Patent Application
20130034578
The invention relates to the field of virology, more specifically the invention relates to the field of influenza virus vaccines.
Influenza is an infectious disease that is caused by three types of influenza viruses, types A, B and C, which belong to the family of Orthomyxoviridae. Influenza viruses are RNA viruses consisting of seven negative single-stranded RNA-segments encoding nine proteins (influenza C), or eight negative single-stranded RNA-segments encoding eleven proteins (influenza A and B) (Chen and Deng, 2009). Birds are thought to be the natural host for all influenza A viruses, but many subtypes may also infect mammals, including humans. Symptoms of influenza include fever, headache, chills, muscle pains and soar throat, symptoms comparable with the common cold. However, influenza can lead to life-threatening complications, such as pneumonia, and death in high-risk groups such as young children, the elderly, and immune compromised or chronically ill individuals. In the past, influenza pandemics, such as the 1918/1919 Spanish Flu pandemic (H1N1 subtype) and the 1968/1969 Hong Kong Flu (H3N2 subtype), have been responsible for the death of millions of people (Khanna et al., 2008).
The influenza viral surface proteins hemagglutinin (HA) and neuraminidase (NA) are the basis for serologically different influenza subtypes. Currently 16 HA (H1-H16) and 9 NA (N1-N9) serotypes have been identified, which are used to classify influenza viruses (e.g. H3N2). Antigenic drift, due to one or more mutations causing amino acid substitution(s), may cause minor antigenic changes in HA and NA, resulting in the escape of immunity of a host. Additionally, a process called antigenic shift results in the formation of new virus subtypes (reassortants) through combination of HA and NA from different influenza virus subtypes. These alterations in HA and/or NA may in part be a result of selection pressure (Chen and Deng, 2009).
HA is present in the viral membrane. It must be cleaved by host proteases to yield two polypeptides, HA1 and HA2, in order to be infectious. Following cleavage, the newly exposed N-terminal of the HA2 polypeptide then acts to mediate fusion of the viral with the host cell membrane, which allows the virus to initiate infection of the host cell (Skehel and Wiley, 2000). HA is a homotrimer composed of three structurally distinct regions, a globular head consisting of anti-parallel beta-sheets which contains the receptor binding site and the HA1/HA2 cleavage site, a triple-stranded, coiled-coil, alpha-helical stalk, and a globular foot consisting of anti-parallel beta-sheets. In
NA catalyses the hydrolysis of terminal sialic acid residues of glycoproteins of the host cell, thereby preventing binding of HA to these proteins. NA thus facilitates release of the virus from a cell and hence spreading of the virus. NA is a homotetramer, anchored to the viral membrane by its N-terminus. In
Influenza virus infections are most prevalent in winter and seasonal influenza vaccines are developed each year. These vaccines contain two influenza A viruses (H1N1 and H3N2) and one influenza B virus (Khanna et al., 2008). New compositions need to be developed each year to protect against the predicted circulating strains. Even though they may have high homology, a specific vaccine may not protect against different strains from the same influenza A subtype. In addition to seasonal human influenza vaccines, several influenza vaccines for treatment of animals exist, such as vaccines against avian H5N1 and H9N2 influenza infection in poultry and H1N1 and H3N2 influenza infection in pigs. Currently available influenza vaccines are either live attenuated or inactivated influenza viruses which are generally produced by growing the virus in embryonized chicken eggs. Several disadvantages of this production method exist including the often high-level biocontainment facilities required to manage influenza viruses, the inability to obtain high yields of influenza virus in embryonized chicken eggs, the high specificity for a very limited number of influenza A subtypes, and the inability to use vaccines in individuals (mainly children) with egg allergies (Lipatov et al., 2004).
A number of attempts have been made to develop vaccines based on recombinant proteins derived from HA or NA. However all of these vaccines have limitations with respect to efficiency and efficacy of treatment. Wei et al. (J Virol. 2008) describe vaccination of mice with recombinant H5N1 HA proteins. Plasmids for expression of the recombinant H5N1 HA proteins contain nucleic acid sequences encoding a trimerization domain and a His-tag, which are both removed after purification of the proteins. To be effective the mice had to be immunized more than once (week 0 and 3) with relatively high amounts (20 μg) of protein. In Song et al. (PLoS One. 2008) and WO 2007/103322 immunization of mice with two doses of a composition comprising the globular head of HA fused to TLR5 ligand flagellin is described. Additionally, in Song et al. (PLoS One. 2008) rabbits are immunized with two doses of 5 or 15 μg HA fused to flagellin. In Saelens et al. (Eur J Biochem. 1999) three doses are used of a composition comprising recombinant HA expressed in P. pastoris and Ribi adjuvant, an oil-in-water emulsion, for immunizing mice, and WO 95/18861 describes the use of three doses of a composition comprising neuraminidase for immunizing mice. Kong et al. (Proc Natl Acad Sci USA. 2006) need three doses to immunize mice with plasmid DNA expressing an influenza virus hemagglutinin, as is also described in WO 2007/100584, WO 2008/112017 and WO 2009/092038.
It is an object of the present invention to provide improved immunogenic compositions for eliciting a protective immune response against an influenza virus. The invention therefore provides an immunogenic composition comprising at least one recombinant multimeric ectodomain of an influenza virus protein or part thereof fused to a streptavidin affinity tag, and a pharmaceutically acceptable diluent.
An “immunogenic composition” is herein defined as a composition that is capable of eliciting an immune response when administered to an individual. The elicited immune response can be humoral, cellular or a combination thereof and includes, but is not limited to, the production of antibodies, B cells, and T cells. An immune response as used herein is preferably directed specifically to one or more immunogens of a composition according to the invention.
An immunogenic composition of the present invention can be administered to an individual by any technique known in the art including, but not limited to, intramuscular (IM), intradermal (ID), subcutaneous (SC), intracranial (IC), intraperitoneal (IP), or intravenous (IV) injection, transdermal, oral, intranasal, or rectal administration, and combinations thereof. Furthermore administration of an immunogenic composition according to the invention can be performed individually, such as by injection or oral or nasal administration, or collectively, such as by supplementing drinking water or by spraying onto animals or into the air above animals, for example farm animals. In a preferred embodiment an immunogenic composition according to the invention is used for eliciting an immune response. It is preferred that the immunogenic composition is used as a vaccine.
An “individual” is herein defined as a human or an animal, preferably an animal that can be infected by influenza virus, such as birds, including but not limited to poultry. Individuals include but not limited to chickens, ducks, geese, turkeys, swans, emus, guinea fowls and pheasants, humans, pigs, ferrets, seals, rabbits, cats, dogs and horses. In a preferred embodiment of the invention an individual is a mammal, such as a human, a pig or a horse, or a bird. In one embodiment of the invention said mammal is a human.
A “multimeric ectodomain” is herein defined as an extracellular part of a surface protein which surface protein is composed of at least two (non-covalently bound) subunits. Said surface protein can be any surface protein of an influenza virus. By using a multimeric ectodomain of an influenza virus surface protein or part thereof the correct secondary, tertiary and quaternary structure of said influenza virus surface protein is obtained and/or preserved. An “influenza protein” is herein defined as any protein encoded by the influenza viral genome. “A pharmaceutically acceptable diluent” as used herein is defined as any solution, substance or combination thereof that has no biologically or otherwise unwanted activity, meaning that it can be administered to an individual together with other components of an immunological composition without causing a substantial adverse reaction.
Throughout the application a recombinant multimeric ectodomain of an influenza protein or part thereof fused to a streptavidin affinity tag is further also referred to as a recombinant multimeric ectodomain or part thereof according to the invention.
