calculated in the primary and secondary interfaces where the total score is calculated according to the measurements of experiments 1 and 3, if defined. Stot(s')=Nstab(s')-Nnab(s')-Nendo(s')

[0392] Missing values ​​in experiment 2 are set to 0 (normalized mean). If we do not define the primary and secondary interfaces, we calculate the scores for all variants that satisfy the constraints defined above. Higher scores are considered better.

[0393] Identifying hot spots Hotspots are identified by sorting loci by variants with the highest scores. Loci with negative scores are discarded. The threshold gamma (range 0-1) represents the proportion of loci with the highest non-negative total scores to be excluded. Set gamma=0.40. Gamma=0.25 corresponds to more hits but less specific, and gamma=0.75 corresponds to more specific but fewer hits.

[0394] Selection of mutant libraries Selection of mutant libraries for secondary validation can be done by two strategies: exploitation and exploration. The end user can decide to either fully explore the loci with the highest ranks or combine high-ranking mutants from all loci with top ranks.

[0395] We propose to first explore: take the highest ranking variant from each locus, and then exploit: explore lower ranking variants at each locus in the subsequent library.

[0396] In silico coding import pandas as pd import numpy as np import matplotlib as mpl import matplotlib.pyplot as plt import matplotlib.patches as patches #Load data DMS = pd.read_csv("Deep_Mutational_Scan_RBD_stability_ACE2binding.csv") dms2 = pd.read_csv("Tableofmutation_antibody-escape_fraction_scores.csv") # Identify missing sites missing_sites = np.setdiff1d(np.unique(DMS['site_SARS2'].to_numpy()), np.unique(dms2['site'].to_numpy()), ) print(f"Number of sites missing nAb data, {len(missing_sites)}: "+ str(missing_sites)) missing_sites2 = np.setdiff1d(np.unique(dms2['site'].to_numpy()), np.unique(DMS['site_SARS2'].to_numpy()) ) print(f"Number of sites missing ACE2 data, {len(missing_sites2)}: "+ str(missing_sites2)) # Primary and secondary interfaces determined based on nAb RBD crystal structures: # 6XC2, 6XCN,6XDG, 6XE1, 6XKQ,7CDI, 7CDJ, 7JMO, 7JMP,7JMW,7JV2,7JV6,7JVA,7JW0,7K90,7LX5. from itertools import chain interface_residues = [(443, 463), (469, 507)] secondary_interface_residues = [(403, 410), (417, 423)] total_interface_range = list(chain(*[range(*r) for r in interface_residues+secondary_interface_residues])) # pre-proccessing antibody_condition, mutation, escape = (dms2['condition'].to_numpy(),dms2['wildtype'].to_numpy()+dms2['site'].to_numpy().astype('str')+dms2['mutation'].to_numpy(), dms2['mut_escape'].to_numpy()) unique_abc = np.unique(antibody_condition) all_mutations = DMS['mutation'].to_numpy() all_expression = np.zeros(len(all_mutations)*len(unique_abc)) all_ace_binding = np.zeros(len(all_mutations)*len(unique_abc)) all_escape = np.zeros(len(all_mutations)*len(unique_abc)) j=0 for abc in unique_abc: idx_condition = antibody_condition==abc for i,m in enumerate(all_mutations): candidate=DMS.loc[np.where(DMS['mutation'].to_numpy()==m)[0]] all_expression[i+j*len(all_mutations)] = candidate['expr_avg'].to_numpy() all_ace_binding[i+j*len(all_mutations)] = candidate['bind_avg'].to_numpy() condition_selection = np.where(((mutation==m)*(idx_condition)))[0] if len(condition_selection)>0: all_escape[i+j*len(all_mutations)] = escape[condition_selection[0]] else: all_escape[i+j*len(all_mutations)] = np.nan j=j+1 finite_values = ~(~np.isfinite(all_expression)+~np.isfinite(all_ace_binding)) all_expression = all_expression[finite_values] all_ace_binding = all_ace_binding[finite_values] all_mutations = np.tile(all_mutations, len(unique_abc))[finite_values] all_resid = np.array([int(a[1:-1]) for a in all_mutations]) all_escape = all_escape[finite_values] _all_mutations = DMS['mutation'].to_numpy() all_conditions = np.concatenate([np.tile(abc, len(_all_mutations)) for abc in unique_abc ])[finite_values] def standardize(x): xhat=(x-x[np.isfinite(x)].mean()) return xhat / xhat[np.isfinite(xhat)].std() def compute_score(expression, escape, binding): escape_finite = np.isfinite(escape) escape_impute = np.array(escape) escape_impute[~escape_finite] = 0. return expression - binding - escape_impute #enforce constraints alpha=0.5 beta=1.0 constraints_fulfilled = ((all_expression>-alpha).astype(int)*(all_ace_binding<-beta).astype(int)).astype(bool) #compute score on data fulfilling constraints tot_score=compute_score(standardize(all_expression[constraints_fulfilled]), standardize(all_escape[constraints_fulfilled]), standardize(all_ace_binding[constraints_fulfilled])) #identify hotspots mutation_hotspots = {} for i in np.argsort(tot_score)[::-1]: y = all_mutations[constraints_fulfilled][i] resi = all_resid[constraints_fulfilled][i] if int(y[1:-1]) in total_interface_range: if mutation_hotspots.get(resi): mutation_hotspots[resi].append([y, all_expression[constraints_fulfilled][i], all_ace_binding[constraints_fulfilled][i], tot_score[i]]) else: mutation_hotspots[resi]=[[y, all_expression[constraints_fulfilled][i], all_ace_binding[constraints_fulfilled][i], tot_score[i]]] # Filtering of low-scoring variants max_total_scores = np.array([np.max([l[-1] for l in mutation_hotspots[key]]) for key in mutation_hotspots]) argsort_total_scores = np.argsort(max_total_scores)[::-1] # decending scores by index positive_max_total_scores = max_total_scores[argsort_total_scores]>0. scores_positive_and_sorted = max_total_scores[argsort_total_scores][positive_max_total_scores] gamma = 0.40 bisect_res = 1000 score_thres = np.linspace(0,scores_positive_and_sorted[0],bisect_res)[np.argmin([(gamma-np.mean(threshold>scores_positive_and_sorted))**2. for threshold in np.linspace(0,scores_positive_and_sorted[0],bisect_res)])] print(f"score threshold for gamma={gamma}: ", ) # Diagnostic plot plt.plot(np.linspace(0,scores_positive_and_sorted[0],bisect_res), [np.mean(threshold>scores_positive_and_sorted) for threshold in np.linspace(0,scores_positive_and_sorted[0],bisect_res)]) plt.vlines([score_thres],ymin=0, ymax=[gamma],color='k') plt.hlines([gamma],xmin=0, xmax=[score_thres],color='k') plt.xlabel("score threshold") plt.ylabel(r"$\gamma$") def stringify_top_mutants(mutation_hotspots, key, site_variant_scores): sorted_hits = np.argsort(site_variants_scores)[::-1] hits, counts = np.unique([mutation_hotspots[key][yy][0] for yy in sorted_hits], return_counts=True) return ', '.join([hit+f"({count})" for hit,count in zip(hits,counts) ]) top_mutants=[] for i,key in enumerate(mutation_hotspots.keys()): if max_total_scores[i]>score_thres: site_variants_scores = [variant[-1] for variant in mutation_hotspots[key]] print(key,'\t', stringify_top_mutants(mutation_hotspots, key, site_variants_scores),'\t', np.round(max_total_scores[i],2)) top_mutants.append(mutation_hotspots[key][np.argmax(site_variants_scores)][0]) # generate plot summarizing selected variants in the context of all data and constraints. fig, ax = plt.subplots(4,1, figsize=(4,12), gridspec_kw={'height_ratios': [2,2,1, 1]},constrained_layout=True ) ax[0].scatter(dms2['site'].to_numpy(), dms2['site_max_escape'].to_numpy(), marker='.',alpha=0.1, label="Site for escape mutations") for i,ir in enumerate(interface_residues): if i==0: ax[0].hlines(1.05, *ir, color='k', lw=5, label="primary nAb interface") else: ax[0].hlines(1.05, *ir, color='k', lw=5) for i,ir in enumerate(secondary_interface_residues): if i==0: ax[0].hlines(1.05, *ir, color='r', lw=5, label="secondary nAb interface") else: ax[0].hlines(1.05, *ir, color='r', lw=5) ax[0].vlines(missing_sites, ymin=-0.08, ymax=0.0, color='r',lw=0.7, label="missing site" ) ax[0].set_xlabel("RBD position") ax[0].set_ylabel("Maximum Escape") ax[0].legend() ax[1].scatter(DMS['expr_avg'].to_numpy(), DMS['bind_avg'].to_numpy(),marker='.',c='k',alpha=0.5) ax[1].scatter(all_expression[constraints_fulfilled][np.isin(all_mutations[constraints_fulfilled], top_mutants)], all_ace_binding[constraints_fulfilled][np.isin(all_mutations[constraints_fulfilled], top_mutants)],marker='o',c='r',alpha=0.8) ax[1].set_xlabel("Expression") ax[1].set_ylabel("ACE2 binding") rect = patches.Rectangle((-0.5, -5), 1.5, 4.00, linewidth=0, edgecolor='none', facecolor='y', alpha=0.2) ax[1].add_patch(rect) ax[3].scatter(DMS['expr_avg'].to_numpy(), DMS['bind_avg'].to_numpy(),marker='.',c='k',alpha=0.5) ax[3].scatter(all_expression[constraints_fulfilled][np.isin(all_mutations[constraints_fulfilled], top_mutants)], all_ace_binding[constraints_fulfilled][np.isin(all_mutations[constraints_fulfilled], top_mutants)],marker='o',c='r',alpha=0.8) ax[2].scatter(DMS['expr_avg'].to_numpy(), DMS['bind_avg'].to_numpy(),marker='.',c='k',alpha=0.5) ax[2].scatter(all_expression[constraints_fulfilled][np.isin(all_mutations[constraints_fulfilled], top_mutants)], all_ace_binding[constraints_fulfilled][np.isin(all_mutations[constraints_fulfilled], top_mutants)],marker='o',c='r',alpha=0.8) ax[2].set_xlim(-0.5,1) ax[2].set_ylim(-2.1,-1.2) ax[2].set_xticklabels([]) ax[3].set_xlim(-0.5,1) ax[3].set_ylim(-4.85,-4.6) ax[3].set_xlabel("Expression") ax[3].set_ylabel("ACE2 binding") ax[2].set_ylabel("ACE2 binding") for bm in top_mutants: _idx = (DMS['mutation']==str(bm)).to_numpy().argmax() ax[2].annotate(str(bm), (DMS['expr_avg'][_idx], DMS['bind_avg'][_idx] ), va='center', ha='center') for bm in top_mutants: _idx = (DMS['mutation']==str(bm)).to_numpy().argmax() ax[3].annotate(str(bm), (DMS['expr_avg'][_idx], DMS['bind_avg'][_idx] ), va='center', ha='center') for _,let in zip(ax, ["A","B","C"]): _.text(-0.2,1.05, let, fontsize=16, transform=_.transAxes) #plt.tight_layout() plt.savefig("nAb_RBD_ACE2_DMS_Bloom.pdf") RBD_WT_AA_Seq=''.join(DMS['wildtype'].to_numpy().reshape(-1,21)[:,0]) # algorithms and functions to generate csv of top-ranked variants dna2aa = { 'ATA':'I', 'ATC':'I', 'ATT':'I', 'ATG':'M', 'ACA':'T', 'ACC':'T', 'ACG':'T', 'ACT':'T', 'AAC':'N', 'AAT':'N', 'AAA':'K', 'AAG':'K', 'AGC':'S', 'AGT':'S', 'AGA':'R', 'AGG':'R', 'CTA':'L', 'CTC':'L', 'CTG':'L', 'CTT':'L', 'CCA':'P', 'CCC':'P', 'CCG':'P', 'CCT':'P', 'CAC':'H', 'CAT':'H', 'CAA':'Q', 'CAG':'Q', 'CGA':'R', 'CGC':'R', 'CGG':'R', 'CGT':'R', 'GTA':'V', 'GTC':'V', 'GTG':'V', 'GTT':'V', 'GCA':'A', 'GCC':'A', 'GCG':'A', 'GCT':'A', 'GAC':'D', 'GAT':'D', 'GAA':'E', 'GAG':'E', 'GGA':'G', 'GGC':'G', 'GGG':'G', 'GGT':'G', 'TCA':'S', 'TCC':'S', 'TCG':'S', 'TCT':'S', 'TTC':'F', 'TTT':'F', 'TTA':'L', 'TTG':'L', 'TAC':'Y', 'TAT':'Y', 'TAA':'_', 'TAG':'_', 'TGC':'C', 'TGT':'C', 'TGA':'_', 'TGG':'W', } aa2dna = { #la dna2aa[key]:key for key in dna2aa.keys() } def translate(seq): prot="" for i in range(0,len(seq),3): prot=prot+dna2aa[seq[i:i+3]] return prot # reference sites RBD_start = 954 RBD_start_Bloom = 954+36 RBD_end = 1623 RBD_end_Bloom = 1623-30 # `wildtype` nucleotide sequence # `2-P variant` nucleotide sequence # Default output: # 1. Nucleotide sequence RBD # 2. Nucleotide sequence full spike (with 2-P mutation) # 3. Aminoacid sequence RBD # 4. Aminoacid sequence full spike (with 2-P mutation) # # return 2 dna seqs, 2 prot seqs # def build_sequences(wt_dna_full_spike, wt_dna_full_spike_2p, rbd_variant): #parse mutation origin_aa, site, target_aa = rbd_variant[0],int(rbd_variant[1:-1]), rbd_variant[-1] site_to_dna = (site-1)*3 target_codon = aa2dna[target_aa] #apply mutation mutated_seq_ = wt_dna_full_spike[:site_to_dna]+target_codon+wt_dna_full_spike[site_to_dna+3:] mutated_seq_2p = wt_dna_full_spike_2p[:site_to_dna]+target_codon+wt_dna_full_spike_2p[site_to_dna+3:] #translate sequences translated_sequence = translate(mutated_seq_[RBD_start:RBD_end]) translated_sequence_2p = translate(mutated_seq_2p) return {"Variant_Nucleic_Acid_sequence_RBD": mutated_seq_[RBD_start:RBD_end], "Variant_Nucleic_Acid_sequence_2-P":mutated_seq_2p, "Variant_Amino_Acid_sequence_RBD":translated_sequence, "Variant_Amino_Acid_sequence_2-P":translated_sequence_2p[:-1]} def aggregate(mut, scor, spike_reference=None, spike_reference_2p=None, thres=0): out = {} sequences = {} for m, s in zip(mut, scor): if s>thres: if out.get(m): out[m].append(s) else: out[m]=[s] _seqs = build_sequences(spike_reference,spike_reference_2p, m) for key in _seqs.keys(): if sequences.get(key): sequences[key].append(_seqs[key]) else: sequences[key]=[_seqs[key]] for key in out.keys(): out[key] = np.round(np.mean(out[key]),3) return {**{"mutant":list(out.keys()), "scores":[out[s] for s in out.keys()]}, **sequences} descending_sort_tot_score = np.argsort(tot_score)[::-1] mutants = all_mutations[constraints_fulfilled][decending_sort_tot_score] full_ranking=pd.DataFrame(aggregate(mutants, tot_score[decending_sort_tot_score], spike_reference=spike_reference_sequence, spike_reference_2p=spike_reference_sequence_2p, thres=score_thres)) full_ranking.to_csv("top-variant-ranking_2.csv")

