Rotavirus particles with chimeric surface proteins

Abstract

The present invention relates to the use of rotavirus particles for displaying a heterologous protein, alone or in complex with another molecule. The invention further relates to methods that employ these modified rotavirus particles to rapidly determine the structure of the heterologous protein or the complex using cryo-electron microscopy (cryo-EM). The invention also relates to a method of immunizing a patient, wherein said method comprises administering to the patient the modified rotavirus particles of the invention.

Claims

1. A chimeric protein complex comprising a trimer-forming rotavirus VP7 surface protein linked to a heterologous protein, wherein the rotavirus VP7 surface protein is linked to the heterologous protein non-covalently by a two-part adapter system, wherein the first part of the adapter system comprises a first adapter polypeptide that is fused to the rotavirus VP7 surface protein optionally via a linker sequence, and the second part of the adapter system comprises a second adapter polypeptide that is fused to the heterologous protein optionally via a linker sequence, wherein the first and the second parts of the adapter system form a stable complex with each other, and wherein the chimeric protein complex is capable of recoating and thereby forming a part of an outer layer of double-layered rotavirus particles in vitro.

2. The chimeric protein complex of claim 1, wherein the first adapter polypeptide and the second adapter polypeptide comprise a heptad repeat sequence.

3. A rotavirus particle comprising the chimeric protein complex of claim 1.

4. A formulation comprising a rotavirus particle of claim 3 and a solution, optionally together with an excipient.

5. A nucleic acid composition comprising: (a) an open reading frame encoding a modified rotavirus surface protein comprising a trimer-forming rotavirus VP7 surface protein, a first adapter polypeptide that is fused to the trimer-forming rotavirus VP7 surface protein optionally via a linker sequence; and (b) an open reading frame encoding a fusion protein comprising a protein that is heterologous to the trimer-forming rotavirus VP7 surface protein, a second adapter polypeptide that is fused to the heterologous protein optionally via a linker sequence, wherein the first and the second adapter polypeptides form a stable protein complex with each other; and (c) optionally a promoter sequence that is operationally linked to the open reading frame of (a) and further optionally a promoter sequence that is operationally linked to the open reading frame of (b).

6. The nucleic acid composition of claim 5, wherein the adapter polypeptide comprises a heptad repeat sequence.

7. A kit comprising: (a) a first nucleic acid encoding a modified rotavirus surface protein comprising a trimer-forming rotavirus VP7 surface protein and a first adapter polypeptide, and a second nucleic acid comprising a nucleotide sequence encoding a second adapter polypeptide and a multiple cloning site, and wherein insertion of a coding region for a heterologous protein in the multiple cloning site yields an open reading frame encoding a fusion protein comprising the heterologous protein and the second adapter polypeptide; or (b) a first nucleic acid encoding a modified rotavirus surface protein comprising a trimer-forming rotavirus VP7 surface protein and a first adapter polypeptide and a second nucleic acid encoding a fusion protein comprising a heterologous protein and a second adapter polypeptide; wherein, in each case (a) and (b), the first adapter polypeptide and the second adapter polypeptide are able to form a stable protein complex, optionally wherein the kit further comprises a rotavirus particle, wherein the particle is from the same species of rotavirus as the rotavirus from which the trimer-forming VP7 surface protein originated or from a different rotavirus species.

8. A method for preparing the rotavirus particle of claim 3, wherein the method comprises propagating a rotavirus particle comprising an outer layer in a cell grown in a culture medium, purifying the rotavirus particle from the culture medium, removing the outer layer from the rotavirus particle to obtain a rotavirus double-layered particle (DLP), and recoating the rotavirus DLP with the chimeric protein complex of claim 1 to yield the rotavirus particle of claim 3.

9. A method for preparing the rotavirus particle of claim 3, wherein the method comprises propagating a rotavirus particle comprising an outer layer in a cell grown in a culture medium, purifying the rotavirus particle from the culture medium, removing the outer layer from the rotavirus particle to obtain a rotavirus DLP, and recoating the rotavirus DLP with a first fusion protein comprising a trimer-forming rotavirus VP7 surface protein, a first adapter polypeptide comprising a heptad repeat sequence, and optionally a linker sequence and mixing the recoated rotavirus DLP with a second fusion protein comprising a trimer-forming heterologous protein, a second adapter polypeptide comprising a heptad repeat sequence, and optionally a linker sequence to yield the rotavirus particle of claim 3.

10. A method for preparing the rotavirus particle of claim 3 comprising mixing a rotavirus particle comprising a first fusion protein comprising a trimer-forming rotavirus VP7 surface protein, a first adapter polypeptide comprising a heptad repeat sequence, and optionally a linker sequence with a second fusion protein comprising a trimer-forming heterologous protein, a second adapter polypeptide comprising a heptad repeat sequence, and optionally a linker sequence to yield the rotavirus particle of claim 3.

11. A method for determining a structure of a heterologous protein, wherein the method comprises the steps of (i) recoating a rotavirus double-layered particle (DLP) with the chimeric protein complex of claim 1 to yield a suspension of rotavirus particles displaying the chimeric protein complex, (ii) freezing the suspension, (iii) imaging the rotavirus particles using cryo-EM to obtain a plurality of micrographs, and (iv) analysing the plurality of micrographs to obtain a three-dimensional model of the chimeric protein complex.

12. A method for determining a structure of a heterologous protein in complex with a molecule, wherein the method comprises the steps of (i) recoating a rotavirus double-layered particle (DLP) with the chimeric protein complex of claim 1 to yield a suspension of rotavirus particles displaying the chimeric protein complex, (ii) adding to the suspension a molecule that specifically binds to the heterologous protein, wherein the molecule forms a complex with the chimeric protein complex, (iii) freezing the suspension, (iv) imaging the rotavirus particles using cryo-EM to obtain a plurality of micrographs, and (vi) analysing the plurality of micrographs to obtain a three-dimensional model of the chimeric surface protein comprising all or part of the heterologous protein complexed to the molecule.

13. The method of claim 12, wherein the molecule is a proteinaceous molecule.

14. The method of claim 13, wherein the proteinaceous molecule is (a) an antibody or fragment thereof, wherein the antibody or fragment specifically binds the heterologous protein; or (b) a cell surface receptor, wherein the heterologous protein is a viral cell entry protein and the proteinaceous molecule is bound by the viral cell entry protein.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Schematic overview of modifications made to (A) the rotavirus VP7 protein and (B) the heterologous protein. HR1 and HR2 form part of a two-part adapter system for non-covalently binding the heterologous protein to the rotavirus VP7 protein. The heterologous signal peptide sequences serve the purpose of achieving high expression levels in the chosen expression systems. The affinity tags allow easy purification. Protease recognition site P.sub.1 can be used to remove the affinity tag A.sub.1 after purification. The second set of affinity and protease recognition sites (A.sub.2 and P.sub.2) can be replaced by suitable linker sequences and may serve as a spacer sequence that may be needed to display large heterologous protein. The trimerization tag is optional and can be used in aiding in trimer formation of some heterologous proteins which then facilitates binding to the trimeric rotavirus VP7 protein. The trimerization tag, particularly coiled-coil based trimerization tags (e.g. GCN4), can also serve as structural modules to extend the space available for a heterologous protein which is displayed on the surface of a rotavirus particle recoated with a modified rotavirus VP7 protein.

(2) FIG. 2: Schematic overview of the complex formed by a modified trimeric rotavirus VP7 protein and a trimeric heterologous protein. The rotavirus VP7 protein is non-covalently bound to the heterologous protein via a two-part adapter system, where one part of the adapter system (HR2) is linked to the rotavirus VP7 protein and the other part of the adapter system (HR1) is linked to the heterologous protein. HR1 and HR2 form a six-helix bundle resulting in a stable complex for non-covalently attaching the heterologous protein to the rotavirus VP7 protein. The chimeric surface protein can become part of the outer layer of the rotavirus particle by in vitro recoating DLPs.

(3) FIG. 3: Electron micrographs of (A) purified DLPs, (B) DLPs recoated with rotavirus VP7 protein and (C) DLPs recoated with VP7 protein displaying influenza virus HA as the heterologous protein. The particles were negatively stained prior to image acquisition.