As used herein an “influenza virus” encompasses influenza virus type A, influenza virus type B and influenza virus type C and all influenza virus subtypes. “Influenza type” as used herein refers to influenza type A, type B or type C. “Influenza strain” as used herein refers to different influenza A viruses, for example H1N1, H1N2, H1N7, H2N2, H3N2, H3N8, H4N8, H5N1, H5N2, H5N9, H6N2, H6N5, H7N2, H7N3, H7N7, H8N4, H9N2, H10N7, H11N6, H12N5, H13N6. “Hemagglutinin” or “HA” as used herein refers to a hemagglutinin protein of influenza virus and may be of any influenza type, A, B or C, and any serotype, for example H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16, including subtypes belonging to the same serotype but with small difference as a result of for example antigenic drift. “Neuraminidase” or “NA” as used herein refers to a neuraminidase protein of influenza virus and may be of any influenza type, A, B or C, and any serotype, for example, N1, N2, N3, N4, N5, N6, N7, N8 or N9, including subtypes belonging to the same serotype but with small difference as a result of for example antigenic drift. “Part thereof' as used herein is defined as any part of a recombinant multimeric ectodomain of an influenza virus protein that is smaller than an entire multimeric ectodomain of an influenza virus protein, but has a similar immunogenic activity.
For example, a part of a recombinant multimeric HA according to the invention comprises a HA2, or a part of a HA2 comprising a highly conserved epitope proximate to the HA1 subunit of HA (Ekiert et al., 2009), or a HA ectodomain lacking the globular head domain. Preferably said epitope of a HA2, part of a HA2 or HA ectodomain lacking the globular head domain comprises amino acids 1-68 of HA fused to amino acids 293-524 of HA (amino acid numbering according to the numbering of HA of influenza strain X31, EMBL-EBI Accession no. P03438). Because said epitope of HA is highly conserved in different influenza virus types subtypes and strains a wide protection against different influenza virus types, subtypes or strains is provided if said part is used in an immunogenic composition according to the invention.
In a preferred embodiment a use of the immunogenic composition according to the invention for the preparation of a prophylactic agent for eliciting an immune response against an influenza virus is provided. Also provided is an immunogenic composition according to the invention for use as a vaccine.
A “streptavidin affinity tag” or “strep-tag” is herein defined as a tag that binds to streptavidin or a derivative thereof, such as Strep-Tactin, comprising at least one peptide consisting of nine amino acids: Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly, or at least one peptide consisting of eight amino acids (also called strep-tag II): Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, wherein Trp is tryptophan, Ser is serine, His is histidine, Pro is proline, Gln is glutamine, Phe is phenylalanine, Glu is glutamine, Lys is lysine, Ala is alanine, Arg is arginine and Gly is glycine. In a preferred embodiment a streptavidin affinity tag comprises two of more of said peptides, each consisting of eight or nine amino acids, more preferably three of said peptides. In one embodiment a streptavidin affinity tag comprises more than three of said peptides. A strep-tag is, for instance, used for isolation and/or purification purposes. The tag binds to streptavidin or a derivative thereof, for instance immobilized on a column. Elution of a protein fused to a strep-tag from the column can be performed using biotin or a derivative or homologue thereof, such as desthio-biotin. The streptavidin affinity tag can be placed at the C- or N-terminus of the protein of interest. A ‘derivative’ as used herein is defined as any variant of streptavidin which has the capacity to be bound by a streptavidin affinity tag, and can thus be used to isolate or purify a protein containing a streptavidin affinity tag. A preferred derivative of streptavidin is Strep-Tactin.
Elution of the bound recombinant proteins can be performed under mild conditions by competition with biotin or a suitable derivative. With mild conditions it is meant that purification can be performed under physiological conditions, such as aqueous solutions, room temperature, neutral or slightly basic pH, and normal pressure.
In a preferred embodiment of the invention a recombinant multimeric ectodomain of an influenza protein or part thereof is selected from the group consisting of a recombinant trimeric influenza hemagglutinin ectodomain or part thereof and a recombinant tetrameric influenza neuraminidase ectodomain or part thereof.
In some embodiments an amino acid sequence of a recombinant multimeric ectodomain or part thereof according to the invention is altered such as by amino acid substitutions, deletions and/or additions that provide proteins functionally equivalent to a recombinant multimeric ectodomain or part thereof according to the invention. Functionally equivalent proteins can be identified by any method known in the art, for example by their ability to induce an immune response against influenza virus in an individual, or in an in vitro assay wherein binding to antibodies is determined. An altered recombinant multimeric ectodomain is provided in an immunogenic composition according to the invention for example because it can be produced at a higher level, compared to a non-altered parent multimeric ectodomain.
Recombinant multimeric ectodomains or parts thereof according to the invention are preferred over monomeric ectodomains or parts thereof of influenza virus proteins because such multimeric ectodomains will result in more effective elicitation of an immune response. Therefore, a lower dosage and a reduced number of administrations of an immunogenic composition comprising such recombinant multimeric ectodomain according to the invention is needed in order to elicit a sufficient immune response, compared to immunogenic compositions comprising a monomeric ectodomain of an influenza virus protein.
An immunogenic composition according to the invention may comprise one recombinant trimeric influenza hemagglutinin ectodomain or part thereof or one recombinant tetrameric influenza neuraminidase ectodomain or part thereof. Such immunogenic composition is particularly suitable for eliciting an immune response in an individual against an influenza virus subtype or strain, or more than one related influenza virus subtypes or strains. In some embodiments however, an immunogenic composition according to the invention comprises a combination of one or more trimeric hemagglutinin ectodomains or parts thereof, and/or one or more tetrameric neuraminidase ectodomains or parts thereof.
Thus for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 trimeric hemagglutinin ectodomains or parts thereof can be combined, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and/or H16, or 2, 3, 4, 5, 6, 7, 8 or 9 tetrameric neuraminidase ectodomains or parts thereof can be combined, such as N1, N2, N3, N4, N5, N6, N7, N8 and/or N9. Additionally, an immunogenic composition according to the invention may comprise a combination of one or more trimeric hemagglutinin ectodomains or parts thereof and one or more tetrameric neuraminidase ectodomains or parts thereof of different influenza subtypes. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 trimeric hemagglutinin ectodomains or parts thereof and 1, 2, 3, 4, 5, 6, 7, 8 or 9 tetrameric neuraminidase ectodomains or parts thereof can be combined, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, N1, N2, N3, N4, N5, N6, N7, N8 and/or N9. It will be clear to a skilled person that the term “one or more ectodomains” encompasses one or more ectodomains from one or more influenza virus subtypes, such as for example Influenza A virus and Influenza B virus. Thus, an immunogenic composition according to the invention can be a multivalent composition with which an improved protection against an influenza virus infection can be achieved as compared to an immunogenic composition comprising one recombinant trimeric influenza hemagglutinin ectodomain or part thereof or one recombinant tetrameric influenza neuraminidase ectodomain or part thereof. Additionally, or alternatively, with a multivalent composition a wider protection against more than one influenza virus type, or influenza virus subtypes or strains can be achieved. Furthermore, an immunogenic composition according to the invention may be less sensitive to antigenic alterations than traditional inactivated or live attenuated influenza virus vaccines.
Other advantages of immunogenic compositions according to the present invention are for example the possibility to produce the recombinant multimeric ectodomains or parts thereof in large quantities in cell culture, and at relatively low costs since no high-level biocontainment facilities are necessary for production of immunogenic compositions according to the invention.
In contrast to prior attempts to produce vaccines based on recombinant influenza virus proteins, only very low amounts of a recombinant multimeric ectodomain or part thereof of an influenza virus protein or part thereof fused to a streptavidin affinity tag according to the invention are necessary for eliciting an immune response in an individual. As is shown in more detail in the examples, amounts as low as 3.75 pg of recombinant protein in ferrets, 5 pg of recombinant protein in swine and 0.4 pg of recombinant protein in chickens are sufficient to elicit a protective immune response in these respective animals upon challenge with influenza virus.
