WO2019226050A2

WO2019226050A2 - Novel viral anti-infective reagents

Info

Publication number: WO2019226050A2
Application number: PCT/NL2019/050300
Authority: WO
Inventors: Frederik Anton De Wolf; Aart Van Amerongen; Hubertus Franciscus Jozef Savelkoul
Original assignee: Wageningen Universiteit; Stichting Wageningen Research
Priority date: 2018-05-24
Filing date: 2019-05-23
Publication date: 2019-11-28
Also published as: WO2019226050A3

Abstract

The invention relates to a protein comprising at least one immune stimulating domain, at least one hydrophilic random coil domain, and at least one Virus Entry inhibitor (VEI) domain. The invention further relates to a nucleic acid molecule encoding said protein, to an expression construct comprising said nucleic acid molecule, to a pharmaceutical composition comprising the protein, and to methods of producing the protein.

Description

Title: Novel viral anti-infective reagents

FIELD

The invention is in the field of anti-infective reagents for protecting against virus infection, especially influenza virus infection.

INTRODUCTION

Virus infections, especially influenza virus infections, are a serious threat to poultry industry due to the socio-economic impact of an outbreak. Likewise, elderly humans are very sensitive to influenza infections with considerable health and welfare consequences. In 2014 influenza infections in avian husbandry caused an economic loss of 45 M€ in The Netherlands. It is a zoonotic disease, and can cause death in humans. As a consequence of major outbreaks, the infection is now endemic requiring high control costs. The movement of migratory birds has caused outbreaks to rapidly emerge in several countries and regions simultaneously, including Europe. Such widespread outbreaks could disrupt the global poultry population and global trade.

Influenza also has a significant economic impact, including lost or reduced productivity in the work place or at home due to incapacitating illness, and increased health-care costs from treating the disease. Every year about 300,000 people, mostly elderly, present with signs of acute influenza infection of which around 150 individuals will eventually succumb. Accordingly, interventions to effectively prevent or treat influenza infections in poultry and humans are urgently needed.

There is thus a need for reagents that provide useful protection against viruses such as com on circulating influenza viruses, preferably after a single dose of the reagent. These tools preferably provide cross -protection against related viruses, allowing their use in priming and protecting an immunologic ally naive population against an outbreak of a novel pandemic strain.

SUMMARY OF THE INVENTION

The invention provides an anti-infective chimeric protein comprising at least one immune stimulating domain, at least one hydrophilic random coil domain, and at least one Virus Entry Inhibitor (VEI) domain. Said anti-infective chimeric protein has proven activity against the most vital and most invariant (conserved) part of influenza virus, i.e.. the cell attachment domain. This ensures activity against a large range of virus variants/serotypes and thus, is bound to have high impact based on a range of veterinary and medical applications.

Said at least one immune stimulating domain preferably comprises at least one interferon domain, preferably an interferon-a and/or interferon gamma domain, most preferably a human interferon alpha 1 domain. The at least one Virus Entry Inhibitor (VEI) domain of an anti-infective chimeric protein according to the invention preferably comprises a PDZ interacting domain, preferably from an avian or human influenza virus. Said at least one Virus Entry Inhibitor (VEI) domain preferably is from H1N1 or H7N1 influenza virus.

A preferred VEI domain of an anti-infective chimeric protein according to the invention is or comprises the C-terminal amino acids ESEV-C-terminus or RSKV- C-terminus.

A further preferred VEI domain of an anti-infective chi eric protein according to the invention interacts with one or more PDZ domain of Scribble, preferably human or avian Scribble.

The invention further provides a nucleic acid molecule encoding an anti- infective chimeric protein according to the invention.

The invention further provides an expression construct comprising the nucleic acid molecule according to the invention.

The invention further provides a vector, preferably a viral vector, encoding the anti-infective chimeric protein according to the invention.

The invention further provides a pharmaceutical composition comprising the anti-infective chimeric protein according to the invention, or the vector according to the invention, and a suitable excipient.

The invention further provides anti-infective chimeric protein according to the invention, for use in a method of treating an individual suffering from a viral infection.

The invention further provides a method of producing an anti-infective chimeric protein according to the invention comprising expressing a nucleic acid encoding an anti-infective chimeric protein according to the invention in a relevant cell and isolating the thus produced protein. Said relevant cell preferably is a yeast cell, preferably a cell of a methylotrophic yeast such as Pichia pastoris (now termed Komagatae.Ua phaffii ) .

In a preferred method of producing an anti-infective chimeric protein according to the invention, the at least one hydrophilic random coil domain is used for purification of the protein.

FIGURE LEGENDS

Figure 1. Schematic representation of gel formation by triblock copolymers.

(a) Triblock copolymer with (Pro-Gly-Pro)9 end blocks and random coil mid block.

(b) Detail of a (Pro-Gly-Pro)9 end block in triple-helical form and of a trimeric knot in the gel. Three mid blocks originate from the knot, each of which also has a (Pro- Gly-Pro)9 block at its other end (not shown for clarity) (c) Gel consisting of triblock copolymers. The circles represent the time -averaged space occupied by the random coiled mid blocks. The dark bars represent a random subset of trimeric knots formed between three neighboring chains.

Figure 2. Production of protein polymers (a) SDS-PAGE: lane 1, molecular weight marker; lane 2, culture supernatant of TR4T; lane 3, culture supernatant of TP4T; lane 4, purified TR4T; lane 5, purified TP4T. For culture supernatants, 5 pL was loaded, and for purified proteins, ~20 pg. (b) MALDI of purified TR4T and TP4T. Singly and doubly charged molecular ions are indicated.

DETAILED DESCRIPTION

Definitions

The term“anti-infective reagent”, as is used herein, refers to a

multifunctional chimeric protein that comprises a Virus Entry Inhibitor (VEI) domain, an immune stimulating domain and a hydrophilic, random coil domain.

The term“immune stimulating domain^", as is used herein, refers to a secreted protein, preferably a cytokine, that enhances a cellular immune responses. Such cytokines include type 1 cytokines such as TNF-alpha (TNFu), interleukin-2 (IL2), interferon, IL-12 and TNF-beta (TNFh).

The term“interferon”, as is used herein, refers to a member of the type I interferon family, including IFN-a, IFN-B, IFN-w, IFN-k, and IFN e/x, for which IFN-u is a prototype member, and/or a Type 2 interferon gamma (IFNy).

Type I interferons bind to the IFN-u/B receptor. Type I interferon is expressed rapidly after infection and plays a key role in innate defense against pathogens, especially viral pathogens. In addition, Type I interferons may serve as a link between the innate response and the adaptive immune response, providing both antiviral activity and immunostimulatory functions.