[0397] Example Data Input Mutation scanning (MS) data (https: / / www.sciencedirect.com / science / article / pii / S0092867420310035 https: / / science.sciencemag.org / content / sci / early / 2021 / 01 / 22 / science.abf9302.full.pdf) was used to correlate escape mutation loci to structural epitopes. nAbs: Identification of primary and secondary interacting epitopes by RBD-SARS-CoV-2 structures of the following neutralizing antibodies: REGN10933, REGN10987(6XDG), C144 7K90, P2B-2F6&P2B-2F11(https: / / www.nature.com / articles / s41467-020-20501-9), CC12.1 6XC2, S2H13 7JV6, Full Spike;7JV2, CC12.3 6XC4, C105 6XCN, CV30 6XE1, S2A4 7JVA, S304 7JW0, CV07-250 6XKQ, COVA1-16Fab7JMW, COVA2-39 7JMP, COVA2-04 7JMOWNb2 and WNb10 7LX5.

[0398] Example Results The following results are example outcomes of the analysis outlined above for the receptor binding domain of the spike protein of the SARS-CoV-2 virus: Thresholds: alpha=0.5, beta=1.0, gamma=0.40.

[0399] [Table 6]

[0400] [Table 7]

[0401] Figure 2 is a visual analysis of the results for SARS-CoV-2 RBD. It shows that the published SARS-CoV-2 RBM mutant screen and bioinformatic analysis using published neutralizing antibody structures identify RBM mutants with desirable properties. Some amino acid positions can be mutated to distinct residues to minimize ACE2 receptor binding, maximize protein expression, and minimally disrupt binding of published neutralizing antibodies.

[0402] FIG. 2A) Graphical representation of neutralizing antibody (nAb) maximal escape for distinct RBD positions (locuses, dots) compared to primary and secondary interfaces (black and grey lines) identified by the crystal structure. Missing data are indicated by faint vertical lines.

[0403] FIG. 2B) Scatter plot of expression (x-axis) versus ACE2 binding (y-axis). All data are represented by black dots. The variants with the highest scores are represented by grey dots for each top locus.

[0404] Figure 2C) is an expanded view of the grey box in the lower right of Figure 2B), showing the candidate amino acid exchanges identified by screening. [Example 3]

[0405] In vitro determination of the binding avidity of ACE2, various monoclonal nABs, and various SARS-mRBDs with single, double, and triple amino acid substitutions by use of ELISA - Figures 4A-C, 5A-D Single, double, and triple distinct point mutations were introduced into the SARS-CoV-2 RBD. Experiments were performed as described in Example 4.

[0406] FIG. 3A) All mutations preserved binding to the neutralizing antibody S309 as determined by ELISA.

[0407] FIG. 3B) The majority of the mutants largely or completely abrogated binding to ACE2 in ELISA.

