WO2022167396A1

WO2022167396A1 - Method for identifying emerging pathogen mutations enabling host cell entry and immune evasion

Info

Publication number: WO2022167396A1
Application number: PCT/EP2022/052280
Authority: WO
Inventors: Günter Roth
Original assignee: Biocopy Gmbh
Priority date: 2021-02-02
Filing date: 2022-02-01
Publication date: 2022-08-11

Abstract

The present invention concerns a method for identifying pathogen mutations enabling host cell entry and/or immune evasion, the method comprising (a.) providing (i.) one or more host cell receptors or fragments thereof (potentially) involved in host cell entry by the pathogen; (ii.) multiple mutants of one or more pathogen surface proteins or fragments thereof (potentially) involved in host cell entry of the pathogen and/or (potentially) recognized by the host's immune system; and (iii.) pathogen inhibiting immune interactors directed against the one or more pathogen surface proteins; (b.) bringing the one or more host cell receptors or fragments thereof into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants that bind to the host cell receptors (binding mutants), and (c.) bringing the pathogen inhibiting immune interactors into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors (immune evasion mutants). In preferred embodiments, the mutants identified as binding mutants and immune evasion mutants (emerging mutants) are isolated and arranged on a protein array, and/or are analyzed individually for binding to one or more host cell receptors and/or pathogen inhibiting immune interactors.

Description

METHOD FOR IDENTIFYING EMERGING PATHOGEN MUTATIONS ENABLING HOST CELL ENTRY AND IMMUNE EVASION

Description

The present invention describes a systematic approach to identify emerging mutations of pathogens that are for example not covered by an existing vaccine, and the method of the invention may be used to adapt or modify existing vaccines to cover potentially occurring bypass mutants.

The present invention concerns a method for identifying pathogen mutations enabling host cell entry and/or immune evasion, the method comprising (a.) providing (i.) one or more host cell receptors or fragments thereof (potentially) involved in host cell entry by the pathogen; (ii.) multiple mutants of one or more pathogen surface proteins or fragments thereof (potentially) involved in host cell entry of the pathogen and/or (potentially) recognized by the host’s immune system; and (iii.) pathogen inhibiting immune interactors directed against the one or more pathogen surface proteins; (b.) bringing the one or more host cell receptors or fragments thereof into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants that bind to the host cell receptors (binding mutants), and (c.) bringing the pathogen inhibiting immune interactors into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors (immune evasion mutants). In preferred embodiments, the mutants identified as binding mutants and immune evasion mutants (emerging mutants) are isolated and arranged on a protein array, and/or are analyzed individually for binding to one or more host cell receptors and/or pathogen inhibiting immune interactors.

Background of the invention

It is well known that pathogens, primarily bacteria and viruses, suffer errors within their genomic code (DNA or RNA) during replication and reproduction due to their high replication number, count, short life cycle, high rate of propagation and multiplication. These errors are commonly referred to as mutations. In many cases, these mutations are detrimental to the pathogen and lead to the extinction of that mutation. However, some mutations allow novel capabilities, such as altered binding behaviour, and in extreme cases can allow the pathogen to switch from one species to another. This mechanism is well known for many viruses, e.g. measles, which is thought to have originally been a disease of cattle. Such pathogens that change their host population/host species are also referred to as emergent pathogens. Such changes of the host species are made possible by one or more essential mutations. However, it is difficult to predict which mutation can lead to an emergent pathogen. A current example is the emerging infectious disease COVID-19 caused by the virus SARS-CoV2. To date, not even the species of origin has been precisely identified.

The occurrence of mutations and their inheritance has already been described centuries ago and could be explained mechanistically after the structural description of DNA in 1953 by Watson and Crick. Since then, the mutation mechanisms have been elucidated in detail (Bose et al., Chemical and UV Mutagenesis, Methods Mol BioL; 1373: 111-5, 2016), although some of the mutations are also due to pure chance, when a wrong base is simply incorporated during DNA replication. In particular, viral and bacterial DNA polymerases can have error rates of one base in 10.000 (McInerney et al., Error rate comparison during polymerase chain reaction by DNA polymerase, Research Article | Open Access, 2014). This means that, for example, a Corona virus with about 30.000 bases could already contain an average of three errors/mutations per replication. In the usual case, mutations are inhibitory, i.e. the mutant virus gains a disadvantage in its replication or function leading to disappearance of the mutant virus.

A mutant virus can be successful if the mutation results in an advantage in terms of stability, infectivity or replication speed, for example, and if this mutant can thus replicate faster and better as compared to the wild-type virus. This appears to have happened in the case of COVID-19 with the SARS-CoV2 mutant D614G, which has replaced the original pathogen from Wuhan (Korber et al., Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID- 19 Virus, Volume 182, Issue 4, Pages 812-827. e19, 2020). If the pathogen acquires the ability to switch from one species to another and then infect, replicate and infect further individuals of this species, this is called an emergent pathogen. This process is particularly common in viruses (Dennehy, Evolutionary ecology of virus emergence, Ann. N.Y. Acad. Sci. ISSN 0077-8923, 2017).

Provided that a pathogen is sufficiently well identified and characterized, it may be possible that a vaccine can be developed. For this purpose, characteristic proteins or molecules of the pathogen or an attenuated or inactivated form of the pathogen can be administered to an individual leading to the induction of a specific immune response against the pathogen, mostly mediated by T and B cells. Thus, the spread of the pathogen can be strongly contained and in particularly successful cases, this can lead to the extinction of the pathogen, as achieved for the smallpox virus (since 1980 at the latest, the world has been considered free of smallpox according to the WHO). This can only be achieved if a vaccine characterizes the pathogen very well and the immune system reacts sufficiently well to the vaccination.

Furthermore, in order to rapidly contain an emerging infectious disease, vaccination coverage should be rapid, and there should not be too many infected people. Furthermore, rate of spreading of the pathogen is dependent on its infectivity (low infectivity is associated with slow horizontal transmission), the inactivation of the pathogen outside the host, and the pathogens ability to move freely between species (as these then serve as reservoirs for the virus). In the case of measles, for example, the bat can serve as a reservoir. Furthermore, measles virus is extremely infective/contagious as well as long-lasting outside the host under certain environmental conditions, so that even if all people were vaccinated, it will likely take about 50 years for measles to die out.

Thus, if the pathogens can continue to multiply in the main host or in other species during the vaccination phase, there is a purely statistical chance that a “bypass mutant” will develop, which can then cause an infection despite vaccination. Such immune-evasive mutations are widely known, especially for HIV and influenza, so that in the case of HIV to date no universal vaccine is available and in the case of influenza an annually adapted vaccine is necessary.

However, it can currently not be predicted which mutations of a pathogen are most likely to lead to generation of a bypass mutant. However, targeting such regions has the potential to build a longterm vaccine (Cicin-sain et al., Targeted deletion of regions rich in immune-evasive genes from the cytomegalovirus genome as a novel vaccine strategy, JOURNAL OF VIROLOGY, p. 13825-13834, 2007).

The occurrence of a mutation with the potential to evade an existing vaccine is purely random and not predictable, as even the leading vaccine company Moderna stated on 26.01.2020 (https://www.sciencemag.org/news/2021/01/vaccine-20-moderna-and-other-companies-plan-tweaks- would-protect-against-new; doi: 10.1126/science.abg7691), where they announced, that they will make a second generation vaccine covering the upcoming described mutants. This statement inherently shows that there is currently no way to predict the potential of upcoming evasion mutants, meaning that this is beyond state of the art and beyond knowledge even of the most leading vaccine producers in the world.

Predicting at which time, at which geographical location or at which position of the genome of a pathogen an emerging mutation will occur is almost impossible. It has been shown that tropical (warm and humid) locations, as well as simultaneous animal and human congregations with parallel poor hygienic conditions lead to increased mutation rates. Therefore, many emergences are due to animal markets, which represent an entry point for pathogens with respect to the human species.

Genomic sequences of a pathogen that mutate preferentially can be identified retrospectively by genetic analysis of the resulting phylogenetic trees of the respective pathogen (Bush, Predicting adaptive evolution, NATURE REVIEWS GENETICS, 2001 , p. 387-392). However, this analysis is limited to the frequency of a mutation in relation to the sequence, but not to its molecular effectiveness. Furthermore, it has been shown that mutations can even give rise to new organelles within cells (Ji et al., Construction of a highly error-prone DNA polymerase for developing organelle mutation systems, Nucleic Acids Research, 2020, Vol. 48, No. 21). Thus, it is empirically possible to let mutations happen and then examine them for their function. However, this is contra-productive in the case of a pathogen, since one then generates a new pathogen and only find out that it is potentially dangerous after it infects a species.

The infectivity/infection rate of a pathogen can be roughly estimated based on two parameters. First, how fast the pathogen can penetrate a cell, and second, how transmissible it is from one host to another. The dangerousness of a pathogen correlates on the one hand with its infectivity, and on the other hand with its lethality and/or the severity of an infection. For example, Ebola, with about 30% lethality, is dangerous after infection, but the infection rate is low, so Ebola has not yet become a worldwide phenomenon. In contrast, pathogens such as herpes, EBV or CMV are highly infective, but are mostly associated with relatively mild disease courses and are sometimes even completely inconspicuous. Therefore, pathogens are particularly dangerous when they have a high infection rate as well as a high risk/lethality, such as measles or COVID-19.

With respect to an emergent pathogen’s lethality or the severity of an infection, no statement can be made per se. On the other hand, generic statements can be made about the infection rate if, for example, the pathogen carries molecules that facilitate entry and/or exit from the host organism. In the case of entry, these are interacting molecules that exhibit binding affinity. In the case of COVID- 19, this is the S protein of the virus, which interacts with the ACE2 receptor of the human host cell (Kumar and Khodor, Pathophysiology and treatment strategies for COVID-19, Transl Med 18:353, The fact that bypass mutants could render vaccines ineffective particularly via a detour through another host mechanism was noted with concern in late 2020 (Rambaut et al., Preliminary genomic characterization of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations). In addition, it occurred that COVID-19 was transmitted from a human host to mink and back to a human. Concern was expressed in the general media that this could circumvent the newly developed vaccines from Moderna, Biontech and AstraZeneca. This concern was further supported experimentally by showing that certain mutations in the S protein of SARS-CoV-2 were nevertheless not inhibited by neutralizing antibodies after infection and healing had occurred (Andreano et al., SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma, 2020).

Occurrence of evasion mutants as a result of vaccination is generally accepted and regulated surveillance is recommended as the best countermeasure (Mas et al., Antigenic and sequence variability of the human respiratory syncytial virus F glycoprotein compared to related viruses in a comprehensive dataset, Vaccine, 2018, p. 6660-6673). Thus, again, the clear state of the art is that a genetic drift should be monitored. A prediction of the genetic drift has not been realized so far.

In order to generate a generic, universally protective vaccine, scientist are attempting to perform structural averaging of various circulating mutants. Such approaches are based on the assumption that the immune system can then always address this structure, which should always be similar, via cross-reactivity, and therefore a corresponding vaccine should be effective against all potential mutations of the respective pathogen. In the case of cowpox to smallpox this approach was successful, but in the case of e.g. influenza research continues without success (Deng and Wang, A perspective of nanoparticle universal influenza vaccines, ACS Infectious Diseases, 2018). Very often it turns out that already a single amino acid, and thus potentially a single DNA base, decisively determines the functionality in terms of binding ability or enzymatic activity of the encoded protein.

To date, the corresponding prediction algorithms are too weak to even generate the chance of predicting a potentially dangerous mutant. The main experimental approach to address this problem is through cultivation, propagation and evolution of the respective pathogen under various influencing factors, as described by Johnston and Cannon (Construction of mutant strains of Neisseria gonorrhoeae lacking new antibiotic resistance markers using a two gene cassette with positive and negative selection, Gene, 1999). However, this is an evolutionary and randomized process that works with living pathogens and thus carries the risk of release of the generated mutants.

Doud et al. (Complete mapping of viral escape from neutralizing antibodies", PLOS PATHOGENS, vol. 13, no. 3, 13 March 2017, page e1006271) describe a method for identifying influenza virus HA escape mutants in which a library of HA virus mutants is incubated with or without neutralizing antibodies and subsequently used to infect cells. The viral RNA is isolated from the infected cells and sequenced to detect differential selection by the antibodies. This method has the disadvantage that it must use infective viral particles, which are potentially dangerous. Furthermore, the binding of the HA to the host cell receptor is not directly assessed, but the viral RNA serves as an indirect measure for viral infectivity in the presence of the antibodies. Since the readout of this method is based on viral replication/detection of viral RNA in the infected cells, the method has an amplification bias, and it is not known how viral entry/receptor binding is affected by the mutation in comparison to viral replication in the cell. In another article, Doud et al. describe a similar approach with the same disadvantages ("How single mutations affect viral escape from broad and narrow antibodies to H1 influenza hemagglutinin", NATURE COMMUNICATIONS, vol. 9, no. 1 , 1 December 2018). Also, Wu et al. ("Different genetic barriers for resistance to HA stem antibodies in influenza H3 and H1 viruses", SCIENCE, vol. 368, no. 6497, 19 June 2020, pages 1335-1340) examine the impact of influenza HA mutations on the effect of neutralizing antibodies by deep mutational scanning, facing the same disadvantages.