Immunogenic compositions according to the invention can be administered in a single dose or in multiple doses. However, as opposed to other vaccines based on recombinant influenza proteins, protection against an influenza virus infection is achieved after one administration of an immunogenic composition according to the invention. Thus, immunogenic compositions according to the present invention are more effective than other immunogenic compositions based on recombinant influenza proteins known in the art.
A strep-tag is not likely to influence protein function, structure, folding, secretion, and has a low immunogenicity. In addition, elution of a strep-tag containing protein is achieved under mild conditions. Without being bound by theory, it is believed that a combination of at least one recombinant multimeric ectodomain according to the invention and the use of a streptavidin affinity tag results in the advantages of the present invention over immunogenic composition comprising influenza proteins known in the art, possibly because of the mild conditions that can be employed during the purifications of proteins and, hence, the good conservation of the proteins' antigenicity and the more effective elicitation of an immune response.
In some embodiments of the invention a trimerization domain is used to provide a trimeric recombinant influenza hemagglutinin ectodomain or part thereof. The term “trimerization domain” as used herein is defined as a domain that mediates the formation of a trimer out of three monomeric proteins or parts thereof. Suitable trimerization domains include, but are not limited to, a trimerization sequence from bacteriophage T4 fibritin and an artificial trimerization domain (GCN4-pII) based on the yeast GCN4 coiled coil protein. In a preferred embodiment, trimerization of a recombinant influenza hemagglutinin ectodomain or part thereof is provided by a GNC4-based trimerization domain. Said GCN4-based trimerization domain preferably comprises the amino acid sequence
In some embodiments of the invention a tetramerization domain is used to provide a tetrameric recombinant influenza neuraminidase ectodomain or part thereof. The term “tetramerization domain” as used herein is defined as a domain that mediates the formation of a tetramer out of four monomeric proteins or parts thereof. Suitable tetramerization domains include, but are not limited to, the Sendai virus phosphoprotein tetramerization domain and a tetramerization domain (GCN4-pLI) derived by mutation from the yeast GCN4 dimerization domain. In a preferred embodiment, tetramerization of a recombinant influenza neuraminidase ectodomain or part thereof is provided by a GCN4-based tetramerization domain. A GCN4-based tetramerization domain preferably comprises the amino acid sequence
In some embodiments an immunogenic composition according to the invention comprises an adjuvant and/or a carrier or other excipient known in the art. “An adjuvant” is defined herein as a substance added to an immunological composition in order to enhance the immune response of an individual to a particular antigen at either the cellular of humoral level. Suitable adjuvants include, but are not limited to aluminium and calcium salts such as aluminium phosphate, aluminium hydroxide, aluminium potassium sulphate (alum) and calcium phosphate, organic adjuvants such as Squalene, oil-based adjuvants such as complete and incomplete Freund's adjuvant, RIBI Adjuvant System® (RAS®), Stimune (Specol)®, TiterMax®, Montanides, MF-59, AS03, QS21, Adjuvant 65, saponin, MDP, and Syntex Adjuvant Formulation (SAF)®, monophosphoryl lipid A, GerbuO Adjuvant, immune-stimulating complexes (ISCOMs), cytokines such as Interferon-gamma, immunostimulatory nucleic acid sequences such as CpG oligonucleotides, liposomes, virus-like particles, surfactants such as hexadecylamine, polyanions such as pyran and dextran sulfate. A preferred adjuvant is Stimune (Specol). Further preferred adjuvants for use in humans are aluminium phosphate or hydroxide, calcium phosphate, ISCOMs, AS03 or MF59.
A preferred adjuvant is ISCOMs. ISCOM technology has several advantages over other adjuvants. ISCOMs stimulate both humoral and cell-mediated immune responses. ISCOMs is a highly efficient adjuvant, enabling further reduction of the amounts of a recombinant multimeric ectodomain or part thereof according to the invention. A preferred ISCOMs is ISCOM Matrix-M. Without being bound to theory it is possible that the use of ISCOMs in a method or immunogenic composition according to the present invention may even enhance the advantages of the present invention over immunogenic composition comprising influenza proteins known in the art, such as a lower dosage and a reduced number of administrations of an immunogenic composition according to the invention.
As used herein “a carrier” or “a scaffold” is defined as a molecule such as a peptide, polypeptide, protein, protein complex, or chemical compound that is able to associate with any component of immunogenic compositions as defined herein for the purpose of transport of said component, presentation to the immune system, and/or enhancing the immunogenicity of said immunogenic composition. Suitable carriers or scaffolds include but are not limited to a biological scaffold such as a virus, a bacteria, a virus-like particle and a bacterial remnant such as a bacterial wall skeleton, a chemical scaffold such as an array of chemically linked streptavidin units or derivatives thereof, a biochemical multivalent linker, a diphtheria or tetanus toxoid, Keyhole limpet hemocyanin (KLH), serum albumin (such as BSA), hemocyanin, albumine, non-toxic mutant diphtheria toxin CRM197, outer membrane protein complex (OMPC) from Neisseria meningitidis, the B subunit of heat-labile Escherichia coli, a virus-like particle assembled from recombinant coat protein of bacteriophage Qb and cellulose beads.
The present invention further provides a vector comprising an expression cassette comprising a nucleic acid sequence encoding a signal sequence, a nucleic acid sequence encoding a recombinant ectodomain of an influenza protein or part thereof, a nucleic acid sequence encoding a trimerization or tetramerization sequence, and a nucleic acid sequence encoding a streptavidin affinity tag.
A vector according to the invention is particularly suitable for expression and production of a recombinant multimeric ectodomain or part thereof according to the invention. Therefore, in a preferred embodiment, a use of a vector according to the invention for the preparation of an immunogenic composition according to the invention for eliciting an immune response against an influenza virus is provided. Also provided is a vector according to the invention for use as a vaccine.
The term “vector” as used herein is defined as a nucleic acid molecule, such as a plasmid, bacteriophage or animal virus, capable of introducing a heterologous nucleic acid sequence into a host cell. A vector according to the invention allows the expression or production of a protein or part thereof encoded by the heterologous nucleic acid sequence in a host cell. Vectors suitable in a method according to the invention include, but are not limited to, pCD5 (Pouyani and Seed, 1995) and pCAGGS (Niwa et al., 1991). A “host cell” is a cell which has been transformed, or is capable of transformation, by a nucleic acid molecule such as a vector. The term “transformation” is herein defined as the introduction of a foreign nucleic acid into a recipient cell. Transformation of a recipient cell can result in transient expression of a recombinant protein by said cell, meaning that the recombinant protein is only expressed for a defined period of time. Alternatively, transformation of a recipient cell can result in stable expression, meaning that the nucleic acid is introduced into the genome of the cell and thus passed on to next generations of cells. Additionally, inducible expression of a recombinant protein can be achieved. An inducible expression system requires the presence or absence of a molecule that allows for expression of a nucleic acid sequence of interest. Examples of inducible expression systems include, but are not limited to, Tet-On and Tet-Off expression systems, hormone inducible gene expression system such as for instance an ecdysone inducible gene expression system, an arabinose-inducible gene expression system, and a Drosophila inducible expression system using a pMT/BiP vector (Invitrogen) which comprises an inducible metallothioneine promoter.
A “signal sequence” is herein defined as a signal peptide that directs transport of a protein or part thereof to the endoplasmic reticulum of a eukaryotic cell—the entry site of the secretory pathway. A signal sequence can be located N-terminal or C-terminal of the protein or part thereof. Signal peptides can be cleaved off after insertion of the protein into the endoplasmic reticulum. For example, in one embodiment a pMT/BiP vector for expression of recombinant protein in Drosophila cells comprises a BiP signal sequence. In another embodiment, a pCD5 vector for expression of recombinant protein in mammalian cells comprises the signal sequence
An expression cassette preferably comprises a promoter/enhancer which allows for initiation of transcription of nucleic acid sequences further contained within the expression cassette. Examples of promoters include, but are not limited to, RAmy3D and CaMV promoters for expression in plant cells, polyhedrin, p10, IE-0, PCNA, OplE2, OplE1, and Actin 5c promoters for expression in insect cells, and beta-actin promoter, immunoglobin promoter, 5S RNA promoter, or virus derived promoters such as cytomegalovirus (CMV), Rous sarcoma virus (RSV), and Simian virus 40 (SV40) promoters for expression in mammalian cells. A preferred vector is provided by the pCD5 expression vector for expression of recombinant protein in mammalian cells comprising a CMV promoter. A further preferred vector is provided by a pCAGGS expression vector for expression of recombinant protein in mammalian cells comprising a CMV enhancer/chicken beta-actin (CAG) promoter.