(4) FIG. 4: (A) Coomassie-stained acrylamide gel after SDS-PAGE. Lane 1 shows the molecular weight marker (MW). Lane 2 was loaded with the purified DLPs used in the recoating reaction (DLP). Lane 3 was loaded with the purified VP7-HA protein complex (pro) used for recoating the DLPs. Lane 4 was loaded with the input mixture of DLPs, modified VP7 protein and HA protein for the recoating reaction (inp). Lanes 5-7 was loaded with the bands observed after purification of the recoated particles on a CsCl gradient (bands 1 and 2 and top band). Lanes 8-14 are loaded with the same samples in the same order as lanes 1-7, but no reducing agent was added to the samples prior to SDS-PAGE. (B) Rotavirus DLPs recoated with the VP7-HA protein complex on a CsCl gradient. Bands 1 and 2 and top band correspond to the samples loaded on lanes 5-7 in panel A. The positions where recoated particles and DLPs would typically migrate are indicated by dotted arrows.

(5) FIG. 5: Cross-section through a three-dimensional reconstruction of rotavirus DLP recoated with a modified VP7 protein (A) displaying HA, (B) displaying HA with bound ScFv fragments of antibody CR6261, and (C) displaying HA with bound Fab fragments of antibody CR6261. CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1/HA2. The reconstruction is based on images acquired by performing cryo-EM on recoated DLPs.

(6) FIG. 6: Superposition of a DLP (white) onto a three-dimensional reconstruction of a DLP recoated with modified rotavirus VP7 protein and influenza virus HA as the modified heterologous protein. The reconstruction is based on images acquired by performing cryo-EM on recoated DLPs.

(7) FIG. 7: Detail of three-dimensional reconstruction of a rotavirus particle displaying influenza virus HA bound to Fab fragments of antibody CR6261. The rotavirus particles were prepared by recoating DLPs with a modified VP7 protein containing an adapter sequence (HR2). The HA protein was non-covalently bound to the modified VP7 protein via an C-terminally fused HR1 heptad repeat sequence which forms a six-helix bundle with the HR2 heptad repeat sequences of the VP7 protein. CR6261 binds at the membrane proximal end of each HA subunit (HA1 and HA2). The reconstruction is based on images acquired by performing cryo-EM on recoated DLPs.

MODES FOR CARRYING OUT THE INVENTION

Example 1: Construction of Expression Vector for Modified Recombinant VP7, HA and HIV Gp140 Proteins

(8) The wild type VP7 gene of rhesus rotavirus (RRV) G3 strain was cloned into a pFastBacDual vector (Invitrogen) between the BamH I and Not I restriction sites via standard procedures. A Kozak sequence (GCCACC; SEQ ID NO: 19) was designed at the 5 end before the start codon of the VP7 coding sequence. Modifications of the VP7 coding sequence were carried out by inverted PCR. Primers of appropriate annealing temperatures were designed containing the relevant modifications at the ends of the primers. FIG. 1A shows the various modifications that were added to the rotavirus coding sequence.

(9) The final protein encoded by the modified VP7 coding sequence has the following features: The first 21 amino acids encode the honeybee melittin signal sequence for expression of the modified protein in insect cells. The VP7 protein signal peptide has been removed. Other signal sequences can be used as appropriate, such as the HIV consensus signal sequence or the signal peptide of human tissue plasminogen activator (htPA) for expression in human cells. (b) Amino acid residues 22 to 33 form an optional affinity tag (Strep-tag II plus a linker for more efficient signal peptide cleavage) for protein purification purposes and can be replaced by any other affinity tags, such as His-tag, HA-tag, FLAG-tag, etc. (c) Amino acid residues 34 to 40 form a TEV protease recognition sequence for cleavage of the affinity tag and can be replaced by the recognition sequences of any other proteases, such as PreScission protease (i.e. Rhinovirus 3C protease), factor Xa, enterokinase, thrombin, furin, etc. (d) Amino acid residues 41 to 136 are the sequences of rhesus rotavirus VP7 N-terminal portion (VP7 amino acid residues 51 to 146). (e) Amino acid residue 137 is a one-amino acid linker and could be replaced by another appropriate linker sequence. (f) Amino acid residues 138 to 165 are part of the C-heptad repeat of HIV gp41 HXB2 strain and could be replaced by a heptad repeat sequences from another retrovirus, paramyxovirus, etc. as long as replacement sequence is compatible to the heptad repeat sequences used in the modified HA or gp140 protein constructs (see below). (g) Amino acid residues 166 to 187 are a factor Xa protease recognition sequence followed by a protein C-tag flanked by linkers; this modification is optional and could be replaced by other sequences, such as a designed epitope or simply a linker sequence such as GGSGGSGGSGGSGGS (SEQ ID NO: 20) or GGSGGSGGSGGSGGSGG (SEQ ID NO: 21). Presenting an epitope can be useful for packing antibody fragments recognising the epitope to further stabilise the displayed assemblies (including the heterologous protein) for the purpose of structural studies. Like I said, designing for structural studies is way more complex than designing for presenting antigens. (h) Amino acid residues 188 to 367 are the C-terminal portion of rhesus rotavirus VP7 (VP7 amino acid residues 147 to 326). The modified VP7 protein encoded by this modified coding sequence has the amino acid sequence of SEQ ID NO: 22.

(10) Alternative constructs were prepared containing some of the modifications described in the preceding paragraph. For example, some of these constructs include an additional epitope tag which is recognised by anti-HIV antibody 2F5 (e.g. SEQ ID NO: 29 and 31). In other constructs, the C-heptad repeat of the HIV gp41 HXB2 strain was replaced with the C-heptad repeat of Nipah virus (e.g. SEQ ID NO: 32-41).

(11) The length of heptad repeat sequence was varied in some constructs (e.g. SEQ ID NO: 27 and 28). Similarly, the linker connecting the C-heptad repeat sequence and the remainder of the VP7 protein coding sequence was shortened in some of the constructs (see e.g. SEQ ID NO: 24-26) The different variants are summarised in Table 1. The modified VP7 protein sequence of SEQ ID NO: 22 is included as reference sequence. The sequences in Table 1 are shown from N-terminus to C-terminus.

