# Patent

## Title
Recombinant neuraminidase and uses thereof

## Abstract

A recombinant neuraminidase based on amino acid sequence (SEQ ID NO: 1) of wild-type pH1N1-NA (A/Texas/05/2009) influenza virus is provided. The recombinant neuraminidase of the present invention has an ectodomain with an amino acid sequence identical to SEQ ID NO: 1 and replaced at specific positions 149, 344, 365 and 366 residue(s) with corresponding amino acids of other influenza viruses. The recombinant neuraminidase may incur cross-protective immunity and be used as universal influenza vaccine.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to recombinant neuraminidase and uses thereof, particularly to recombinant neuraminidase capable of being used as universal influenza vaccine and uses thereof.
2. Description of the Prior Art
Members of the Orthomyxoviridae family, influenza A viruses are enveloped viruses containing a single strand, 8-segment negative sense RNA genome typically encoding 11-12 viral proteins. Influenza A virus subtypes have been classified based on the antigenic properties of hemagglutinin (HA) and neuraminidase (NA) glycoproteins, respectively designated as H1-H16 and N1-N9. One recent report describes H17N10 and H18N11 identified from fruit bats. According to phylogenetic analyses, N1-N9 can be classified as belonging to group 1 (including N1, N4, N5 and N8) or group 2 (including N2, N3, N6, N7 and N9). To date, only N1, N2 and N9 subtypes are known to trigger human epidemics.
NA, an enzymatic protein with a tetrameric complex structure, is capable of cleaving sialic acid linkages on cell surfaces, thereby facilitating viral release from infected cells. NA also contributes to viral transmission and infection by destroying decoy receptors on cilia, mucins, and cellular glycocalyx. NA immunogenicity was first observed in human subjects immunized with a NA-specific inactivated vaccine. The use of recombinant NA (rNA) proteins expressed in yeast or insect cells elicits protection against lethal virus challenges in immunized mice. Ferrets immunized with rNA proteins exhibit a distinctive type of protection in addition to that provided by HA immunization alone. NA-inhibiting (NI) antibodies are known to limit virus spreading and to mitigate clinical symptoms of IAV infection. Mice immunized with a reverse-genetic reassortant H1N1 virus containing seasonal influenza virus NA exhibit cross-reactive NI antibodies and reduced mortality from pH1N1 virus challenges. Live attenuated influenza vaccines (LAIVs) for seasonal H1N1, H3N2 and pH1N1 strains have been reported as inducing cross-reactive NI antibodies to H5N1 viruses in ferrets, and NI antibodies elicited by a seasonal trivalent influenza vaccine have been reported as providing cross-protective immunity against lethal H5N1 challenges, also in ferrets. Further, NA-based virus-like particles (VLPs) containing NA, M1 and M2 have been shown to elicit more potent NI antibodies and to confer cross-protective immunity against H5N1 and pH1N1 viral challenges in mice. NI antibodies have also been detected in humans vaccinated with an H5N1 inactivated vaccine, as well as in humans exposed to natural infections. NA immunogenicity and cross-protective mechanisms remain unclear.
SUMMARY OF THE INVENTION
The present invention is directed to providing a recombinant neuraminidase that may incur cross-protective immunity and be used as universal influenza vaccine.
According to one embodiment of the present invention, a recombinant neuraminidase comprises an ectodomain provided with an amino acid sequence at least 95% identical to SEQ ID NO: 1, wherein the amino acids at positions 365 and 366 is replaced from IS to TN.
According to another embodiment of the present invention, a polynucleotide encoding the above-mentioned recombinant neuraminidase is provided.
According to still another embodiment of the present invention, a recombinant influenza virus comprises the above-mentioned recombinant neuraminidase or polynucleotide encoding the same.
According to yet another embodiment of the present invention, an influenza virus vaccine comprises the above-mentioned recombinant neuraminidase or polynucleotide encoding the same, wherein the influenza virus vaccine may be an inactivated vaccine, an attenuated influenza, a virus-like particle vaccine or a subunit vaccine.
Other advantages of the present invention will become apparent from the following descriptions taken in conjunction with the accompanying drawings wherein certain embodiments of the present invention are set forth by way of illustration and examples.



BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing aspects and many of the accompanying advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed descriptions, when taken in conjunction with the accompanying drawings, wherein:
FIGS. 1A to 1D illustrate construction, expression and characterization of soluble recombinant NA (rNA) proteins;
FIGS. 2A to 2E illustrate NA-specific IgG antibodies induced by rNA immunization;
FIGS. 3A to 3H illustrate NA-inhibiting antibodies elicited by H5N1-rNA and pH1N1-rNA immunization against H5N1, pH1N1, H3N2 and H7N9 virus;
FIG. 4 illustrates the corresponding IC50 values elicited by H5N1-rNA and pH1N1-rNA immunization against homologous and heterologous viruses;
FIG. 5 illustrates detection of H5N1-rNA and pH1N1-rNA specific antibodies secreting B-cells in spleens;
FIGS. 6A to 6E illustrate H5N1-rNA and pH1N1-rNA protective immune responses against different virus challenges;
FIGS. 7A to 7E illustrate body weight recovery tied to H5N1-rNA and pH1N1-rNA immunization responses against different virus challenges;
FIG. 8 illustrates the mutated sites and comparison result between pH1N1 and H5N1;
FIG. 9 illustrates the top view and side view of pH1N1 NA;
FIGS. 10A to 10D illustrate the increased NA-inhibiting antibody curves produced by WT and group 1 mutant pH1N1-rNAs immunizations against different virus strains;
FIG. 11 illustrates the corresponding IC50 values against the homologous pH1N1 viruses and the heterosubtypic H5N1, H3N2 and H7N9 viruses;
FIGS. 12A and 12B illustrate the amino acid alignments of different groups neuraminidase (NA);
FIGS. 13A to 13D illustrate increased NA-inhibiting antibody curves produced by WT and group2 mutant pH1N1-rNAs immunizations against different virus strains;
FIG. 14 illustrates the corresponding IC50 values against the homologous pH1N1 viruses and the heterosubtypic H5N1, H3N2 and H7N9 viruses;
FIGS. 15A to 15C illustrate plaque morphologies, virus titers and NA enzymatic activity of PR8 viruses, pH1N1/PR8 viruses, and pH1N1/PR8 viruses with the I365T/S366N mutation.



DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention provides a recombinant neuraminidase, which is based on the sequence list (SEQ ID NO: 1) of emerging wild-type influenza virus pH1N1-NA (A/Texas/05/2009). The recombinant neuraminidase of the present invention comprises an ectodomain provided with an amino acid sequence identical to SEQ ID NO: 1.
Refer to Table 1, which illustrates corresponding amino acids at specific position 149344365 and 366 of pH1N1H5N1H3N2H7N9. The present invention is directed to providing substitution at specific positions with corresponding amino acids of H5N1, H3N2, and H7N9 so as to incur cross-protective immunity. To be specific, the amino acid sequence comprises one of the following amino acids: (i) V replacing I at position 149; (ii) Y or H replacing N at position 344; and (iii) TA, TN or ED replacing IS at positions 365 and 366.







TABLE 1







Corresponding amino acids at specific position 149,


344, 365 and 366 of pH1N1, H5N1, H3N2, H7N9









Position












Strain
149
344
365, 366







A/Texas/05/2009(H1N1)
I
N
IS



A/Vietnam/1203/2004(H5N1)
V
Y
TN



A/Udorn/307/1972(H3N2)
I
H
ED



A/Shanghai/02/2013(H7N9)
I
N
TA










That is, the recombinant neuraminidase of the present invention may obtained by site-directed mutagenesis at mutations of NA gene using a plasmid encoding wild-type pH1N1 gene (SEQ ID NO: 1) as the template.
The sequences of H5N1, H3N2 and H7N9 of the present invention are listed in Seq ID: NO.9; Seq ID: NO.10; Seq ID: NO.11.
It is known that 94.6% of amino acids in the NA ectodomain of influenza virus are conserved. Those skilled in the art of the present invention may appreciate that some amino acids in the NA ectodomain of influenza virus may be replaced without resulting in obvious influences in protein structure or function. Therefore, the recombinant NA ectodomain of the present invention may be provided with an amino acid sequence at least 95%, preferable 97% or 99% identical to SEQ ID NO: 1.
Referring to Table 2, in one specific embodiment, the amino acid sequence of the ectodomain is selected from the group consisting of Seq ID NOs: 2 to 4 and 6 to 8.