An expression cassette according to the invention which is intended for the expression of a trimeric influenza hemagglutinin ectodomain or part thereof preferably comprises from 5′ to 3′: a nucleic acid sequence encoding a signal sequence; a nucleic acid sequence encoding a recombinant ectodomain of influenza hemagglutinin or part thereof; a nucleic acid sequence encoding a trimerization sequence, preferably a GCN4-based trimerization sequence, and a nucleic acid sequence encoding a streptavidin affinity tag. A vector according to the invention which is intended for the expression of a tetrameric influenza neuraminidase ectodomain or part thereof preferably comprises from 5′ to 3′: a nucleic acid sequence encoding a signal sequence; a nucleic acid sequence encoding a streptavidin affinity tag; a nucleic acid sequence encoding a tetramerization sequence, preferably a GCN4-based tetramerization sequence, and; a nucleic acid sequence encoding a recombinant ectodomain of influenza hemagglutinin or part thereof.
A recombinant multimeric ectodomain or part thereof according to the invention is for instance expressed in a prokaryotic cell, or in a eukaryotic cell, such as a plant cell, a yeast cell, a mammalian cell or an insect cell. Additionally a recombinant multimeric ectodomain or part thereof according to the invention can be expressed in plants. Prokaryotic and eukaryotic cells are well known in the art. A prokaryotic cell is for instance E. coli. Examples of plants and/or plant cells include, but are not limited to, corn (cells), rice (cells), duckweed(cells), tobacco (cells, such as BY-2 or NT-1 cells), and potato(cells). Examples of yeast cells are Saccharomyces and Pichia. Examples of insect cells include, but are not limited to, Spodoptera frugiperda cells, such as Tn5, SF-9 and SF-21 cells, and Drosophila cells, such as Drosophila Schneider 2 (S2) cells. Examples of mammalian cells that are suitable for expressing a recombinant multimeric ectodomain or part thereof according to the invention include, but are not limited to, African Green Monkey kidney (Vero) cells, baby hamster kidney (such as BHK-21) cells, Human retina cells (for example PerC6 cells), human embryonic kidney cells (such as HEK293 cells), Madin Darby Canine kidney (MDCK) cells, Chicken embryo fibroblasts (CEF), Chicken embryo kidney cells (CEK cells), blastoderm-derived embryonic stem cells (e.g. EB14), mouse embryonic fibroblasts (such as 3T3 cells), Chinese hamster ovary (CHO) cells, mouse myelomas (such as NSO and SP2/0), quail muscle cells (QM5), and derivatives of these cell types. In a preferred embodiment mammalian HEK293T cells or S2 insect cells are used for production of a recombinant multimeric ectodomain or part thereof according to the invention.
Further provided by the invention is a method for the preparation of an immunogenic composition according to the invention, comprising providing a vector according to the invention, transforming a host cell with said vector, culturing said host cell in a culture medium, thereby allowing expression of the recombinant multimeric ectodomain of an influenza protein or part thereof fused to a streptavidin affinity tag, and isolating said recombinant multimeric ectodomain of an influenza protein or part thereof fused to a streptavidin affinity tag.
When using a method according to the invention for expression of a recombinant multimeric ectodomain or part thereof according to the invention in a host cell said multimeric ectodomain or part thereof is preferably secreted into the culture medium and can be isolated from said culture medium. Isolation of said multimeric ectodomain or part thereof is for instance performed by centrifuging or filtrating the culture medium to remove cells and cell remnants, and purification using any method known in the art, for instance gel electrophoresis or chromatography methods, such as affinity chromatography or high performance liquid chromatography. In a preferred embodiment a recombinant multimeric ectodomain or part thereof according to the invention is purified using the fused streptavidin affinity tag, for instance using purification columns with streptavidin or Strep-Tactin Sepharose (IBA Germany) and an elution buffer for displacing the Strep-tag protein from the column comprising biotin or a derivative or homologue thereof.
In some embodiments a vector according to the invention is derived from an animal virus such as, but not limited to, vaccinia virus (including attenuated derivatives such as the Modified Vaccinia virus Ankara, MVA), Newcastle Disease virus (NDV), adenovirus or retrovirus. It is preferred that an animal virus that used as a vector according to the invention is attenuated. Any of these vectors is particularly suitable for expression of a recombinant multimeric ectodomain or part thereof according to the invention in a host in which an immunogenic response is intended to be elicited. An advantage of the use of an animal virus such as NDV is that it can be efficiently grown in embryonated chicken eggs at low costs and in sufficiently high amounts. In a preferred embodiment, after propagation of the animal virus in embryonated chicken eggs isolated virus is subsequently used to infect eukaryotic cells. Infected cells are then used for production of a recombinant multimeric ectodomain or part thereof according to the invention. Examples of cells that are suitable for expressing a recombinant multimeric ectodomain or part thereof according to the invention are mammalian cells as described above. In another preferred embodiment, after for example propagation of the animal virus in embryonated chicken eggs, the isolated virus is used to directly infect cells of a host in which an immunogenic response is to be elicited. One advantage of infecting a host in which an immunogenic response is to be raised with a vector according to the invention is that administration is very efficiently carried out, for instance by spraying onto animals or into the air above animals. The invention therefore further comprises an immunogenic composition comprising the vector according to the invention, and a pharmaceutically acceptable diluent.
A vector according to the invention for use in an immunogenic composition according to the invention preferably comprises an expression cassette comprising a promoter that is suitable for initiation of transcription in cells of the intended host. Examples of suitable promoters for expression of at least one recombinant multimeric ectodomain or part thereof according to the invention in avian hosts include, but are not limited to, avian beta-actin promoter or a chicken RNA pol I promoter. Examples of suitable promoters for expression of at least one recombinant multimeric ectodomain or part thereof according to the invention in mammalian hosts include, but are not limited to, beta-actin promoter, immunoglobin promoter, 5S RNA promoter, or virus derived promoters such as cytomegalovirus (CMV), Rous sarcoma virus (RSV) and Simian virus 40 (SV40) promoters for mammalian hosts.
In some embodiments a nucleic acid sequence encoding a recombinant ectodomain or part thereof according to the invention is altered such as by nucleic acid substitutions, deletions and/or additions that enable expression of proteins functionally equivalent to a recombinant multimeric ectodomain or part thereof according to the invention. Functionally equivalent proteins can be identified by any method known in the art, for example by their ability to induce an immune response against influenza virus in an individual, or in an in vitro assay wherein binding to antibodies is determined.
In some embodiments a nucleic acid sequence encoding a trimeric influenza hemagglutinin ectodomain or part hereof, and/or a nucleic acid sequence encoding a tetrameric influenza neuraminidase or part thereof is inserted into a vector. In another embodiment nucleic acid sequences encoding more than one trimeric influenza hemagglutinin ectodomain or part hereof, and/or tetrameric influenza neuraminidase or part thereof or combinations thereof are inserted into a vector. In yet another embodiment an immunogenic composition according to the invention comprises separate vectors in each of which a nucleic acid sequence encoding a trimeric influenza hemagglutinin ectodomain or part hereof, and/or a tetrameric influenza neuraminidase or part thereof is inserted.