(12) TABLE-US-00002 TABLE 1 Description of construct Sequence signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 22 (G)-HR2 (HIV, 28 a.a.)-linker 3 (Factor Xa-linker 3a (SGG)-Protein C tag-linker 3b (SGG))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 23 (G)-HR2 (HIV, 28 a.a.)-linker 3 (Factor Xa-linker 3a (SGG)-Protein C tag-linker 3b (G))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 24 (G)-HR2 (HIV, 28 a.a.)-linker 3 (linker 3a (SGG)-Protein C tag-linker 3b (SGG))- VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 25 (G)-HR2 (HIV, 28 a.a.)-linker 3 (linker 3a (SGG)-Protein C tag-linker 3b (G))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 26 (G)-HR2 (HIV, 28 a.a.)-linker 3 (linker 3a (G)-Protein C tag-linker 3b (G))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 27 (G)-HR2 (HIV, 42 a.a.)-linker 3 (linker 3a (SGG)-Protein C tag-linker 3b (SGG))- VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 28 (G)-HR2 (HIV, 42 a.a.)-linker 3 (linker 3a (G)-Protein C tag-linker 3b (G))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 29 (G)-HR2 (HIV, 28 a.a.)-linker 3 (linker 3a (G)-HIV antibody 2F5 epitope-linker 3b (SGG)-Protein C tag-linker 3c (SGG))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 30 (G)-HR2 (HIV, 28 a.a.)-linker 3 (linker 3a (G)-HIV antibody 2F5 epitope-linker 3b (G)-Protein C tag-linker 3c (G))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 31 (G)-HR2 (HIV, 28 a.a.)-linker 3 (linker 3a (G)-HIV antibody 2F5 epitope-linker 3b (GS)-Factor Xa-linker 3c (SGG)-Protein C tag-linker 3d (SGG))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 32 (G)-HR2 (Nipah, 30 a.a.)-linker 3 (linker 3a (K)-Factor Xa-linker 3b (SGG)-Protein C tag-linker 3c (SGG))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 33 (G)-HR2 (Nipah, 30 a.a.)-linker 3 (linker 3a (K)-Factor Xa-linker 3b (SGG)-Protein C tag-linker 3c (G))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 34 (G)-HR2 (Nipah, 30 a.a.)-linker 3 (linker 3a (SGG)-Protein C tag-linker 3b (SGG))- VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 35 (G)-HR2 (Nipah, 30 a.a.)-linker 3 (linker 3a (SGG)-Protein C tag-linker 3b (G))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 36 (G)-HR2 (Nipah, 30 a.a.)-linker 3 (linker 3a (G)-Protein C tag-linker 3b (G))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 37 (G)-HR2 (Nipah, 30 a.a.)-linker 3 (HIV antibody 2F5 epitope (14 a.a.)-linker 3a (SGG)-Protein C tag-linker 3b (SGG))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 38 (G)-HR2 (Nipah, 30 a.a.)-linker 3 (HIV antibody 2F5 epitope (14 a.a.)-linker 3a (G)- Protein C tag-linker 3b (G))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 39 (G)-HR2 (Nipah, 30 a.a.)-linker 3 (linker 3a (G)-HIV antibody 2F5 epitope (9 a.a.)- linker 3b (SGG)-Protein C tag-linker 3c (SGG))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 40 (G)-HR2 (Nipah, 30 a.a.)-linker 3 (linker 3a (G)-HIV antibody 2F5 epitope (9 a.a.)- linker 3b (G)-Protein C tag-linker 3c (G))-VP7 (147-326) signal (honeybee melittin)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 SEQ ID NO: 41 (G)-HR2 (Nipah, 30 a.a.)-linker 3 (linker 3a (G)-HIV antibody 2F5 epitope (9 a.a.)- linker 3b (GS)-Factor Xa-linker 3c (SGG)-Protein C tag-linker 3d (SGG))-VP7 (147-326) signal (htPA)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 (G)-HR2 SEQ ID NO: 42 (HIV, 28 a.a.)-linker 3 (Factor Xa-linker 3a (SGG)-Protein C tag-linker 3b (SGG))- VP7 (147-326) signal (htPA)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 (G)-HR2 SEQ ID NO: 43 (HIV, 28 a.a.)-linker 3 (linker 3a (G)-Protein C tag-linker 3b (G))-VP7 (147-326) signal (htPA)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 (G)-HR2 SEQ ID NO: 44 (Nipah, 30 a.a.)-linker 3 (linker 3a (K)-Factor Xa-linker 3b (SGG)-Protein C tag- linker 3c (SGG))-VP7 (147-326) signal (htPA)-linker1 (EDSA)-strep tag II-TEV-VP7 (51-146)-linker 2 (G)-HR2 SEQ ID NO: 45 (Nipah, 30 a.a.)-linker 3 (linker 3a (G)-Protein C tag-linker 3b (G))-VP7 (147-326)

(13) DNAs consisting of both the vector and the modified genes were generated by PCR. The PCR products were gel purified and subjected to T4 polynucleotide kinase treatment to generate phosphorylated ends. Blunt end ligation was then carried out for the resulting DNA by incubating the DNA with appropriate amounts of T4 ligase for 2 hours or overnight at room temperature. The ligation products were then treated with Dpn I for 30 minutes at 37 C. before being used to transform DH5 cells via standard protocols.

(14) Typically three to four colonies were picked and overnight cell cultures were grown to prepare plasmid DNA using the Miniprep Kit (Qiagen). The plasmids were examined by agarose gel electrophoresis and the correct sequences were confirmed by DNA sequencing. Plasmids of correct sequences were used to transform DH10Bac competent cells via standard procedures. Two to three white colonies were selected for each construct and overnight cell cultures were grown for the extraction of recombinant bacmid DNA by isopropanol/ethanol precipitation (Solution I: 15 mM Tris, pH 8.0, 10 mM EDTA, and 100 g/mL RNase A; Solution II: 0.2 M NaOH and 1% SDA; and Solution III: 3 M potassium acetate, pH 5.5; all filter-sterilized). The purified bacmids were examined by PCR using the M13 primers and the correct DNAs were used to transfect monolayers of sf9 cells in 6-well plates, each well seeded with 1 million cells. P1 viruses were harvested 5 days post transfection and P2 viruses were produced by infecting sf9 cells (density of 1.52 million/mL) with 0.050.1% P1 viruses. P2 viruses were harvested 57 days post infection and were used for protein expression.

(15) The modified VP7 protein constructs described above can be used to display trimer-forming heterologous proteins with correspondingly modified trimer-forming heterologous protein (see FIG. 1B for a schematic overview of suitable modifications). As shown in FIG. 2, the HR2 heptad repeat sequences of the modified VP7 protein and the HR1 heptad repeat sequences of the correspondingly modified trimer-forming heterologous protein form a stable complex via a six-helix bundle which surfaces as an adaptor for non-covalently mounting the heterologous protein on the rotavirus VP7 protein. Influenza A haemagglutinin (HA) protein and the gp140 fusion protein of HIV-1 were chosen as examples for trimer-forming heterologous proteins.

(16) The HA gene of influenza A virus H1N1 Solomon Islands 2006 was cloned into a pFastBac LIC vector (Life Technologies) by means of ligation independent cloning (LIC) method. The pFastBac LIC vector was created by inserting a LIC site in a pFastBac1 vector (Invitrogen). A Kozak sequence (SEQ ID NO: 19) was designed at the 5 end of the start codon before the coding sequence. Modification in the coding sequence of the HA gene were introduced by inverted PCR.

(17) The final protein encoded by the modified HA coding sequence has the following features: (a) The first 38 amino acids encode the Baculovirus gp64 signal peptide for expression of the modified protein in insect cells. The HA protein signal peptide has been removed. Other signal sequences can be used as appropriate, such as the HIV consensus signal sequence or the signal peptide of human tPA for expression in human cells. (b) Amino acid residues 42 to 47 form an optional affinity tag (His-tag plus a linker for more efficient signal peptide cleavage) for protein purification purposes and can be replaced by any other affinity tags, such as strep-tag, HA-tag, FLAG-tag, etc. (c) Amino acid residues 48 to 54 form a TEV protease recognition sequence for cleavage of the affinity tag. A linker sequence is also included. The TEV protease recognition sequence can be replaced by the recognition sequence of any other proteases, such as PreScission protease (i e Rhinovirus 3C protease, GE Healthcare, Life Sciences), factor Xa, enterokinase, thrombin, furin, etc. (d) Amino acid residues 55 to 381 are the sequences of the HA1 portion of the hemagglutinin of influenza A Solomon Islands 2006 strain, which could be modified/mutated as appropriate or be replaced by that of any other influenza strains as shown below. (e) Amino acid residues 382 to 386 are the recognition sequence of enterokinase engineered between HA1 and HA2 for cleavage purposes. This site is optional and could be replaced by recognition sequences of Factor Xa, TEV protease, or others as appropriate. (f) Amino acid residues 387 to 565 are the sequence of HA2 portion of HA. (g) Amino acid residues 566 to 595 are a trimerization tag from bacteriophage T4 fibritin (Foldon), which is optional and can be replaced by any other trimerization tag or linker sequences. (h) Amino acid residues 596 to 629 are part of the N-heptad repeat of HIV gp41 HXB2 strain and could be replaced by other suitable heptad repeat sequences e.g. from any other retrovirus, paramyxovirus, etc. The numbering of the HA amino acid sequences used in the description of the modified HA coding sequence is according to GenBank ID ABU50586.1 (incorporated herein by reference in the version available on the date of the filing of the priority application). The amino acid sequence of this construct is shown in SEQ ID NO: 46.