[0408] FIG. 3C) The specificity irrelevant negative control antibody 9B9 is not bound by the RBD mutant, except for non-specific binding.

[0409] In Figure 3, the abbreviations stand for the following: RBD-L: RBD-L455R (SEQ ID NO: 55) RBD-G4: RBD-G496R (SEQ ID NO: 95) RBD-G5: RBD-G502R (SEQ ID NO: 15) RBD-AG5: RBD-A475R-G502R (SEQ ID NO: 96) RBD-G4G5: RBD-G496R-G502R (SEQ ID NO: 97) RBD-AG4G5: RBD-A475R-G496R-G502R (SEQ ID NO: 98) RBD-LAG4: RBD-L455R-A475R-G496R (SEQ ID NO: 99)

[0410] This example was carried out as described in Example 4, except that 9B9 was used as a control antibody. [Example 4]

[0411] In vitro determination of the binding affinity of ACE2 and various monoclonal nABs to SARS-wtRBD and various SARS-mRBD by using ELISA - Figures 4A-F and 6; Determination of EC50 values ​​for different monoclonal nABs and ACE2 considering SARS-wtRBD and different SARS-mRBD - Figure 5 In Figure 4, we experimentally confirm that bioinformatically screened RBD mutants L455R (SEQ ID NO: 55) and G502R (SEQ ID NO: 15) reduce ACE2 binding, while preserving the binding of RBD-specific neutralizing antibodies. Similar to Figure 6, here additional G502 mutants G502D (SEQ ID NO: 5), G502Y (SEQ ID NO: 20), G502K (SEQ ID NO: 9), G502S (SEQ ID NO: 16), G502P (SEQ ID NO: 13), G502E (SEQ ID NO: 2), G502A (SEQ ID NO: 3), G502V (SEQ ID NO: 18), G502N (SEQ ID NO: 12), G502Q (SEQ ID NO: 14), G502T (SEQ ID NO: 17), G502M (SEQ ID NO: 11), G502H (SEQ ID NO: 7), G502L (SEQ ID NO: 10) and G502F (SEQ ID NO: 6) were experimentally screened. While G502R completely abrogated ACE2 binding in this ELISA-based assay setup, L455R did not reduce ACE2 binding as well. In addition, the G502R mutation preserved the binding of all tested neutralizing antibodies of four distinct classes, namely class I (C12.1, P2C-1F11, REGN10933, C144), class II (C144, S2H13, P2B-2F6, REGN10987), class III (S309), and class IV (EY6A). No reduction in binding of neutralizing antibodies was observed with the G502R mutant. Moreover, the RBD mutant does not nonspecifically recognize the control antibodies VRC01 (anti-HIV GP140) or 4A8 (anti-SARS-CoV spike N-terminal domain). The RBD mutants A475R+G496R, A475R, and F456A reduce ACE2 binding, but neutralizing antibody binding is not preserved as well as L455R and G502R.

[0412] Figure 5 shows the effective concentration in μg / ml at which 50% of binding is achieved (EC50) for the indicated neutralizing antibodies or ACE2-hFcg1. As shown in Figure 5, lower EC50 values ​​indicate higher binding affinity. ~ indicates no binding was detected.

[0413] ACE2-hFcg1 was cloned by fusing recombinant human ACE2 Q18-V739 fragment to human IgG1-Fc portion (E99-K330 portion, where the first amino acid is G encoded by J-CH1 fusion). The RBD containing the signal peptide spanning amino acids M1-Q14 and R319-F541 of the Wuhan SARS-CoV-2 mutant (GenBank, MN908947.3) was complemented upstream of the C-terminal hexahistidine tag with the Twin-Strep tag sequence (WSHPQFEKGGGSGGGSGGSAWSHPQFEK) and cloned into the pcCDNA3.1 vector. Amino acid mRBD modifications G502R, L455R, A475R, G496R, G502D, G502Y, G502K, G502S, G502P, G502E, G502V, G502N, G502Q, G502T, G502M, G502H, G502L, and G502F were introduced by PCR mutagenesis to generate RBD mutants with abrogated ACE2 binding (T. Zhou et al., 2020b). Similarly, RBD mutations N501Y, K417N, and E484K were generated in the novel coronavirus variant. The heavy and light chain sequences of the following monoclonal antibodies were cloned into Oxford Genetics' IgG1 heavy chain and kappa or lambda light chain expression vectors: EY6A (D. Zhou et al., 2020a), P2B-2F6 and P2C-1F11 (Ge et al., 2021), REGN10933 and REGN10987 (Hansen et al., 2020), CC12.1 (Rogers et al., 2020), C144 (Robbiani et al., 2020), VRC01 (Wu et al., 2010), S2H13 (Piccoli et al., 2020), S309 (Pinto et al., 2020), and 4A8 (Chi et al., 2020).

[0414] For recombinant protein production, the plasmids were used to transfect HEK293 cells, which were grown in culture medium at 37°C in a humidified 8% CO2 incubator. Cells were grown to a density of 2.5 million cells per mL, transfected with PEI (4 μg / mL in cell suspension) and DNA (1200 ng / ml in cell suspension), and cultured for 3 days. Supernatants were collected and proteins were purified by His SpinTrap columns (Cytiva, 95056-290) or protein G columns according to the manufacturer's instructions. Eluted proteins were transferred by buffer exchange into phosphate-buffered saline (PBS) using Amicon Ultra-4 centrifugal filter units (Millipore, UFC805008) with a 50 kDa cutoff. Protein concentration was determined by His-tag specific ELISA using mouse anti-His tag antibody (Abcam, #ab18184) and alkaline phosphatase conjugated goat anti-mouse IgG Fc antibody (Southern Biotech, cat#SBA-1033-04) as detection reagents. Protein production was confirmed by SDS-PAGE and Western blot using mouse anti-His antibody (Abcam, #ab18184) and IRDye800CW donkey anti-mouse antibody (Li-Cor Biosciences, #925-32212).

[0415] Recombinant RBD was immobilized on high-binding 96-well ELISA plates (Corning, CLS3690) at 4 μg / ml in PBS (Sigma Aldrich, MO, USA) overnight at +4°C. Plates were blocked with 1% BSA in PBS (Thermo Fisher, Gibco, MA, USA) for 1 h at room temperature. Recombinant antibodies and ACE2-hFcg1 were diluted to the indicated serial dilutions in PBS with 1% BSA, added to the coated plates and incubated for 1 h at room temperature. Plates were developed with anti-human IgG alkaline phosphatase (AP) conjugated antibody (Southern Biotech, cat#2040-04) diluted 1:500 in PBS with 1% BSA. Bicarbonate buffer containing 4-nitrophenyl phosphate disodium salt hexahydrate substrate (SIGMA, Cat No. S0942-50TAB) was added and the absorbance at 405 nm was measured using a Cytation5 device (BioTek). Between all indicated incubation steps, plates were washed three times with PBS containing 0.1% Tween-20. [Example 5]

[0416] In vivo determination of B cell frequency following immunization with either SARS-wtRBD or various SARS-mRBD using a mouse model with controlled sACE2 levels - Figure 7 FIG. 7 shows that in an in vivo control mouse model, masking of the RBD by soluble ACE2 reduced the amount of B cells producing antibodies (competitive antibodies, cAbs) that interfere with the RBD-ACE2 interaction during immunization. Moreover, FIG. 7 shows that the RBD mutants G502R (SEQ ID NO: 15) (abbreviated as RBD-G5 in FIG. 7) and L455R (SEQ ID NO: 55) (abbreviated as RBD-L in FIG. 7) can abrogate the masking of the RBD by soluble ACE2 during in vivo immunization. Thus, the introduced mutations improved the generation of B cells producing antibodies that interfere with the RBD-ACE2 interaction. Moreover, FIG. 7 shows that the two RBD mutants G502R and L455R can be successfully combined in one immunization to block the masking of soluble ACE2. Moreover, Figure 7 shows that masking of the RBD also occurs during immunization with the full spike, which is a high-frequency SARS-CoV-2 vaccine antigen and is licensed for therapeutic use.

[0417] Immunization scheme: Mice were immunized with RBD (wild type RBD [SEQ ID NO: 1], RBD mutant G502R [SEQ ID NO: 15], and RBD mutant L455R [SEQ ID NO: 55]) alone or with RBD and sACE2. Spleens were analyzed 4-21 days after booster immunization. Single cell technology (DropMap) was used to analyze antibodies secreted into droplets by single B cells. The percentage of B cells producing antibodies capable of competing with ACE2 binding to the RBD is shown. For each condition, N=3 mice were immunized.

[0418] Recombinant RBD proteins were produced as described in Figures 4 and 5 (see Example 4).

[0419] Recombinant mhACE2 was constructed by introducing the following mutations into the wild-type S19-V739 murine ACE2 fragment: L20T, N24Q, N30D, N31K, Q34H, K60Q, S63N, E64N, Y73L, E74K, K78T, T79L, S82M, F83Y, T90N, P91L, I92T, I93V, S103N, H353K, N368D, and R387A. MuranACE2 containing an N-terminal His tag was cloned into a mammalian expression pcDNA3.1 vector containing a signal peptide (SP) (MGWSCIILFLVATATGVHS) and a molecular tag (His tag) consisting of eight histidine moieties attached to the N-terminus of ACE2 via a GSSGSSGSSGSS linker. It was experimentally determined that a C-terminal His tag was not suitable for efficient protein purification because it was likely to be cleaved by proteases such as ADAM17 expressed by HEK293 cells, therefore, a His tag was introduced between the SP and the N-terminus of the ACE2 fragment.