Thus, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleic acid sequences encoding a trimeric hemagglutinin ectodomain or part thereof can be combined in one vector, such as nucleic acid sequences encoding H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and/or H16. Alternatively 2, 3, 4, 5, 6, 7, 8 or 9 nucleic acid sequences encoding a tetrameric neuraminidase ectodomain or part thereof can be combined in one vector, such as nucleic acid sequences encoding N1, N2, N3, N4, N5, N6, N7, N8 and/or N9. Additionally, an immunogenic composition according to the invention may comprise a combination of one or more nucleic acid sequences encoding a trimeric hemagglutinin ectodomain or part thereof and one or more nucleic acid sequences encoding a tetrameric neuraminidase ectodomain or part thereof of different influenza subtypes. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleic acid sequences encoding trimeric hemagglutinin ectodomains or parts thereof and 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleic acid sequences encoding tetrameric neuraminidase ectodomains or parts thereof can be combined, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, N1, N2, N3, N4, N5, N6, N7, N8 and/or N9. It will be clear to a skilled person that the term “one or more ectodomains” encompasses one or more ectodomains from one or more influenza virus subtypes, such as for example Influenza A virus and Influenza B virus. This way, an immunogenic composition according to the invention may comprise a multivalent composition with which a wider protection against more than one influenza virus type, or influenza virus subtypes or strains is achieved. Furthermore, such an immunogenic composition may be less sensitive to antigenic alterations than traditional inactivated or live attenuated influenza virus vaccines.
Further provided by the invention is a method for eliciting an immune response against an influenza virus in an individual comprising administering to said individual an immunogenic composition according to the invention.
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
Based on H3 numbering, a cDNA clone encoding amino acid residues corresponding to residues 18 to 523 of the HA from A/Viet Nam/1203/2004 (H5N1) (Genbank accession no. ABW90137.1) was synthesized using human-preferred codons by GenScript USA Inc. The HA ectodomain encoding cDNA was cloned into the pCD5 expression vector for efficient expression in mammalian cells (Zeng et al., 2008). The pCD5 vector had been modified such that the HA-encoding cDNA was cloned in frame with DNA sequences coding for a signal sequence, a GCN4 isoleucine zipper trimerization motif (ELIKRMKQIEDKIEEIESKQKKIENEIARIKKIKLVPRGSLE; (Pancera et al., 2005) and the Strep-tagII (WSHPQFEK; IBA, Germany).
Protein expression and purification. pCD5 expression vector containing the HA ectodomain-encoding sequence was transfected into HEK293S GnTI(−) cells (Reeves et al., 1999) using polyethyleneimine (PEI) in a 1:5 ratio (μg PEI: μg DNA). At 6 h post transfection, the transfection mixture was replaced by 293 SFM II expression medium (Invitrogen), supplemented with sodium bicarbonate (3.7 g/liter), glucose (2.0 g/liter), Primatone RL-UF (3.0 g/liter, Kerry Bio-Science, Zwijndrecht, The Netherlands), penicillin (100 units/ml), Streptomycin (100 μg/ml), glutaMAX (Gibco), and 1.5% DMSO. Tissue culture supernatants were harvested 5-6 days post transfection. HA protein expression and secretion was confirmed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by western blotting using a mouse anti-Strep-tag antibody (IBA, Germany). Secreted HA proteins were purified using Strep-tactin sepharose beads according to the manufacturer's instructions (IBA, Germany). The concentration of purified protein was determined by using a Nanodrop 1000 spectrophotometer (Isogen Life Sciences) according the manufacturer's instructions.
Biological characterization of recombinant HA. Oligomerization status of the HA protein was determined by analyzing the elution profile using a Superdex200GL 10-300 column (U-Protein Express). Functionality of the soluble HA trimer (sHA3) was assessed using a fetuin solid phase assays (Lambre et al., 1991). 1 μg/ml fetuin per well was used to coat 96-well nunc maxisorp plates. When indicated in the figure legend fetuin was treated with Vibrio Cholera derived neuraminidase (VCNA) followed by extensive washing steps. sHA3 was pre-complexed with horseradish peroxidase (HRP)-linked anti-Strep-tag antibody (2:1 molar ratio) for 30 min at 0° C. prior to incubation of limiting dilutions on the fetuin-coated plates (60 min, room temperature [RT]). HA-binding was subsequently detected using tetramethylbenzidine substrate (BioFX) in an ELISA reader (EL-808 [BioTEK]), reading the OD at 450nm.
Vaccination-Challenge Experiments in Chickens
SPF White Leghorn chickens (Lohmann) of 6 weeks old were used. The birds were housed in a climatized room under Bsl-3 conditions. In the first experiment, one group of 10 chickens was immunized twice (on day 0 and 21) by i.m. injection (0.5 mL) of 10 μg sHA3 adjuvanted with Stimune (ID—Lelystad, Lelystad, The Netherlands). As a challenge control, a group of 10 chickens received an equal volume of PBS in Stimune (mock vaccinated). Three weeks after the vaccination, the birds were challenged by infection with 105 TCID50 of A/Vietnam/1194/04 virus (0.1 ml intranasally, i.n. and 0.1 ml intratracheally, i.t., UniProt Taxonomy ID: 284217) and monitored over a period of 14 days for signs of disease. Blood samples were collected at 3 weeks after each immunization (on d21 and d42) and on day 14 post infection (p.i.). Trachea and cloaca swabs were taken from each chicken on day 2, 4 and 7 p.i.
In the next experiment, a total of 70 one-day-old layer hens (White Leghorn) purchased from a local breeder was used. The chickens were vaccinated against NDV (Newcastle Disease virus) and IBV (Infectious Bronchitis virus) at the age of one day according to the farm's routine. At the age of 6 weeks, the birds were transported to the Biosafety level (Bsl-) 3 facility and allocated to 7 experimental groups of 10 birds each. Six groups were immunized either by a single intramuscular injection of 10, 2 or 0.4 μg sHA3 antigen on day 21 or twice (on day 0 and 21) with the same doses. As a challenge control, one group of 10 birds was mock-vaccinated (on day 0 and 21) with PBS in Stimune. Four weeks after vaccination (day 49), chickens were challenged as above and observed daily for clinical signs during 14 days. Blood samples were taken before vaccination, after the first (day 21) and second (day 49) vaccination and on day 14 p.i. Trachea and cloaca swab samples were taken on the same days as described above.
Virus Detection Assay
Trachea and cloaca swabs were stored in cold tryptose broth medium. The medium was clarified by low-speed centrifugation and the supernatant was harvested, aliquoted and stored at −70° C. Upon freeze-thawing, trachea and cloaca swabs sampled from the same bird on the same day were pooled, RNA was extracted and the presence of viral RNA was assayed using quantitative RT-PCR assay (Taq Man PCR) directed against the M1 gene, encoding an influenza virus matrix protein. First, cDNA was synthesized using primer 5′-CACTGGGCACGGTGAGC-3′, then part of the M1 gene was amplified by running 45 cycles of Light Cycler PCR using primer 5′-CTTCTAACCGAGGTCGAAACGTA-3′ as the reverse primer in the presence of the TaqMan fluorescent probe 5′-6FAM-TCAGGCCCCCTCAAAGCCGA-X-ph. Negative and positive control samples were tested in parallel. The lower limit of detection was determined to be approximately 500 TCID50. Some samples gave inconclusive results, meaning that they gave only a very weak signal (fluorescence <0.07) after >31 cycles.
Hemagglutination Inhibition Assay
Heat-inactivated immune sera from chicken blood samples were tested for HI activity with 1% chicken red blood cells and 4 HAU (hemagglutinating units) of A/Vietnam/1194/04 according to standard methods. Red button formation was scored as evidence of hemagglutination. Antibody titers were expressed as the reciprocal of the highest serum dilution showing HI.
Results
Expression, Purification, and Characterization of the H5 Trimers.