(18) Alternative constructs were prepared containing some of the modifications described in the preceding paragraph. For example, the trimerization domain was omitted from of the alternative constructs (e.g. SEQ ID NO: 48 and 49). In some constructs, the enterokinase recognition sequence was replaced by the Factor Xa recognition sequence (e.g. SEQ ID NO: 52). This construct also did not include a linker between the HA coding sequence and the C-heptad repeat sequence. In other constructs, both the trimerization domain and the enterokinase recognition sequence were omitted (SEQ ID NO: 51). The N-heptad repeat of Nipah virus was sometimes used in place of the N-heptad repeat of HIV gp41 HXB2 strain (see e.g. SEQ ID NO: 47 and 49). The length of heptad repeat sequence was varied in some constructs (e.g. SEQ ID NO: 54, 56 and 58). In other constructs, the length of the linker connecting the HA coding sequence to the N-heptad repeat sequence was varied (see e.g. SEQ ID NO: 56 and 58). Modified HA genes of H3 Wisconsin 2005 (see SEQ ID NOs: 60 and 61) and H5 Vietnam 2004 (see SEQ ID NOs: 62 and 63) were codon optimized and synthesized by GeneArt (Life Technologies) before they were included in modified constructs. The numbering of the HA amino acid sequences used in the descriptions of these constructs is according to GenBank IDs AAT73274.1 and ACV49644.1 (both incorporated herein by reference in the versions available on the date of the filing of the priority application), respectively. The structure of these modified HA coding sequences is summarised in Table 2. The modified HA protein sequence of SEQ ID NO: 46 is included as reference sequence. The sequences in Table 2 are shown from N-terminus to C-terminus.

(19) TABLE-US-00003 TABLE 2 Description of construct Sequence signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 46 (GYLLE)-HA (18-339, i.e. HA1)-enterokinase (DDDDK)-HA (344-519, i.e. HA2)- linker 3 (RSL)-Foldon (T4 Fibritin C terminal bit)-HR1 (HIV, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 47 (GYLLE)-HA (18-339, i.e. HA1)-enterokinase (DDDDK)-HA (344-519, i.e. HA2)- linker 3 (RSL)-Foldon (T4 Fibritin C terminal bit)-HR1 (Nipah, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 48 (GYLLE)-HA (18-339, i.e. HA1)-enterokinase (DDDDK)-HA (344-519, i.e. HA2)- HR1 (HIV, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 49 (GYLLE)-HA (18-339, i.e. HA1)-enterokinase (DDDDK)-HA (344-519, i.e. HA2)- linker 3 (RS)-HR1 (Nipah, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 50 (GYLLE)-HA (18-519)-HR1 (HIV, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 51 (GYLLE)-HA (18-519)-linker 3 (RS)-HR1 (Nipah, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 52 (GYLLE)-HA (18-339, i.e. HA1)-Factor Xa (IEGR)-HA (344-519, i.e. HA2)-HR1 (HIV, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 53 (GYLLE)-HA (18-339, i.e. HA1)-Factor Xa (IEGR)-HA (344-519, i.e. HA2)-linker 3 (RS) HR1 (Nipah, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 54 (GYLLE)-HA (18-339, i.e. HA1)-Enterokinase (DDDDK)-HA (344-511, i.e. HA2)- linker 3 (IGE)-HR1 (HIV, 39 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 55 (GYLLE)-HA (18-339, i.e. HA1)-Enterokinase (DDDDK)-HA (344-511, i.e. HA2)- linker 3 (IGEARQ)-HR1 (Nipah, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 56 (GYLLE)-HA (18-339, i.e. HA1)-Enterokinase (DDDDK)-HA (344-519, i.e. HA2)- linker 3 (RSI)-HR1 (HIV, 38 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 57 (GYLLE)-HA (18-339, i.e. HA1)-Enterokinase (DDDDK)-HA (344-519, i.e. HA2)- linker 3 (RSIRQ)-HR1 (Nipah, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 58 (GYLLE)-HA (18-339, i.e. HA1)-Enterokinase (DDDDK)-HA (344-519, i.e. HA2)- linker 3 (RSIKKLIGE)-HR1 (HIV, 39 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 59 (GYLLE)-HA (18-339, i.e. HA1)-Enterokinase (DDDDK)-HA (344-519, i.e. HA2)- linker 3 (RSIKKLIGEARQ)-HR1 (HIV, 34 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 60 (GYLLE)-HA (17-341, i.e. HA1)-Enterokinase (DDDDK)-HA (347-522, i.e. HA2)- linker 3 (RSL)-HR1 (HIV, 38 a.a.) signal (Baculovirus gp64 signal peptide)-linker 1 (ADP)-6xHis tag-TEV-linker 2 SEQ ID NO: 61 (GYLLE)-HA (17-341, i.e. HA1)-Enterokinase (DDDDK)-HA (347-522, i.e. HA2)- linker 3 (RSL)-Foldon (T4 fibritin C-terminal bit)-HR1 (HIV, 35 a.a.) signal (Baculovirus gp64 signal peptide)-HA (17-20)-6xHis tag-TEV-HA (21-345, SEQ ID NO: 62 i.e. HA1)-Enterokinase (DDDDK)-HA (346-521, i.e. HA2)-linker (RSL)-HR1 (HIV, 38 a.a.) signal (Baculovirus gp64 signal peptide)-HA (17-20)-6xHis tag-TEV-HA (21-345, SEQ ID NO: 63 i.e. HA1)-Enterokinase (DDDDK)-HA (346-521, i.e. HA2)-linker (RSL)-Foldon (T4 fibritin C-terminal bit)-HR1 (HIV, 35 a.a.)

(20) Each of the three modified HA genes were subcloned into pFastBacDual (Life Technologies) between the Sal I and Not I sites. Recombinant baculoviruses bearing the modified HA genes were created following similar procedures as those described for VP7.

(21) Modified gp140 genes of HIV-1 clade A 1992 Uganda 037.8 serotype and clade C 1997 were codon optimized and synthesized by GeneArt (Life Technologies). The modified genes were subcloned into pFastBacDual (Life Technologies) between the Sal I and Not I sites.

(22) The final protein encoded by the modified gp140 coding sequence of the HIV-1 clade A strain has the following features: (a) The first 21 amino acids encode the honeybee melittin signal peptide for expression of the modified protein in insect cells. The gp140 signal peptide has been removed. Other signal sequences can be used as appropriate, such as the HIV consensus signal sequence or the signal peptide of human tPA for expression in mammalian/human cells. (b) Amino acid residues 22 to 29 form an optional affinity tag (His-tag plus a linker for more efficient signal peptide cleavage) for protein purification purposes and can be replaced by any other affinity tags, such as strep-tag, HA-tag, FLAG-tag, etc. (c) Amino acid residues 30 to 36 form a TEV protease recognition sequence for cleavage of the affinity tag and can be replaced by the recognition sequences of any other proteases, such as PreScission protease (i.e. Rhinovirus 3C protease, GE Healthcare, Life Sciences), factor Xa, enterokinase, thrombin, furin, etc. (d) Amino acid residues 37 to 685 is the gp140 coding sequence of HIV strain 1992 Uganda 037.8 which could be modified/mutated as appropriate or be replaced by that of any other HIV/SIV strains. (e) Amino acid residues 686 to 694 are a linker and could be replaced by any appropriate other linker sequences. (f) Amino acid residues 695 to 721 are a trimerization tag from bacteriophage T4 fibritin (Foldon), which is optional or can be replaced by any other trimerization tag or linker sequences. (g) Amino acid residues 722 to 755 are part of the N-heptad repeat of HIV gp41 HXB2 strain and could be replaced by other suitable heptad repeat sequences e.g. from any other retrovirus, paramyxovirus, etc. The amino acid sequence of this construct is shown in SEQ ID NO: 64.

(23) Alternative constructs were prepared containing some of the possible modifications indicated in the preceding paragraph. For example, some of these constructs do not include the trimerization tag, and the linker sequence between the gp140 coding region and the N-heptad repeat has been shortened (e.g. SEQ ID NO: 66 and 67). Other constructs are adapted for expression in mammalian cells by replacement of the signal peptide (e.g. SEQ ID NO: 65 and 67). In some constructs, the N-heptad repeat of the HIV gp41 HXB2 strain has been replaced with the N-heptad repeat of Nipah virus (SEQ ID NO: 68-71 and 76-79), in others, the gp140 coding sequence from HIV-1 clade A 1992 Uganda 037.8 serotype has been replaced with the gp140 coding sequence from HIV-1 clade C 1997 (SEQ ID NO: 72-79). The numbering for the clade A and C gp140 is according to GenBank ID AAB05027.1 and AF286227.1 (both incorporated herein by reference in the versions available on the date of the filing of the priority application), respectively.

(24) The different variants are summarised in Table 3. The modified gp140 protein sequence of SEQ ID NO: 64 is also included as reference sequence. The sequences in Table 3 are shown from N-terminus to C-terminus.