[0420] Despite the RBD, the wild-type full spike (SEQ ID NO: 259) containing the 2-P mutation (K986P and V987P substitutions) was used as the immunogen. After cloning the full-length spike M1-Q1208 of the Wuhan SARS-CoV-2 mutant (GenBank, MN908947.3) into the pcCDNA3.1 vector, the amino acid sequence SGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH* containing the protease recognition site, GGGS linker, trimerization domain, and hexahistidine tag was cloned.

[0421] In Figure 7, the right three bars showing full spike, low ACE2 spike, and high ACE2 spike correspond to Figure 1G, where low ACE2 has 18% RBD binding and high ACE2 has 100% RBD binding.

[0422] BALB / cJRj mice (6-8 weeks old at initiation, all female) were purchased from Janvier Labs and housed in the animal facility of ETH Zurich throughout the experiment. After 2 weeks of acclimation, mice were immunized on the indicated days (primary immunization day 0, booster immunization day 28) with RBD-His6 (final amount 3 μg / immunization, diluted in sterile PBS) adsorbed 1:1 to Alhydrogel® adjuvant 2% (vac-alu-250, InvivoGen) for at least 2 hours. Where indicated, muranACE2 (final amount 9.2, 1.8 or 0.37 μg / immunization, diluted in sterile PBS), neutralizing mouse IgG2b (final amount 13.5, 2.7 or 0.54 μg / immunization, 40592-MM57, Sino Biological Co., Ltd., diluted in sterile PBS), SuperMuran ACE (final amount 0.37 μg / immunization, diluted in sterile PBS), NoMuran ACE2 (final amount 9.2 μg / immunization, diluted in sterile PBS), or full-length spike protein (final amount 3 μg / immunization, diluted in sterile PBS) were added to the antigen and adjuvant mixture immediately prior to injection (<10 min). Mice were immunized intraperitoneally with a total volume of 100 μl of each antigen / adjuvant mixture. All immunizations were performed in triplicate. At the indicated time points after the boost, spleens were harvested and IgG-SC were prepared and mounted as described elsewhere (Bounab et al., 2020). Briefly, unprocessed cells from B cell lineages were extracted using Miltenyi's Pan B cell purification kit II. Cells were kept on ice or at 4°C throughout the experiment. In parallel, whole blood was drawn from mice, allowed to clot, and serum was later purified and stored at 4°C before titration. All experiments described in this study were verified and approved by the Cantonal Ethics Committee of Zurich under license number ZH215 / 19.

[0423] A droplet-generating polydimethylsiloxane microfluidic chip and a 2D observation chamber were fabricated as described elsewhere (Bounab et al., 2020), and this assembly was used to generate droplets of approximately 50 pL in volume. The emulsion was directly introduced into the 2D observation chamber, which was then loaded onto an inverted fluorescence microscope (Ti2 Eclipse, Nikon) to complete the filling of the chamber, and data were generated as described below.

[0424] Purified cells from the B cell lineage were harvested by centrifugation (400g, 5 min, 4°C) and resuspended to a final concentration of 2x106 cells / ml in staining solution (Hanks' Balanced Salt Solution plus 5μM Cell Trace Violet, both from Thermo Fisher) to allow selection of droplets containing B cells during analysis. Immediately before mounting, cells were harvested (400g, 5 min, 4°C) and washed once with droplet medium consisting of phenol red-free RPMI1640 (cat.no.11835030, Thermo Fisher). Droplet medium further contained 5% Knockout Serum Replacement (Thermo Fisher), 0.5% recombinant human serum albumin (cat.no. A9986, Sigma Aldrich), 25 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) pH 7.4, 1× penicillin-streptomycin, and 0.1% Pluronic F-127 (all supplied by Thermo Fisher). Cells were resuspended in droplet medium to obtain a final inclusion mass λ (average number of cells per droplet) of 0.2–0.4; i.e., approximately 20–30% of the droplets contained one cell.

[0425] Paramagnetic nanoparticles were prepared as previously described (Bounab et al., 2020). Beads were prepared several hours in advance and nanoparticles were thoroughly resuspended by pipetting dropwise before each measurement. The final concentrations of reagents in the droplet were: wtRBD-His6 25 nM, AlexaFluor750 human ACE2-hFc (10108-H02H, Sino Biological, labeled in-house) 25 nM, AlexaFluor488-labeled anti-His6 (ab237336, Abcam) 30 nM, and AlexaFluor647-labeled anti-IgG Fc (315-606-046, Jackson Immuno Research) 75 nM.

[0426] Images of droplets and encapsulated cells were acquired using a 10x objective (NA 0.45, Nikon) to obtain an array of 10 × 10 images after 1 h of incubation at room temperature. This allowed imaging of approximately 4–50 000 droplets per experiment, resulting in 10–20 000 cells analyzed. Excitation light was provided by an LED source (SOLA light engine, Lumencor Inc.) and emitted fluorescence was recorded using appropriate bandpass filters (DAPI, FITC, Cy5, and Cy7 filter sets, all Semrock), camera settings (Orca Flash4, Hamamatsu) at room temperature (25 °C), and ambient oxygen concentration. To align the line of beads vertically during measurements (for data analysis), two strong neodymium magnets (BZX082, K&J Magnetics) were placed on either side of the 2D observation chamber.

[0427] The acquired data were analyzed using a custom-made Matlab script (Mathworks, a version can be found on GitHub at https: / / github.com / LCMD-ESPCI / dropmap-analyzer). In detail, brightfield images were used to detect and isolate droplets. DAPI images were used to confirm the presence of live cells in each droplet, and all other fluorescent channels confirmed the relocation of fluorescence to the bead line (Bounab et al., 2020). The resulting raw data was exported to Excel (Microsoft) and droplets containing live cells were sorted. Droplets containing cells were further sorted for increased relocation of anti-IgG (threshold 1.3, corresponding to 1.1 nM IgG1 or 2.6 nM IgG2a / b). After the presence of IgG-secreting cells was ensured, the rearrangements of Alexa488 (RBD, antigen) and Alexa750 (ACE2, competitor) in the same droplets were analyzed, and the ratio of Alexa488 / 750 (RBD / ACE2 rearrangements) was determined and plotted against the rearrangement of Alexa488. At this stage, 50–600 IgG-secreting cells were selected in each sample. Competing antibodies were identified as those with a ratio of RBD / ACE2 rearrangements higher than x?(Alexa488-rearranged blank droplets / Alexa750-rearranged blank droplets) + 0.02. Using this threshold, calibration samples with cAbs were reliably identified as cAbs (93 ± 8%, N = 6), while the number of misidentified cAbs remained low (1.4 ± 0.5%, N = 6). Unless otherwise stated, the average frequency of identified cAbs in measurements of individual mice is presented (N = 3), with standard deviations shown in Figure 7. At low frequencies there were no significant differences, but all frequency measurements, especially high nAb and high muranACE2, exceeded the levels of the negative control experiment (1.1±1.1% without added RBD). [Example 6]

[0428] In silico modeling of epitope masking by soluble ACE2 for the newly emerged SARS-CoV-2 variant containing the N501Y substitution - Figure 8A and B Figures 8A and B show that ACE2 masking of the RBD is increased for newly emerged viral variants.

[0429] In silico modeling of epitope masking was expressed as %RBD binding under distinct ACE2 serum concentrations. The N501Y mutation that arose in the newly emerged SARS-CoV-2 viruses from the UK and South Africa increased affinity and therefore masking by ACE2. Masking effect, here represented by 36% RBD binding, increases with decreasing affinity (Kd) as shown in the correlation.

[0430] Thus, masking capacity correlates with the affinity of the RBD for the receptor, with novel variants with increased affinity causing increased masking at low sACE2 serum concentrations. [Example 7]

[0431] In vitro determination of the binding affinity of DPP4 and various monoclonal nABs to either MERS-wtRBD or various MERS-mRBD by use of ELISA - Figure 9 Figure 9 shows that MERS-CoV RBD mutations D510A (SEQ ID NO: 195), E536R (SEQ ID NO: 198), D537K (SEQ ID NO: 196), and D539K (SEQ ID NO: 197) abrogate binding to the DPP4 receptor to different degrees, with D510 and D539 being the most effective receptor binding abolished mutations. Moreover, Figure 9 shows that the binding of MERS-CoV neutralizing antibodies 4C2h, D12, LCA60, and MERS4V2 is maintained by D510A, which binds equally well to RBD-D510A compared to RBD-WT. The D171K mutation preserved three of the four neutralizing antibodies, albeit with lower binding affinity. Thus, the D510A and D171K RBD mutants were selected for animal immunization.