In order to express a soluble, trimeric HA ectodomain (sHA3) in mammalian cells, the H5 ectodomain-coding sequence was first cloned into the appropriate expression vector. In the pCD5 vector used, HA-sequence was preceded by a signal peptide-encoding sequence, to allow efficient secretion of the recombinant protein, and followed by sequences coding for the GCN4 isoleucine-zipper trimerizaton motif (Pancera et al., 2005) and the Strep-tagll (
Efficacy of sHA3 Vaccine Against a Lethal H5N1 Infection in Chickens
To assess the protective efficacy of sHA3 in chickens, ten chickens were vaccinated twice (on day 0 and 21) intramuscularly (i.m.) with 10 μg of Stimune-adjuvanted sHA3 and challenged 3 weeks later by intranasal (i.n.)/intratracheal (i.t.) inoculation of a lethal dose of H5N1 A/Vietnam/1194/04 virus. In addition, 10 birds were mock-vaccinated to serve as challenge controls. As shown in
In the second vaccination-challenge experiment, chickens were vaccinated with 10, 2 or 0.4 ug of sHA3 either twice or by a single injection and challenged four weeks later by infection of a lethal dose of A/Vietnam/1194/04 as described above. A booster vaccination with a dose of 0.4 ug of sHA3 was sufficient to protect 90% of the chickens against mortality, while all chickens survived when a booster vaccination of 2 or 10 μg was administered (
We also analyzed whether vaccination with sHA3 decreased or prevented virus shedding. To this end trachea and cloaca swabs were taken from the vaccinated and challenged chickens from the experiment shown in
Materials and Methods
Influenza A Viruses
Influenza virus A/Netherlands/602/2009 was isolated from the first case of a laboratory confirmed 2009-A/H1N1 infection in The Netherlands by inoculation of 11-day old embryonated chicken eggs (Munster et al, 2009). Virus stocks of influenza virus A/Netherlands/602/2009 (H1N1) were prepared by infecting confluent Madin-Darby Canine Kidney (MDCK) cells. After cytopathologic changes were complete, culture supernatants were cleared by low speed centrifugation and stored at −70° C. Infectious virus titers were determined in MDCK cells as described previously (Rimmelzwaan et al., 1998). All experiments with these viruses were performed under Bio Safety Level (BSL)-3 conditions.
Preparation of HA and NA Antigens
Human-codon optimized genes encoding the hemagglutinin (HA) ectodomain (a.a. 17-522,
Protein Expression and Purification
HEK293T cells were transfected with the pCD5 expression vectors containing the HA and NA encoding sequences using polyethyleneimine (PEI) in a 1:5 ratio (μg DNA:μg PEI). At 6 h post transfection, the transfection medium was replaced by 293 SFM II expression medium (Invitrogen), supplemented with sodium bicarbonate (3.7 g/liter), glucose (2.0 g/liter), Primatone RL-UF (3.0 g/liter, Kerry Bio-Science, Zwijndrecht, The Netherlands), penicillin (100 units/ml), streptomycin (100 μg/ml), glutaMAX (Gibco), and 1.5% DMSO. Tissue culture supernatants were harvested 5-6 days post transfection and HA and NA ectodomains were purified from the culture medium using Strep-Tactin affinity chromatography (IBA GmbH). HA and NA protein expression and purification was confirmed by western blotting using a Strep-Tactin-HRP conjugate (IBA GmbH) (data not shown) and SDS-PAGE analysis. Oligomerization of the proteins was determined by gel filtration chromatography and by blue-Native-PAGE analysis.
Ferrets
Healthy young adult outbred female ferrets (Mustela putorius fur; between 6 and 12 months old) were supplied by Schimmel, Uddel, The Netherlands. Ferrets were screened for the presence of antibodies against circulating influenza A/H1N1 and A/H3N2 viruses and the swine-origin 2009 A(H1N1) influenza virus [2009 A(H1N1) influenza virus] by hemagglutination inhibition assay. Ferrets that were tested negative for these viruses were used in this experiment. An independent animal ethics committee approved the experimental protocol before the start of the experiments.
Immunizations and Infections
Thirty-six seronegative ferrets were divided into six groups of 6 ferrets and vaccinated twice with the following formulations: 3.75 μg trimeric HA+3.75 μg tetrameric NA in PBS (group 1); 3.75 μg trimeric HA in Iscom Matrix-M (Isconova, Uppsala, Sweden; group 2); 3.75 pg tetrameric NA in Iscom Matrix-M (group 3); 3.75 pg trimeric HA+3.75 μg tetrameric NA in Iscom Matrix-M (group 4); PBS (group 5); Iscom Matrix-M (group 6). Vaccinations were performed with an interval of 20 days under anesthesia with ketamine in the quadriceps muscles of the hindleg in a total volume of lml. Ferrets were housed in groups and received food and water ad-libitum. At 32 days after the last vaccination, the animals were anesthesized with ketamine/medetomidine (reversed with atipamezole), weighed and subsequently challenged intratracheally with 1×106 TCID50 of influenza A/NL/602/09 (H1N1) in a volume of 3 ml Phosphate Buffered Saline (PBS). After infection, ferrets were monitored three times daily for the development of clinical signs. Before infection and two and four days after infection, nose and throat swabs of each ferret were collected while ferrets were anesthesized with ketamine. Four days after infection, animals were weighed and subsequently killed by exsanguination while under anesthesia with ketamine and medetomidine. Necropsies were performed according to standard procedures.
Serology
Serum samples were collected before vaccination, at the day of second vaccination (day 20) and at the day of challenge (day 52). Sera were stored at '120° C. until use. Sera were tested for the presence of anti-HA antibodies using a hemagglutination inhibition assay (HI-assay) with 1% turkey erythrocytes and for the presence of virus neutralizing antibodies using a micro virus neutralization assay (VN-assay) as described previously (Frank et al., 1980 and Palmer et al., 1975). Sera were tested for the presence of antibodies reactive with influenza A/NL/602/09 (nH1N1). For this purpose, reverse genetics viruses were produced. The titers obtained with these viruses were comparable with those against the wild-type strains (data not shown). Positive control serum specific for influenza A/NL/602/09 (H1N1) was obtained from a ferret infected with this virus (Munster et al., 2009). Sera were also tested for the presence of neuraminidase inhibiting (NI) antibodies using a fetuin-based assay (Lambre et al., 1991). In brief, 96-well Nunc MaxiSorp plates were coated overnight at 4° C. with 100 μl of 5 μg/ml fetuin. Sixty-μl volumes of serially diluted serum samples were incubated with an equal volume of S2 cell culture supernatant containing NA (40× diluted in PBS-Ca/Mg [0.901 mM/0.493 mM], incubated for 30 minutes at 37° C. and then 100 μl of the mixture was added to the fetuin-coated plates. After one hour incubation at 37° C., the plates were washed and neuraminidase activity was subsequently measured using a peroxidase-labelled peanut agglutinin (Sigma, Zwijndrecht, The Netherlands) (2.5 μg/ml). Plates were incubated for 1 h at room temperature, washed after which 100 μl of peroxidase substrate (TMB) was added to each well. After 5 minutes, reaction was stopped by the addition of 100 μl of 0.3 M phosphoric acid and OD values were measured at 450 nm using an ELISA reader (EL-808 [BioTEK]).