(25) TABLE-US-00004 TABLE 3 Description of construct Sequence signal (honeybee melittin signal peptide)-linker1 (ED)-6xHis tag-TEV-HIV clade A SEQ ID NO: 64 gp140 (27-675)-linker2 (linker 2a (SR)-Factor Xa-linker 2b (GSG))-Foldon (T4 Fibritin C-terminal bit)-HR1 (HIV, 34 a.a.) signal (HIV consensus signal peptide)-linker1 (ED)-6xHis tag-TEV-HIV clade A SEQ ID NO: 65 gp140 (27-675)-linker2 (linker 2a (SR)-Factor Xa-linker 2b (GSG))-Foldon (T4 Fibritin C-terminal bit)-HR1 (HIV, 34 a.a.) signal (honeybee melittin signal peptide)-linker1 (ED)-6xHis tag-TEV-HIV clade A SEQ ID NO: 66 gp140 (27-675)-linker2 (GSG)-HR1 (HIV, 34 a.a.) signal (HIV consensus signal peptide)-linker1 (ED)-6xHis tag-TEV-HIV clade A SEQ ID NO: 67 gp140 (27-675)-linker2 (GSG)-HR1 (HIV, 34 a.a.) signal (honeybee melittin signal peptide)-linker1 (ED)-6xHis tag-TEV-HIV clade A SEQ ID NO: 68 gp140 (27-675)-linker2 (linker 2a (SR)-Factor Xa-linker 2b (GSG))-Foldon (T4 Fibritin C-terminal bit)-HR1 (Nipah, 32 a.a.) signal (HIV consensus signal peptide)-linker1 (ED)-6xHis tag-TEV-HIV clade A SEQ ID NO: 69 gp140 (27-675)-linker2 (linker 2a (SR)-Factor Xa-linker 2b (GSG))-Foldon (T4 Fibritin C-terminal bit)-HR1 (Nipah, 32 a.a.) signal (honeybee melittin signal peptide)-linker1 (ED)-6xHis tag-TEV-HIV clade A SEQ ID NO: 70 gp140 (27-675)-linker2 (S)-HR1 (Nipah, 34 a.a.) signal (HIV consensus signal peptide)-linker1 (ED)-6xHis tag-TEV-HIV clade A SEQ ID NO: 71 gp140 (27-675)-linker2 (S)-HR1 (Nipah, 34 a.a.) signal (honeybee melittin signal peptide)-linker1 (ED)-6xHis tag-TEV-Linker2 SEQ ID NO: 72 (AENLWV)-HIV clade C gp140 (31-667)-linker3 (S)-Foldon (T4 Fibritin C-terminal bit)-HR1 (HIV, 34 a.a.) signal (HIV consensus signal peptide)-linker1 (ED)-6xHis tag-TEV-Linker2 SEQ ID NO: 73 (AENLWV)-HIV clade C gp140 (31-667)-linker3 (S)-Foldon (T4 Fibritin C-terminal bit)-HR1 (HIV, 34 a.a.) signal (honeybee melittin signal peptide)-linker1 (ED)-6xHis tag-TEV-HIV clade C SEQ ID NO: 74 gp140 (31-667)-linker2 (SGI)-HR1 (HIV, 34 a.a.) signal (HIV consensus signal peptide)-linker1 (ED)-6xHis tag-TEV-HIV clade C SEQ ID NO: 75 gp140 (31-667)-linker2 (SGI)-HR1 (HIV, 34 a.a.) signal (honeybee melittin signal peptide)-linker1 (ED)-6xHis tag-TEV-Linker2 SEQ ID NO: 76 (AENLWV)-HIV clade C gp140 (31-667)-linker3 (S)-Foldon (T4 Fibritin C-terminal bit)-HR1 (Nipah, 32 a.a.) signal (HIV consensus signal peptide)-linker1 (ED)-6xHis tag-TEV-Linker2 SEQ ID NO: 77 (AENLWV)-HIV clade C gp140 (31-667)-linker3 (S)-Foldon (T4 Fibritin C-terminal bit)-HR1 (Nipah, 32 a.a.) signal (honeybee melittin signal peptide)-linker1 (ED)-6xHis tag-TEV-Linker2 SEQ ID NO: 78 (AENLWV)-HIV clade C gp140 (31-667)-linker3 (S)-HR1 (Nipah, 34 a.a.) signal (HIV consensus signal peptide)-linker1 (ED)-6xHis tag-TEV-linker2 SEQ ID NO: 79 (AENLWV)-HIV clade C gp140 (31-667)-linker3 (S)-HR1 (Nipah, 34 a.a.)

Example 2: Preparation of a Stable VP7-HA Complexes

(26) To produce HA-VP7 complexes, Hi5 cells at the density of 210.sup.6/mL were infected with 0.5% VP7 baculovirus and 1.0% HA baculovirus in the presence of 5.0% heat inactivated FBS (Sigma-Aldrich). The medium was harvested 5 days post infection by spinning down the cells at 4000g for 45 minutes. The supernatant was diafiltrated against 8 liters of 1TNC (20 mM Tris, pH 8.0, 100 mM NaCl, and 1.0 mM CaCl.sub.2; supplemented with 0.02% sodium azide) using a filter of 10 KDa cutoff in a Cogent M Tangential Flow Filtration System (Millipore). The buffer exchanged samples were supplemented with 1.0 mM PMSF and clarified by centrifugation for 1 hour at 10,000 RPM in a JA10 rotor (Beckman). The protein complexes were purified from the supernatant by a StrepTactin column (IBA), followed by a NiNTA column (Qiagen), and a Superose 6 column (Amersham)

(27) The purified HA-VP7 complexes were treated with 0.002% (w/w) enterokinase at 4 C. for 48 hours. The cleavage of HA0 into HA1 and HA2 was examined by SDS-PAGE. Enterokinase was inactivated by adding 1EDTA-free complete protease inhibitor tablet. The sample was supplemented with a redox buffer (10:1 molar ratio of GSH and GSSG) at a final concentration of 0.20.5 mM and treated with TEV protease (1/50200, w/w) either at room temperature for 4 hours or at 4 C. for overnight. Removal of the tags was also examined by SDS-PAGE.

Example 3: Preparation of a Stable HIV Gp140-VP7 Complexes

(28) The modified VP7 and gp140 genes encoding the proteins of SEQ ID NOs: 22, 26, 32 and 36 and SEQ ID NOs: 64, 66, 68 and 70, respectively were subcloned into pVRC8400 and pVRC-IRES-Puro vectors between Sal I and Not I for expression in mammalian cells. The signal peptide for the VP7 gene were changed to a human tPA signal peptide and the signal peptide for the gp140 gene were changed to the HIV consensus signal peptide sequence. The resulting modified VP7 proteins and modified gp140 proteins have the amino acid sequences of SEQ ID NOs: 42-45 and SEQ ID NOs: 65, 67, 69 and 71, respectively. Co-expression and complex formation were tested for the following combinations of modified VP7 protein and modified gp140 protein: SEQ ID NOs. 42+65, SEQ ID NOs. 42+67, SEQ ID NOs. 43+65, SEQ ID NOs. 43+67, SEQ ID NOs. 44+69, SEQ ID NOs. 44+71, SEQ ID NOs. 45+69, SEQ ID NOs. 45+71.

(29) HIV gp140-VP7 complexes were expressed by co-transfecting 293T cells with a plasmid hosting the gp140-HR1 gene and another hosting the VP7-HR2 gene. Transient transfection was done by following standard protocols using polyethylenimine (PEI, 25 KDa, linear or branched). For higher protein yield, stable cell lines were selected against puromycin. In brief, 70% confluent 293T cells were transfected with a mixture of Lipofectoamine 2000 (Invitrogen) and the pVRC-IRES-puro version of the two plasmids (2:1 mass ratio of lipofectoamine to total DNA) following standard procedures. At 20 hours post transfection, the medium was replaced with DMEM (Gibco) supplemented with 10% Fetal Bovine Serum (Atlas), 1% GlutaMAX (Gibco), 100 unites/mL penicillin/streptomycin (Gibco), and 2 g/mL puromycin (Gibco). The medium was kept fresh by changing it every three to four days until colonies form (visible to the naked eye). Individual colonies were picked and cultured in 24-well plates for small-scale expression tests. Colonies harbouring both genes were selected based on the detection of both proteins on western blots. The colony with the highest yield of the complex was scaled up and saved as stocks in liquid nitrogen. The cells were scaled up in puromycin-containing media and protein expression could be carried out using either adherent or suspension cell cultures in 293 Freestyle medium

(30) (Gibco) supplemented with 5% heat inactivated FBS (Sigma-Aldrich). The medium was harvested after 5 days by spinning down the cells at 4000g for 45 minutes. Diafiltration and protein purification were carried out following the same procedures as described for the HA-VP7 complexes.