[0432] For cloning of human DPP4 and monoclonal antibodies, an IgH signal peptide (SP) sequence with the sequence MGWSCIILFLVATATGVHS was placed in front of the protein N-terminus to facilitate protein secretion. Non-antibody proteins contained either an 8xHis tag or a 6xHis tag on the N- or C-terminus for purification purposes. Gibson Assembly cloning was performed using Gibson Assembly Master Mix (New England Biolabs, #E2611) according to the manufacturer's instructions. E. coli chemically competent for plasmid transformation was generated in-house and transformed according to the NEB transformation protocol (New England Biolabs, #E1601). Isolation of plasmids from bacterial cultures was performed using the PureYield™ Plasmid Miniprep System (Promega, #A1222) and PureYield™ Plasmid Midiprep System (Promega, #A2496) according to the manufacturer's instructions. The sequence of the expression cassette was verified by DNA sequencing (LGC Genomics GmbH).

[0433] The gene for recombinant DPP4 with an N-terminal 8×His tag (hDPP4-N′His) containing the S38-P766 fragment of human DPP4 (Uniprot, P27487) was synthesized by GenScript and cloned into the SP-containing mammalian expression pSF vector (Oxford Genetics) using Gibson Assembly.

[0434] MERS-CoV RBD amino acids G372-L588 were cloned upstream of the Twin Strep tag sequence (WSHPQFEKGGGSGGGSGGSAWSHPQFEK) before a GGGS linker followed by a C-terminal 8xHis tag. This was introduced into the mammalian expression vector pcDNA3.1+ containing the signal peptide spanning amino acids M1-Q14 of the Wuhan SARS-CoV-2 mutant (GenBank, MN908947.3), AviTag™, and a GG linker. Four amino acid modifications, D510A, E536R, D171K, and D539K, were introduced by PCR mutagenesis (see Table 4) to generate six RBD mutants with abrogated DPP4 binding using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, #E0554) according to the manufacturer's instructions.

[0435] [Table 8]

[0436] The synthetic V(D)J inserts of monoclonal antibodies LCA60, 4C2h, D12, and MERS-4V2 were generated individually by overlap PCR (see Tables 4-5). PCR products were recovered using ProNex beads (Promega, #NG2002) according to the manufacturer's instructions. The PCR products were then cloned into pSF expression vectors containing SP and Ig kappa constant region R1-C107 (light chain) or IgG1 H constant region A1-K330 (heavy chain) using Gibson Assembly.

[0437] [Table 9-1] [Table 9-2] [Table 9-3]

[0438] [Table 10]

[0439] The concentration ELISA aims to calculate the amount of protein produced using a purchased recombinant protein of similar size as a positive control. Two of the three concentration ELISAs used in this project are DPP4-ELISA (measures the concentration of all human DPP4 proteins) and His-ELISA (measures the concentration of all His-containing proteins) (see Table 6).

[0440] Finally, the protein concentration of the self-produced proteins was estimated by fitting the absorbance at 280 nm measured on a spectrophotometer (Biozym, #31DS-11FXPLUS) between the theoretical extinction coefficients (ProtParam tool in Expasy, Swiss Institute of Bioinformatics, see Table 7). With this in mind, 96-well half-area plates (Greiner Bio-One GmbH, #675061) were coated with serial dilutions of the self-produced proteins with a starting concentration of 1000 ng / ml for the DPP4 concentration ELISA or 200-fold diluted samples for the His concentration ELISA. Standard proteins for ELISA were coated on the same plates as shown in Table 7. The coated plates were incubated overnight at 4°C.

[0441] [Table 11]

[0442] [Table 12]

[0443] Plates were washed three times with PBS containing 1% Tween (PBS-T) in an automated washer (Biotek, EL406). Plates were blocked with 25 μl of PBS containing 1% BSA for 1 h at RT. Plates were washed three times with PBS-T and 25 μl of primary antibody (Table 7) was added to each well and incubated for 1 h at RT. Plates were washed three times with PBS-T and 25 μl of secondary antibody (Table 7) was added to each well and incubated for 1 h at RT. Plates were washed three times with PBS-T and 50 μl of 4-nitrophenyl phosphate disodium salt hexahydrate substrate (pNPP) (Sigma, #S0492-50TAB) was added. Exactly 30 min after addition of pNPP, the absorbance of the wells was measured at 405 nm and 620 nm (BioTek, Cytation5).

[0444] Correlation between the values ​​obtained in the positive controls and these actual concentrations was used to calculate the concentrations of the samples.

[0445] The concentration of the self-made antibody is determined using ELISA. Goat anti-IgG human antibody (Southern Biotech, #2040-04) is used to coat the plates using 25 μl of 2 μg / ml. The plates are incubated overnight at 4° C.

[0446] Plates were washed 3 times with PBS-T and blocked with 25 μl of PBS with 1% BSA for 1 h at RT. Plates were washed 3 times with PBS-T and 25 μl of serial dilutions of samples and positive control (Southern Biotech, #0150-01) were added at starting dilutions of 1:200 and 2 μg / ml, respectively, and incubated for 1 h at RT. Plates were then washed 3 times with PBS-T and 25 μl of goat anti-human IgG (Southern Biotech, #2040-04) was added and incubated for 1 h at RT. Plates were washed 3 times with PBS-T and 50 μl of pNPP (Sigma, #S0492-50TAB) was added. Exactly 30 min after addition of pNPP, the absorbance of the wells was measured at 405 nm and 620 nm (BioTek, Cytation5).

[0447] Binding of Twin Strep-tagged MERS-CoV-RBD and MERS-CoV spike to DPP4 was tested by ELISA. 96-well half-area plates (Greiner Bio-One GmbH, #675061) were coated with 25 μl of 4 μg / ml homemade DPP4 diluted in PBS and incubated overnight at 4°C.

[0448] Plates were then washed 3 times with 100 μl / well PBS-T (Biotek, EL406). Plates were blocked with 25 μl PBS with 1% BSA for 1 h at RT. Plates were washed 3 times with 100 μl / well PBS-T and 25 μl of serial dilutions of MERS-CoV-RBD or MERS-CoV spike protein were added to each well and incubated for 1 h at RT. Serial dilutions were generated by 3-fold dilutions with a starting concentration of 50 μg / ml. Plates were washed 3 times with PBS-T and 25 μl of AP-conjugated streptavidin (Southern Biotech, #0150-01) at 2 μg / ml was added to each well and incubated for 1 h at RT. Plates were washed 3 times with PBS-T and 50 μl of pNPP (Sigma, #S0492-50TAB) was added. Exactly 30 minutes after addition of pNPP, the absorbance of the wells was measured at 405 nm and 620 nm (BioTek, Cytation 5).

[0449] An increase in absorbance at 405 nm indicated DPP4 binding to the Twin Strep-tagged proteins of the sample.

[0450] HEK293 were cultured in suspension at 37°C in a humidified 8% CO2 incubator. For protein production, cells were grown to a density of 2x106 cells per ml, transfected with 1mg / ml PEI (Polysciences Inc., #23966-1) and 38μg of plasmid DNA, and cultured for 3 days. After this, cells were centrifuged at 4000g for 10 minutes at RT and the supernatant was collected. The supernatant was filtered using a 0.45μm polyethersulfone membrane. Proteins were purified from the resulting supernatant using a His Spin Trap™ column according to the manufacturer's instructions (Cytiva, #95056-290). Eluted proteins were transferred to phosphate-buffered saline (PBS) by buffer exchange. Buffer exchange included six successive concentration and dilution steps by centrifugation at 4000g for 4-14 min at 4°C using Amicon Ultra-4 centrifugal filter units with 10 kDa or 50 kDa cut-off for RBD protein or hDPP4-N'His and monoclonal antibodies, respectively (Millipore, #UFC801008, #UFC805008). Protein concentrations were determined by ELISA for all proteins. Protein production was confirmed by SDS-PAGE and Western blot. [Example 10]

[0451] Pull-down assay of human DPP4 and rabbit DPP4 from serum using either MERS-wtRBD or various MERS-mRBDs - Figure 10 Figure 10 shows that MERS-CoV RBD mutations D510A (SEQ ID NO: 195), D537K (SEQ ID NO: 196), and D539K (SEQ ID NO: 197) abrogate pull-down of human DPP4 and rabbit DPP4 from serum. Thus, the D510A, D537, and D539 mutations are suitable for undermasking of MERS-CoV RBD by serum DPP4.

[0452] Soluble DPP4 was pulled down from 200 μl of serum using MERS-RBD-TwinStrep conjugated with streptavidin magnetic beads (IBA Life Science, #2-4090-010). First, 120 μl of streptavidin magnetic beads were placed in a 1.5 ml tube. The streptavidin magnetic beads were separated from the liquid using a magnetic rack. The liquid was removed and the precipitated beads were washed twice with 1 ml of 1× wash buffer (IBA Life Science, #2-1003-100) and twice with 1 ml of PBS. After washing, the beads were resuspended in 50 μl of PBS.

[0453] TwinStrep containing proteins were conjugated with streptavidin magnetic beads (Starlab, #S8012-0000) by adding 24 μg of the desired protein and incubating the solution at 37°C for 1 hour with shaking at 1400 rpm. After 1 hour, the beads were washed 3 times with 1 ml PBS, pelleted using a magnetic rack, and the supernatant exchanged. Blocking of the beads was necessary to occupy potential space of the streptavidin magnetic beads. Blocking of the beads was performed by incubating the beads in 1 ml 1% BSA-PBS for 1 hour at RT with shaking at 1400 rpm.