BAUM et al. ("Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies", SCIENCE, 15 June 2020, page eabd0831) describe a method for identifying or generating SARS-Cov-2-S escape mutants that are no longer inhibited by certain antibodies. Here, multiple S mutants are expressed on VSV particles, and the particles are used to infect cells in the presence of S-specific antibodies. The RNA of the infected cells is sequenced. Similar to the method of Doud et al., Baum et al. also use viral particles for infection of cells and determine viral RNA for identifying escape mutants. Again, the method requires potentially harmful infective viral particles and does not enable direct binding studies between the pathogen surface protein and the cellular receptor are possible and the readout will contain a replication bias.

Accordingly, all these assays are based on competitive infection and replication of a pool of viral mutants in presence of inhibiting or neutralizing antibodies, while none of the described methods enables a direct assessment of the binding kinetics of the pathogen surface protein mutants to the host cell receptor, and how this is influenced the presence of inhibitory immune receptors such as antibodies.

In the field of biotechnology and technical biology, methods have been developed that allow screening of millions and more molecular interactions between potential binding partners, for example between proteins. Such techniques, as for example phage display (US 8685893B2) and ribosome display (DE60115405T2) enable identifying previously unknown interactions, and studying known interactions, for example by modifying one of the binding partners by introducing various mutations, by screening multiple molecules and/or mutations of molecules provided in a library.

Furthermore, microarrays offer the possibility of arranging many molecules, for example various (potential) antigens of a pathogens, on a small area for analysing an immune response against such antigens, which may be the result of a vaccination (Nakajima et al., Protein Microarray Analysis of the Specificity and Cross-Reactivity of Influenza Virus Hemagglutinin-Specific Antibodies, Clinical Science and Epidemiology, 2018; Furman and Davis, New approaches to understanding the immune response to vaccination and infection, Vaccine. 33(40): 5271-5281 , 2015; Gallerano et al., HIV microarray for the mapping and characterization of HIV-specific antibody responses, Royal society of chemistry, 2015; Jiang et al., SARS-CoV-2 proteome microarray for global profiling of COVID-19 specific IgG and IgM responses, Nature Communication, 2020). This approach can be used to estimate how well a known vaccine protects against known mutants of the respective pathogen, and to establish antibody profiles against pathogens. However, nothing can be deduced about newly emerging mutants.

Accordingly, there is a need in the art for a method that enables evaluation of a high number of mutants of a pathogen for their potential to (i) infect a certain host, and at the same time (ii) to evade from an existing immune response or available treatment against the pathogen. Such a method should avoid the risk of generating new mutants of the pathogen that may represent a potential public health threat.

Summary of the invention

In light of the prior art the technical problem underlying the invention was the provision of a method for identifying emerging pathogen mutations enabling host cell entry and immune evasion. The resulting method should enable a systematic investigation of existing and future mutations of an already known pathogen or a potentially emergent pathogen.

This problem is solved by the feature of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

The invention therefore relates to a method for identifying pathogen mutations enabling host cell entry and/or immune evasion, the method comprising a. Providing i. one or more host cell receptors or fragments thereof; ii. multiple mutants of one or more pathogen surface proteins or fragments thereof; and iii. pathogen inhibiting immune interactors directed against the one or more pathogen surface proteins; b. Bringing the one or more host cell receptors or fragments thereof into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants that bind to the host cell receptors (binding mutants), and c. Bringing the pathogen inhibiting immune interactors into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors (immune evasion mutants).

In embodiments, the invention relates to a method for identifying pathogen mutations enabling host cell entry and/or immune evasion, the method comprising a) Providing i) one or more host cell receptors or fragments thereof (potentially) involved in host cell entry by the pathogen; ii) multiple mutants of one or more pathogen surface proteins or fragments thereof, which are preferably (potentially) involved in host cell entry of the pathogen and/or preferably (potentially) recognized by the host’s immune system; and iii) pathogen inhibiting immune interactors directed against the one or more pathogen surface proteins; b) Bringing the one or more host cell receptors or fragments thereof into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants binding to the one or more host cell receptors (binding mutants), and c) Bringing the pathogen inhibiting immune interactors into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors (immune evasion mutants).

The present invention describes a systematic approach to identify mutations of pathogens that can lead to emergence of the respective pathogen, for example mutations of a pathogen that are not covered by an existing active and/or passive vaccine and/or known therapeutic immune interactors, such as immune receptors including therapeutic antibodies (also therapeutic single-chain antibodies) or DARPins, affimers, affibodies and other molecular binders , and the invention may be used to adapt existing vaccines/immune receptors to cover potentially occurring bypass mutants even before they come into existence in the future. Furthermore, the invention is applicable to pathogens which are known to circulate in one species but are so far still harmless for a second species (preferably humans). However, mutations of such pathogens may render them more dangerous for the second species, and the present invention can be used to identify such mutations, again even before they occur in the circulating pathogen. The present invention makes it possible to develop a vaccine against the emergence of new diseases (emergent pathogens) or to prevent the spread of these diseases before they have really emerged in the core. In this way, emergent diseases can be contained not only before they spread, but also before the come into existence.

The present invention is based on the entirely surprising and innovative combination of process steps that enables the identification of potentially dangerous emerging mutants of a pathogen that may not be present in a host population yet, but which may arise in the future. Therefore, the method of the invention enables preparation for potential future public health threats before they occur. Furthermore, the method enables testing whether a therapeutic or prophylactic agent that already exists will provide protection or will be effective against mutant variants of a pathogen that may arise in the future. Accordingly, based on the results of the method of the invention, one could modify existing therapeutic/prophylactic agents to also cover potential future variants of the pathogen.

The present invention is based on the concept of a double assessment of mutants of one or more pathogen surface proteins that are critical for host cell infection and therefore infectivity of the respective pathogen. The method of the invention is preferably used for intracellular pathogens that require entry into a host cell for reproduction and for inducing disease in the host. This concerns primarily viruses, which have to enter a host cell to highjack the host cell machinery for viral replication. However, the method can also be used for other pathogens, in particular intracellular bacteria and parasites and even prions that enter host cells through engagement of pathogen surface molecules with a with one or more molecules on the host cell surface, which are termed host cells receptors in the context of the invention.

The invention uses this principle of host cell entry through interaction between pathogen surface proteins and host cell receptors to identify mutations of the pathogen surface proteins involved in host cell entry that may lead to an emergent version of the pathogen. An emergent mutant pathogen as used herein relates to versions pathogens with mutated surface proteins that still enable engagement with the host cell receptor but which are no longer recognized by the immune system of the host or by a therapeutic agent, such as a therapeutic immune receptor. Accordingly, such a mutant can lead to rapid spreading in the host since infectivity is maintained (or even enhanced) by the mutation, but the mutated pathogen is no longer contained by the immune system of the host (which may have been vaccinated with a vaccine effective against the wildtype variant of the pathogen) or which is not sufficiently inhibited by available treatment agents. Furthermore, an emergent mutant pathogen may also be a pathogen that can now enter a different host species due to a mutation of one or more surface proteins affecting host cell entry, which was not possible for the wildtype variant that gave rise to the mutant. This would lead to a mutant pathogen that can enter and potentially spread in a new host species (preferably human) which may not have any specific immune response against this pathogen and for which there are no therapeutic agent available.

Accordingly, the present invention makes it possible to on the one hand screen pathogens that are already present in a certain host species, such as human, for emerging mutations that may occur in the future which are still infective for the host, but which are no longer contained by available pathogen inhibiting immune interactors. Furthermore, it is possible to screen mutants of pathogen that are not yet circulating in a given host species, such as humans, but which may gain the ability to enter this host through mutations of pathogen surface molecules involved in host cell entry. Furthermore, such mutants that gain access to the new host species can be analysed for binding and inhibition of host cell entry by available immune interactors.

As used herein “pathogen inhibiting immune interactors directed against the one or more pathogen surface proteins” comprise also immune interactors that bind to glycosylation patterns or other posttranslational modification that may be present on the pathogen surface protein and that can be recognized by the host’s immune system. Furthermore, in embodiments the immune interactors can be directed against lipopolysaccharides or cell wall teichoic acids on a pathogens surface, such as on the surface of a bacterium, wherein the mutations may affect the structure and/or recognition by the immune interactors. The skilled person understands that mutation of the pathogen surface protein can lead to modification or change of posttranslational modification (such as glycosylation) of the pathogen surface molecule, which can contribute to a changed binding behaviour and/or even molecular retargeting, by the immune interactors and/or by the host cell receptor.

In the context of the method of the invention it is understood that mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors comprise mutants that are not bound by the pathogen inhibiting immune interactors (which are preferably known or expected to bind to the wildtype variant of the pathogen surface protein), mutants still binding to the one or more host cell receptors in presence of the immune interactors (wherein preferably it is known that the immune interactors inhibit binding of the wildtype variant of the pathogen surface protein to the host cell receptor), and mutants whose activity is not inhibited by the immune interactors (wherein preferably it is known that the immune interactors inhibit the activity of the type variant of the pathogen surface protein). In this context, the term activity preferably relates to the activity of the pathogen surface molecule to mediate host cell entry, for example through an enzymatic activity. Further, decreased binding is understood as a lower binding affinity of the immune interactors to the mutant as compared to the wildtype of the pathogen surface molecule. A decreased inhibition is understood as a lower activity of the mutant pathogen surface protein to mediate host cell entry as compared to the wildtype, and/or a lower enzymatic activity of the mutant pathogen surface protein to mediate host cell entry as compared to the wildtype.

In embodiments, isolating and/or identifying mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors relates to isolating and/or identifying mutants not bound by the pathogen inhibiting immune interactors and/or mutants still binding to the one or more host cell receptors (in the presence of the immune interactors) and/or mutants whose activity is not inhibited (in presence of the immune interactors).

The present invention makes it conceivable to generate and screen mutations of individual molecules of already known pathogens and to measure their interaction with the host cell and additionally determine whether one of the mutations bypasses an existing vaccine or treatment agent, for example, or whether an emergence in the sense of a species change can take place.

The skilled person understands that the method of the present invention can be used in multiple scenarios depending on the knowledge about the pathogen and the interaction of the pathogen with a host cell. For example, when it is not known, which host cell receptor is used by a pathogen to enter the host cell, multiple candidate host cell receptors or even all surface molecules potentially acting as host cell receptors could be tested for binding to one or more potentially interacting pathogen surface molecules for identifying host cell receptors that interact with pathogen surface proteins. In embodiments, the one or more host cell receptors can be provided by providing whole cells comprising all cell surface receptors and all surface molecules potentially acting as host cell receptors.

In other cases, where the host pathogen interaction is already well described, as for example for SARS-CoV2, where the S protein of the virus is known to mediate host cell entry via ACE2 on the host cell, one could limit the analysis to assessing the interaction between ACE2, for example only human ACE2 or ACE2 version from various species, and multiple mutants of the S protein.

From these two examples, where on the one hand neither the host cell receptor is known nor the interacting pathogen surface molecule or on the other hand both molecules are well characterized, the skilled person can deduct many variants of the method of the present invention, which depend on the pathogen and the knowledge about its interaction with its hosts.

If the pathogen’s present host provides a given molecule for interaction and host cell entry, the candidate molecules of a potential future host that may enable species barrier crossing events can be narrowed by a skilled person for example to such molecules of the potential future host, which (a) have a similar chemical structure or (b) a homologous function in the novel host. This may limit the number of molecules to be screened on the host site. Similarly, in case of intra-host evasion the skilled person can deduce a list of molecules of the host which are similar or have homologous sequences to the known interacting molecule, e.g. like a switch from EGFR1/HER1 to HER2, 3 or 4. This example also shows that many receptors of cells can be assigned to molecular families with high similarity. It is utmost likely that a pathogen may change with high probability to such a different family member, which means quite often that the pathogen switches tissues which are attacked first.

Therefore, in embodiments of the invention the one or more host cell receptors can comprise members of or even the whole protein family of a known receptor and/or molecules with a homologous binding site for analysing binding of the provided mutants of the pathogen surface protein/molecule.

For identifying binding mutants of a pathogen surface protein in the context of the present invention, the skilled person is aware of multiple techniques that can be used. For example, one of the binding partners to be assessed (the one or more host the receptors or the mutants of the one or more pathogen surface proteins) can be coupled to a solid phase, such as a sepharose column, microbeads or a microarray or any other solid phase known to be used in biotechnological interaction studies, and the other binding partner can be provided in solution to freely bind to the solid phase could component, if there is a binding affinity. The non-binders can be washed off the solid phase comprising the coupled molecules and subsequently the binding mutants can be identified by known techniques. For examples, all binding mutants can be eluted from the solid phase and can subsequently be identified by suitable techniques, which can include mass spectrometry or sequencing, depending on the specific features of an embodiment.

For example, in case of using a display library to study multiple mutants of a pathogen surface protein, the binding mutants can be identified by sequencing analysis, for example after eluting them form the solid phase comprising the coupled host cell receptors. Furthermore, mass spectrometry can be employed to identify binders on the protein/amino acid level.

Furthermore, in embodiments where mutants of a known sequence are spotted on a protein array and a host cell receptor in solution is brought into contact with the microarray, for example by incubating the solution comprising the host cell receptor with the microarray, the receptor can comprise a fluorescent or other suitable label and binding of the receptors to certain spots of the array corresponding to a specific mutant can be identified for example by fluorescent analysis.

Additionally, in the context of the present invention the provided mutants of the pathogen surface protein are also analysed for binding to provided pathogen inhibiting immune interactors.