Example 4: Preparation of Fabs and Fvs

(31) Monoclonal antibody CR6261 recognises a highly conserved helical region in the membrane-proximal stem of the influenza HA protein [67]. Structure factors and coordinates for the CR6261 sHgL Fab fragment were deposited with the Protein Data Bank under accession 4EVN (incorporated herein by reference in the version available on the date of the filing of the priority application) and have been described previously [68].

(32) The coding sequence for the antigen-binding fragment (Fab) and the single chain variable fragment (scFv) of monoclonal antibody CR6261 were cloned into the pVRC-IRES-Puro vector between Sal I and Not I following standard procedures as described above. The human tPA signal peptide was used for efficient secretion in these constructs in mammalian expression systems. The amino acid sequences of the final constructs are shown in SEQ ID NOs: 81-83. An scFv of monoclonal antibody CR6261 for expression in bacterial cells was also prepared (see SEQ ID NO: 80).

(33) Fabs were expressed by transient transfection of 293T cells in roller bottles. For each roller bottle, 125 g each of the purified plasmid DNA hosting the heavy and light chain genes were mixed in 12.5 mL unsupplemented DMEM (Gibco) and incubated in the hood for 15 minutes. PEI (500 m, linear or branched) was also diluted in 12.5 mL unsupplemented DMEM and incubated in the hood for 15 minutes before being added drop by drop and mixed well into the DNA mixture. The total of 25 mL mixture was incubated in the hood for an extra 15 minutes to allow a DNA-PEI complex to form before being added to cells at 5070% confluence in roller bottles. The cells were incubated for over 5 hours with the DNA-PEI complex at 37 C., and the medium was replaced by 250 mL of 293 FreeStyle medium (Gibco) supplemented with 100 units/mL penicillin/streptomycin. Medium was harvested 5 days post transfection, and Fabs were purified using a CaptureSelect Lc Kappa or Lc Lambda affinity resin, followed by size exclusion chromatography on an S200 column (Amersham)

(34) ScFvs were expressed either in 293T cells as secreted proteins or in E. coli as inclusion bodies, from which protein was then extracted and refolded. The same transient transfection procedures as described above were followed for mammalian cell expression. For the E. coli expression, protein expression was induced for about 5 hours using 1 mM IPTG at cell density of 0.60.8 O.D.600 nm. The cells were harvested, washed with 1PBS, and lysed by sonication on ice. The inclusion bodies were extracted after removing the soluble fractions of the cells by centrifugation for 15 minutes at 20000g. Inclusion bodies were dissolved in 100 mM Tris pH 7.5, 8 M urea, and 10 mM -mercaptoethanol, clarified by centrifugation (20 minutes at 40000g), and the supernatant was purified on a NiNTA column. The eluted sample, also in the denatured form, was diluted into the refolding buffer (100 mM Tris pH 7.5, 1 M arginine, 500 mM NaCl, 10% glycerol, and 1 mM EDTA) drop by drop at 4 C. to a final protein concentration of lower than 0.1 mg/mL. The refolded sample was dialyzed against 20 mM Tris, pH 7.5, 100 mM NaCl four times, each time 20 volumes. The dialyzed sample was then clarified by centrifugation at 10,000 rpm in a JA10 rotor (Beckman) and the supernatant was passed through a NiNTA column to concentrate the protein. The eluted fractions were then combined, concentrated, and further purified on an S200 size exclusion column (Amersham)

Example 5: DLP Preparation

(35) Rhesus rotavirus serotype G3 was amplified by infecting MA-104 cells. Briefly, MA104 cells were grown in M199 medium (Gibco) supplemented with 10% fetal bovine serum (HyClone Laboratories), 10 mM HEPES, pH 7.0, 2 mM L-glutamine, and 100 units/mL penicillin/streptomycin. The cells were maintained and scaled up to a 10-stack cell culture chamber (Corning) or roller bottles. When the cells became confluent, the medium was replaced by serum-free MA199 supplemented with 1 g/mL porcine pancreatic trypsin (Sigma-Aldrich) for rotavirus inoculation and amplification. The medium of infected MA104 cells was harvested about 3642 hours post infection and stored at 80 C. for future use.

(36) The frozen medium of infected MA104 cells was thawed at 4 C. overnight. Cell debris was cleared by low-speed centrifugation at 3000g for about 10 minutes. The resulting supernatant was filtered through Whatman Filter paper to remove residual cell debris before it was further passed through a 0.45 m ExpressPlus filter unit (Millipore) under vacuum. The virus particles were then pelleted at 45,000 rpm for 1 h at 4 C. in a 45Ti rotor (Beckman)

(37) The pellet was resuspended in a total of 10 mL of 1TNE buffer (20 mM Tris, pH 8.0, 100 mM NaCl, and 1 mM EDTA), briefly sonicated, and extracted twice with Freon 113 (Sigma-Aldrich). The aqueous phase was recovered and concentrated into about 1 mL in an Amicon centrifugal filtration device (100 KDa cutoff) at 3000g for about 10 minutes. The concentrated sample was resuspended by pipetting up and down a few times and layered over a preformed CsCl gradient in 1TNE (1.26 to 1.45 g/mL density as determined by refractometry). Samples were centrifuged at 55000 rpm in an SW 55Ti rotor (Beckman) at 4 C. for about 2 hours. The DLP band was collected and dialyzed overnight at 4 C. against 1TN buffer (20 mM Tris, pH 8.0, 100 mM NaCl) supplemented with 0.02% sodium azide.

(38) The DLPs were negatively stained and visualised using electron microscopy (EM; FIG. 3A).

Example 6: Recoating of DLPs with HA-VP7 Complex

(39) The DLPs obtained in Example 5 and the modified rotavirus VP7 protein prepared in Example 1 were mixed and incubated at 4 C. for at least 1 hour in the presence of 510 mM CaCl.sub.2. The resulting particles were negatively stained and visualised using EM. A single VP7 layer covering each particle was observed confirming that the modified VP7 protein was able to recoat DLPs (FIG. 3B).

(40) Recoating of DLPs with the HA-VP7 complex obtained in Example 2 was carried out at 4 C. for at least 1 hour by mixing them at a molar ratio of 1:1.5 to 1:2 in the presence of 510 mM CaCl.sub.2. The recoated rotavirus particles were negatively stained and visualised using EM. In addition to the single VP7 layer covering the DLP, a further layer surrounding the recoated particles was observed suggesting that the HA-VP7 complex correctly assembled on the surface of the DLPs (FIG. 3C).

(41) To confirm that the additional layer was formed by the HA protein, the recoated particles were loaded onto a preformed CsCl gradient in 1TNC (density 1.25 g/mL to 1.45 g/mL) and centrifuged in a SW60 rotor (Beckman) at 58,000 rpm for 2 hours. The resulting bands were collected, dialyzed against 1TNC, and examined by SDS-PAGE. As a control, DLPs, the HA-VP7 complex and the mixture of both as used in the recoating reaction were also loaded on the gel in separate lanes. After separation, separate bands corresponding to the VP2 and VP6 proteins of the DLPs, the VP7 protein and the HA protein were observed at the correct stoichiometry in the sample that corresponded to the recoated particles (FIG. 4). This confirmed that the further layer surrounding the recoated particles was composed of HA protein at full occupancy.

Example 7: Three-Dimensional Image Reconstruction of HA-VP7-Antibody Complex

(42) High-resolution cryo-EM has the potential to replace X-ray crystallography as the method of choice for analysing large molecular complexes, in particular in areas where rapid structural elucidation of a large number of molecular complexes is needed. One such area is influenza vaccine design, where antigenic drift leads to rapid changes in the sequence of the immunodominat surface antigen, the HA protein. Broadly protective monoclonal antibodies have been previously identified and many of these antibodies recognise conserved epitopes that are not subjected to rapid change. To investigate their suitability as protective agents against newly emerging influenza strains, we need to screen these broadly neutralising anti-HA antibodies against different variants of the HA protein.