IFNy, or type II interferon, is a cytokine that is critical for innate and adaptive immunity against viral, bacterial and protozoal infections. IFNy is able to inhibit viral replication directly. IFNy binds to a heterodimeric receptor consisting of Interferon gamma receptor 1 (IFNGR1) and Interferon gamma receptor 2

(IFNGR2).

The term“interferon domain”, as is used herein, refers to an active part of an interferon that is able to bind to its interferon receptor and to activate this receptor. Interferon is reported to stimulate production of interferon-induced transmembrane (IFITM) proteins which inhibit virus entry and cell-cell fusion of several viruses, including coronavirus, HIV-1, influenza and Ebola viruses (Li et a , 2015. J Biol Chem 290: 4248-4259).

The term“Type 1 interferon domain”, as is used herein, refers to an active part of a Type 1 interferon that is able to bind to the IFN-u/B receptor and activate this receptor. The term“Type 2 interferon domain”, as is used herein, refers to an active part of a Type 2 interferon that is able to bind to the heterodimeric receptor IFNy receptor and activate this receptor.

The term Virus Entry Inhibitor (VEI) domain, as is used herein, refers to proteineous domain that inhibits attachment and/or entry of a virus to a cell. Said VEI domain preferably is or comprises the receptor and/or co-receptor interacting domain of a virus. For example, said VEI domain may comprise a conserved GPG[RQ] motif (Haqqani and Tilton, 2013. Antivir Res 98: 158-170) from the crown of the third variable loop region of HIV gp!20 protein that provides virus attachment to CCR5 or CXCR4 receptors. As an alternative, or in addition, said VEI domain is or comprises a conserved KKTK consensus motif that attaches to heparin sulfate proteoglycans on a cell surface (Zhang and Bergelson, 2005. J Virol 79: 12125-12131). As an alternative, or in addition, said VEI domain is or comprises a Arg-Gly-Asp (RGD) motif of adenovirus, and/or alternative motifs such as RGG, KGD, KGE, GGG, YGD, LDV and SDI for interaction with inte grins on the cell surface (Azab et ah, 2013. J Virol 87: 5937-5948). A preferred VEI domain is or comprises a DLXXL or RGDLXXL consensus sequence of foot-and-mouth disease virus VP1 capsid protein binding to inte grins (Berryman et ah, 2013. J Virol 87: 8735-8744). As an alternative, or in addition, said VEI domain is or comprises a I.FCF[ [)K|, L[LI][DEN][LF][DE] and/or PWXXW motif for binding to clathrins (Lemmon and Traub, 2012. Traffic 13: 511-519). As an alternative, or in addition, said VEI domain is or comprises a PDZ interacting domain comprising a |8T|CF motif, a FCF motif, and/or a [ϋE]CF motif (James and Roberts, 2016. Pathogens 5: 8). Herein above, the single letter amino acid code is used, wherein X denotes any amino acid residue and F denotes a hydrophobic amino acid residue.

The term“PDZ”, as is used herein, is an abbreviation for post-synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlgl), and zonula occludens-I protein (zo-1). A PSD95/DLG/ZO-1 (PDZ) domain is one of the most widely distributed protein-protein interaction domains. The domain is an evolutionarily conserved domain of approximately 90 amino acids folded into six 6- sheets and two u-helices.

The term“PDZ-interacting domain”, as is used herein, refers to a domain that is capable of interacting with a PDZ domain. A PDZ-interacting domain preferably is a class I interacting domain, comprising the consensus sequence (S/T)CF wherein X denotes any amino acid and F denotes a hydrophobic amino acid. Said consensus sequence preferably is present in the C-terminal region of the anti- infective chimeric protein, most preferably at the carhoxy terminus of the anti- infective chimeric protein.

The term“random coil domain”, as is used herein, refers to a domain that lacks a secondary and tertiary structure. Said random coil domain preferably is biocompatible and has an overall hydrophilic character. A preferred random coil domain in an anti-infective, chimeric protein does not induce and/or stimulate an immune response against the at least one hydrophilic, random coil domain.

Examples of a hydrophilic random coil domains are collagen-like (GXX)n repeats, wherein n is an integer from 3-400, preferably from 30-140, silk-like

[(GAGAGA)mGE]n, [(GAGAGA)mGH]n and/or [(GAGAGA)mGK]n repeats, wherein m is an integer from 1-5, preferably about 3, and n is an integer from 5-60, elastin-like (VPGXG)n repeats, wherein n is an integer from 10-200, preferably from 40-100, (XiP)n repeats (Qi et ah, 2007. Peptide Sci 90: 28-36) and/or (P)n, wherein Xi denotes a hydrophilic amino acid residue preferably threonine and/or serine, and n is an integer from 10-200. In addition, gelatin and/or collagen amino acid sequences, without hydroxylated proline residues, and//or randomized (GXX)n repeats such as (GXX)n(XGX)_m(XXG)₀, wherein each of n, m and o is an integer from 10-200, can be used as a random coil domain as is described in Werten et ah, 1999 (Werten et ah, 1999. Yeast 15, 1087-1096) and in Werten et ah, 2009 (Werten et ah, 2009. Biomacromolecules 10: 1106-1113). Examples of suitable random coil domains are known in the art, for example P” -block domains as described in Werten et ak, 2001. Protein Engin 14: 446-454, and R” -block domains as described in Werten et ak, 2009. Biomacromolecules 10: 1106-1113). Herein above, the single letter amino acid code is used, wherein X denotes any amino acid residue. Said random coil domain preferably provides both flexibility regarding movement of flanking domains with respect to each other and with respect to the cell-bound receptor proteins to which they should bind. Said random coil domain can in addition be used for purification of the anti-infective, chimeric protein.

The term“hinge domain”, as is used herein, refers to a part of a protein that resides in between two independent domains of that protein and that provides flexibility regarding folding of the two independent domains. A hinge region preferably is free of cysteine residues. A random coil domain may function as a hinge region in that it also provides flexibility regarding folding of the two neighboring domains.

The term“individual”, as is used herein, refers to a bird or mammal, preferably an avian species such as a chicken, goose, duck or turkey, or a human.

Anti infective

The present invention is directed to an anti-infective, chimeric protein comprising at least one Virus Entry Inhibitor (VEI) domain, at least one immune stimulating domain and at least one hydrophilic, random coil domain. These anti- infectives consist of safe and highly innovative modular proteins incorporating several modules, each with a different function.

In a preferred anti-infective, chimeric protein according to the invention, said at least one Virus Entry Inhibitor (VEI) domain is or comprises at least one PDZ interacting domain.