These immune interactors comprise molecules that are known to bind to the wildtype of one or more pathogen surface molecules that are assessed in the context of the invention and to inhibit efficient infection and/or replication of the wildtype in the host. These immune interactors can for example comprise therapeutic or diagnostic antibodies (monoclonal or polyclonal), antisera of a host that already went through an infection with the respective pathogen or a serum from a subject that has been vaccinated with a vaccine that is effective against the wildtype version of the pathogen, or any other known immune receptor or sample comprising such immune receptors. One example of immune interactors would be a monoclonal antibody that is known to block an otherwise occurring interaction between the host cell receptor and the pathogen surface molecule by binding to the pathogen surface molecule. However, in view of the present disclosure, the skilled person can deduct further examples of immune interactors to be examined in the context of the method of the invention, depending on the specific pathogen and host that are assessed.

In the context of the invention, a mutant of a pathogen surface molecule is considered an immune evasion mutant if it is no longer bound by the immune interactor known to bind to the wild type version of the pathogen surface molecule.

The skilled person is aware of multiple ways of assessing binding between the pathogen inhibiting immune interactors and the pathogen surface protein and therefore for identifying immune evasion mutants that are (in contrast to the wildtype version) no longer bound by the assessed immune interactors.

For example, similar to the assessment of binding to the host cell receptor, one of the binding partners to be assessed (the immune interactors or the mutants of the one or more pathogen surface proteins) can be coupled to a solid phase, such as a sepharose column, microbeads or a microarray or any other solid phase known to be used in biotechnological interaction studies, and the other binding partner can be provided in solution to freely bind to the solid phase could component, if there is a binding affinity. If the immune interactors are coupled to the solid phase, the non-binding mutants (immune evasion mutants) can be washed off the solid phase and can be identified by suitable techniques, which can include mass spectrometry or sequencing, depending on the specific features of an embodiment.

For example, in case of using a display library to study multiple mutants, the immune evasion mutants can be identified by sequencing analysis. Furthermore, mass spectrometry can be employed to identify non-binding immune evasion mutants.

Furthermore, in embodiments where pathogen surface protein mutants of a known sequence are spotted on a protein array and immune interactors in solution are brought into contact with the microarray, for example by incubating the solution comprising the host cell receptor with the microarray, the immune interactors can comprise a fluorescent or other suitable label and binding of the immune interactor to certain spots of the array corresponding to a specific mutant can be identified, for example by fluorescent analysis.

In other embodiments, mutants not bound by the pathogen inhibiting immune interactors and/or binding to the one or more host cell receptors in presence of the pathogen inhibiting immune interactors can be achieved by bringing the immune interactors in contact with the mutants of the pathogen surface protein and subsequently bringing the mixture into contact with the one or more host cell receptors, which preferably solid phase coupled to a solid phase, and to identify those mutants that still bind to the host cell receptors despite being preincubated with the immune interactors known to block interaction between the wildtype version of the pathogen surface protein and the host cell receptors. Accordingly, in such embodiments immune evasion mutants can be identified as those mutants that display differential binding to the host cell receptor in presence (after preincubation with immune interactors) and absence of the immune interactors.

Accordingly, there are different ways of identifying emerging mutants according to the present invention. In embodiments, the binding of the assessed mutants to the host cell receptors and to the immune interactors can be assessed directly, for example by incubating the mutants with solid phase could receptors and with solid phase coupled immune interactors. These two steps can be performed each with the full set of mutants provided in the method of the present invention, or they can be performed subsequently to each other, wherein for example first all provided mutants are assessed for binding to the host cell receptor, and the isolated binding mutants from this step are subsequently analysed for binding to the immune interactors, or the other way around. Such a sequential assessment of the binding behaviour of the mutants can be advantageous, since the number of mutants to be assessed in the second binding assessment is reduced. Alternative embodiments, where a differential binding of the mutants to the host cell receptors in presence of or after preincubation with the immune interactors is assessed can also be performed. Such embodiments can be advantageous if not only binding of the immune interactors, but also inhibition of binding to the host cell receptor through binding to the immune interactors is to be assessed. However, in most cases binding of immune interactors is a good measure for determining potential inhibition of the pathogen.

In preferred embodiments of the invention, the mutants identified as binding mutants and immune evasion mutants (emerging mutants) are isolated and arranged on a protein array. Such embodiments are useful, since a large number of potential emerging mutants (for example up to 10^A6 identified candidates) that have gained or maintained the ability to bind to the host cell receptor and that are no longer bound and/or blocked by the immune interactors as assessed in a first screening round from a huge pool of mutants, which can of up to 10^A15 mutants present in a display library, can be analysed in a more exact way and still efficient way (in parallel) by arranging them on a protein microarray. For example, a large number of potential emerging mutants identified in a first screening round can be reanalysed for binding to the host cell receptor and also to host cell receptor variants (for example form variant present in one species or variants from different species), wherein binding can be measured not only in a binary way (binding or not-binding) but also the specific binding affinity could be assessed. The same is true for analysing the interaction with the immune interactors in more detail, and also other immune interactors can be tested on the identified candidate emerging mutants present on the array. For example, if the antiserum has been used as immune interactor in the initial screening, the specific binder(s) among the multiple components of the antiserum can be identified using the microarray. Accordingly, the method of the invention can comprise the subsequent analysis steps performed with the identified emerging mutants that can be arranged on a microarray.

In a preferred embodiment of the method of the invention, the emerging mutants are analysed individually for binding to one or more host cell receptors and/or pathogen inhibiting immune interactors. Such individual analysis can be performed using protein microarrays, which comprise the different identified emerging mutants in distinct locations, such as distinct spots of the array. Accordingly, each identified candidate emerging mutant can be analysed individually but still in parallel to the other candidates, which allows reanalysis/verification/in depth characterization of a large number of emerging mutants identified in the initial screening.

In embodiments, the multiple mutants of the one or more pathogen surface proteins or fragments thereof are provided in solution in a display library, such as a phage display library, a ribosome display library, a bacterial display, yeast display or ribosome display.

Display techniques are a well known and established way of screening a large number of mutants or variants of a protein, here the one or more pathogen surface molecule and in certain embodiments potentially also multiple potential host cell receptors, for binding to a target molecule. Using such techniques, it is possible to screen a large number of mutants for binding to a target, for example, 10^A2, 10^A3, 10^A4, 10^A5, 10^A6, 10^A7, 10^A8, 10^A9, 10^A10, 10^A1 1 , 10^A12, 10^A13, 10^A14, 10^A15, or more mutants.

Display techniques are advantageous, since they enable measuring interactions between a large number of variants of a protein or fragments of a protein, which preferentially comprise a known or suspected binding/interaction region of the protein, and a known or potential binding partner, here an interaction between one or more pathogen surface proteins and/or mutants thereof and one or more (known or potential) host cell receptors. So the interaction can be measured on the protein level.

However, it is great advantage that subsequent identification of candidates can be performed on the nucleic acid level by sequencing to deduct the sequence of the identified mutants of interest.

In embodiments, the multiple mutants of the one or more pathogen surface proteins or fragments thereof are provided immobilized on a solid surface, such as a protein microarray. Corresponding or similar embodiments of the invention have already been described above in the context of a subsequent analysis of the emerging mutants identified in an initial screening. However, the method of the invention can also start from the beginning with the provision of (a selection of) multiple mutants of one or more pathogen surface proteins or fragments thereof on a microarray, wherein each mutant is provided in a distinct and preferably known location of the array.

Accordingly, in embodiments the invention relates to for identifying pathogen mutations enabling host cell entry and/or immune evasion, the method comprising a. Providing i. one or more host cell receptors or fragments thereof; ii. multiple mutants of one or more pathogen surface proteins or fragments thereof immobilized on a protein microarray; and iii. pathogen inhibiting immune interactors directed against the one or more pathogen surface proteins; b. Bringing the one or more host cell receptors or fragments thereof into contact with the protein microarray of multiple mutants of the one or more pathogen surface proteins, and identifying mutants that bind to the host cell receptors (binding mutants), and c. Bringing the pathogen inhibiting immune interactors into contact with the protein array of multiple mutants of the one or more pathogen surface proteins, and identifying mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors (immune evasion mutants).

Preferably, each of the multiple mutants of the one or more pathogen surface proteins or fragments thereof is provided in a distinct and preferably known location on the array. Preferably, the sequence of each of the multiple mutants is known, so the determined binding properties of each mutant with respect to the one or more host cell receptors or fragments thereof and the pathogen inhibiting immune interactors can be assigned to the respective mutation of the pathogen surface protein.

Preferably, the multiple mutants of the one or more pathogen surface proteins or fragments thereof provided on the protein array have been selected/isolated from a larger pool of mutants of the one or more pathogen surface proteins or fragments.

In embodiments, the multiple mutants of the one or more pathogen surface proteins or fragments thereof of the protein array have been identified and isolated from a larger pool of multiple mutants of the one or more pathogen surface proteins or fragments thereof as mutants that bind to the host cell receptors by bringing the one or more host cell receptors of fragments thereof into contact with the larger pool of multiple mutants of the one or more pathogen surface proteins or fragments thereof.

In embodiments, the multiple mutants of the one or more pathogen surface proteins or fragments thereof of the protein array have been identified and isolated from a larger pool of multiple mutants of the one or more pathogen surface proteins or fragments thereof as mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors by bringing the pathogen inhibiting immune interactors into contact with the larger pool of multiple mutants of the one or more pathogen surface proteins or fragments thereof.

In embodiments, the multiple mutants of the one or more pathogen surface proteins or fragments thereof of the protein array have been identified and isolated from a larger pool of multiple mutants of the one or more pathogen surface proteins or fragments thereof as mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors, and as mutants that bind to the host cell receptors.

Accordingly, in embodiments the invention relates to a method for identifying pathogen mutations enabling host cell entry and/or immune evasion, the method comprising a. Providing i. one or more host cell receptors or fragments thereof; ii. multiple mutants of one or more pathogen surface proteins or fragments thereof; and iii. pathogen inhibiting immune interactors directed against the one or more pathogen surface proteins; b. Bringing the one or more host cell receptors or fragments thereof into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants that bind to the host cell receptors (binding mutants), and/or c. Bringing the pathogen inhibiting immune interactors into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors (immune evasion mutants), d. Arranging identified binding mutants and/or immune evasion mutants and/or emerging mutants on a protein array, wherein preferably the location and sequence of each mutant on the protein array is known, e. Using the protein array for individually analyzing binding of the mutants of the protein array to the one or more host cell receptors and/or to the pathogen inhibiting immune interactors. Accordingly, in embodiments the method of the invention can be used for performing a high throughput screening of the binding properties of individual mutants of the pathogen surface proteins with respect to the host cell receptor and/or to the pathogen inhibiting host interactors. For example, in case of SARS-CoV-2, multiple mutants of the S-protein of the virus can be provided on the protein array and the binding of each of the mutants to antibodies, for example antibodies present in the serum of individuals that have been vaccinated against SARS-CoV-2 or an inhibiting monoclonal antibody, can be assessed, as well as the binding of each of the mutants to ACE2 can be assessed and analyzed.

This method makes it possible for the first time to assess the specific binding properties of mutated pathogen surface proteins to relevant interaction partners in high numbers and in parallel. The selection of the mutants does not have to be limited to tens or hundreds of mutants, but it is possible to individually assess the binding properties of multiple hundred thousand or millions of mutants in parallel.

The mutants of the microarray can be selected from an even larger pool of mutants of the one or more pathogen surface proteins or fragments thereof, which can be for example provided in solution in a display library, such as a phage display library, a ribosome display library, a bacterial display, yeast display or ribosome display. From such a display library mutants can be selected that for example with respect to their binding to the host cell receptor or to the pathogen inhibiting immune interactors or with respect to both. For example, the array may comprise only mutants that were isolated from a display library as being capable of binding to the host cell receptor. In addition or alternatively, the array comprise mutants that are no longer bound or bound to a weaker/lesser extend by pathogen inhibiting immune interactors that are known to bind to the wild type version of the pathogen surface protein.

The steps of the method of the invention where the mutants or the one or more pathogen surface proteins or fragments thereof are brough into contact with the host cell receptors or with the pathogen inhibiting immune interactors may also be referred to as binding assessment steps. In other words, the method of the invention comprises a step of assessing the binding of the mutants to the host cell receptors or fragments thereof and a step of assessing the binding of the mutants to the pathogen inhibiting immune interactors.

In embodiments of the method of the invention, step b. comprises assessing binding of the multiple mutants of one or more pathogen surface proteins or fragments on the protein microarray to the host cell receptors or fragments thereof to the one or more host cell receptors or fragments thereof, and step c. comprises assessing the binding of the multiple mutants of one or more pathogen surface proteins or fragments on the protein microarray to the pathogen inhibiting immune interactors.

In embodiments, as a result of the method of the invention the user receives information about the binding behavior of multiple individual mutants of the pathogen surface protein with respect to their binding to the one or more host cell receptors and to pathogen inhibiting immune interactors. Accordingly, using it is possible to assess whether and how a certain kind of amino acid exchange, deletion or insertion at a given position influences the binding properties of a pathogen surface protein. Based on the binding information generated in the method of the invention it is therefore possible to assess and predict the risk potential of each of the analyzed mutant in case it occurs in a circulating pathogen.