[0454] After the blocking step, the beads were washed 3 times with 1 ml of PBS and finally resuspended in 120 μl of PBS. 120 μl of beads were transferred to a 2 ml tube. 200 μl of serum was added to the 2 ml tube. The 2 ml tube was placed in a 50 ml tube with paper towels to ensure stability inside. The 50 ml tube was incubated at 4° C. overnight with rotation.

[0455] After overnight incubation, the 2 ml tubes are placed on a magnetic rack and the serum is collected for further analysis. The precipitated beads are washed 3 times with 1 ml PBS and stored for further analysis.

[0456] The enzyme activity of soluble DPP4 (sDPP4) in serum was measured as follows.

[0457] First, 10 μL of serum sample was added to 80 μL of assay buffer (50 mM TrisHCl, pH 9.0 at 37°C) preheated to 37°C in a 96-well plate (Sigma, #M0812-100EA). Second, the plate was incubated at 37°C for 15 minutes. Third, the substrate Gly-Pro p-nitroanilide (Biozol, #BAC-4025614.0250) was freshly prepared to a concentration of 5 mM in assay buffer. Then, 10 μL of substrate was added to each sample, and the samples were measured by absorbance at 405 nm (BioTek, Cytation5) in a kinetic measurement at 37°C for 2 hours with measurements every 15 minutes. To relate absorbance to the production of p-nitroaniline (pNA) and subsequent concentration of DPP4, standards of pNA (Santa Cruz Biotechnology Inc, #sc-272000A) and recombinant DPP4 (Sigma-Aldrich Chemie, #D3446) were included on the plate and prepared as shown in Tables 9 and 10.

[0458] [Table 13]

[0459] [Table 14]

[0460] Analysis of the DPP4 activity assay is performed entirely using RStudio.

[0461] First, the average value of the two blank values ​​is subtracted from all values ​​in a time-dependent manner.Then, the measurement value at time zero is subtracted from all samples to avoid sample fluorescence interfering with further calculations.

[0462] An interceptless linear regression of known concentrations of p-nitroaniline (pNA) versus absorbance at 405 nm is performed. The data selected for this analysis are those belonging to the 45 minute time point. The resulting slope is then used to measure the amount of pNA released by all samples during the analysis.

[0463] The activity (nmol pNA / min) of the control DPP4 samples is then analyzed. The highest concentration used for DPP4 (0.3 μg / ml) shows a linear increase in absorbance from 15 to 60 min. Thus, optimal substrate cleavage occurs at high as well as low concentrations of DPP4 at 45 to 60 min. Later time points are not optimal for high concentrations of DPP4. Optimal substrate cleavage means that the r-squared of the linear regression of DPP4 concentration vs. activity is higher than 0.96. However, samples containing small amounts of DPP4 should be measured at later time points, in which case the high concentration control DPP4 sample must be filtered out until optimal substrate cleavage is achieved.

[0464] An intercept-free linear regression of known concentrations of DPP4 versus pNA released upon optimal substrate cleavage is performed and the slope is used to determine the concentration of active DPP4 (aDPP4) in the sample. [Example 11]

[0465] In vitro validation of in silico scoring – Figure 13 In Figure 13, the abrogation of ACE2 binding and preservation of nAb epitopes of selected RBD mutants is confirmed in vitro. wtRBD and three representative RBD mutants were tested by ELISA for binding to ACE2-hFcg1, class 1 and class 2 nAbs (Figure 13a) and class 3 and class 4 nAbs (Figure 13b). Anti-spike NTD4A8 antibody and anti-MERS-CoV antibody LCA60 acted as negative controls. Residual nAb binding and % ACE2 binding are presented relative to WT. Optical density OD at wavelength 405 nm.

[0466] To further validate the in silico scoring findings in vitro, high (G502E, total score 4.79) and moderate (L455R, total score 3.05) scoring RBD mutants were again evaluated, as in Examples 3 and 4, although there was also a mutant with a negligible score (E484K, total score -2.93). All three were recombinantly produced and tested by ELISA for binding to ACE2 and neutralizing antibodies (nAbs) of distinct binding classes.

[0467] Therefore, recombinantly expressed SARS-CoV-2 RBD, whole spike or spike NTD were coated on high-binding 96-well ELISA plates (Corning, #CLS3690) at 10 μg / ml in PBS (Sigma Aldrich, MO, USA) overnight at +4°C. Plates were blocked with PBS / 1% BSA (Thermo Fisher, Gibco, MA, USA) for 1 h at room temperature. Serum and proteins were diluted in successive dilutions in PBS / 1% BSA, added to the coated plates and incubated for 1 h at room temperature. Plates were developed with anti-human IgG alkaline phosphatase (AP) conjugated antibody (Southern Biotech, #2040-04) or anti-rabbit IgG-AP conjugated antibody (Jackson Immuno Research, #111-056-003), both diluted 1:500 in PBS / 1% BSA. Bicarbonate buffer containing 4-nitrophenyl phosphate disodium salt hexahydrate substrate (Sigma, #S0942-50TAB) was added and absorbance at 405 nm was measured with a Cytation5 device (Agilent BioTek). Plates were washed three times with PBS / 0.05% Tween-20 between all indicated incubation steps. IgG titers (ED50) were determined by nonlinear regression sigmoidal curve fitting performed with R (stats package). For curve fitting, the upper and lower plateaus of S309 (RBD) or 4A8 (NTD) reference antibodies were applied to all respective samples.

[0468] The high-scoring mutant G502E completely abrogated ACE2 binding, while maintaining the binding of all nAbs tested. The moderate-scoring mutant L455R partially abrogated ACE2 binding (71% abrogated compared to WT), but also attenuated the interaction with three of the six class 1 and class 2 nAbs, namely P2C-1F1125, REGN1093326, and C14427. Class 3 and class 4 nAbs were not affected by the L455R mutation. The low-scoring mutant E484K did not significantly affect receptor binding, while the binding of three of the six class 1 and class 2 nAbs was reduced or abolished (Figures 13A and 13B). Thus, the in vitro results confirm the in silico scoring.

[0469] To further test a broader set of RBD mutants in vitro, we focused on three selected nAbs from class 1 and class 2, namely CC12.128, P2C-1F1125, and REGN1093326, whose epitopes overlap with the ACE2 binding interface. The binding data well reflected the gradient scores obtained from the in silico ranking (Figure 13C).

[0470] In addition to Example 4, FIG. 6, since a significant number of G502 amino acid exchanges scored highly in silico, all possible amino acid exchanges at the G502 position were tested in vitro, and all mutants showed high potency, except for G502K, G502L, and G502F, which showed only slight reductions in nAb or ACE2 binding. [Example 12]

[0471] Loss of receptor binding reduces the membrane fusion ability of SARS-CoV-2 spike - Figure 14A, B Loss of receptor binding interferes with antigen internalization - Fig. 14C, D Figure 14 shows (A) the formation of VeroE6 syncytia from technical replicates in triplicate and (B) HEK293T cell-cell fusion events from technical replicates in duplicate, with the two locations determined by imaging at 10x magnification after 48 hours. The number of syncytia detected and the average number of nuclei per syncytium are represented by violin plots showing the median and the 75% and 25% percentiles.

[0472] Figure 14C and Figure 14D show representative images of maximum intensity Z-projections of live cell spinning disk confocal microscopy of SARS-CoV-2 spike internalization kinetics into VeroE6 cells pretreated with 1 μM heparin (D) or medium only (C) for the indicated times. In all images, the cytoplasm was stained with CellTracker™ CMFDA; cells were labeled in green and shown in grey in Figure 14C,D. Spike AF647 protein was labeled in magenta and shown in white in Figure 14C,D. HIV-1 glycoprotein gp140BG505 served as a control antigen.

[0473] Surface expression of fusion-competent antigens may induce side effects through the formation of syncytia, which contribute to COVID-19 tissue damage. To demonstrate that abrogation of receptor binding efficiently blocks fusion, transmembrane spike mutants were expressed either in HEK293T cells that were subsequently mixed with ACE2-eGFP-transfected HEK293T cells, or in VeroE6 cells that endogenously express ACE2. VeroE6 syncytium formation and HEK293T cell-cell fusion assays were performed.

[0474] In detail, to determine the membrane fusion ability of SARS-CoV-2 mutants determined in silico, various SARS-CoV-2 spike full-length proteins were transfected in triplicate with pMAX-GFP reporter plasmid (from the copepod P. plumata, kindly provided by A. Lanzavecchia) supplemented with PEI at a 1:1 ratio to transfect VeroE6 cells. SARS-CoV-1 full-length spike protein acted as a control. After 48 hours, cells were stained with NucBlue™ LIVE / READY™ Hoechst 33342 Reagent and imaged using phase contrast, DAPI, and GFP channel / filters on a Cytation5 device for syncytium formation observation. Pictures taken at 10x magnification were used for image presentation and for counting syncytia and nuclei with ImageJ. Each replicate was counted three times and averaged.