Virus Titers in Lungs and in Throat and Nose Swabs
Samples of all lobes of the right lung and of the accessory lobe of infected ferrets were collected and snap frozen on dry ice with ethanol and stored at −70° C. until further processing. Lung samples were weighed and subsequently homogenized with a FastPrep-24 (MP Biomedicals, Eindhoven, The Netherlands) in Hank's balanced salt solution containing 0.5% lactalbumin, 10% glycerol, 200 U/ml penicillin, 200 μg/ml streptomycin, 100 U/ml polymyxin B sulfate, 250 μg/ml gentamycin, and 50 U/ml nystatin (ICN Pharmaceuticals, Zoetermeer, The Netherlands) and centrifuged briefly. After collection of nose and throat swabs, swabs were stored at −70° C. in the same medium as used to homogenize lung samples. Quadruplicate 10-fold serial dilutions of throat, nose and lung samples were used to infect MDCK cells as described previously (Rimmelzwaan et al., 1998). HA activity of the culture supernatants collected 5 days post inoculation was used as indicator of infection. The titers were calculated according to the Spearman-Karber method and expressed as log TCID50 per gram for lung tissue or per ml for swabs (Karber, 1931).
Statistical Analysis
Significance among animal groups was analyzed by ANOVA and Tukey test subsequent to ANOVA. Differences were considered significant at P<0.05.
Results
Antibody Responses Induced by Immunization with Soluble HA and NA
Multimeric, soluble versions of the HA ectodomain (sHA; a.a.17-522) and the neuraminidase head domain (sNA; a.a.75-469) of the pandemic 2009 A(H1N1) influenza virus, expressed in mammalian HEK293T cells and purified from the cell supernatant (
Protection Against Clinical Signs After Infection with 2009 A(H 1N1) Influenza Virus
Vaccinated ferrets were challenged intratracheally with 106 TCID50 2009 A(H1N1) virus 5 weeks after the boost. No clear signs of disease were observed during the immunization period or after the challenge inoculation with 2009 A(H1N1) influenza virus. Yet, loss of body weight became obvious after the inoculation in the PBS and IMM control groups as well as in the non-adjuvanted sHA+sNA vaccine group (
Histopathological changes were located in multifocal to coalescing lesions with more than 50% of the lungs affected in the unprotected control ferrets inoculated with PBS or adjuvant only (IMM) (
Protection of Vaccine Against Virus Replication
To measure the effect of vaccination with low doses of 3.75 μg trimeric HA and/or tetrameric NA on the severity of the infection induced by the challenge inoculation with 2009 A(H 1N1) influenza virus, virus titers were determined in lungs, throat and nose at day 4 after inoculation. The virus replicated efficiently in the lungs of control ferrets (
High viral loads in the nose were observed both in the control animals (PBS and IMM groups) after inoculation with 2009 A(H1N1) influenza virus. Viral loads in the nose were considerably reduced in the animals immunized with either adjuvanted sHA or sNA or the unadjuvanted sHA+sNA combination. Animals immunized with the adjuvanted combination of sHA and sNA antigens had the highest and significant reduction of nose viral titers (
Cross-Neutralizing Antibody Responses Induced by Immunization with Soluble HA and NA
The sera of ferrets collected before virus challenge were tested for cross-neutralizing antibodies using HI and NI assays against different other H1N1 influenza strains. Cross-neutralizing antibodies inhibiting hemagglutination against A/NL/386/86, A/NL/25/80 and A/NewYersey/8/76 H1N1 influenza virus were present in the sera of sHA−IMM and sHA+sNA−IMM immunized animals (
Materials and Methods
Cells and Viruses
Quail muscle (QM5) cells (Antin & Ordahl, 1991) were cultured in QT35 complete medium (Life Technology) and VERO cells (ATCC CCL-81) were cultured in Dulbecco's modified Eagle's medium (Invitrogen). The media were supplemented with 5% fetal bovine serum and antibiotics. Recombinant fowlpox virus fpEFLT7 (Britton et al., 1996; herein designated FPV-T7), providing stable expression of bacteriophage T7 RNA polymerase in both avian and mammalian cells, was propagated in primary chicken embryo liver cells. Influenza virus A/Vietnam/1194/04 H5N1 (kindly provided by Dr. Alan Hay from the WHO Influenza Centre at the National Institute for Medical Research, London) was propagated in 9-day-embryonated specific pathogen-free (SPF) chicken eggs to produce a virus stock.
Construction of Full-Length Newcastle Disease Virus (NDV) cDNAs
Full-length cDNA of NDV Herts, pFL-Herts (de Leeuw et al. 2005), with the unique restriction sites AscI and FseI flanking the F gene, was used as the backbone for the insertion of the H5 HA gene between the P and M genes. To this end, the unique restriction sites XbaI and PmeI were created in the P-M intergenic region by site-directed mutagenesis. Subsequently, a synthetic linker (GCTCTAGATAAGAAAAAATACGGGTAGAAGTTTAAACGGCGCGCCGG) encoding the sequence of an NDV end-start box (bold) was inserted between the XbaI and PmeI site (underlined) of the full-length construct.
To modify the polybasic cleavage site of the F0 protein into the monobasic motif (GRQGR↓L; creating FAPBC), the F gene of Herts was amplified from pFL-HertsAF using the primer pairs pAscl(Herts) (5′ TTGGCGCGCCCCAGGTGCAAGATGGGC 3′) together with N043 (5′ TTTACTAGTTTACGAAAAGTATTGGATTTGTGCCCC 3′), and N044 (5′ GGAGGGAGACAGGGACGCCTTATAGGTGCCATTATCG 3′) together with pFsel(Herts) (5′ CCCGATTGAGGGCCGGCCTCCCC 3′). To fuse both PCR fragments, a second PCR was performed using the primers pAscl(Herts) and pFsel(Herts). The resulting PCR product was restricted with AscI and FseI and inserted between the AscI and FseI site of pFL-Herts creating pFL-HertsAPBC.
Viral RNA was extracted from H5N1 A/Vietnam/1194/2004 (NIBRG14) virus using a High Pure Viral RNA kit (Roche). From the extracted viral RNA, cDNA was generated using Superscript II (Invitrogen) and primer CAI085 (5′ AGCAAAAGCAGG 3′) matching the non-coding region of each segment. Subsequently the HA gene was PCR amplified using primer pMT036 (AGCTTTGTTTAAACAATGGAGAAAATAGTGCTTCTTTTTGC; PmeI site underlined) and pMT038 (CCGGCGCGCCGTTTAAACTTAAATGCAAATTCTGCATT GTAAC; PmeI site underlined). HA cDNA was subcloned into the pGEM-Teasy plasmid (Promega). This vector was digested with PmeI and the HA gene was cloned into pFL-HertsAPBC generating pFL-HertsΔPBC-H5.
To create pFL-HertsΔPBC-H53, the pCD5-sH53 expression vector was used (Cornelissen et al. 2010). In this vector, the H5-sequence was preceded by a signal peptide-encoding sequence and followed by sequences coding for the GCN4 isoleucine-zipper trimerizaton motif (Harbury et al. 1993) and the Strep-tag II. The vector was digested with PmeI and the liberated insert was ligated with the PmeI-digested pFL-HertsΔPBC. The pCD5 vector had been generated such that a PmeI restriction site was present upstream of the signal peptide sequence and downstream of the Strep-tag, and the size of the genome still complied with “the rule of six” (de Leeuw et al. 1999) after insertion of the PmeI-digest.
All plasmids were generated according to standard procedures (Sambrook et al., 1989). Sequence analysis of modified regions in all constructs generated by PCR was performed to verify that no inadvertent changes had been introduced.
Recovery of Recombinant NDV
Recombinant NDV was generated by reverse genetics using the FPV-T7 RNA polymerase system as described (Peeters et al., 1999). Briefly, VERO cells grown to subconfluency in 6-well dishes were infected with FPV-T7 and transfected with the full-length construct and the helper plasmids expressing NP, P and L. The supernatant was harvested after 6 days of incubation at 37° C., clarified by low-speed centrifugation and filtered through a 0.2 μm filter. The filtrate was used to infect fresh VERO cell cultures. After incubation for 3 days in medium containing 5% allantoic fluid, the supernatant was collected and inoculated (0.2 ml) into the allantoic cavity of 9-to-11 day-embryonated SPF chicken eggs to amplify virus. After 3 days, allantoic fluid was harvested, clarified and aliquoted. Virus recovered from pFL-Herts APBC-H53 was designated rNDV-H53 and virus recovered from pFL-Herts ΔPBC-H5 was designated rNDV-HA. Virus titers (TCID50 ml−1) of the harvests were determined in QM5 cells by the end-point dilution method and calculated according to Reed and Munch (Reed and Munch, 1938. A simple method of estimating fifty percent endpoints. Am J Hyg 27: 493-497).