Furthermore, using the results of such embodiments of the method of the invention providing binding information of multiple individual mutants it is possible to deduct information concerning individual mutations or mutation combinations of the pathogen surface protein that may not have been comprised by the protein array with respect to their potential qualify as binding mutants, immune evasion mutants and/or emerging mutants.

Using the binding results of hundreds or thousands of individual mutants of the protein array, such as mutants that comprise one or more amino acid point mutations/exchanges and/or one or more amino acid deletions and/or insertions as compared to a wild type/reference sequence, it is possible to generate a prediction model for to binding properties of practically each possible mutant of the respective pathogen surface protein.

Accordingly, embodiments the method of the invention relating to the individual analysis of binding properties of multiple mutants of the pathogen surface protein provided on a protein microarray comprise a step of deducting (or generating) from binding data generated by assessing the binding of the multiple mutants of the protein array to the one or more host cell receptors or fragments thereof and/or to the pathogen inhibiting immune interactors a prediction model for binding properties of mutants of the one or more pathogen surface proteins concerning binding to the one or more host cell receptors and/or to the pathogen inhibiting immune interactors.

In embodiments, the prediction model uses and/or is based on artificial intelligence analysis of the binding data. In embodiments, the prediction model uses and/or is based on machine learning using the binding data of the multiple mutants of the protein microarray.

In embodiments, the prediction model also uses structural data of the one or more pathogen surface protein and of mutants thereof.

In embodiments, the prediction model is used to predict the occurrence and/or infectivity and/or immune evasion of mutants of the pathogen surface protein.

Similarly, in embodiments, the host cell receptors and/or the immune interactors can be provided immobilized on a solid surface, such as a protein microarray.

In a preferred embodiment, more than 10^A3, preferably more than 10^A6, most preferably more than 10^A9 mutants of the one or more pathogen surface proteins or fragments thereof are provided and subsequently assessed for their binding behaviour, either as endpoint or in terms of kinetics. This enables to randomly screen a large number of potentially occurring mutations, and not only analysis of few mutants that may already be known. The method therefore allows an unbiased approach in terms of selecting mutants to be analysed, since the number of mutations to be screened is not a limiting factor.

In embodiments, the steps of bringing the multiple mutants of the one or more pathogen surface proteins into contact with the one or more host cell receptors or fragments thereof or with the pathogen inhibiting immune interactors can involve an assessment of the binding behavior of the mutants, either as endpoint or in terms of kinetics. This would also include embodiments, where binding of the mutants can be assessed by Biacore measurements or other label free techniques known to the skilled person.

In embodiments of the method of the invention, the multiple mutants of the pathogen surface protein are selected from a group comprising - with respect to their coding nucleic acid sequence - nucleotide exchange mutants with preferably 1 - 30 or more changes, nucleotide insertion mutants, nucleotide deletion mutants and/or frameshift mutants, as compared to the coding nucleotide sequence of the pathogen surface protein of a circulating variant of the respective pathogen (wildtype). A skilled person can deduct corresponding AS mutants. Preferably, the mutant are nucleotide exchange mutants that lead to amino acid (AS) exchanges, or deletion or insertions mutants that lead to AS deletions or insertions. Such point mutations may have impact locally to the binding behaviour of neighbouring AS but also by structural destabilization and reorientation may also have a global effect to the binding behaviour. Again, a prediction of such changes is not possible for more than 10 or 100 mutants using state of the art techniques. Currently, if a distinct mutation is known this can be simulated, but for a complete pool of simulation only quantum computing may rise hope to bring solutions (https://physicsworld.com/a/quantum-approach-reveals- faster-protein-folding/), but is still not applicable for a complete pool. Mutations which may induce a frame shift, an elongation or truncation of the protein may mainly have a global effect on the molecule’s structure and will massively change the binding behaviour with a high chance that the novel mutation may also bind to a complete other molecule of the host or even another host. It is most probable that point mutations change intra-host binding and frame shift, truncation and elongation of proteins are more likely to provide inter-species emergence.

Accordingly, the mutants to be screened in the context of the present invention comprise amino acid (AS) exchange mutants, amino acid deletion mutants, amino acid insertion mutants and frame shift mutants (also including truncations and elongations) that have a completely different AS sequence from the location of the mutation (nucleotide deletion/insertion leading to a shift of the reading frame). In embodiments, the location of the mutations of the surface protein can be limited to a certain domain or region of the surface protein, for example a region that is known or suspected of mediating interaction with a host cell receptor and/or that is preferentially addressed by known immune interactors, such as known antibodies or antisera against the pathogen.

In embodiments, not the full-length pathogen surface protein and mutants thereof are provided, but only relevant fragments of the pathogen surface protein, for example fragments known to comprise a region or domain decisive for host cell receptor engagement, such as for example the receptorbinding domain (RBD) or the spike protein or SARS-CoV-2.

In preferred embodiments, the mutants have no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 AS mutations with respect to a reference variant/wildtype variant of the pathogen surface protein or the assessed fragments thereof. Higher numbers of mutations are very unlikely. In embodiments, the AS mutations of the provided mutants can be distributed across the whole sequence of the mutant, or the mutations can be focussed in one or several distinct regions.

There are multiple well described techniques known in the art that can be used to establish mutant libraries to be used in the context of the invention. cDNA libraries are known to the skilled person since decades (P., Clark, David (2009). Biotechnology: applying the genetic revolution. Pazdernik, Nanette Jean. Amsterdam: Academic Press/Elsevier. ISBN 9780121755522. OCLC 226038060). cDNA libraries can then be introduced into a carrier system enabling to display the library either intra-cellular or even extracellular on the surface of the carrier system, like in the yeast2hybrid (see for example Young KH (February 1998). "Yeast two-hybrid: so many interactions, (in) so little time". Biology of Reproduction. 58 (2): 302-11), which is known to the skilled person since more than 20 years. Using such display techniques, all molecules of the host or different hosts can be provided. For the pathogen surface molecule, the same can be done. Therefore, mutations libraries of a (potentially) interacting molecules, regardless of whether a molecule is from a host or from a pathogen, can be generated. This knowledge is not only available for the skilled person, but commercialized broadly, for example by the company GenScript (https://www.genscript.com/synthetic_library.html), where site directed mutations (exactly defined to the DNA base position, 20 variants), or walking scans (one point mutation after the other, 20 times the randomized base pairs, so 20X) or randomized libraries (generated here via synthesis to 10^A 11 different mutants including deletions) are accessibly to anyone with enough money. However, the screening is to be done by the respective costumer. Sloning described and patented already in 2003 how a triplet mutation library can be made, so that any AS can be addressed with high quality (US7262031 B2) and already since 1991 the skilled person knows how to make PCR fragments and generate with them a cloning library (EP0738779B1). Therefore, the skilled persons has tools in hand and can even hire companies to generate such libraries, comprising in case of ribosome or phage display up to 10^A11 to 10^A15 mutants. For example, the antibody library from MorphoSys known as HuCal library contains more than 4*10^A10 different mutants made from distinct building blocks (https://www.bio-rad-antibodies.com/hucal-antibody-technology.html). They introduced, with large effort, but finally on 6 different positions several thousand mutations each and refined them for functionality like glycosylation etc, which then multiplies up to more than 10^A10 different mutants (Knappik A et al. (2000). Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol. 296:57-86; Prassler J et al. (2011). HuCAL PLATINUM, a synthetic Fab library optimized for sequence diversity and superior performance in mammalian expression systems. J Mol Biol.

413:261-78). This library was 2000 unprecedented, but in 2020 it is a simple matter of time and money and accessible to any skilled person of the field, even as a service order. Screening against a given target is stat of the art, but finding of evasion mutations is even here not possible without additional developments like revealed in the invention.

In preferred embodiments, a known interacting pathogen surface molecule of the pathogen will be either randomly mutated over the whole interacting molecule to screen the mutations against the interacting molecule of the host or even all (surface) molecules of the host. In further preferred embodiments, where the interacting pathogen surface molecule is known and detailed knowledge of the molecule’s structure, for example base on NMR measurements or crystallography, is available, a directed screening for example of a known binding site can be performed by inserting mutations in preferred regions/portions/domains of the molecule, for example after careful molecular modelling base by base in the areas where the interacting molecule seemingly has strong molecular interactions with the host molecule. Furthermore, all libraries can be generated with the above- mentioned techniques. And even after a first library has been generated a second library may be generated from the finding of the first library (so called panning or evolutionary selection, see for example Ehrlich GK, Berthold W, and Bailon P. Phage display technology. Affinity selection by biopanning. Methods in molecular biology. 2000. 147:195-208).

In further embodiments of the method of the invention, the host cell receptors of fragments thereof are coupled to a solid phase for bringing them into contact with the multiple mutants of the one or more pathogen surface proteins or fragments thereof, such as an affinity column, a resin, a column, beads, or a microarray. In such embodiments, the mutants of the pathogen surface protein can be in solution and can be run over or incubated with the solid phase coupled receptors, and the nonbinders can flow through, in case of a column, or washed off the surface or separated from the surface by other established techniques.

In further specified embodiments, the two or more host cell receptors or fragments thereof are spatially ordered and separated on the solid phase or alternatively in a randomized mix. For example, the case of using more than one potential host cell receptors or fragments thereof, the different receptors can be in different locations of a solid phase, which would enable subsequent determining of which mutant binds to which receptor. This is particularly advantageous in cases where there are several host cell receptor candidates and preferentially also several pathogen surface protein candidates, since such embodiments would enable determining which pathogen surface protein (mutant) can bind to which host cell receptor candidate, since there may be more than one interactions couples present in the reaction. Spatial separation of the different solid phase coupled receptors would enable identifying such couples. A random mix of the different solid surface coupled host cell receptors without spatial separation between the different receptors however can be easier to produce and can be sufficient for certain applications.

Similarly, embodiments can be envisioned with more than one pathogen surface proteins to be included in the method, wherein the mutants of each of the surface proteins are provided in distinct and differentiable locations.

Furthermore, embodiments can be envisioned with more than one pathogen surface proteins to be included in the method, wherein the mutants of each of the surface proteins are provided as random mixture distributed across a solid surface enabling avidity effects between different molecules of the host with one or more molecules of the pathogen.

In further embodiments of the method of the invention, the pathogen inhibiting immune interactors are coupled to a solid phase for bringing them into contact with the multiple mutants of the one or more pathogen surface proteins or fragments thereof, such as an affinity column, a resin, a column, beads, or a microarray. In such embodiments, the mutants of the pathogen surface protein can be in solution and can be run over or incubated with the solid phase coupled immune interactors, and the non-binders can flow through, in case of a column, or washed off the surface or separated from the surface by other established techniques.

In further specified embodiments, two or more immune interactors are spatially ordered and separated on the solid phase or alternatively in a randomized mix. For example, the case of using more than one distinct immune interactors, such as two different antibodies, the different antibodies can be in different locations of a solid phase, which would enable subsequent determining of which mutant binds to which antibody. This is particularly advantageous in cases where, for example, different (therapeutic) antibodies are tested for their ability to bind to mutants of a pathogen surface protein, since such embodiments would enable determining which pathogen surface protein mutant can bind to which immune interactor/antibody. Spatial separation of the different solid phase coupled receptors would enable identifying such couples. A random mix of the different solid surface coupled immune interactors without spatial separation between the different immune interactors however can be easier to produce and can be sufficient for certain applications.

In embodiments of the method of the invention, the provided one or more host cell receptors or fragments thereof are from a species so far not known to be infected by the pathogen (potential host). Such embodiments are particularly advantageous for identifying potential mutations of a pathogen that is currently circulating in a different host population, preferably not a human population, but which is suspected of being able to make a species shift through mutation. Accordingly, a surface protein of such a pathogen known or suspected to be involved in host cell entry, could be screened for mutations enabling entry into, for example, human host cells.

In a preferred embodiment of the method of the invention, the pathogen is an intracellular pathogen, preferably a virus.

In specific embodiments of the invention, the host cell receptor is ACE2 and the pathogen is a SARS-CoV1 or SARS-CoV2. In an even more specific embodiment, only the SARS-CoV1 or SARS- CoV-2 S-protein or fragements thereof is used as a pathogen surface protein.

In one embodiment, the method as described herein wherein the inhibiting immune interactors directed against the one or more pathogen surface proteins are selected from the group comprising a) Therapeutic and diagnostic immune receptors directed against the one or more pathogen surface proteins; and/or b) serum from a subject having immunity against the circulating pathogen, such as a subject vaccinated against the pathogen, a subject that went through an infection with the pathogen, or a subject that has immunity due to cross-reactive immune receptors, or a subject from another species which cannot be infected by the pathogen but gains antibodies against it.

The method of the present invention can be used to assess the effectiveness of existing therapeutic immune interactors, such as the therapeutic monoclonal antibodies (e.g. monoclonal antibody (mAb) cocktail REGN-COV2 and/or Eli Lilly’s LY-CoV555 human IgG 1 mAb targeting the spike (S) glycoprotein in case of SARS-Cov-2), or the immune interactors comprised by an immune response triggered by an available vaccine (such as the vaccines of BioNTech-Pfizer, AstraZeneca or Moderna against SARS-CoV-2), against mutants of the pathogen surface protein (for example the S- protein of SARS-CoV-2).

In preferred embodiments, an identified binding mutant, immune evasion mutant or emerging mutant is used for designing a preventive, curative or symptomatic treatment agent, such as a vaccine or a therapeutic immune receptor, against the pathogen carrying the emerging mutation.