A further preferred anti-infective, chimeric protein comprises at least one immune stimulating domain that can stimulate an antiviral immune response, at least one hydrophilic, random coil domain, and at least one Virus Entry Inhibitor (VEI) domain, preferably at least one PDZ interacting domain.

Such a preferred anti-infective, chimeric protein may comprise an immune stimulating domain, a hydrophilic, random coil domain, and at least two VEI domains such as a conserved GPG[RQ] motif and a PDZ interacting domain preferably comprising the consensus sequence (S/T)Xd>.

A further preferred anti-infective, chimeric protein may comprise an immune stimulating domain, a hydrophilic, random coil domain, and at least two VEI domains such as a conserved KKTK consensus motif and a PDZ interacting domain preferably comprising the consensus motif (S/T)CF. A further preferred anti-infective, chimeric protein may comprise an immune stimulating domain, a hydrophilic, random coil domain, and at least two VEI domains such as a conserved RGD consensus motif and/or alternative motifs such as RGG, KGD, KGE, GGG, YGD, LDV and SDI, and a PDZ interacting domain preferably comprising the consensus motif (S/T)CF.

A further preferred anti-infective, chimeric protein may comprise an immune stimulating domain, a hydrophilic, random coil domain, and at least two VEI domains such as a conserved DLXXL or RGDLXXL consensus motif and a PDZ interacting domain preferably comprising the consensus motif (S/T)CF.

A further preferred anti-infective, chimeric protein may comprise an immune stimulating domain, a hydrophilic, random coil domain, and at least two VEI domains such as a conserved EFCF[ϋE], L[LI] [DEN] [LF] [DE] and/or PWXXW consensus motif and a PDZ interacting domain preferably comprising the consensus motif (S/T)CF .

A further preferred anti-infective, chimeric protein may comprise an immune stimulating domain, a hydrophilic, random coil domain, and at least two VEI domains such as a conserved GPG[RQ] motif and a conserved EFCF|DE],

L[LI] [DEN] [LF] [DE] and/or PWXXW consensus motif; a conserved KKTK consensus motif and a conserved EFCF[ϋE], L[LI] [DEN] [LF] [DE] and/or PWXXW consensus motif; a conserved RGD consensus motif and/or alternative motifs such as RGG, KGD, KGE, GGG, YGD, LDV and SDI and a conserved L®X®[DE], L[LI] [DEN][LF] [DE] and/or PWXXW consensus motif, a conserved DLXXL or RGDLXXL consensus motif and a conserved EFCF|DE], L[LI] [DEN] [LF] [DE] and/or PWXXW consensus motif; two conserved EFCF|DE], L[LI][DEN] [LF][DE] and/or PWXXW consensus motifs, a conserved GPG[RQ] motif and a conserved KKTK consensus motif, a conserved RGD consensus motif and/or alternative motifs such as RGG, KGD, KGE, GGG, YGD, LDV and SDI and a conserved KKTK consensus motif, a conserved DLXXL or RGDLXXL consensus motif and a conserved KKTK consensus motif; two conserved KKTK consensus motifs, a conserved LFXF[DE], L[LI] [DEN] [LF] [DE] and/or PWXXW consensus motif and a conserved RGD consensus motif and/or alternative motifs such as RGG, KGD,

KGE, GGG, YGD, LDV and SDI; a conserved L®X®[DE], L[LI] [DEN] [LF] [DE] and/or PWXXW consensus motif, a conserved DLXXL or RGDLXXL consensus motif and a conserved RGD consensus motif and/or alternative motifs such as RGG, KGD, KGE, GGG, YGD, LDV and SDI; a conserved L®X®[DE],

L[LI] [DEN][LF] [DE] and/or PWXXW consensus motif and a conserved RGD consensus motif and/or alternative motifs such as RGG, KGD, KGE, GGG, YGD, LDV and SDI; two conserved RGD consensus motifs and/or alternative motifs such as RGG, KGD, KGE, GGG, YGD, LDV and SDL

Said at least one PDZ interacting domain preferably is from a virus. Said protein can be used to block or hamper attachment of said virus to a PDZ domain- comprising receptor on a cell surface, while simultaneously inducing or stimulating an immune response against the virus by the cell or in the vicinity of the cell by binding of the at least one immune stimulating domain to its receptor.

Said chimeric protein provides a safe, powerful and versatile anti-infective, which can be near 100 % effective in the prevention and/or curing of a broad range of virus infections, especially (avian) influenza viral infections. The number, nature and lay-out of bioactive modules can be straightforwardly varied by design. This means that the design can be modified with minimal effort to confer activity against other viral diseases of animals and humans.

Said at least one immune stimulating domain preferably is or comprises two immune stimulating domains, three immune stimulating domains, four immune stimulating domains, five immune stimulating domains or more than five immune stimulating domain.

Said at least one immune stimulating domain preferably is or comprises an interferon domain, two interferon domains, three interferon domains, four interferon domains, five interferon domains or more than five interferon domains.

Said at least one immune stimulating domain preferably is or comprises an interferon alpha amino acid sequence, more preferably a human interferon alpha 1 amino acid sequence and/or a chicken interferon A1/A2 amino acid sequence.

Said protein may comprise one interferon domain, two type 1 interferon domains, three type 1 interferon domains or more than three type 1 interferon domains.

Said at least one immune stimulating domain preferably comprises an interferon alpha amino acid sequence and/or an interferon gamma amino acid sequence, more preferably a human interferon alpha 1 amino acid sequence and/or a human interferon gamma amino acid sequence. As an alternative, said at least one immune stimulating domain comprises a chicken interferon alpha amino acid sequence, preferably a chicken interferon A1/A2 amino acid sequence and/or a chicken interferon gamma amino acid sequence.

A preferred at least one PDZ interacting domain comprises the consensus sequence (S/T)X<I>, wherein X denotes any amino acid and F denotes a hydrophobic amino acid. The consensus region preferably is present in the carboxy terminal region of the chimeric protein, preferably at the carboxy terminus.

Said at least one PDZ interacting domain is preferably from an avian or human virus, preferably a avian or human influenza virus. Said at least one viral PDZ interacting domain preferably comprises the non-structural protein 1 (NS1), or a C-terminal part thereof comprising the C-terminal PDZ domain at amino acid residues 227-230 of human NS1 and/or at amino acid residues 222-225 of chicken NS1.