[0475] HEK293 cells were seeded in duplicate and divided into two groups. The donor group was transfected with various SARS-CoV-2 full-length spike proteins as well as SARS-CoV-1 full-length spike control proteins by PEI. The acceptor group was transfected with human ACE2-eGFP plasmid by PEI. The next day, both groups of cells were mixed together in a 1:1 ratio and seeded into 24-well plates. After 24 hours, the cells were treated and analyzed as described for VeroE6 cells.

[0476] Although comparable levels of spike protein were expressed on the cell surface, G502E and G502R completely abrogated membrane fusion activity, whereas mutants L455R and E484K reduced the ability to fuse cells (Fig. 14A,B). Similar to abrogation of receptor binding, substitutions that stabilize the spike with two prolines (2P) strongly blocked the membrane fusion activity of the spike. Thus, the data suggest that abrogation of receptor binding represents an alternative strategy to prefusion stabilization to avoid vaccine-induced syncytia formation.

[0477] Spike binding to the ACE2 receptor, which induces ACE2 internalization and clearance, promotes tissue damage. Therefore, we further addressed whether the G502E mutant could abrogate cellular uptake of the spike by live-cell imaging.

[0478] In detail, to visualize the internalization of SARS-CoV-2 complete spike and RBD proteins, 40,000 VeroE6 cells were seeded in wells of 8-well Ibidi glass-bottom slides (Ibidi, #80827). The next day, cells were washed once with PBS and nuclei were stained with NucBlue™ LIVE / READY™ Hoechst 33342 Reagent (Thermo Fisher, Life Technologies, #R37605) for 15 min at 37°C according to the manufacturer's instructions. After another PBS wash, cells were stained with pre-warmed CellTracker™ Green CMFDA solution (Invitrogen, Thermo Fisher, #C2925, working concentration 8 μM) and incubated for 30 min at 37°C. The CMFDA solution was then removed and the released dye was quenched by adding FCS-containing medium for 5 min. Upon another wash with PBS, cells were treated with 10 μg / ml RBD or 20 μg / ml spike solutions of SARS-CoV-2, as well as SARS-CoV-2 NTD or HIV-1 gp140 BG505 isolated complete spike control proteins, respectively. After 90 min of incubation at 37°C, cells were washed once with PBS and fixed with 4% PFA + 20% sucrose in PBS for 20 min at 4°C. Thereafter, cells were washed and kept in PBS. Images of fixed samples were acquired on a Zeiss Laser Scanning confocal microscope (LSM780). For detection, a photomultiplier tube was used. The system was controlled by Zeiss ZEN2010 software (Carl Zeiss Microscopy). Single- and multicolor confocal imaging of fixed samples was performed in sequential mode with the following fluorophore-specific excitation (Ex.) and emission filter (EmF.) settings: Hoechst (Ex.: 405 nm, EmF.: 415–480 nm), CMFDA (Ex.: 488 nm, EmF.: 490–578 nm), Alexa Fluor647 (Ex.: 633 nm, EmF.: 638–735 nm).Images were acquired with a PL APO DIC M27 63x / 1.40NA oil immersion objective (Carl Zeiss Microscopy). A z-stack from bottom to top of the cell was performed over 8 μm at 0.39 μm intervals. Contrast adjustment, z-projection, and orthogonal projections were performed using FIJI software (https: / / imagej.net / software / fiji / ). Each condition was performed in duplicate.

[0479] Live imaging of RBD and spike protein absorption was performed by incubating VeroE6 cells with pre-warmed CellTracker™ Green CMFDA solution (working concentration 8 μM) for 30 min at 37°C. Afterwards, the CMFDA solution was removed and the released dye was quenched by adding FCS-containing medium for 5 min. Upon another PBS wash, cells were pretreated with either 1 μM porcine heparin (Sigma-Aldrich, #H3393-50KU) or medium for only 1 h. Cells were imaged in live cell imaging buffer Fluorobrite DMEM medium (Gibco by Thermo Fisher, #A1896701) supplemented with 10% FCS and 1% penicillin-streptomycin. Solutions of SARS-CoV-2 spike WT and G502E were added to the respective wells to a final concentration of 20 μg / ml. SARS-CoV-2 RBD WT and G502E were added to a final concentration of 10 μg / ml. Dual-color luminescence live imaging was performed with a Nikon Spinning Disk Confocal Microscope equipped with a Perfect Focus System, Yokogawa CSU-X-1, an automatic stage, and a Nikon TiE with a heatable humidified chamber. The system was controlled by Nikon NIS Elements software (NIS5.02.01 (build 1270)). Cells were imaged with an OKOLAB system controlled at 37 °C and 5% CO2. A 40x NA0.95 air objective was used for image acquisition and an EMCCD camera (Andor AU-888) for detection. Images were acquired with 1024x1024 pixels and a total size of 163x163 μm in 16-bit format. The cell line CMFDA was excited with a 488 nm laser (150 mW) and AF647-labeled proteins with a 640 nm laser (100 mW). Laser power and exposure time were kept the same throughout one experiment. In each well of a μ-slide 8-well glass-bottom Ibidi chamber, five different positions were imaged. A z-stack (11 slices, 0.6 μm step size) was set for all positions using a Nikon TiZ-Drive.For time-lapse, the first image (0 min) was acquired before addition of labeled protein, the second immediately after this addition, and then continuously every 30 min for at least 12 h. Contrast adjustment, z-projection, and time point selection were performed using FIJI (https: / / imagej.net / software / fiji / ). Selected time points of separately treated wells are shown. Each condition was performed in triplicate.

[0480] Fluorescently labeled SARS-CoV-2 spike 2P protein was adsorbed and internalized by VeroE6 cells within 90 min (Figure 14C). The G502E mutant exhibited significantly reduced spike uptake within hours.

[0481] The monomeric RBD of SARS-CoV-2 has recently been reported to possess one binding site that recognizes cellular heparan sulfate (HS), and the trimeric complete spike protein possesses nine, which may contribute to viral uptake. Therefore, spike internalization in the presence of heparin was evaluated. Indeed, heparin treatment reduced spike uptake as well as the number of fluorescent cells (Figure 14D). Similar results were observed for the G502E spike. Thus, the G502E mutation abolishes ACE2-mediated uptake while maintaining partial spike internalization through other receptor interactions, e.g., HS. Taken together, this data suggests that antigen design according to the methods disclosed herein may have positive implications for vaccine safety and efficacy by preventing cell-cell fusion, removing functional receptors from the cell surface, and reducing antigen uptake.

Claims

1. 1. A method for designing and / or obtaining an active ingredient for a vaccine composition, comprising: (i) providing a mutated viral receptor binding domain (VIRUS-mRBD) or a fragment thereof, comprising one or more mutations in a wild-type viral receptor binding domain (VIRUS-wtRBD); (ii) VIRUS-mRBD or a fragment thereof a) Reduced binding of the viral receptor-binding domain to the receptor (RBD receptor) (VIRUS-RBD receptor) compared to VIRUS-wtRBD determining whether (iii) if a) is satisfied, selecting the VIRUS-mRBD or a fragment thereof as the active ingredient; A method comprising:

2. In step (ii), the VIRUS-mRBD or a fragment thereof is b) binding to anti-Virus-wtRBD neutralizing antibodies (Virus-wtRBD-nAB), and c) Where appropriate, protein and / or peptide stability determining whether to indicate In step (iii), when a) and b) are satisfied simultaneously, and optionally a), b), and c) are satisfied simultaneously, selecting the VIRUS-mRBD or a fragment thereof as the active ingredient; The method of claim 1 further comprising:

3. step (i) comprises providing a library of VIRUS-mRBDs or fragments thereof, each of which comprises one or more mutations in a VIRUS-wtRBD, wherein the mutations in the VIRUS-mRBDs or fragments thereof contained in the library are at least partially different; Step (ii) a) Reduced binding to the VIRUS-RBD receptor compared to VIRUS-wtRBD The method is carried out by screening a library for a VIRUS-mRBD or a fragment thereof that exhibits The method according to claim 1 or 2, wherein step (iii) comprises selecting, from the library, one or more VIRUS-mRBDs or fragments thereof that satisfy a) as active ingredients.

4. Step (ii) a) reduced binding to the VIRUS-RBD receptor compared to VIRUS-wtRBD; and b) binding to VIRUS-wtRBD-nAB, and c) Where appropriate, protein and / or peptide stability The method is carried out by screening a library for a VIRUS-mRBD or a fragment thereof that exhibits The method of claim 1, wherein step (iii) comprises selecting, from the library, one or more VIRUS-mRBDs or fragments thereof that simultaneously satisfy a) and b) and, optionally, c) as active ingredients.

5. Step (iii) is to identify one or more VIRUS-mRBDs or fragments thereof from the library. a) their ability to reduce binding to the VIRUS-RBD receptor compared to VIRUS-wtRBD; and b) Where appropriate, their ability to bind to VIRUS-wtRBD-nAB, and c) and, where appropriate, their ability to indicate the stability of proteins and / or peptides. and scoring the selecting, based on the scoring, from the library one or more VIRUS-mRBDs or fragments thereof having the highest score for a) or the highest score for a combination of a) and b) and, if appropriate, c) as active ingredients; The method of claim 1 further comprising:

6. The method of claim 1, wherein steps (i) and (ii) are performed in vitro and / or the scoring in step (iii) is performed in silico.