Intracellular Immunostaining
Expression of the HA protein by recombinant NDV in QM5 cells was analyzed by intracellular immunostaining as described (Wensvoort et al., 1986). For this, the cells were permeabilized, fixed with 3% paraformaldehyde and incubated with monoclonal antibody (mAb) 15A6 anti-H5 (1:1000; Santa Cruz). Anti-F mAb 8E12A8C3 (1:5000; CVI, Lelystad) was used for detection of NDV F protein. A rabbit-anti-mouse-HRPO conjugate (1:2000; IBA Germany) was used as the secondary antibody. Mock-infected cell monolayers served as negative controls.
Production and Purification of sH53
QM5 cells grown to 90% confluency in T75 flasks were infected with rNDV-sH53 at an MOI of 3. After 72 h of incubation at 37° C., the supernatant was collected (10 ml) and clarified by low-speed centrifugation (10 min at 2500 rpm). The pH of the harvest was adjusted to 7.8 and Avidin was added until a final concentration of 3 μg/ml. After 4 h, recombinant sH53 protein was purified using Strep-tactin sepharose beads as described by the manufacturer (IBA, Germany). The purification product (15 μl) was subjected to SDS-PAG electrophoreses (12% Bis-Tris) using MOPS running buffer (Invitrogen) and proteins were stained with Blue stain reagent (Thermo Scientific). The sH53 protein concentration was determined with a nano-drop spectrophotometer and also estimated on gel using a series of bovine serum albumin (BSA) of known amounts as reference.
Intracerebral Pathogenicity Index
The standardized Office International des Epizooties (OIE) chicken pathogenicity test was conducted under BSL3 conditions and after approval by the Animal Ethics Committee. Ten one-day-old SPF chickens were inoculated intracerebrally with 0.1 ml of a 1/10 dilution of the egg-grown NDV-HertsΔPBC virus and observed for clinical signs every day over a period of 10 days. The birds received a score of 0 if they appeared normal, 1 if they were sick, 2 in case of severe illness and 3 if they were death. The intracerebral pathogenicity index (ICPI) was calculated as the mean score per bird per observation as described in the European Community Council Directive 92/66/EEC (1992). An index of 3.0 means that all birds died within 24 hours.
Vaccination of Chickens and Homologous Virus Challenge
The animal study was conducted under BSL3 conditions and after approval by the Animal Ethics Committee. SPF White Leghorn chickens (Charles River Laboratories, Germany), 6 weeks of age, were used in this study. Ten chickens per group were vaccinated by the intramuscular (i.m.; 0.5 ml) route with 107 TCID50 of vaccine virus. In addition, one group of 10 chickens was vaccinated i.m. with 5 μg of recombinant sH53(NDV) protein adjuvated with Stimune (Prionics, Lelystad). Another group of 10 chickens received an equal volume of PBS in Stimune (i.m.) to serve as mock-vaccinated control. Three weeks after the vaccination, all birds were challenged by intranasal (0.1 ml) and intratracheal (0.1 ml) inoculation of in total 105 TCID50 of H5N1 A/Vietnam/1194/04 virus. The chickens were monitored during the next 10 days for signs of disease and mortality. They received a score of 0 if they appeared normal, 1 if they were sick, 2 in case of severe illness and 3 if they were killed or dead, and were inspected twice a day as long as severe illness was scored. Birds that were reluctant to move in response to manipulation or were unable to reach the feeding and water bin were euthanized. The clinical index was calculated as described above for the ICPI. Blood samples were collected at 3 weeks after immunization.
HI Antibody Detection
Heat-inactivated immune sera prepared from chicken blood samples were serially diluted in cell culture medium in twofold steps. The samples were tested for hemagglutination inhibition (HI) activity using 1% chicken red blood cells and 4 hemagglutinating units (HAU) of H5N1 A/Vietnam/1194/04. For determination of NDV-specific HI antibodies 8 HAU of Herts/33 virus were used. Red button formation was scored as evidence of hemagglutination. Antibody titers were expressed as the reciprocal of the highest serum dilution showing HI.
Results
Virulence of HertsAPBC Virus
The cleavage site of the F0 protein is the major determinant of virulence of NDV (de Leeuw et al, 2005). In velogenic NDV strains, such as Herts/33, the cleavage site typically has a polybasic amino acid motif, which can be cleaved by intracellular proteases allowing the virus to grow in multiple organs. In order to generate attenuated virus, the polybasic cleavage site (PBC) of the F0 gene of pFL-Herts was modified into a monobasic cleavage motif. To confirm the attenuated nature of the virus recovered from this construct, its pathogenicity was tested in vivo. To this end, ten chickens received 0.1 ml of virus by intracerebral injection. Calculation of the clinical scores after 10 days of observation revealed an ICPI of 0.04, demonstrating that modification of the PBC indeed had turned the virus into a lentogenic pathotype.
Propagation of rNDV-sH53 and Expression of sH53 Protein
rNDV-sH53 virus could be readily recovered upon transfection of pFL-HertsΔPBC-H53 into VERO cells and grown up to high titers in embryonated chicken eggs and in the QM5 cell line. The allantoic fluid of infected eggs yielded a titer of 8.5 and the supernatant of QM5 cells a titer of 7.5 log TCID50 ml−1. Egg-cultured rNDV-HA vaccine virus had a titer of 7.5 log TCID50 ml−1.
To confirm expression of HA, QM5 cells grown in 6-well dishes were infected with egg-cultured rNDV-sH53 vaccine virus or mock-infected. As controls for protein expression, cells were infected with wt NDV (Herts) or rNDV-HA. At 2 days p.i. (post infection), the monolayers were subjected to immunostaining using F- and HA-specific mAbs. As shown in
Production and Purification of NDV-Expressed sH53 Protein
sH53 protein was purified from the supernatant of NDV-sH53-infected QM5 cells by using Strep-tactin beads. The purification product was analyzed by SDS-PAGE followed by Blue staining. As
Vaccination of Chickens with NDV-sH53 or sH53 Protein
To examine the immunogenicity of sH53 (NDV) and the potential of NDV-sH53 as a live vaccine, ten chickens were vaccinated intramuscularly (i.m.). To examine the protective efficacy of sH53 protein produced in NDV-sH53-infected QM5 cells, one group of ten chickens was vaccinated i.m. with Stimune-adjuvanted protein. Another 10 birds were mock-vaccinated to serve as challenge controls. Three weeks after the vaccination, all chickens were challenged with a lethal dose of the homologous H5N1 virus. As expected on basis of the ICPI, none of the chickens showed clinical signs upon vaccination.
The percentage of chickens surviving the infection and clinical scores per group observed after the challenge are shown in
Blood samples were collected 3 weeks after the immunization and assayed for the presence of HI antibodies directed against NDV and influenza virus. The HI assay revealed that i.m. vaccination was able to generate an immune response. HI antibody titers detected after i.m. vaccination with NDV-sH53 or sH53 protein reached levels between 12 and 256 (
All chickens that were vaccinated with rNDV-H53 showed an immune response to NDV, whereas the chickens that received an i.m. injection of sH53 only developed influenza HI antibody titers, as expected. NDV HI antibody titers of the NDV-sH53-vaccinated groups reached the same level as the titers of the NDV-HA-vaccinated groups (
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| Number | Date | Country | Kind |
|---|---|---|---|
| 1015952.8 | Apr 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL2011/050234 | 4/8/2011 | WO | 00 | 10/18/2012 |