(43) The feasibility of such an approach using cryo-EM analysis was tested. DLPs were recoated with modified VP7 protein alone or with a HA-VP7 complex as described in Example 6. HA-VP7-antibody recoated particles were obtained by mixing the purified HA-VP7 complexes with the Fabs or scFvs of monoclonal antibody CR6261 prepared in Example 4 at a molar ratio of 1:1.52.0. The mixture was incubated at 4 C. for 30 minutes. The complexes were purified by loading the mixture after the end of the incubation period on a Superose 6 column. The purified complexes were then used to recoat DLPs following the same procedures as described in Example 6. Alternatively, the antibody fragments were added directly to particles recoated with HA-VP7 complexes at a molar ratio of 1:1.21.5 either before or after the CsCl gradient purification step.

(44) Cryo-grids were prepared with a Vitrobot Automated plunger (FEI). Quantifoil Holey carbon grids were glow discharged and left at room temperature overnight before use. For each grid, 4 L sample at a concentration equivalent to 5 mg/mL rotavirus DLPs was applied to one side surface of the grid. During plunging, the chamber moisture was maintained close to 100% and the temperature at around 22 C. The grid was then blotted for 4 seconds from both sides with filter paper, immediately followed by plunging into liquid ethane for vitrification. The grids were then stored in liquid nitrogen before being used for data collection.

(45) Data were collected on a Tecnai F30 electorn microscrope (FEI) operated at 300 kV. The optical system was aligned using standard procedures (beam shift, beam tilt, eucentric height, pivot points, rotational center, astigmatism, etc.). The image acquisition procedures involved finding the desired imaging area, adjusting defocus (between 0.6 and 3.0 m), testing grid drift rate (less than 2 per second), and the final image exposure. These procedures were semi-automatically achieved using the program SerialEM (Cryo-electron Microscopy Facility, University of ColoradoBoulder). The data were recorded as movies on a K2 Summit direct detection camera (Gatan) using super resolution mode (pixel size: 0.99 ). The movie protocol of reference 24 was used. The dose rate on the sample was 3 electrons/.sup.2 per second. Each frame recorded 0.5 second exposure and the final movie consisted of 24 frames with an accumulated dose of 36 electrons/2.

(46) The frames of each movie were aligned using IMOD (Cryo-electron Microscopy Facility, University of ColoradoBoulder) scripts based on image cross correlation. The aligned frames were simply averaged for initial image processing. As the resolution improved later, however, the averaged images from the first 13 frames (or the best series of frames) of each movie, which would correspond to 20 electron dose, were used. Particle images were picked using e2boxer.py of the EMAN2 image processing suite [69]. Images with obvious defects, aberrations, abnormal focus, contamination, overlap, or large sample drifts were manually excluded after visual inspection. The particle coordinates were used to excise image stacks of individual particles with 16001600 pixel dimensions using proc2d of the EMAN2 image processing suite [69]. Defocus values were determined using the program CTFFIND3 [23].

(47) The structure refinement and reconstructions were carried out using the program FREALIGN [40]. The initial orientation search was performed using 4 binned data and a previously calculated map of the VP7 recoated particles (7RP) (see reference 1) as a 3-dimensional reference. The initial alignment parameters of the excised particles, determined by a systematic search (mode 3) at a 1 angular interval, were further refined against the latest reconstructions (mode 1) until there was no further improvement in resolution. During alignment, a radial shell mask between 220 and 400 was applied to retain the density corresponding to the rotavirus portion and to exclude density corresponding to the RNA and HA spikes. The images were also low pass filtered (up to 15 ) to avoid possible overfitting in the alignment process. The alignment parameters were then used to calculate the reconstructions of the entire particle (within the radial shell of 600 or between the radii of 220 and 600 for the protein contents).

(48) Subsequently, 2 binned and later unbinned images were used to further refine the alignment parameters following similar strategies to those used for the 4 binned data, i.e. an initial systematic search (mode 3) followed by multiple cycles of angular and positional refinement (mode 1) until no further improvement in resolution was observed. During each stage of refinement, the shell mask between 220 and 400 was applied and the data were restricted to the most reliable resolution (up to 10 for 2 binned data and up to 8 for unbinned data). Reconstructions were calculated for the entire particle within the radial shell of 600 .

(49) FIG. 5A shows a cross-section through a three-dimensional reconstruction of rotavirus DLP recoated with modified VP7 protein only. FIG. 5B shows a cross-section through a three-dimensional reconstruction of rotavirus DLP recoated with an HA-VP7 complex as described in Example 6. FIG. 6 shows a superposition of a DLP onto the three-dimensional reconstruction of the recoated particle shown in FIG. 5B. FIG. 5C shows a cross-section through a three-dimensional reconstruction of rotavirus DLP recoated with modified VP7 protein displaying HA with bound ScFv fragments of antibody CR6261.

(50) To assess the reliability of refinement, different sets of refinement were carried out by masking out different regions of the particle, and the alignment parameters were used to calculate reconstructions for the omitted regions that were not included during refinement. For example, alignment parameters obtained by refining the HA portion (by applying a shell mask between 425 and 600 ) were used to calculate the reconstruction for the rotavirus portion (by applying a shell mask between 220 and 400 ). The resulting map contained densities clearly representing VP2, VP6, and VP7, suggesting minimum model bias. The T=13 quasi-equivalent arrangement of the rotavirus VP6 and VP7 layers clearly extended to the adaptor and HA portion of the particle. Local 13-fold averaging was not applied but can be performed to further improve the resolution. Rigid body fitting of the rotavirus VP2, VP6, and VP7, the six-helix bundle, the HA, and the Fabs into the electron density maps was performed using the UCSF chimera [47].

(51) An example of the level of detail that can be achieve by further refining the reconstructions obtained from cryo-EM as described in the preceding paragraph is shown in FIG. 7. A detail of the three-dimensional reconstruction of a rotavirus particle displaying influenza virus HA bound to Fab fragments of antibody CR6261 is shown. Some secondary structures can be visualised in the calculated map. As expected, the variable domain of CR6261 is seen to interact with both HA1 and HA2.

Example 8: Immunisation Studies with HA-VP7 Protein Complexes Mounted on Rotavirus DLPs

(52) Female 6-8-week-old BALB/c mice are immunised intramuscularly (IM) three times at three-week intervals with one of three different formulations, each formulated in 100 l of a calcium-containing buffer. Group 1 receives of formulated HA-VP7 protein complexes mounted on rotavirus DLPs, group 2 receives purified HA, and group 3 receives purified HA mixed with VP7-recoated DLPs. A fourth group of mice is mock-injected with 100 l buffer. Experiments can include various doses for each of the groups. Equivalent amounts of antigen can be used to compare immune response to equivalent doses of antigen in various formats. Alternatively, equivalent amounts of total protein can be used to compare immune responses to various amounts of total protein.

(53) One or more groups can include an adjuvant. Adjuvants can be particularly useful in stimulating an immune response in the mice in group 2 receiving purified HA antigen.

(54) Each vaccine group contains eight mice, and the buffer alone control group contains five mice. Blood is drawn at days 1, 20, 41, 56 and 84 from administration of the first injection.

(55) Mice are monitored after immunisation for body weight change, injection site reactions, and other clinical observations. The antibody responses to the administered antigens are analysed using methods known in the art to analyse antibody specificity (e.g. ELISA assay and competition ELISA assay against HA and rotavirus proteins including VP7 protein) and neutralization activity (e.g. influenza haemagglutination inhibition assay, see e.g. references 70 and 71).

(56) Results demonstrate a substantially higher immune response to the HA-VP7 protein complexes mounted on rotavirus DLPs as compared to the purified HA, including purified HA formulated with adjuvant. Competition ELISA demonstrate that antibody responses to the HA-VP7 protein complexes are predominantly to the HA portion of the complex rather than the VP7 or rotavirus portion of the complex. Results demonstrate substantially higher neutralization antibody titres as a result of immunization with the HA-VP7 protein complexes mounted on rotavirus DLPs as compared to the purified HA, including purified HA formulated with adjuvant. These results demonstrate the increased immunogenicity of flu antigens when mounted on rotavirus VP7-coated DLP as compared to flu antigens not mounted on VP7-coated DLPs.