A preferred at least one PDZ interacting domain is from H INI or H7N1 influenza virus. Said at least one PDZ interacting domain preferably is or comprises the amino acid sequence ESEV or RSKV in the C-terminal part of the protein. Preferred amino acid sequences of the at least one PDZ interacting domain are provided in Table 1. Table 1. Preferred amino acid sequences of a PDZ interacting domain

An at least one PDZ interacting domain in a protein according to the invention preferably interacts with one or more PDZ domain of Scribbled, preferably human and/or avian Scribble. Scribble protein was identified as a scaffold protein that is involved in cell polarization processes. The protein binds to many other proteins, including influenza NS1 protein and papillomavirus E6 protein. Assays for detecting binding to Scribble are known to a person skilled in the art. Suitable assays are describe, for example, in Liu et ah, 2010. J Virol 84: 11164-11174 and Thomas et ah, 2011. Virol J 8: 25, which are incorporated herein by reference.

The at least one immune stimulating domain, at least one hydrophilic random coil domain and the at least one Virus Entry Inhibitor (VEI) domain are separated by at least one hydrophilic random coil region. Said hydrophilic random coil region provides flexibility and may be used for purification by providing selective, differential precipitation of the chimeric anti-infective protein resulting from changes in pH, temperature or salt strength. In addition, the one or more hydrophilic random coil regions may stimulate secretion of the chimeric anti- infective protein. For example, a chimeric anti-infective protein comprising one or more elastin-like (VPGXG)n repeats, wherein n is an integer from 10-200, preferably from 40-100, may specifically be precipitated by an increase in temperature above 25 °C, preferably above 30 °C, preferably above 35 °G. The increase in temperature may be executed in addition to an increase in salt concentration, for example by addition of sodium chloride.

A chimeric anti-infective protein may in addition comprise a hinge region. A preferred hinge region is a linker polypeptide comprising from 1 to about 60 amino acid residues, preferably from 5 to about 40 amino acid residues, most preferred about 15 amino acid residues such as 10 amino acid residues, 11 amino acid residues, 12 amino acid residues, 13 amino acid residues, 14 amino acid residues, 15 amino acid residues, 16 amino acid residues, 17 amino acid residues, 18 amino acid residues, 19 amino acid residues or 20 amino acid residues. Preferred examples of such amino acid sequences include Gly-Ser linkers, for example of the type (Glyx Sery)z such as, for example, (Gly4 Ser)3, (Gly4 Ser)7 or (Gly3 Ser2)3, as described in WO 99/42077, and the GS30, GS15, GS9 and GS7 linkers described in, for example, WO 06/040153 and WO 06/122825, as well as hinge -like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678). A most preferred hinge region is or comprises a (Gly4 Ser)3 linker. It is noted that a prolonged hinge region, comprising more than 40 amino acids in total, is hydrophilic and may also be used for purification of the chimeric anti-infective protein, for example by precipitation after addition of ammonium sulfate, similar to an hydrophilic random coil domain.

A chimeric anti-infective protein comprising one or more immune stimulating domains such as one or more interferon domains, including type 2 and/or type 1 interferon domains, one or more VEI domain such as one or more PDZ interacting domains, which are separated by at least one hydrophilic random coil domain, may additionally comprise at least one hinge region. Said hinge region may be present in between the individual one or more immune stimulating domains, such as in between the interferon domains and/or in between the individual VEI domains such as in between individual PDZ interacting domains.

As an alternative, or in addition, an amino acid sequence which allows efficient isolation and/or purification of the protein may be present at the N- terminus and/or C-terminus of the chimeric anti-infective protein. Said amino acid sequence preferably comprises at least one tag sequence, preferably repeats of a tag sequence such as a tandem repeat. A preferred tag is selected from HIS, GBP,

CYD, Strep II, V5, FLAG and heavy chain of protein C peptide tags. A preferred tag is the V5 tag, comprising 14 amino acids (GKPIPNPLLGLDST), but which may be used with a shorter 9-amino acid (IPNPLLGLD) sequence. Said at least one tag sequence may be separated from the chimeric anti-infective protein by a recognition and cleavage sequence for an endoprotease. Said endoprotease preferably is a ubiquitous endoprotease such as a subtilisin family member. Said recognition sequence may comprise two adjacent basic amino acid residues such as, for example, the amino acid sequence KR and/or RK. As an alternative, said endoprotease is enterokinase, having the recognition/cleavage sequence

DDDDK | X, and/or a TEV protease with a preferred recognition/cleavage sequence E(N/X)LYFQ | (S,G,A), where X could be any amino acid residue. In addition any other endoprotease that is known in the art to be suitable for this purpose may be employed.

Production of anti infective

A protein according to the invention may be produced using prokaryotic cells or eukaryotic cells, preferably mammalian cells such as CHO cells or HEK cells, fungi such as filamentous fungi, or yeasts such as Saccharom.yc.es cerevisiae or me thy lo trophic yeast such as Pi chi a past oris. Advantages of eukaryotic expression systems are: They are more compatible with (longer) repeating amino acid and DNA sequences such as in poly (GXX), poly (GAGAGAGE), which often result in unwanted recombination and adaptation of the gene in bacteria: They provide efficient protein secretion resulting in high titers which simplifies processing and reduces reprocessing costs; They provide relatively little secretion of other proteins and in particular only limited secretion of proteases that may break down the product. Moreover, eukaryotic cells often carry out desirable post translational modifications that may resemble posttranslational modifications that occur in mammalian cells.

Production of a protein according to the invention in prokaryotic cells, preferably Escherichia coli, may be performed as described in Arbabi-Ghahroudi et a , 2005. Cancer Metastasis Rev 24: 501-519). Production of proteins in bacteria such as E. coli can be performed by secretion of the protein into the periplasmic space, as is known to a person skilled in the art.

A further preferred host cell for production of a protein comprises

mammalian cells such as fibroblasts, Chinese hamster ovary cells, mouse cells, kidney cells, human retina cells, or derivatives of any of these cells. A most preferred cell is a human cell such as, but not limited to, Hek293 and PER.C6.

Production of proteins in filamentous fungi is preferably performed as described by Joosten et a , 2005. J Biotechnol 120: 347-359. Methods of producing proteins in Saccharomyces cerevisiae are known in the art, for example as described by van der Laar et ah, 2007. Biotech Bioeng 96: 483-494; or Frenken et a , 2000. J Biotechnol 78: 11—21.

A most preferred method of protein production is by expression in a methylotrophic yeast strain. Methylotrophic yeast are those yeast genera capable of utilizing methanol as a carbon source for the production of the energy resources necessary to maintain cellular function and containing a gene for the expression of alcohol oxidase. Typical methylotrophic yeasts include members of the genera Pichia, Hansenula, Torulopsis, Candida, and Karwinskia. These yeast genera can use methanol as a sole carbon source. In a more preferred embodiment, the methylotrophic yeast strain is Pichia pas tor is (Komagataella phaffii). Pichia in particular is beneficial in this respect, when compared to other fungi, as it is a GRAS-status production organism and no endotoxin is produced. Furthermore,

FDA has approved the presence of killed Pichia, or remnants thereof, in the final product to a level of 10% (w / w).