It is a great advantage of the method of the present invention that it enables the identification of potentially dangerous emerging mutants in a laboratory environment without requiring the generation of the full pathogen before a corresponding mutant pathogen develops and spreads in the present host population, so one can develop new and adjust existing preventive and therapeutic agents before such an event. In another preferred embodiment, the method as described herein, wherein the identification of the binding mutants and/or the immune evasion mutants occurs by means of mass spectrometry analysis, sequencing analysis, by an inhibition assay.

In another preferred embodiment, identifying and potentially verifying the binding mutants and/or immune evasion mutants is achieved via an inhibition assays, especially in an competitive or noncompetitive inhibition assay, where binding of the mutants is assessed in the presence of protective sera or immune reagents protecting against the wildtype of the pathogen. For example, in the presence of protective sera or immune reagents protecting against the wildtype of the pathogen, the mutant do or do not bind onto a microarray yielding the different mutants of the host cell receptor. For the detection of the binding in a preferred embodiment SPR (surface plasmon resonance), SCORE (single color reflectometry) are used for direct detection or traditional fluorescence for indirect detection via staining with a second interacting immune molecule.

Detailed description of the invention

The present invention concerns a method for identifying pathogen mutations enabling host cell entry and immune evasion, the method comprising (a.) providing (i.) one or more host cell receptors or fragments thereof (potentially) involved in host cell entry by the pathogen; (ii.) multiple mutants of one or more pathogen surface proteins or fragments thereof (potentially) involved in host cell entry of the pathogen and/or (potentially) recognized by the host’s immune system; and (iii.) pathogen inhibiting immune interactors directed against the one or more pathogen surface proteins; (b.) bringing the one or more host cell receptors or fragments thereof into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants that bind to the host cell receptors (binding mutants), and (c.) bringing the pathogen inhibiting immune interactors into contact with the multiple mutants of the one or more pathogen surface proteins, and isolating and/or identifying mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors (immune evasion mutants).

Pathogen

As used herein “pathogen” refers to organism that can cause disease in a host organism. The different types of pathogens and the severity of the diseases that they cause are very diverse. A pathogen is an infectious agent and brings disease to its host. As with any organism, pathogens prioritize survival and reproduction. The human body’s immune system acts as a defence against pathogens. The body can easily fight off some pathogens, but others are potentially fatal. As used herein, a pathogen is preferably selected from a group consisting of bacteria, viruses, fungi, pirons, protists and parasitic worm.

Pathogens cause a variety of different diseases, with some being more severe than others. Mammalian and in particular human bodies are nutrient-rich and can provide a pathogen with an ideal environment in which to grow and multiply. Diseases resulting from bacterial pathogens include but not limited to tuberculosis, meningitis, food poisoning, gonorrhoea, typhoid and chlamydia. Diseases resulting from viral pathogens include but not limited to influenza, rotaviruses, measles, mumps, HIV, the common cold caused by coronaviruses, COVID-19 caused by SARS-CoV-2. Diseases resulting from fungi included but not limited to asthma, skin and nail infections, lung infections, such as pneumonia, bloodstream infections and meningitis. Diseases resulting from protozoa include but not limited to dysentery, malaria, African trypanosomiasis or sleeping sickness. The diseases relating to parasitic worm include but not limited to lymphatic filariasis, onchocerciasis and schistosomiasis.

In preferred embodiments, the pathogen of the invention is a coronavirus, more preferably a betacoronavirus, most preferably SARS-CoV1 and SARS-CoV2.

In general, pathogens can be classified to intracellular pathogen and extracellular pathogen on the basis of their site of replication and dependence on host cells.

In preferred embodiments, the method of the invention relates to intracellular pathogens. Intracellular pathogen includes facultative intracellular pathogens and parasites and obligate intracellular pathogens.

Facultative intracellular pathogens comprise, but are not limited to, Bartonella henselae, Francisella tularensis, Listeria monocytogenes, Salmonella Typhi, Brucella, Legionella, Mycobacterium, Nocardia, Neisseria, Rhodococcus equi, Yersinia, Staphylococcus aureus.

Obligate intracellular pathogens include, without limitation , viruses, certain bacteria, including, Chlamydia, and closely related species, Rickettsia, Coxiella, certain species of Mycobacterium such as Mycobacterium leprae, Certain protozoa, including, Apicomplexans (Plasmodium spp., Toxoplasma gondii and Cryptosporidium parvum), Trypanosomatids (Leishmania spp. and Trypanosoma cruzi), certain fungi, Pneumocystis jirovecii. In the context of the invention, obligate intracellular pathogens are preferred.

Host cell receptors and pathogen surface proteins

As used herein “host cell” refers to a cell that is invaded by or capable of being invaded by a pathogen, preferably an intracellular pathogen, such as a virus. The host cell could be found in animal or plant and can be isolated from the host for in vitro analysis.

As used herein “host cell receptor” refers to a molecule on the surface of a host cell (a cell surface protein of a host cell) which is used by the pathogen, such as virus, as an attachment and entry receptor. Host cell receptor can be glycosylated proteins. In embodiments, the host cell receptor can be a sugar molecule or a lipid on the surface of a host cell. In embodiments, certain pathogens can bind to more than one host cell receptor with potentially more than one pathogen surface proteins.

Cell surface molecules that can function as host cell receptors of pathogens (and in particular intracellular pathogens such as viruses). In embodiments, host cell receptors can comprise the molecules comprised by the cluster of differentiation (CD). The cluster of differentiation (also known as cluster of designation or classification determinant and often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules providing targets for immunophenotyping of cells. CD molecules can act in numerous ways, often acting as receptors or ligands important to the cell. CD molecules can often initiate a signal cascade upon engaging with a ligand, altering the behaviour of the cell, for example through initiation of a conformational change or a signal cascade. Some CD proteins do not or not only play a role in cell signalling, but have other functions, such as cell adhesion. CD for humans is numbered up to more than 370 by now. Host cell receptors of pathogens include but are not limited to a-Dystroglycan, Transferrin receptor for arenaviruses, HBGA for Norovirus, Hsp70 for Japanese encephalitis virus, sialic acid for Influenza A, Nephrin B2 for Henipahvirus, DC-SIGN for Bunyavirus, TIM-1 for Hepatitis A virus, CD155 for Polivirus, ICAM-1 Rhinovirus, LDLR for Rhinovirus, LSTs for John Cunningham virus, GM1 for SV40 polyomavirus, JAM for Reovirus, Laminin receptor for Sindbis virus, Nectin-1/2 or HVEM for Herpes simplex virus 1 and 2, SLAM or Nectin-4 for Measles virus, PSGL-1 or SR-B2 for Enterovirus 71 , Glut-1 or Neuropilin-1 for human T-cell leukemia virus, CAR and av integrins for Adenovirus, TIM-1 and NPC1 for Ebola virus, CD81 and SR B1 (claudin-1 and occludin) CD21 and MHC-II for Epstein-Barr virus. DAF and CAR for Coxsackievirus, angiotensin-converting enzyme 2 (ACE2) for SARS-Coronavirus preferably SARS-CoV1 or SARS-CoV2. Further receptors of pathogens and in particular intracellular pathogens have been described in the art and can be identified by the skilled person, for example in the literature.

Intracellular pathogens can engage with one or more cell surface molecules to facilitate entry. Some pathogens, preferably virus particles, use single-receptor species; others use alternative molecules, either of which is sufficient for virus entry, whereas other viruses require a specific combination of receptors.

In the context of the invention, possible pathogens include influenza virus and coronavirus. Host receptor for influenza virus as used herein includes but not limited to Siaa2,3/2,6Gal Receptor, Siaa2,3Gal Receptors, Siaa2,3/2,6Gal Receptors, Neu5,9Ac2, Neu5,9Ac2 and Neu5Gc9Ac Receptors. For coronavirus, described host receptors include, without limitation, Neu5,9Ac2 Receptors, Aminopeptidase N (APN; CD13), a2,3Neu5Gc, fAPN, pAPN, NL63, hACE2 229E, hAPN, 4-O-Ac-Sias, 4,5-di-N-acetylneuraminic acid a-methylglycoside (a-4-N-Ac-Sia), S1-NTD, CEACAM1 , 9-O-Ac-Sias, 4-O-Ac-Sias, 9-O-acetylated sialoglycans, 7,9-di-O-acetyl Sia, 9-O-Ac-Sias, Porcine NCAM, S1-NTD 9-O-Ac-Sia, Neu5,9Ac2, Neu5,9Ac2, RsACE2, Host ACE2, hACE2, DDP4, hDDP4 (CD26), a2,3-linked sialic acids type 1 lactosamines, Nonsialylated type 2 poly-N-acetyl- lactosamines, pAPN in porcine alveolar macrophages.

In embodiments of the invention, the pathogen can be herpes-simplex virus, human papilloma virus, chronic paralysis virus, porcines circoviurs, rotavirus, bluetongue virus, bovine virus, rhinovirus, meales virus, CDV, HIV, HTLV-1 , Hepatitis-B-virus, DHBV. Host cell receptors to be used in the context of the invention therefore include but are not limited to Integrin, HVEM, Nectinl Z2, Integrin, GFR, CD63, CD151 , TfR, Integrin, HSC70, Integrin, TfR, CD63, LDLR, CD46, Nectin4, CD150, CD46, Nectin4, CD150, GLUT1 , ASGPR, NTCP, P80, HSC70, HSC60, P120.

Host cell receptors of the invention further include CD antigens selected from group consist of CD1a, CD1 b, CD1c, CD1d, CD1e, CD2-CD101 , CD105, CD117, CD120a, CD120b, CD127, CD132, CD133, CD134, CD148, CD152, CD154, CD171 , CD235a.

Host cell receptor further include proteins of the human surfaceome of 2886 proteins which are identified with a surfaceome predictor SURFY known in the art. The surfaceome of total 2886 proteins is listed in the research article “the in silico human surfaceome” from Bausch-Fluck et al. in 2018 (PNAS November 13, 2018 115 (46) E10988-E10997).

As used herein the term “host cell entry” refers to the earliest stage of infection in the life cycle of pathogen, as the pathogen comes into contact with the host cell and introduces material into the cell. The way of how a pathogen enters a host cell is different depending on the type of pathogen. In one embodiment the pathogen is a virus with a naked capsid which enters the cell by attaching to the attachment factor (host cell receptor) located on a host cell, making a hole in the membrane of the host cell and inserting the viral genome. In another embodiment, the pathogen is an enveloped virus which attaches to an attachment factor located on the surface of the host cell and then the fusion event occurs. The fusion event is when the virus membrane and the host cell membrane fuse together allowing a virus to enter. It does this by attachment - or adsorption - onto a susceptible cell which holds a receptor that the virus can bind to. The receptors/proteins of the viral envelope effectively become connected to complementary receptors on the cell membrane. This attachment causes the two membranes to remain in mutual proximity, favouring further interactions between surface proteins. This is also the first requisite that must be satisfied before a cell can become infected. Satisfaction of this requisite makes the cell susceptible. Viruses that exhibit this behaviour include many enveloped viruses such as HIV, Herpes simplex virus, coronavirus.

To enter host cells, coronaviruses first bind to a cell surface receptor for viral attachment, subsequently enter endosomes, and eventually fuse viral and lysosomal membranes. A virus surface-anchored spike protein mediates coronavirus entry. SARS-CoV S1 contains a receptorbinding domain (RBD) that specifically recognizes angiotensin-converting enzyme 2 (ACE2) as its receptor. The RBD constantly switches between a standing-up position for receptor binding and a lying-down position for immune evasion. To fuse membranes, SARS-CoV spike needs to be proteolytically activated at the S1/S2 boundary, such that S1 dissociates and S2 undergoes a dramatic structural change. These SARS-CoV entry-activating proteases include cell surface protease TMPRSS2 and lysosomal proteases cathepsins. These features of SARS-CoV entry contribute to its rapid spread and severe symptoms and high fatality rates of infected patients.

As used herein the term “pathogen surface protein” relates to a protein present of the surface of a pathogen, which may be involved in enabling host cell entry by the pathogen. Furthermore, due to their exposition pathogen surface proteins can be targets of the host immune response and may therefore be recognized by various immune receptors, such as antibodies, T cell receptors, and/or innate immune receptors. Pathogen surface molecules can allow a pathogen to adhere to host cells and or host tissue, to invade non-phagocytic epithelia and endothelial cells, and/or to evade immune response. The pathogen surface proteins serve also as good targets for developing new vaccines. For example, generation of an effective immune response, for example antibodies specific for a pathogen surface protein involved in host cell entry, may block the attachment of the pathogen to the host cell surface and therefore prevent a productive infection of the host.

Accordingly, pathogen surface proteins, in particular in case of viruses, are often involved in host cell entry and are often the target of the host’s immune response against the virus. Therefore, a viral surface protein of unknown function is potentially involved in host cell entry and is also a potential target for immune interactors, such as immune receptors.

Pathogen mutation

As used herein the term “pathogen mutations” relates to an alteration in the nucleotide sequence of the genome of the pathogen, including but not limited to insertion mutation, deletion mutation, substitution mutation in nucleic acid sequences. Some mutations occur by chance, when a wrong base is simply incorporated during DNA or RNA replication. In particular, viral and bacterial DNA polymerases can have error rates of one in 10,000 bases. This means, e.g. a corona virus with 30,000 bases could already contain an average of three errors/mutations per replication. In some embodiment, the mutation is inhibitory, i.e. the mutation in the virus affects the replication or function of the virus in an unfavourite way and consequently it cannot be expressed and thus disappears. In another embodiment, the mutation affects the stability, infectivity or replication rate in a favourite way so that such pathogen could replicate faster and more accurate. In a preferred embodiment, the mutant could be SARS-CoV-2 with mutation D614G which help the virus spread all over the world.