7. - the VIRUS-mRBD is a CORONA-mRBD according to the invention, more preferably a SARS-mRBD and / or a MERS-mRBD according to the invention, - the VIRUS-wtRBD is a CORONA-wtRBD according to the invention, more preferably a SARS-wtRBD (SEQ ID NO: 1) and / or a MERS-wtRBD (SEQ ID NO: 194) according to the invention, - the VIRUS-RBD receptor is a CORONA-RBD receptor, more preferably ACE2 and / or DPP4, - the VIRUS-wtRBD-nAB is a CORONA-wtRBD-nAB according to the invention, more preferably a SARS-wtRBD-nAB and / or a MERS-wtRBD-nAB according to the invention, The method according to claim 1, wherein the Virus-mSpike is a Corona-mSpike according to the invention, more preferably a SARS-mSpike and / or a MERS-mSpike according to the invention.

8. A virus-mRBD or a fragment thereof obtained as an active ingredient by the method of claim 1.

9. A mutant coronavirus receptor binding domain (mRBD) (CORONA-mRBD) or a fragment thereof, which has reduced binding affinity to a coronavirus RBD receptor (CORONA-RBD receptor) compared to a wild-type coronavirus receptor binding domain (CORONA-wtRBD).

10. The CORONA-mRBD or fragment thereof of claim 9, which exhibits binding to an anti-CORONA-wtRBD neutralizing antibody.

11. The CORONA-mRBD or fragment thereof of claim 9 or 10, which reduces cell-cell fusion.

12. The CORONA-mRBD or fragment thereof of claim 9, which reduces cellular antigen uptake and / or receptor internalization.

13. (A) the coronavirus is SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), CORONA-mRBD is a mutant receptor binding domain of SARS-CoV-2 (SARS-mRBD) or a fragment thereof; the wild-type receptor-binding domain is SARS-wtRBD (the wild-type receptor-binding domain of SARS-CoV-2); the RBD receptor is ACE2 (angiotensin converting enzyme 2), or (B) the coronavirus is MERS-CoV (Middle East Respiratory Syndrome coronavirus), CORONA-mRBD is a mutated receptor binding domain of MERS-CoV (MERS-mRBD) or a fragment thereof; the wild-type receptor-binding domain is MERS-wtRBD (the wild-type receptor-binding domain of MERS-CoV); The CORONA-mRBD or fragment thereof according to claim 9, wherein the RBD receptor is DPP4 (dipeptidyl peptidase 4).

14. (A) the SARS-mRBD or fragment thereof comprises an amino acid sequence or fragment thereof that includes one or more substitutions of amino acid residues at positions selected from G184, Y187, L137, Y171, F138, Q180, F168, Y131, or S55 of SARS-wtRBD of SEQ ID NO: 1, wherein the SARS-mRBD or fragment thereof has 85% or greater amino acid sequence identity to SEQ ID NO: 1, excluding the substitutions; and / or (B) The CORONA-mRBD or fragment thereof of claim 9, wherein the MERS-mRBD or fragment thereof comprises an amino acid sequence or fragment thereof containing one or more substitutions of amino acid residues at positions selected from L140, D144, E170, D171, or D173 of the MERS-wtRBD of SEQ ID NO: 194, wherein the MERS-mRBD or fragment thereof has 85% or more amino acid sequence identity to SEQ ID NO: 194, excluding the substitutions.

15. (A) SARS-mRBD or a fragment thereof - the substitution of the amino acid residue at position G184 is selected from G184A, G184C, G184D, G184E, G184F, G184H, G184I, G184K, G184L, G184M, G184N, G184P, G184Q, G184R, G184S, G184T, G184V, G184W or G184Y; - the substitution of the amino acid residue at position Y187 is selected from Y187A, Y187C, Y187D, Y187E, Y187G, Y187I, Y187K, Y187L, Y187M, Y187N, Y187Q, Y187R, Y187S, Y187T or Y187V; - the substitution of the amino acid residue at position L137 is selected from L137D, L137E, L137K, L137R or L137Y; - the substitution of the amino acid residue at position Y171 is selected from Y171A, Y171C, Y171E, Y171I, Y171K, Y171L, Y171M, Y171N, Y171P, Y171Q, Y171R, Y171S, Y171T or Y171V; - the substitution of the amino acid residue at position F138 is selected from F138A, F138C, F138E, F138G, F138I, F138K, F138N, F138Q, F138R, F138S, F138T, F138W or F138Y; - the substitution of the amino acid residue at position Q180 is selected from Q180C, Q180D, Q180I, Q180K, Q180L or Q180V; - the substitution of the amino acid residue at position F168 is selected from F168C, F168D or F168E; - the substitution of the amino acid residue at position Y131 is selected from Y131A, Y131C, Y131D, Y131E, Y131F, Y131G, Y131H, Y131I, Y131L, Y131M, Y131N, Y131P, Y131Q, Y131S, Y131T, Y131V or Y131W, and / or - the substitution of the amino acid residue at position S55 is S55N, and / or (B) MERS-mRBD or a fragment thereof, - the substitution of the amino acid residue at position L140 is L140A, - the substitution of the amino acid residue at position D144 is D144A, - the substitution of the amino acid residue at position E170 is E170R, - the substitution of the amino acid residue at position D171 is D171K, and / or 10. The CORONA-mRBD or fragment thereof according to claim 9, wherein the substitution of the amino acid residue at position D173 is D173K.

16. (A) the SARS-mRBD or fragment thereof comprises an amino acid sequence or fragment thereof comprising a substitution of one of the amino acid residues at a position selected from G184 or L137 of SARS-wtRBD of SEQ ID NO: 1, wherein: - the substitution of the amino acid residue at position G184 is selected from G184E, G184R or G184D; - the substitution of the amino acid residue at position L137 is L137R, and / or (B) the MERS-mRBD or fragment thereof comprises an amino acid sequence or fragment thereof comprising a substitution of one of the amino acid residues at a position selected from D144 and D171 of the MERS-wtRBD of SEQ ID NO: 194, wherein: - the substitution of the amino acid residue at position D144 is D144A, 10. The CORONA-mRBD or fragment thereof according to claim 9, wherein the substitution of the amino acid residue at position D171 is D171K.

17. (A) the SARS-mRBD or a fragment thereof comprises an amino acid sequence selected from SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:5, SEQ ID NO:20, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:13, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:11, SEQ ID NO:7, or SEQ ID NO:10, SEQ ID NO:55, or SEQ ID NO:30; (B) The CORONA-mRBD or a fragment thereof of claim 9, wherein the MERS-mRBD or a fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 195 or SEQ ID NO:

196.

18. A coronavirus mutant spike protein (CORONA-mSpike) or a fragment thereof, comprising the CORONA-mRBD or a fragment thereof described in claim 9.

19. A polypeptide or protein comprising the CORONA-mRBD of claim 9 or a fragment thereof, or CORONA-mSpike or a fragment thereof comprising the CORONA-mRBD of claim 9 or a fragment thereof.

20. - a CORONA-mRBD or a fragment thereof according to claim 9, - CORONA-mSpike or a fragment thereof, comprising the CORONA-mRBD or a fragment thereof according to claim 9, or - A polypeptide or protein comprising the CORONA-mRBD or a fragment thereof according to claim 9, or a CORONA-mSpike or a fragment thereof comprising the CORONA-mRBD or a fragment thereof according to claim 9. A nucleic acid comprising a nucleotide sequence encoding

21. As an active ingredient, - one or more CORONA-mRBDs or fragments thereof according to claim 9, and / or one or more CORONA-mSpikes or fragments thereof comprising the CORONA-mRBD or fragments thereof according to claim 9, and / or - one or more polypeptides or proteins comprising a CORONA-mRBD or a fragment thereof according to claim 9 or a CORONA-mSpike or a fragment thereof comprising a CORONA-mRBD or a fragment thereof according to claim 9, and / or - a CORONA-mRBD or a fragment thereof according to claim 9, CORONA-mSpike or a fragment thereof comprising the CORONA-mRBD or a fragment thereof according to claim 9; or A polypeptide or protein comprising the CORONA-mRBD of claim 9 or a fragment thereof, or CORONA-mSpike or a fragment thereof comprising the CORONA-mRBD of claim 9 or a fragment thereof. one or more nucleic acids comprising a nucleotide sequence encoding 10. A vaccine composition comprising:

22. For the prevention and / or treatment of a disease caused by a coronavirus in a subject, One or more CORONA-mRBDs or fragments thereof according to claim 9, and / or One or more CORONA-mSpikes or fragments thereof comprising the CORONA-mRBD or fragments thereof according to claim 9, and / or One or more polypeptides or proteins comprising the CORONA-mRBD or a fragment thereof according to claim 9, or a CORONA-mSpike or a fragment thereof comprising the CORONA-mRBD or a fragment thereof according to claim 9, and / or The CORONA-mRBD or a fragment thereof according to claim 9 . CORONA-mSpike or a fragment thereof comprising the CORONA-mRBD or a fragment thereof according to claim 9; or A polypeptide or protein comprising the CORONA-mRBD of claim 9 or a fragment thereof, or CORONA-mSpike or a fragment thereof comprising the CORONA-mRBD of claim 9 or a fragment thereof. and / or one or more nucleic acids comprising a nucleotide sequence encoding 22. The vaccine composition of claim 21.

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