A preferred Pichia is GS115, available for example from Thermo Fisher Scientific (Pittsburgh, PA 15275), and wild type Pichia strains that are rendered auxotrophic by functional deletion of a marker protein. Methods for protein production in P. pastoris are known in the art. Preferred methods are described in Werten et al., 1999. Yeast 15, 1087-1096), Werten et a , 2001. Protein Engin 14: 446-454 and Werten et al., 2009. Biomacromolecules 10: 1106-1113.

A protein according to the invention is preferably produced by the provision of a nucleic acid encoding said protein to a cell of interest. Therefore, provided herein is a nucleic acid encoding a protein according to the invention. Said nucleic acid, preferably DNA, is preferably produced by recombinant technologies, including the use of polymerases, restriction enzymes, and ligases, as is known to a skilled person. Alternatively, said nucleic acid is provided by artificial gene synthesis, for example by synthesis of partially or completely overlapping oligonucleotides, or by a combination of organic chemistry and recombinant technologies, as is known to the skilled person. Said nucleic acid is preferably codon-optimised to enhance expression of the protein in the selected cell or cell line. Further optimization preferably includes removal of cryptic splice sites, removal of cryptic polyA tails and/or removal of sequences that lead to unfavourable folding of the mRNA. The presence of an intron flanked by splice sites may encourage export from the nucleus. In addition, the nucleic acid preferably encodes a protein export signal for secretion of the protein out of the cell into the periplasm of prokaryotes or into the growth medium, allowing efficient purification of the protein.

Said nucleic acid molecule encoding said protein, preferably a codon- optimized nucleic acid, preferably is present in an expression construct allowing optimal expression of the protein in a host cell. Said nucleic acid molecule preferably is optimized for expression in a methylotrophic yeast, preferably in Pichia pastoris . Said expression construct preferably comprises a protein export signal for secretion of the protein out of Pichia pastoris into the growth medium, allowing efficient isolation and purification of the protein from the growth medium. A preferred construct for expression in P. pastoris is pPIC or a pPIC derivative, available for example from Thermo Fisher Scientific (Pittsburgh, PA 15275).

Said infective chimeric protein according to the invention in a host cell preferably is produced in a fermenter or fermentation chamber. Said fermenter preferably is a stirred tank bioreactor, preferably a continuous stirred-tank reactor (CSTR), also known as backmix reactor.

The invention further provides a method of producing a protein of the invention, the method comprising expressing a nucleic acid encoding a protein of the invention in a relevant cell and recovering the thus produced protein from the cell or, preferably, from the supernatant. The nucleic acid, preferably a vector comprising an expression construct, is preferably provided to a cell by transfection or electroporation. The nucleic acid is either transiently, or, preferably, stably provided to the cell. Methods for transfection or electroporation of cells with a nucleic acid are known to the skilled person. A cell that expresses high amounts of the protein may subsequently be selected. This cell is grown, for example in roller bottles, in fed-batch culture in a Continuous Stirred Tank Reactor, or in continuous perfusion culture. An intermediate production scale is provided by an expression system comprising disposable bags and which uses wave-induced agitation (Birch and Rancher, 2006. Advanced Drug Delivery Reviews 58: 671- 685).

Methods for purification of proteins are known in the art and are generally based on chromatography, such as protein A affinity and ion exchange, to remove contaminants. In addition to contaminants, it may also be necessary to remove undesirable derivatives of the product itself such as degradation products and aggregates. Suitable purification process steps are provided in Berthold and Walter, 1994. Biologicals 22: 135- 150. Said purification process may involves affinity binding to a tag that is comprised in the protein, preferably a V5 tag.

Said methods preferably include bulk precipitation of the secreted product from the growth medium or from the disrupted cells by means of the elastindike region and/or the use of tags or hinge regions. At least the elastindike region responds to stimuli such as pH shift, temperature increase, and/or salt addition by precipitation, allowing differential precipitation of the antidnfective chimeric protein. The multi-block (multi -domain) chimeric proteins do not denature under harsh conditions such as elevated temperatures, as do globular proteins. As a result, anti-infective chimeric proteins are easy to handle in downstream processes under elevated or even extreme conditions. Preferred methods are described in Werten et ak, 1999. Yeast 15: 1087-1096, Werten et ah, 2001. Protein Engin 14: 446-454 and Werten et ah, 2009. Biomacromolecules 10: 1106-1113.

Further provided is a host cell comprising a nucleic acid or vector that encodes a protein according to the invention. Said host cell may be grown or stored for future production of a protein according to the invention.

In vivo expression of anti infective

Further provided is a vector, preferably a viral vector, encoding the protein according to the invention. Said vector preferably additionally comprises means for high expression levels such as strong promoters, for example of viral origin (e.g., human cytomegalovirus) or promoters derived from genes that are highly expressed in a cell such as a mammalian cell (Running Deer and Allison, 2004. Biotechnol Prog 20: 880-889; US patent No: 5888809).

Said vector preferably is a viral vector, preferably a viral vector that is able to transduce mammalian cells such as avian and human cells.

For expression in humans, said viral vector preferably is a recombinant adeno-assoeiated viral vector, a herpes simplex virus-based vector, or a lentivirus- based vector such as a human immunodeficiency virus-based vector. Said viral vector most preferably is a retroviral-based vector such as a lenti virus -based vector such as a human immunodeficiency virus-based vector, or a gamma-retrovirus- based vector such as a vector based on Moloney Murine Leukemia Virus (MoMLV), Spleen-Focus Forming Virus (SFFV), Myeloproliferative Sarcoma Virus (MPSV) or on Murine Stem Cell Virus (MSCV). A preferred retroviral vector is the SFG gamma retroviral vector (Riviere et ah, 1995. PNAS 92: 6733-6737).

Retroviruses, including a gamma-retrovirus-based vector, can be packaged in a suitable complementing cell that provides Group Antigens polyprotein (Gag)- Polymerase (Pol) and/or Envelop (Env) proteins. Suitable packaging cells are human embryonic kidney derived 293T cells, Phoenix cells (Swift et ah, 2001. Curr Protoc Immunol, Chapter 10: Unit 10 17C), PG13 cells (Loew et ah, 2010. Gene Therapy 17: 272-280), and Flp293A cells (Schucht et ah, 2006. Mol Ther 14: 285- 92). For expression in human and avian species such as chicken or goose, a recombinant Newcastle disease virus preferably is used as a viral vector. Methods for generation of recombinant Newcastle disease virus are known in the art, for example as described in Zhao and Peeters, 2003. J Gen Virol 84: 781-8.