As used herein “mutant” refers to a biological entity comprising one or more mutation(s) with respect to a reference, which may be termed the wildtype version. A mutant can be multicellular animal, fungus, virus, unicellular microorganism, such as protists, bacteria, archaea and any polypeptides or fragments thereof which comprises one or more mutation(s). Pathogens and polypeptides expressed by the pathogens or polypeptides of the pathogen expressed by the host comprising one or more mutations are preferred mutants in the context of the present invention. A surface protein of a pathogen which comprises one or more mutations is a more preferred embodiment of a mutant.

In general, a mutation is an alteration in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mitosis, or meiosis or other types of damage to DNA (such as pyrimidine dimers caused by exposure to ultraviolet radiation), which then may undergo error-prone repair (especially microhomology-mediated end joining), cause an error during other forms of repair, or cause an error during replication (translesion synthesis). Mutations may also result from insertion or deletion of segments of DNA due to mobile genetic elements.

In the context of the present invention, the term “multiple mutants of one or more pathogen surface proteins” is understood to comprise more than one variant of a pathogen surface marker protein. This can comprise also a wild-type variant of the pathogen surface marker protein or one or more circulating variants of a pathogen surface marker protein. Preferably, this term comprises a wild-type variant of the pathogen surface protein or fragment thereof, so that the wild-type is present in the method as a reference point to which the determined properties of a mutant can be compared. Accordingly, the it is understood that the method of the invention comprising the provision of multiple mutants of one or more pathogen surface proteins or fragments thereof comprises embodiments, wherein one or more mutants and/or one or more wild-types of one or more pathogen surface proteins or fragments thereof are provided. As used herein, the term “wildtype” refers to a reference sequence of a pathogen surface protein, which is preferably a variant of the protein that has already been described in the art and which is preferably already present in the host population. Since there may be multiple variants of a pathogen circulating in the host population at the same time, these variants can be understood as representing different wildtypes.

In the context of the present invention, a fragment of a host cell receptor or a fragment of a pathogen surface protein is understood as relating to a portion or fragment of the respective protein, that is sufficient to assess binding to a binding partner. Preferably, the fragment is a domain or region of the protein that is known or suspected of mediating interaction with a binding partner and that can be expected to fold in a similar conformation as in the context of the full-length protein. Preferably, protein fragments to be used in the context of the invention have minimum length of at least about 6 AS, such as 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, more preferably about 20, 25, 30, 35, 40, 50, 60 or more AS.

Preferably, protein fragments to be used in the context of the invention have minimum length of at least about 6 AS, and in case of application with an MHC or HLA protein a preferred size of 6, 7, 8, 9, 10, 11 or 12 AS, for binding with TCR (t cell receptors). In further embodiments, protein fragments of preferably about 20, 25, 30, 35, 40, 50, 60 or more AS, or even the whole protein is used, if applied with sera for binding with antibodies.

As used herein “insertion mutant” shall mean a mutant protein or corresponding coding nucleic acid sequence with one or more extra nucleotide(s), which may be added/inserted to the nucleic acid (preferably DNA/RNA) sequence, for example during replication.

The term “deletion mutant” refers to a mutant protein or corresponding coding nucleic acid sequence comprising one or more mutation(s) in which a part of a nucleic acid sequence is lost, for example during replication. Deletion mutants comprise deletions of a single base or base pair of DNA/RNA or loss of larger sequence fragments, such as 2, 3, 4, 5, 6, 7, 8, 9 10, 11 , 12 or more bases or base pairs. In other words, deletion mutants comprise any number of nucleotides deleted from a sequence, from a single base to an entire piece of chromosome.

The term “nucleotide exchange mutant” refers to a biological entity comprising one or more mutation(s) in a nucleic acid sequence in which one or more nucleotides/ bases of a sequence are exchanged for another nucleotide/base, for example during replication. Nucleotide exchange mutants includes one or more changes one or more changed bases in comparison to the reference (wildtype) sequence, preferably 1-30 changes.

As used herein the term “nucleic acid” shall mean any nucleic acid molecule, including, without limitation, DNA, RNA, hybrids or modified variants thereof or mutant thereof.

In the context of the present invention, multiple mutants of a pathogen surface protein or fragments thereof are assessed. The skilled person is aware of methods that enable provision of such multiple mutants, for example in form of a mutant library. Mutant libraries can be used in the context of display screening techniques and have been described in the art. Libraries include site-directed mutagenesis libraries, combinatorial libraries, randomized and degenerated libraries and further examples. Many examples of mutant libraries for used in screening methods have been described (Sidhu et al. Biomol Eng 2001 Sep;18(2):57-63. doi: 10.1016/s1389-0344(01)00087-9; Gera et al. Methods 2013 Mar 15;60(1 ): 15-26. doi: 10.1016/j.ymeth.2012.03.014; Akiba et al. Protein Eng Des Sei 2019 Dec 31 ;32(9):423-431 . doi: 10.1093/protein/gzaa006; Qiu et al. Nat Methods 2018 Nov;15(11):889-899. doi: 10.1038/s41592-018-0189-6. Epub 2018 Oct 30; Schmohl et al. J Pept Sci 2017 Jul;23(7-8):631 -635. doi: 10.1002/psc.2980. Epub 2017 Feb 10).

In regard to the knowledge about the pathogen-host interaction several libraries can be designed and generated. In case a crystal structure is known, dedicated point mutations in the binding site potentially changing binding behavior or substrate specificity (change to bind another human molecule) can be done, either as localized random or computer aided designed approach. If only the molecule is known, then more likely a pure randomized approach either by synthesis or error prone PCR is applied here. 1

Immune interactors

As used herein “pathogen inhibiting immune interactors” refers to immune interactors able to recognise the pathogen and/or pathogen surface protein and preferably affecting the interaction of a pathogen surface molecule with one or more host cell receptors. It is preferred that the provided immune interactors are known or suspected to bind to the wildtype version of the provided one or more pathogen surface molecule and preferably also to interfere with binding of the pathogen surface molecule to the host cell receptor.

Such immune interactors can comprise or be immune receptors, immune cells comprising/carrying immune receptors, including lymphocytes such as, dendritic cells, T-cells and B-cells. Immune interactors further comprise immune receptors such as antibodies, TCRs, chimeric antigen receptors, engineered immune receptors or molecules that bind to pathogen surface molecules. Furthermore, immune interactors can comprise blood or plasma or serum or antiserum samples, preferably antiserum, more preferably antiserum produced from a host, preferably a mammal, immunized with the pathogen surface protein or pathogen or a vaccine against the pathogen.

Furthermore, the term immune interactors comprises artificial molecules interacting with the pathogen as they might to stimulate the immune system or provoke an enhanced immune reaction against the pathogen and/or may slow down or disadvantage the pathogens progression in terms of diffusion, cell entry or growth enabling an advantage to the immunesystem. Such artificial immune interactors may be artificial binders or antibodies as single chain antibodies, nanobodies, DARPINs, affimers, or even small molecules, which may inhibit specifically the host pathogen entry or the replication of the pathogen in the host.

As used herein “immune receptor” is understood shall mean a protein or receptor, which can be located on a cell membrane but can also be soluble, and which binds to a substance (for example, a cytokine or, as in the present case, to a pathogen surface molecule) and which may cause a response in the immune system. Immune receptor comprises pattern recognition receptors (PRRs), killer activated and killer inhibitor receptor (KARs and KI Rs), complement receptor, Fc receptors, B cell receptors, antibodies, T cell receptors, cytokine receptors. The term “cross-reactive immune receptor” refers to immune receptors engaged in a reaction with a first antigen/pathogen and/or an immune response to a first antigen/pathogen and which can additionally bind/react to another antigen/pathogen, which may be structurally similar to the first antigen/pathogen and which is by nature applied to interact with many occurring mutants, which is counter-acted by evasion of said mutations.

As used herein “treatment agent” shall include but is not limited to vaccine, therapeutic immune receptor, therapeutic mRNA/DNA, chemical (small) compound active against the pathogen carrying emerging mutation and which can therefore by used for a therapeutic or preventive treatment. As used herein, “vaccine” comprises “active vaccines” and “passive vaccines”.

As used herein “active vaccine” is a biological preparation that provides active acquired immunity to a particular infectious disease. “Active vaccine” includes inactivated vaccine, attenuated vaccine, toxoid, subunit vaccine, conjugate vaccine, heterologous vaccine, RNA/mRNA vaccine, DNA vaccine. In preferred embodiments, a vaccine induces an immune response against the one or more pathogen surface proteins that are assessed in the method of the present invention. As used herein “passive vaccine” is a material, which is interacting with the pathogen and either marking it, so that the immune system can easier target it, or providing a disadvantage to the pathogen resulting in reduced binding to the host, reduced host entry or reduced replication in the host. Quite often “passive vaccine” is addressing via binding and inhibition surface proteins of the pathogen, “passive vaccine” can be therefore be considered as subspecies of “immune interactors”. Artificial or other immune interactors, such as antibodies, can be used in the context of a passive vaccination. Accordingly, the method of the invention can be used to assess multiple mutants of such surface proteins for binding/recognition by immune interactors that are induced in response to the active vaccine or that can be introduced into the host, for example as a passive vaccine, to find out whether a respective mutant represents a potential threat due to immune evasion gained by the mutation.

Identification of emerging mutants

As used herein “immune evasion” refers to a statistically random but evolutionary probability enhanced strategy used by pathogenic organisms to evade a host’s immune response to maximize their probability of being transmitted to a fresh host or to continue growing. In this process, pathogens use specific mechanisms or mutations to evade recognition by the immune system. For example, in presence of specific antibodies directed against a pathogen surface protein that block, inhibit or attenuate host cell entry by the pathogen, there is a selective pressure for pathogens that have gained mutations of the pathogen surface molecule that are no longer recognized by the host’s immune system by still enable attachment to the host cell receptor for host cell entry.

As used herein “emerging mutant” relates to a mutated form of a pathogen surface protein which is identified as a binding mutant as well as an immune evasion mutant.

In the context of the present invention, it is assessed whether a certain mutant form of a pathogen surface protein can bind to one or more host cell receptors of a certain species. If in the context of the method of the invention the mutant is determined to bind to the provided host receptor, it considered a “binding mutant”. In embodiments, where binding of a wildtype variant of the pathogen surface molecule is occurring, a binding mutant can be a mutant version of the wildtype that despite the mutation still shows binding to the host cells receptor, either stronger binding, about the same binding, or even weaker binding, as long as binding does still occur. Furthermore, in embodiments, where a wildtype variant of the pathogen surface molecule does not bind to the provided host cell receptor, for example because the wildtype variant cannot infect the host species of the provided host cell receptor, a binding mutant is a mutated version of the wildtype pathogen surface molecule that has due to the mutation gained the ability to bind to the host cell receptor from a species so far not being infected by the respective pathogen. In summary, binding mutants in the sense of the invention are variants/mutants of the assessed pathogen surface molecule that bind to one or more of the provided host cell receptors in the context of the method of the invention.

In other words, a mutant which has maintained the (known) ability of a wildtype form of the pathogen surface protein to bind to the host cell receptor, or a mutant which has gained an improved binding in comparison to the wildtype, for example stronger binding/more efficient binding/ binding with a higher affinity as compared to a wildtype, or, if the wildtype did not bind at all to the receptor, gained the ability to bind, can be considered binding mutant. Furthermore, a binding mutant can also be a mutant that binds to the host cells receptor less efficiently than the wildtype, but still with a detectable affinity that would be considered by a skilled person to be sufficient for host cell entry.

In addition to being considered a binding mutant, an emerging mutant of the invention is also considered an immune evasion mutant. As used herein, an “immune evasion mutant” comprises a mutant that is not bound by a provided pathogen inhibiting immune interactor and/or can still bind to the one or more host cell receptors in the presence of the provide immune interactors. In the latter case, an immune evasion mutant is also an emerging mutant since it can bind to the receptor in presence of the immune interactor. Furthermore, immune evasion mutants comprise mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors, mutants not bound by the pathogen inhibiting immune interactors, mutants still binding to the one or more host cell receptors (in the presence of the immune interactors), and mutants whose activity is not inhibited (in presence of the immune interactors).

In preferred embodiments, the provided pathogen inhibiting immune interactors are known to bind to the wildtype variant of the assessed pathogen surface protein and are preferably also known or suspected to inhibit or decrease binding to the one or more host cell receptors. Accordingly, the wildtype variant of the immune pathogen surface protein is bound by the provided immune interactors and preferably cannot bind to the host cell receptor (or at least to a lesser extend) when bound by the immune interactors.

When assessing one or more mutants of the respective wildtype variant of the pathogen surface protein, there are different options with respect to the binding behaviour between the mutants and the immune interactors. There can be binding mutants that are also bound by the immune interactors and for which binding of the immune interactors may also inhibit or decrease binding to the provided host cell receptor. For these mutants, the immune interactors can affect binding of the mutant to the host cell receptor and such mutants are therefore not considered immune evasion mutants and consequently also not emerging mutants.