(57) All animal experiments are performed in accordance with Institutional Animal Care and Use Committee protocols.

Example 9: Immunisation Studies with HA-VP7 Protein Complexes Mounted on Rotavirus DLPs with and without Adjuvant

(58) Female BALB/c mice are immunized by two bilateral 50 l intramuscular injections in the rear quadriceps on days 0, 21, and 42 with 0.3-15 g of purified antigen H1N1 hemagglutinin (HA) protein complexes as set out in Table 4 below.

(59) The complexes are formulated with PBS or adjuvant 1-2 hours prior to immunization. The formulated subunit vaccines are kept on ice until administration.

(60) Mice are monitored after immunisation for body weight change, injection site reactions, and other clinical observations. Serum samples are obtained from the animals by retro-orbital sinus bleeds on day 20 (3 weeks post first immunization) and day 41 (3 weeks post second immunization) and from bleed-outs of euthanized animals on day 63 (3 weeks post third immunization).

(61) The antibody responses to the administered antigens are analysed using methods known in the art (e.g. ELISA assay, neutralization assay). Results demonstrate a substantially higher immune response to the HA-VP7 protein complexes mounted on rotavirus DLPs as compared to the purified HA, including purified HA formulated with adjuvant or purified HA formulated with, but not mounted on VP7-coated DLPs.

(62) All animal experiments are performed in accordance with Institutional Animal Care and Use Committee protocols.

(63) TABLE-US-00005 TABLE 4 Group IM Vaccine (dose) N 1 A/Sol Monobulk (0.3 g) 5 2 7RP (0.3 g) 5 3 7RP (3 g) 5 4 7RP (15 g) 5 5 HA + 7RP (0.3 g) 5 6 HA + 7RP (3 g) 5 7 HA + 7RP (15 g) 5 8 HA-7RP (0.3 g) 5 9 HA-7RP (3 g) 5 10 HA-7RP (15 g) 5 11 A/Sol HA (0.3 g) 5 12 A/Sol HA (3 g) 5 13 A/Sol HA (15 g) 5 A/Sol monobulk is lipid-oligomerized trimerized, recombinant HA. 7RP is VP7 recoated particles without HA. HA + 7RP is VP7 recoated particles on which HA cannot be mounted + trimerized recombinant HA. HA-7RP is VP7 recoated particles mounted with trimerized, recombinant HA. A/Sol HA is trimerized recombinant HA.

(64) Each of the references, patents, and patent applications cited herein and listed below is hereby incorporated by reference in its entirety as if each were incorporated individually. Each Accession number cited herein is incorporated herein by reference in the version available on the date of the fling of the priority application on which the instant application is based.

(65) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

(66) [1] Chen et al. (2009) Proc Natl Acad Sci USA 106(26):10644-8 [2] Trask & Dormitzer (2006) J Virol 80(22):11293-304 [3] Kobayashi et al. (2007) Cell Host Microbe 1(2):147-157 [4] Chandran et al. (1999) J Virol 73(5):3941-50 [5] Chandran et al. (2001) J Virol 75(11):5335-42 [6] Yan et al. (2011) J Virol 85(15):7483-95 [7] Miyazaki et al. (2008) J Virol 82(22):11344-53 [8] Zhang et al. (2010) Cell 141(3):472-82 [9] Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition, C D C, 2009 [10] Yang et al. (2000) J Virol 74(10):4746-54 [11] Meier et al. (2004) J Mol Biol 344(4):1051-69 [12] Wu & Wong (2005) J Biol Chem 280(24):23225-31 [13] Beckett et al. (1999) Protein Sci 8(4):921-9 [14] Chen et al. (1997) Cell 89(2):263-73 [15] Zhu et al. (2003) Protein Eng 16(5):373-9 [16] Yu et al. (2002) J Gen Virol 83(Pt 3):623-9 [17] Xu et al. (2004) J Biol Chem 279(29):30514-22 [18] http://partsregistry.org/Protein_domains/Linker [19] U.S. Pat. No. 4,769,326 [20] Dormitzer et al. (2001) J Virol 75(16):7339-50 [21] Dormitzer et al. (2000) Virology 277(2):420-8 [22] Mohd Jaafar et al. (2005) J Gen Virol 86(Pt 4):1147-57 [23] Mindell & Grigorieff (2003) J Struct Biol 142(3):334-47 [24] Campbell et al. (2012) Structure 20(11):1823-8 [25] Settembre et al. (2011) EMBO J. 30(2):408-16 [26] Chen & Grigorieff (2007) J Struct Biol 157(1):168-73 [27] Wriggers et al. (1999) J Struct Biol 125(2-3):185-95 [28] Jiang et al. (2001) J Mol Biol 308(5):1033-44 [29] Topf et al. (2005) J Struct Biol 149(2):191-203 [30] Tama et al. (2004) J Struct Biol 147(3):315-2 [31] Topf et al. (2008) Structure 16(2):295-307 [32] Trabuco et al. (2009) Methods 49(2):174-80 [33] Schroder et al. (2007) Structure 15(12):1630-41 [34] Zhang et al. (2010) Nature 463(7279):379-83 [35] Baker et al. (2006) PLoS Comput Biol 2(10):e146 [36] DiMaio et al. (2009) J Mol Biol 392(1):181-90 [37] Topf et al. (2006) J Mol Biol 357(5):1655-68 [38] Zhu et al. (2010) J Mol Biol 397(3):835-51 [39] Liang et al. (2002) J Struct Biol 137(3):292-304 [40] Grigorieff (2007) J Struct Biol 157(1):117-25 [41] Cheng et al. (2010) J Mol Biol 397(3):852-63 [42] Jiang et al. (2008) Nature 451(7182):1130-4 [43] Ludtke et al. (2008) Structure 16(3):441-8 [44] Yu et al. (2008) Nature 453(7193):415-9 [45] Ludtke et al. (1999) J Struct Biol 128(1):82-97 [46] Baker et al. (2007) Structure 15(1):7-19 [47] Pettersen et al. (2004) J Comput Chem 25(13):1605-12 [48] Emsley & Cowtan (2004) Acta Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126-32 [49] Emsley et al. (2010) Acta Crystallogr D Biol Crystallogr 66(Pt 4):486-501 [50] Baker et al. (2011) J Struct Biol 174(2):360-73 [51] Zhu et al. (2010) J Mol Biol 397(3):835-51 [52] Jones et al. (1991) Acta Cystallogr A 47 (Pt 2):110-9 [53] Brnger et al. (1998) Acta Cystallogr D Biol Crystallogr 54(Pt 5):905-21 [54] Li & Zhang (2009) Proteins 76(3):665-76 [55] Brnger (1992) Nature 355(6359):472-5 [56] Laskowski et al. (1993) J Appl Cryst 26:283-91 [57] Whittle et al. (2011) Proc Natl Acad Sci USA 108(34):14216-21 [58] Aoki et al. (2009) Science 324(5933):1444-7 [59] Krause et al. (2011) J Virol 85(20):10905-8 [60] Bommakanti et al. (2010) Proc Natl Acad Sci USA 107(31):13701-6 [61] Chakraborty et al. (2006) Biochem J 399(3):483-91 [62] Bhattacharyya et al. (2010) J Biol Chem 285(35):27100-10 [63] Ward et al. (1989) Nature 341:544-546 [64] Bird et al. (1988) Science 242:423-426 [65] Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883 [66] Council of Europe (2014) European pharmacopoeia [67] Ekiert et al. (2009) Science 324(5924): 246-251 [68] Lingwood et al. (2012) Nature 489(7417):566-70 [69] Tang et al. (2007) J Struct Biol 157(1):38-46 [70] Salk et al. (1945) Am J Hyg 42:57-93 [71] de Jong et al., Haemagglutination-inhibiting antibody to influenza virus. In Brown et al. (eds.) (2003) Laboratory Correlates of Immunity to InfluenzaA Reassessment. Basel, Switzerland: Karger, 63-73