Viral expression in vivo preferably is directed at targeting the epithelial lining of the respiratory tract.

As an alternative, non-viral gene therapy may be used for in vivo expression of a protein according to the invention in relevant cells such as the epithelial lining of the respiratory tract. Non-viral delivery may be provided by, for example, nude DNA, liposomes, polymerizers and molecular conjugates, as is known to a skilled person. Minicircle DNA vectors free of plasmid bacterial DNA sequences may be generated in bacteria and may express a nucleic acid encoding a protein according to the invention at high levels in vivo.

Medicament comprising anti infective

The invention further provides a protein according to the invention or a vector according to the invention, for use as a medicament, preferably a

medicament for treatment of a viral infection, preferably influenza.

For example, viruses comprising a PDZ-interacting domain include influenza virus, SAKS coronavirus, neurotropic rabies virus, flavivirus, hepatitis viruses B and C, Kaposi sarcoma herpesvirus, human T cell leukaemia virus type 1, high-risk human papillomaviruses, human immunodeficiency virus typo 1, and adenovirus type 9. An anti-infective chimeric protein comprising at least one immune stimulating domain, at least one hydrophilic random coil domain, and at least one Virus Entry Inhibitor (VEI) domain and in which at least one of the VEI domains is a PDZ-interacting domain may be used to prevent subsequent infection with one or more of the above indicated viruses, and/or to ameliorate symptoms in an individual or even treat an individual that is suffering from an infection with one or more of the above indicated viruses.

A protein according to the invention or a vector according to the invention is preferably used for prophylactic administration or therapeutic administration in avian species or humans that are suffering from a viral infection, preferably influenza. Thus, a protein according to the invention or a vector according to the invention may be administered to an individual that is suspected of suffering from a viral infection, or may be administered to an individual already evidencing a viral infection in order to lessen signs and symptoms of said viral infection.

The administration of a protein according to the invention or a vector according to the invention is preferably provided in an effective amount to an individual in need thereof. An effective amount of a protein according to the invention or a vector according to the invention is a dosage large enough to produce the desired effect in which the symptoms of the viral infection are ameliorated or the likelihood of a viral infection is decreased. A therapeutically effective amount preferably does not cause adverse side effects. Generally, a therapeutically effective amount may vary with the individual's age, condition, and sex, as well as the extent of the infection and can be determined by one of skill in the art.

The dosage may be adjusted by the individual physician in the event of any complication. A therapeutically effective amount may vary from about 0.01 mg/kg to about 500 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose

administrations daily, for one or several days. Preferred is administration of the protein according to the invention or a vector according to the invention for 2 to 5 or more consecutive days in order to effectively treat a viral infection.

A protein according to the invention or a vector according to the invention can be administered by injection or, preferably, by inhalation. Inhalation may be effected by, for example, a portable inhaler and/or the use of aerosols, for example by employing an inhalation nebulizer including an aerosol generator.

If required, a bioadhesive film comprising the protein and/or vector according to the invention may be generated in situ, as is described in US5912007. For this, the protein and/or vector is administered in the presence of an ionic polysaccharide and a cross-linking agent. These components are provided in a carrier which is a confectionery material. Following dissolution through salivation of the carrier, the cross-linking agent may polymerize the ionic polysaccharide to form a film on the buccal epithelial cells in the upper respiratory tract. In addition, domains may be incorporated that form a sticky, self-healing gel that can provide adherence to a mucus layer and/or the buccal epithelial cells in the upper respiratory tract, and/or domains that can interact with extracellular matrix collagen by triple helix hybidization. For example, at least two (Pro-Gly-Pro)n domains, whereby n = 9-12, in a protein chain that are interspersed with a hydrophilic random coil domain may form trimers that interconnect the three chains and form nodes in a gel (Werten et a , 2009. Biomacromolecules 10: 1106-1113). Said trimers may include naturally occurring collagen from an extracellular matrix (Luo et a , 2017.

Biomacromolecules 18: 2539-2551; Wan et ah, 2008. Biomacromolecules 9: 1755- 1763). In addition, polysaccharide chains interacting with the mucous layer can optionally be added, either by chemical addition or by natural glycosylation.

A protein according to the invention or a vector according to the invention may be administered to avian species such as chicken by spray formation, for example by employing sprinklers or backpack sprayers. As is known to a person skilled in the art, the size of the droplets may be controlled for an effective spray vaccination for example by controlling spray pressure and the use of calibrated nozzles.

Preparations for administration to the respiratory tract include sterile aqueous or non- aqueous solutions, suspensions, and emulsions. Examples of non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The invention further provides a pharmaceutical composition comprising a protein according to the invention or a vector according to the invention. A pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier. A carrier, as used herein, means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient.

The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration.

Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts buffers, stabilizers, solubilizers, and other materials which are well known in the art.

The invention further provides the protein according to the invention or a vector according to the invention for use in a method for treatment of a viral infection.

The invention further provides a method of treating an individual suffering from a viral infection, said method comprising providing a protein according to the invention or a vector according to the invention to an individual in need thereof to thereby treat the individual.

The invention further provides use of a protein according to the invention or a vector according to the invention in the preparation of a medicament for treating an individual suffering from a viral infection.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate aspects and preferred embodiments thereof, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

The invention will now be illustrated by the following examples, which are provided by way of illustration and not of limitation and it will be understood that many variations in the methods described and the amounts indicated can be made without departing from the spirit of the invention and the scope of the appended claims.

EXAMPLES

Example 1 (taken from Werten et al., 2009. Biomacromolecules 10: 1106-1113)

In this example, a T-C-T triblock is generated in which T = trimer forming (PGP)n and C = hydrophilic random coil. This patent application concerns a triblock of (1) immunostimulatory (IS) domain, (2) hydrophilic random coil and (3) VEI domain. The T-blocks in T-C-T can easily be replaced by other sequences such as one or more IS domains and/or one or more VEI domains.

Experimental Section

Construction of Expression Vectors. Triple helix-forming block T was prepared by PCR using the oligonucleotides T-FW and T-RV (Table 2). The ~0.1 kb product was cloned into vector pCR4-TOPO (Invitrogen), resulting in vector pCR4-TOPOT. The previously described vector pMTL23-P4 (Werten et a , 2001. Prot Eng 14: 447-454) contains a gene encoding the custom-designed, highly hydrophilic 36.8 kDa collagenous protein“P4”. This vector was digested with Drain (5^’ to the P4 gene) and dephosphorylated. Vector pCR4-TOPO-T was digested with lira 111 /Van 91 1.