Furthermore, there will be mutants that do not bind to the host cell receptor and that are also not bound by the immune interactors. Such mutants can be considered immune evasion mutants, because due to the mutation they do no longer bind to the immune interactors. However, since they are no binding mutants, they are also not considered emerging mutants.

Finally, there are binding mutants that in contrast to the wildtype are not bound by the provided immune interactors and/or which can still bind to the host cell receptors in the presence of the immune interactors. These mutants are considered immune evasion mutants in the sense of the invention.

In other words, all mutants that are no longer or clearly weaker bound by the immune interactors that are known to bind to a corresponding wildtype can be considered immune evasion mutants as the cross-reactivity of the immune interactors and the immune receptors have been disabled due to the molecular structure or enzymatic effects of the mutation. Furthermore, all immune evasion mutants that can bind to or interact with host cell receptor can be considered emerging mutants.

In embodiments, a mutant version of a pathogen, preferably a virus, with a mutated version of a pathogen surface molecule involved in host cell entry is considered an emergent pathogen (virus) when it has gained the ability to switch from one species to another species and replicate in that species. In embodiments, spreading of the emergent pathogen in the population of the species is occurring at an unusually rapid rate and with a high incidence. In embodiments, increased binding affinity of the mutant pathogen protein to the host cell receptor can indicate an increased infectivity of the pathogen. In embodiments, the method of the invention comprises the determining of absolute or relative binding affinities of the mutants to the immune interactors and/or the host cell receptors.

Examples of emergent pathogens comprise by the invention are without limitation SARS coronavirus (CoV), SARS-CoV-2, hantaviruses, Ebola and Marburg viruses, Nipah virus, Hendra virus, and human immunodeficiency virus type 1 (HIV-1) and HIV-2, all cross-species host switches of established enzootic viruses that were unknown before their emergences into humans.

An initial level of protection of hosts against viruses occurs at the level of viral entry into the skin or mucosal surfaces or within the blood or lymphatic circulation or tissues. Defences may include mechanical barriers to entry as well as host factors that bind to virion components to prevent infection. For example, glycans or lectins (often called serum or tissue inhibitors) may bind and eliminate incoming viruses. This was seen for human influenza viruses, which may bind to sialylated a-2-macroglobulin in porcine plasma and to alternative sialylated glycoproteins in other animals. Viruses which lack efficient neuraminidase or esterase activity for the glycans of the new hosts may be bound and inactivated, requiring that viruses infecting those hosts rapidly adapt. Galactosyl(a1- 3)galactose is a glycan that is not found in humans but is present on some intestinal bacteria, so that it elicits an antibody response in humans. Virions produced in hosts which have galactosyl(a1- 3)galactose-modified proteins will rapidly be recognized and inactivated by these antibodies when they enter humans, preventing infection.

The initial viral interaction with cells of a new host is a critical step in determining host specificity, and changes in receptor binding often play a role in host transfer. For example, the SARS-CoV was derived from viruses circulating enzootically in a number of bat reservoirs, and the bat-derived viruses interact differently with the angiotensin-converting enzyme 2 (ACE2) receptors of humans and carnivore hosts such as Himalayan palm civets (Paguma larvata), which harbour viruses that are closely related to the human viruses. As another example, feline panleukopenia virus (FPV) changed its host range to infect dogs by binding specifically to the orthologous receptor on the cells of the new host, the canine transferrin receptor. Mammalian and avian influenza viruses bind preferentially to different sialic acids or glycan linkages that are associated with particular hosts. In addition, avian and mammalian viruses infect cells of different tissues and must recognize sialic acids found on cells of the intestinal tracts of waterfowl or in the respiratory tracts of humans or other mammals so that changes in the binding sites can be selected rapidly as the viruses adapt to new hosts. HIV-1 shows some host specificity of binding to the CD4 host receptor and the CCR5 or CXCR4 coreceptors.

Gaining the ability to bind the new receptor effectively may be a complex process and require multiple changes in the virus. For SARS-CoV, the receptor binding motif includes a short region of the S protein which controls specific ACE2 binding; this motif is largely missing from other group 2 CoVs and from related bat CoVs and may have been acquired from a group 1 CoV by recombination with subsequent mutations. In the case of canine parvovirus (CPV), the FPV gained at least two mutations that allowed it to bind effectively to the canine transferrin receptor. The capsid changes were structurally separate in the assembled capsids but acted together to control receptor binding. Viruses that transferred between hosts to gain new host ranges so that they cause outbreak in those new hosts include but are not limited to measles transferred from cattle to humans, smallpox virus from non-human primates or camels to humans, influenza virus from water birds to humans, pigs and horses, CPV from cats or similar carnivores to dogs, HIV-1 old word primates, chimpanzees to humans, SARS-CoV from bats to Himalayan palm civets or related carnivores or humans, Dengue virus from old world primates to humans, Nipah virus from fruit bats to humans (via pigs) or direct bat to human, Marburg virus and Ebola viruses from reservoir host maybe bats to chimpanzees and humans, Myxoma virus from brush rabbit and Brazilian rabbits to European rabbits, Hendra virus from fruit bats to horses and humans, Canine influenza virus from horses to dogs.

In the context of the present invention, the ability of a mutant of a pathogen surface protein to bind to a host cell receptor, and the ability of an immune interactor to bind to the mutant of the pathogen surface protein and to thereby inhibit an interaction of the pathogen surface protein with a host cell receptor is assessed by bringing the potential binding partners into contact with each other. As used herein, “bringing into contact” is understood as meaning co-incubating the potential binding partners under suitable conditions to enable binding. Subsequently, the binding mutants (and potentially the non-binding mutants) are isolated and/or identified. This process can be understood as the assessment/determining and identification of binding partners in the corresponding reaction mix. Such interaction measurements are well known in the art and can be performed in multiple ways, as is known by the skilled person. Depending on the overall experimental setup and the complexity of the mix of potential binders, it is possible to choose a suitable experimental setup for determining/identifying binding partners. This could include one kind of binding partner being immobilized on a solid surface and a further kind of binding partner being randomly distributed in solution.

As for example realized with DNA in the so-called capture SELEX (Stoltenburg et al. Journal of Analytical Methods in Chemistry Volume 2012, Article ID 415697,14 pages, doi: 10.1155/2012/415697) three different DNA-species compete for interaction with each other. In a first step the DNA library is bound onto a surface via a hybridization (interacting) sequence in the middle of the DNA. If the surface is than incubated with a molecular target only those DNA strands which provide a strong interaction with the target get released, as the target binding has to break the hybridization. So only the best binders will be released from the surface.

In embodiments of the invention studying protein interactions using for example phage display, a surface can be covered with the host cell receptor and then the mutations are incubated on the surface. Only those phages providing binding stick to the surface. After a washing step the binders still remain bound on the surface. If now a mixture of immune interactors and/or a given amount of wildtype pathogen protein is provided all mutants which are “weaker” than the wildtype will be washed away. The remaining binders are superior in comparison to the wildtype protein of the pathogen. Carefully removed and amplied the DNA of the phages will allow to reveal the molecular identity of the binders. It has to be tested if the mutant binders are not only stable after having bound to the pathogen receptor, but also if incubated prior to the incubation with immune interactors.

Furthermore, In the state of the art complete interactomes have been revealed via dedicated phage display variants (Sundell et al., BioMed Research International Volume 2014, Article ID 176172, 9 pages, doi. org/10.1155/2014/176172). Furthermore, Yuen et al. showed how small molecule screening can be used to interrogate molecular interactions via microarrays (Methods Mol Biol. 2018;1762:179-197, doi: 10.1007/978-1 -4939-7756-7_10). Uzoma and Zhu gave an overview about which interactions can be addressed by microarray applications (Genomics, Proteomics & Bioinformatics Volume 11 , Issue 1 , February 2013, Pages 18-28).

Techniques for identification and/or isolation of the identified binding mutants, immune evasion mutants are also well established and depend on the system used for studying interaction of the mutants with the potential binders. For example, all binding or non-binding mutant can be collected either by washing them of after incubation (for non-binding mutants), or by eluting the binders with suitable buffers/under suitable conditions after the non-binders have been washed off to remove unspecific mutants and in embodiments also mutants which interacted with the immune interactors preventing them to bind to the host molecules. Identification can be done by sequencing (for example in case of display libraries, such as phage or ribosome display) or by mass spectrometry, which can directly identify the proteins without the detour of the coding nucleic acid sequence.

Immune evasion can be shown by using a neutralization or inhibiting assay, most preferred a binding inhibition assay. In an exemplary embodiment, in case of a neutralization assay the pathogen molecules/mutants can be labelled with a molecular tag or fluorophore or particle and can be incubated with living cells of the host in presence and absence of immune interactors. Wildtype and non-evasive mutants will then be bound by the immune interactors and such will either not bind to the host cells at all or bind to the host cells but will not show activity in terms of enzymatic activity or incorporation into the host cells. Such an assay could be a so called pseudo neutralization assay (see for example products marketed by Berthold Technologies GmbH & Co. KG: https://www.berthold.com/de/bioanalytik/loesungen-sars-cov-2-covid-19-forschung/pseudovirus- neutralization-assays-in-der-sars-cov-2-forschung/), where in the exemplified assay the integration of the pathogen molecule will lead to a luminescence signal of the host cell. A neutralization assay, which shows integration or activity of the pathogen molecule, a binding inhibition assay shows simply that in presence of the immune interactors no binding to the host molecules occurs for the wildtype or non-evasive mutants. Only evasive mutants will bind to the molecules. Corresponding analysis can be done by screening each mutant one after the other, or in a bead-based format as has been described for magnetic beads (Wilson et al, Journal of Biomolecular Screening2015, Vol. 20(2) 292 -298) and fluorescent beads (Suprun et al., ScientificReports (2019) 9:18425, doi.org/10.1038/s41598-019-54868-7), or in the preferred embodiments by presenting distinct mutants on a microarray to analyse binding kinetics directly to one or more host cell molecules with and without the presence of immune interactors. Evasive mutants will then not get affected by the presence of immune interactors, whilst non-evasive mutants and the wildtype will show clear reduced binding. Theory of such assays is known to skilled persons (Xinyi H, Journal of Theoretical Biology, Volume 225, Issue 3, 7 December 2003, Pages 369-376) and applications are described broadly in literature (see for example Wen et al., Intervirology 2017;60:190-195; Beshr et al. MedChemComm Issue 3 2016, doi.org/10.1039/C9MD00165D).

Furthermore, mutants of the pathogen surface protein, host cell receptors and/or immune interactors can be provided on protein arrays. As used herein “protein array” refers to is a device having multiple kinds of proteins linked to a surface in distinct locations which can be used for high- throughput methods used to track the interactions and activities of provided proteins in parallel, and to determine their function, and determining function on a large scale. Detection techniques for identifying an interaction between specific protein spots and provided potential binders and subsequent provision or isolation of interacting proteins of the array are standard techniques in the art. Preparation of protein arrays comprising proteins/peptides, that have been for example identified in a display screening is now a well established techniques (see for example WO2010/100265A1 , WO2012104399A2, W02013/045700A1 , WO2013/113889A1 , WO2013174942A1). In the art is has been shown that pathogen microarrays can be produced (Syafrizayanti et al., Scientific Reports 7:39756, DOI: 10.1038/srep39756) and assayed against a known interacting RNA. Such could be done also for host molecules to identify strong and weak interaction patterns (Syafrizayanti et al., Scientific Reports 7:39756, DOI: 10.1038/srep39756). But such systems are typically limited to the amount of known mutant molecules. The detection of binding is typically done via fluorescence, but to a skilled person many methods are known to detect binding interactions (“Receptor-Ligand Binding Assays”, Konstantin Yakimchuk, DOI //dx.doi.org/10.13070/mm.en.1.199, Cite as MATER METHODS 2011 ;1 :199).

As used herein “display library” relates to the determination of interaction partners of a protein so that the function or the mechanism of the function of that protein may be determined. “Display library” comprises phage display library, cell surface display library, in vitro display library. Phage display library includes but not limited to T4 phage, T4 phage, lambda phage and M13 phage. Cell surface display library includes but not limited to bacterial display, yeast display and mammalian cell display. In vitro display technology includes but not limited to ribosome display, mRNA display and covalent DNA display. Various ways to generate library are known in the art (Sergeeva 2006 Advanced Drug Delivery Reviews 58 (2006) 1622-1654). The skilled person knows a multitude of display methods (see for example Galan et al., Mol. BioSyst., 2016, 12, 2342-2358).

FIGURES

The invention is further described by the following figures. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

Description of the figures:

Figure 1 : Fluorescent data of binding of SARS-CoV2 S-protein mutants. Fluorescent data set of known mutants of S-protein probed with different immune interactors. Differences in binding pattern at e.g. A10. Serum neg shows signal at B2 as this is a EBV protein. Seemingly the person had an EBV infection, whereas the COVID-19 person had not.

Figure 2: Label-free data set of mutants of S-protein probed with different immune interactors. The therapeutic antibody addresses some proteins (white arrow) which are seemingly not important for immunity, whilst others (black arrow) have been addressed by the immune system and the immune interactors, whilst the therapeutic antibody fails. This means that the therapeutic antibody would not help against this mutant and any vaccine based on the target leading to the development of the antibody would fail too. Therefore, it is best if the whole pattern of all mutants will be addressed. Failing spots are needed then for improving existing vaccines.

Figure 3: Schematic representation of a possible workflow of the invention. In the preferred work flow the mutants (1) and a surface presenting the host molecules (2) will be brought into contact (A). Within this contact only the some of the mutants will bind to the host molecules (binding mutants) (4), whilst the non-binders stay in solution (3). The solution is removed (B) and the binders remain. In a elution step (C) the binding mutants (5) are eluted and then incubated with immune interactors

(6) by simple mixing (D). This forms a mixture of mutants with and without immune interactors bound

(7). This mixture is then incubated (E) with another surface presenting host molecules identical to the surface of the previous binding step (2). The now binding mutants (9) evaded the immune interactors (immune evasion mutants), whilst some other mutants failed (8). After removal of the failing mutants (8) the immune evasion mutants can be eluted (F) and finally analysed for their properties. A shortcut workflow may be a direct mixing (G) of the mutants (1) with the immune interactors, wherein the mixture is subsequently brought in contact with the host molecule surface (2). This may lead to a higher proportion of unspecific interactions but can in work as well. Again, all non-binders (8) are removed and in a final elution step (H) the potential evasion mutants are regained and analysed.

EXAMPLES

The invention is further described by the following examples. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration of the invention described herein.

Example 1. Binding inhibition assay shown for known mutants.

Aim:

Screening of S-protein of SARS-CoV2 (pathogen entry molecule into the host) mutants and RBD- sites, as fragments of the S-protein of SARS-CoV2 for interaction with sera and therapeutic antibodies to analyze if there is already an evasive mutation or at least a non-evasive mutation, which has a clearly improved binding against the ACE2 receptor (host molecule entry for the SARS- CoV2 virus) in comparison to the wildtype.

Materials and methods:

Various S-Proteins have been either bought from commercial vendors or produced by standard recombinant production in cells or cell lysates. Protocols for the production of the S-proteins and the RBD sites are published at (see for example Bertoglio et al., bioRxiv, “SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface”, doi.org/10.1101/2020.06.05.135921).

The according proteins have been spotted with a microarray printer, preferably with a Gesim Nanoplotter, and arrayed in replicates to enhance data reproducibility. As microarray surfaces many active binding chemistries are known to the expert and we used our well established PDITC chemistry (see for example Hoffmann et al. RSC Avances Issue 9, 2012, or Whiten et al. “Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells”, ChemBiochem volume 19, Issue 19, 2018).

Proteins were adjusted to a concentration of 200 pg/ml and printed with 2 nl drops on the surface generating a microarray of exemplary 4 blocks, with each block of 4x15 spots with different S- molecules and S-molecule fragments as well as other SARS-CoV 2 related proteins. Then the microarray was applied in a own built SCORE device, which is technical nearly identical to a bscreen device from BERTHOLD TECHNOLOGIES GmbH & Co. KG in terms of detection. It enables for label-free detection of binding between molecules. General assay protocols are described (Kilb et al., ChemBioChem, volume 20, Issue 12, June 14, 2019). For microarray handling the S-protein provider SinoBiological also provides a handbook („Microarray handling"), which is provide with the SinoBiological product Sino Biological's Sinommune™.

Assay:

After blocking of the microarrays with 55 BSA in PBS, the immune interactors have been flushed over the surfaces with the bscreen device. Sera from COVID-19 pos and neg donors as well as therapeutic or diagnostic antibodies have been applied. The antibodies were typically concentrated at 5 pg/ml and sera were diluted 1 :10 in PBS. As following step a human ACE2 receptor was applied either at 5 pg/ml or at 10 pg/ml, but also a concertation series would be possible to refine the kinetic data. Finally staining steps with anti-human antibodies (staining the immune interacting antibodies of the sera or the therapeutic/diagnostic antibodies in one color e.g. red) and an anti-ACE2 antibody (staining bound ACE2 in another color, e.g. green) allows for additional proof if the given mutants have under immune interactors still binding activity.

Results:

As shown in Figure 1 , fluorescence data showed from COVID-19 positive sera that many mutants are addressed, but some molecules are missed for binding e.g. A8, B8, C8 and D8, meaning that no binding immune interactors exist in the sample. Other molecules are bound clearly like e.g. C2, A6 or A10 or weakly like B13. Negative COVID-19 sera showed only marginal binding or to other diseases like EBV (position B2). By comparison it is obvious that both diagnostic antibodies from SinoBiological bind nearly identical in pattern, but shown differences in pattern intensity, e.g. C3 is weaker for MM43 than for MM57. They also miss like the COVID-19 pos sera A8, B8, C8 and D8. But most important is that both antibodies miss the binding of the COVID-19 pos spots like C2, A6 or A10 or B13. In case these antibodies would be applied for therapies they would miss these molecules and may not protect here.

In case of a label-free binding (see Figure 2) also the immune interaction patterns show clear differences. The envisioned therapeutic antibody addresses clearly mutants (white circle), which are only weakly addressed by a COVID-19 pos sera, meaning that these molecules are basically “not important” for the immune system in case of an infection. On the contrary (black arrow) the immune system addresses molecules, which are missed by the therapeutic antibody, meaning that the antibody does not represent a suitable treatment for this mutant and is rendered ineffective.

The data show that distinct mutants show surprising and unpredictable behavior which is even more amplified as the immune system of the host is addressing not all theoretical immune dominant protein parts or molecules of the virus. So a systematic testing of all possible mutations has to be performed. By evolution it is logical that mutants which override the existing diseases need to have an advantage over the old wildtype of the pathogen. This is typically an evasion of immune patterns, better binding or faster infection. The first two can be addressed by this invention.

Discussion:

Even without an extensive screening approach like a phage display it became obvious that a detailed analysis of binding patterns is needed to identify early escape and evasion mutants. The provided data show this for known and in literature described but not yet fully analyzed mutants. The invention covers therefore two important steps, the generation and primary screening of many mutants under conditions which enhance the amplification of evasion mutants against given immune interactors and in a second step the detailed analysis of the strength of the escape. With this knowledge and a reversed analysis which mutant was evading the immune interactors how strongly it will become possible to provide these molecules to vaccine, therapy and diagnostic producers long before these mutants will be originated in nature. We so to say predict and foresee upcoming and uprising mutants before they emerge. This knowledge can be integrated into the development of drugs and therapies, and we will be able to provide defense against diseases before the emerge. We so to say make “future vaccines” to not yet existing diseases and mutants.

Example 2: Applications using Phage display

This example is directed to the S-Protein of SARS-CoV2, but is exemplary also for other pathogens, in particular viruses.

In a first step the DNA encoding the S-protein of SARS-CoV2 is generated, amplified and prepared in cells. Then this DNA is used in an error-prone PCR to generate a number of 10^A5 to 10^A15 different mutants of the S-protein. This can be made in-house or can be purchased like e.g. as so called scaffold library from the company Creative Biolabs. In the next step, preferably by the company Yumab, a phage display will be applied.

For this either whole human cells, fragments of cells, or as known for SARS-COV2, the interacting molecule ACE2 will be coated onto a surface, beads or a column. Then the surfaces are incubated with the phages of the phage library presenting the S-protein mutants. With serial washing steps non-binders will be removed. After said, the remaining binders will be removed with a mild detergent like pH shifted buffer or high salt concentrations and the phages are amplified.

The yet produced new phage pool contains only S-proteins which bind to the ACE2 or the host cells. Aliquots of this pool are then mixed with different immune interactors like sera from COVID-19 negative and COVID-19 positive subjects as well as antibodies for diagnostics and therapeutics against SARS-CoV2. These mixtures are then incubated with the surfaces containing the one or more host cell receptors, such as host cells, parts of them or in the current example ACE2. The now binding phages are evading the immune interactors and still are functional and as such have the potential for being evasion mutants. The DNA from this pools is gathered and purified and then used by BioCopy for their microarray copying approach, meaning that from each DNA strand a digital PCR is made (Wohrle et al. Scientific Reports volume 10, Article number: 5770 (2020)) generating a DNA microarray, which then is used as template to make protein arrays for subsequent analysis (Kilb et al., ChemBioChem, volume 20, Issue 12, June 14, 2019).

In a first step for the analysis one copy of the array is incubated with ACE2 to proof the binding and the activity of the generated proteins and on a second step immune interactors like the sera from COVID-19 or vaccinated persons are applied followed then by ACE2. The mutants which still show binding after this incubation with the immune interactors are lead candidates for immune evasive mutants. These mutant proteins can then be further validated.

Claims

37 CLAIMS

1 . Method for identifying pathogen mutations enabling host cell entry and/or immune evasion, the method comprising a. Providing i. one or more host cell receptors or fragments thereof; ii. multiple mutants of one or more pathogen surface proteins or fragments thereof immobilized on a protein microarray; and iii. pathogen inhibiting immune interactors directed against the one or more pathogen surface proteins; b. Bringing the one or more host cell receptors or fragments thereof into contact with the protein microarray of multiple mutants of the one or more pathogen surface proteins, and identifying mutants that bind to the host cell receptors (binding mutants), and c. Bringing the pathogen inhibiting immune interactors into contact with the protein array of multiple mutants of the one or more pathogen surface proteins, and identifying mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors (immune evasion mutants).

2. The method according to claim 1 , wherein the mutants identified as binding mutants and immune evasion mutants (emerging mutants) are isolated and arranged on a protein array.

3. The method according to any one of the preceding claims, wherein the multiple mutants are analyzed individually for binding to one or more host cell receptors and/or pathogen inhibiting immune interactors.

4. The method according to any one of the preceding claims, wherein the multiple mutants of the one or more pathogen surface proteins or fragments thereof of the protein array have been identified and isolated from a larger pool of multiple mutants of the one or more pathogen surface proteins or fragments thereof as mutants that bind to the host cell receptors by bringing the one or more host cell receptors of fragments thereof into contact with the larger pool of multiple mutants of the one or more pathogen surface proteins or fragments thereof.

5. The method according to any one of the preceding claims, wherein the multiple mutants of the one or more pathogen surface proteins or fragments thereof of the protein array have been identified and isolated from a larger pool of multiple mutants of the one or more pathogen surface proteins or fragments thereof as mutants with decreased binding and/or inhibition by the pathogen inhibiting immune interactors by bringing the pathogen inhibiting immune interactors into contact with the larger pool of multiple mutants of the one or more pathogen surface proteins or fragments thereof.

6. The method according to any one of claims 4-5, wherein the larger pool of the multiple mutants of the one or more pathogen surface proteins or fragments thereof is provided in solution in a display library, such as a phage display library, a ribosome display library, a bacterial display, yeast display or ribosome display. 38 The method according to any one of the preceding claims, wherein each of the multiple mutants of the one or more pathogen surface proteins or fragments thereof is provided in a distinct and preferably known location on the array. The method according to any one of the preceding claims, wherein step b. comprises assessing binding of the multiple mutants of one or more pathogen surface proteins or fragments on the protein microarray to the host cell receptors or fragments thereof to the one or more host cell receptors or fragments thereof, and step c. comprises assessing the binding of the multiple mutants of one or more pathogen surface proteins or fragments on the protein microarray to the pathogen inhibiting immune interactors. The method according to claim 8, comprising deducting from binding data generated by assessing the binding of the multiple mutants of the protein array to the one or more host cell receptors or fragments thereof and/or to the pathogen inhibiting immune interactors a prediction model for binding properties of mutants of the one or more pathogen surface proteins concerning binding to the one or more host cell receptors and/or to the pathogen inhibiting immune interactors. The method according to claim 9, wherein the prediction model uses and/or is based on artificial intelligence analysis of the binding data. The method according to any one of the preceding claims, wherein more than 10^A3, preferably more than 10^A6, most preferably more than 10^A9 mutants of the one or more pathogen surface proteins or fragments thereof are provided. The method according to any one of the preceding claims, wherein the multiple mutants of the one or more pathogen surface proteins are selected from a group comprising - with respect to their coding nucleic acid sequence - nucleotide exchange mutants with preferably 1 - 30 or more changes, nucleotide insertion mutants, nucleotide deletion mutants and/or frameshift mutants, as compared to the coding nucleotide sequence of the pathogen surface protein of a circulating variant of the respective pathogen (wildtype). The method according to any one of the preceding claims, wherein the one or more host cell receptors or fragments thereof are coupled to a solid phase for bringing them into contact with the multiple mutants of the one or more pathogen surface proteins or fragments thereof, such as an affinity column, a resin, a column, beads, or a microarray. The method according to any one of the preceding claims, wherein the pathogen inhibiting immune interactors are coupled to a solid phase for bringing them into contact with the multiple mutants of the one or more pathogen surface proteins or fragments thereof, such as an affinity column, a resin, a column, beads, or a microarray. The method according to any one of the preceding claims, wherein the pathogen is a virus. The method according to any one of the preceding claims, wherein the host cell receptor is ACE2 and the pathogen is a SARS-CoV1 or SARS-CoV2. The method according to any one of the preceding claims, wherein the pathogen inhibiting immune interactors directed against the one or more pathogen surface proteins are selected from the group comprising a. immune receptors, such as therapeutic and diagnostic immune receptors, directed against the one or more pathogen surface proteins; and/or b. serum from a subject having immunity against the circulating pathogen, such as a subject vaccinated against the pathogen, a subject that went through an infection with the pathogen, or a subject that has immunity due to cross-reactive immune receptors, or a subject from another species which cannot be infected by the pathogen but gains antibodies against it. The method according to any one of the preceding claims, wherein the identification of the binding mutants and/or the immune evasion mutants occurs by means of mass spectrometry analysis, sequencing analysis, or by an inhibition assay.