The released T block was ligated into the linearized and dephosporylated vector, to yield vector pMTL23-TP4. This vector was then digested with Van 91 1 (3’ to the P4 gene) and dephosphorylated, and a second DraIII/Van91I digested T block was inserted to yield vector pMTL23-TP4T. The TP4T gene was cloned into P. pastoris expression vector pPIC9 (Invitrogen) via XhoI/EeoRI.

The gene encoding R4 was constructed by concatenating four copies of an R gene monomer. The monomeric gene was designed by randomizing the sequence of the P gene monomer (Werten et ah, 2001. Prot Eng 14: 447-454) in such a way that not every third residue of the encoded protein is glycine, preventing the formation of collagen triple helices while maintaining the same amino acid composition. The R gene monomer was constructed by overlap extension PCR (Ho et ak, 1989. Gene 77: 51-59) using oligonucleotides RA-FW and RA-RV for the 5’ half of the gene, and oligonucleotides RB-FW and RB-RV for the 3’ half (Table 2). The products of these reactions were combined by overlap extension PCR to generate the entire R gene monomer. The monomer was cloned into vector pMTL2344 via XhoI/EcoRI and multimerized via DraIII/Van91I, as described previously for P4 (Werten et ak,

2001. Prot Eng 14: 447-454) resulting in vector pMTL23-R4. The R4 fragment was cloned into expression vector pPIC9 via XhoI/EcoRI. The construction of pMTL23- TR4T and subsequent subcloning into pPIC9 was analogous to the procedures described for TP4T.

The DNA sequences (and translated amino acid sequences) of the XhoI/EcoRI fragments encoding P4,25 R4, TP4T, and TR4T have been deposited in Gen Bank under accession numbers EU834225-EU834228. Although, for clarity, the T blocks are referred to as (Pro-Gly-Pro)9 in the main text, the cloning procedure results in a Gly-Pro-Pro-Gly-Ala extension at the N-terminus, and an Ala-Gly-Gly extension at the C-terminus.

Transformation of P. pastoris. Expression vectors were linearized with Sall to promote integration at the his4 locus rather than the AOX1 locus, thus enabling normal growth on methanol (Gregg et al., 1985. Cell Biol 5: 3376-3385).

Transformation of P. pastoris his4 strain GS11545 and selection of transformants was as described previously (Werten et al., 1999. Yeast 15, 1087-1096).

Fermentation of P. pastoris. Fed-batch fermentations were performed in 2.5-F Bioflo 3000 fermenters (New Brunswick Scientific), essentially as described by Zhang et al (Zhang et al., 2000. Biotechnol Bioeng 70: 1-8). Minimal basal salts medium was used and no protease -inhibiting supplements were added. The pH was maintained at 3.0 throughout the fermentation by addition of ammonium

hydroxide as base. The methanol fed-batch phase for protein production lasted two to three days. A homemade semiconductor gas sensor-controller, similar to that described by Katakura et al. (Katakura et al., 1998. Ferment Bioeng 86: 482-487) was used to monitor the methanol level in the off-gas and to maintain a constant level of ~0.2% (w/v) methanol in the broth. At the end of the fermentation, the cells were separated from the broth by centrifugation for 10 min at 10000 x g (RT) in an SLA-3000 rotor (Sorvall), and the supernatant was microfiltered.

Protein Purification. All centrifugation steps were performed in an SLA- 1500 or SLA-3000 rotor (Sorvall) for 30 min at 20000 x g and resuspension of protein pellets was always in Milli-Q water at 65 °C. As a precaution, the cell-free broth was heated for 30 min at 65 °C to melt possible gel structures formed by the recombinant protein. The pH was raised to 8.0 by addition of sodium hydroxide to allow precipitation of medium salts by centrifugation (RT). The protein was precipitated from the supernatant by addition of ammonium sulfate to 40% of saturation, followed by incubation on ice for 30 min and centrifugation (4 °C). This precipitation procedure was repeated once, and 50 mM sodium chloride and 40% (v/v) acetone were added to the final resuspended pellet. After centrifugation (4 °C), the pellet was discarded and acetone was added to the supernatant up to 80% (v/v). The protein pellet obtained after centrifugation was air-dried, resuspended, and desalted by extensive dialysis against Milli-Q water. The final product was lyophilized.

SDS-PAGE and N-Terminal Protein Sequencing. The NuPAGE Novex system (Invitrogen) was used for SDS-PAGE, with 10% Bis-Tris gels, MES SDS running buffer, and SeeBlue Plus2 prestained molecular mass markers. Gels were stained with Coomassie SimplyBlue SafeStain (Invitrogen). Blotting of proteins for N- terminal sequencing by Edman degradation was as described previously ((Werten et ah, 1999. Yeast 15, 1087-1096). Protein sequencing was performed by Midwest Analytical (St. Louis, MO).

Mass Spectrometry. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was performed using an Ultraflex mass spectrometer (Bruker). Samples were prepared by the dried droplet method on a 600 I Im AnchorChip target (Bruker), using 5 nig/mL 2,5-dihydroxyacetophenone, 1.5 mg/mL

diammonium hydrogen citrate, 25% (v/v) ethanol, and 1% (v/v) trifluoroacetic acid as matrix. Measurements (50 shots at 20 Hz) were made in the positive, linear mode, with the following parameters: ion source 1, 20000 V; ion source 2, 18450 V; lens, 5000 V; pulsed ion extraction, 550 ns. Protein Calibration Standard II

(Bruker) was used for external calibration.

Results

Rationale for Nonhydroxylated Precision Gels. As an example of the proposed concept, two ABA type triblock copolymers designated TP4T and TR4T are described, but many other functional multiblock configurations are conceivable.

The amino acid sequences of all protein polymers described in this work are available through GenBank (accession numbers ACF33476-ACF33479). The triple helix-forming T blocks at both ends of the triblock copolymers consist of (Pro-Gly- Pro)n homopolymeric stretches (Figure la). On the basis of helix melting studies with chemically synthesized (Pro-Pro-Gly)n (Engel et al, 1977. Biopolymers 16: 601-622; Frank et ak, 2001. Mol Biol 308: 1081-1089; Sutoh and Noda, 1974.

Biopolymers 13: 2391-2404) the length of n was tentatively chosen to be nine to provide a melting point in a biomedically relevant range. The middle section of the molecule acts as a hydrophilic spacer (Figure la) and consists of either of two random coiled block variants. The first is P4, a synthetic gelatin-like molecule previously developed by our group (Werten et ak, 2001. Protein Engin 14: 446-454). It is extremely hydrophilic, acts as a cytophilic protein in human cell culture (Rozkiewicz et ak, 2006. Chemistry 12: 6290-6297) and shows outstanding biocomp atibility as a plasma expander (U.S. Patent application US2005/0119170). Despite P4’s collagenous primary structure, it does not form detectable triple helices at 4 °C because of the absence of 4-hydroxyprolines (Werten et ak, 2001. Protein Engin 14: 446-454). It cannot, however, be excluded beforehand that P4 might play a minor role in network formation in the presence of the proline-rich trimer-forming T blocks. Therefore, a second type of mid block, R4, was

constructed. It has the same amino acid composition as P4, but its protein sequence is quasi-random in that it does not have glycine as every third residue. Thus, R4 by definition cannot form triple helices. Because both the end blocks and mid blocks have a defined length and composition, and because trimerization of the mid blocks is unlikely (P4) or impossible (R4), the resulting gels are expected to contain only cross-links made-up of (Pro-Gly-Pro)9 (Figure lb,c). This is in stark contrast with traditional gels prepared from animal gelatin, where all molecules have a different makeup, and the entire chain engages in network formation.

Biosynthesis of Triblock Copolymers. P. pastoris strains expressing TR4T and TP4T as extracellular proteins were constructed and grown in bioreactors. Culture supernatants were analyzed by SDS-PAGE (Figure 2a). The proteins were purified from the cell-free broth essentially by differential ammonium sulfate precipitation, similarly to the purification of P4 (Werten et ah, 2001. Protein Engin 14: 446-454). The purity of the proteins was estimated to be at least 99%, based on amino acid analysis and subsequent linear least-squares fitting to the observed data of (1) the theoretical composition of the respective pure protein and (2) the composition determined for host-derived proteins present in the medium. Purified TR4T and TP4T migrated as single bands in SDS-PAGE (Figure 2a), indicating their purity and intactness. MALDI mass spectrometry confirms this observation and shows that the molecular weight of the proteins is within experimental error of the expected value of 41741 Da (Figure 2b). Clearly, the proteins migrate abberantly in SDS-PAGE, as was demonstrated previously for P4 (Werten et al, 2001. Protein Engin 14: 446-454). N-terminal sequencing of the hands separated by SDS-PAGE further confirmed the identity of the products. A minor fraction (~15%) of the molecules had a single N-terminal Glu-Ala extension, which is known to occasionally occur because of partial processing of the R-factor prepro secretory signal by the P. pastoris dipeptidylaminopeptidase (Werten et ah, 1999. Yeast 15, 1087-1096). Judging from the intensity of the bands in SDS-PAGE, the volumetric productivity of TR4T and TP4T appears comparable to that of P4, which is produced at 3-6 g/L of clarified broth (Werten et ah, 2001. Protein Engin 14: 446- 454).

Example 2

Primary epithelial cells of chicken, the human BEAS2B bronchial epithelial, the gingival/oral Ca9-22/H01Nl cells and Detroit-562 upper airway epithelial cell lines is used for functional testing of cellular activation, proliferation, receptor expression, tight junction genes and protein analysis and barrier function, cytokine synthesis) of the efficacy in vitro of this treatment in poultry and in humans, respectively.

qPCR for detection of responding early genes and multiplex cytokine assays are used for transcriptome analyses of infected epithelial cells and influenza virus. In addition, in vivo testing is performed in chicken.

Constructs that are tested express the anti-infective chimeric proteins depicted in Table 3 in Pichia pastoris. The resultant proteins will be tested in primary epithelial cells of chicken (protein numbers 3 and 4), the human BEAS2B bronchial epithelial, the gingival/oral Ca9-22/H01Nl cells and Detroit-562 upper airway epithelial cell lines (protein numbers 1 and 2).

For this, methanol fed-batch fermentations of transformed Pichia strains at 30°C and pH 3 are performed in 2.5-L Bioflo 3000 hioreactors (New Brunswick

Scientific). At. the end of fermentation, the cells are separated from the broth by centrifugation for 10 min at 10,000 x g (RT) in an SLA-3000 rotor (Sorvall), and the supernatant is microfiltered. Proteins are purified from the supernatant by selectively precipitating twice at 45% ammonium sulfate saturation followed by dialysis, filter sterilizing (0.2 mhi), and lyophilizing.

It will be clear to a person skilled in the art that the domains listed in Table 3 do not necessarily have to be used in that combination but modules indicated in the columns may be combined randomly. In addition, neither the order of the modules is fixed, provided that the first module will start with a methionine.

Claims

1. An anti-infective chimeric protein comprising at least one immune

stimulating domain, at least one hydrophilic random coil domain, and at least one Virus Entry Inhibitor (VEI) domain.

2. The anti-infective chimeric protein according to claim 1, wherein the at least one immune stimulating domain comprising at least one interferon domain, preferably an interferon-a and/or interferon gamma domain.

3. The protein of claim 1 or claim 2, wherein the at least one immune

stimulating domain is or comprises a human interferon alpha 1 domain.

4. The protein of any one of the previous claims, wherein the at least one Virus Entry Inhibitor (VEI) domain comprises a PDZ interacting domain, preferably from an avian or human influenza virus.

5. The protein of any one of the previous claims, wherein the at least one Virus Entry Inhibitor (VEI) domain is from H1N1 or H7N1 influenza virus.

6. The protein of any one of the previous claims, wherein the at least one Virus Entry Inhibitor (VEI) domain is or comprises the C-terminal amino acids ESEV-C- terminus or RSKV-C-terminus.

7. The protein of any one of the previous claims, wherein the Virus Entry Inhibitor (VEI) domain interacts with one or more PDZ domain of Scribble, preferably human or avian Scribble.

8. A nucleic acid molecule encoding the protein according to any one of claims 1-

7.

9. An expression construct comprising the nucleic acid molecule of claim 8.

10. A vector, preferably a viral vector, encoding the protein according to any one of claims 1-7.

11. A pharmaceutical composition comprising the protein according to any one of claims 1-7, or the vector according to claim 10, and a suitable excipient.

12. The protein according to any one of claims 1-7, for use in a method of treating an individual suffering from a viral infection.

13. A method of producing the protein according to any one of claims 1-7, the method comprising expressing a nucleic acid encoding the protein according to any one of claims 1-7 in a relevant cell and isolating the thus produced protein.

14. The method of claim 13, wherein the relevant cell is a yeast cell, preferably a cell of a methylotrophic yeast such as Pichia pastoris (Komagataella phaffii) .

15. The method of claim 13 or claim 14, wherein the at least one hydrophilic random coil domain is used for purification of the protein.