TABLE 2







The ectodomain sequence of recombinant neuraminidases









SEQ ID




NO.
Mutation site(s)
abbreviation





2
I149V
MutA


3
N344Y
MutB


4
I365T/S368N
MutC


6
N344H
MutD


7
I365E/S366D
MutE


8
I365T/S366A
MutF









One of main purposes of the present invention is directed to provide an influenza virus vaccine that may incur cross-protective heterosubtypic immunity. The influenza virus vaccine of the present invention may be an inactivated vaccine, an attenuated influenza, a virus-like particle vaccine or a subunit vaccine, i.e. recombinant NA and expressing vectors, virus-like particles and recombinant influenza virus encoding recombinant NA may be used in vaccine preparation.
In one specific embodiment, the vaccine of the present invention may be delivered to a subject to incur cross-protective immunity against influenza subtypes including emerging human influenza, human seasonal influenza and avian influenza. The pharmacologically active compounds of the present invention may be processed according to conventional pharmaceutical method for preparing pharmaceutical reagents used for delivering to a patient e.g., a mammal (including humans). In another specific embodiment, the delivery may be achieved by approaches for example, but not limited to, subcutaneous, intramuscular, oral administered, spraying or gene gun injection.
The detailed explanation of the present invention is described as follows. The described preferred embodiments are presented for purposes of illustrations and description, and they are not intended to limit the scope of the present invention.
Materials and Methods
Recombinant NA Protein Expression and Purification
cDNA from the NA genes of A/Vietnam/1203/2004 (H5N1) (GI: 145284408) and A/Texas/05/2009 (pH1N1) (GI: 255602223) was separately synthesized with insect cell-optimized codon sequences from Genomics, Inc. The coding sequences of the NA H5N1 and pH1N1 ectodomains with additional N-terminal sequences containing gp67 signal peptides, six-His residues, tetrameric human vasodilator-stimulated phosphoprotein (hvsp) domains, and a thrombin cleavage site were cloned into pFastBac expression vectors. Next, rNA proteins were produced using a Bac-to-Bac insect cell expression system (Invitrogen) according to manufacturer instructions. Briefly, Sf9 cells were infected with recombinant baculoviruses expressing the NA ectodomains of H5N1 and pH1N1 for 48 h prior to collecting supernatants for additional rNA protein purification using nickel-chelated resin affinity chromatography (Tosoh). H5N1-rNA and pH1N1-rNA purity was confirmed by Coomassie blue staining. Anti-His HRP-conjugated antibodies (Affymetrix) were used for Western blotting characterization.
Production and Purification of H5N1 and H7N9 VLPs
H5N1 and H7N9 VLPs were produced using the procedures described previously. Briefly, the H5HA gene of A/Thailand/1(KAN-1)/2004 (H5N1), the H7HA gene of A/Shanghai/2/2013 (H7N9), and the M1 gene of A/WSN/1933 (H1N1) were cloned into separate pFastBacDual vectors (Invitrogen). The NINA gene of A/Vietnam/1203/2004 (H5N1), the N9NA gene of A/Shanghai/2/2013 (H7N9), and the M2 gene of A/WSN/1933 (H1N1) were cloned into separate vectors. For H5N1 VLP production, Sf9 cells were co-infected with Bac-H5HA-M1 and Bac-N1NA-M2 recombinant baculoviruses at MOIs of 3 and 1, respectively. Culture supernatants were harvested and concentrated 72 h post-infection. VLPs were further purified using a 20% sucrose solution and centrifugation at 33,000 rpm for 3 h. H5N1 VLPs were obtained and stored at 4° C. until used for NI assays. For H7N9 VLPs, Sf9 cells were co-infected with Bac-H7HA-M1 and Bac-N9NA-M2 recombinant baculoviruses at MOIs of 3 and 1, respectively, for 72 h. Subsequent steps were the same as for H5N1 VLP production.
Mouse Immunizations
BALB/c mice (6-8 weeks old) purchased from the Taiwan National Laboratory Animal Center were immunized twice intramuscularly with 2 or 20 μg of rNA proteins plus 10 μg CpG and 10% PEGb-PLACL, squalene and Span® 85 (PELC) emulsion over a three-week interval as described in a previous report. Sera samples were collected 2 weeks following the second inoculation; splenocytes were harvested and isolated one week later.
Viral Challenges
For the two-dose immunization strategy, BALB/c mice (6-8 weeks old) were placed in one of 5 groups, with each group consisting of 5 mice immunized with either 2 or 20 μg of H5N1-rNA or pH1N1-rNA proteins plus CpG/PELC or PBS over a three-week interval. Three weeks following the second inoculations, all mice were intranasally challenged with 10 MLD50 of the H5N1 (NIBRG-14, RG-14), pH1N1 (A/California/07/2009, CA/09), or H7N9 (A/Taiwan/01/2013, TW/13) viruses. PBS-immunized mice were used as a mock control. Survival rates and body weights were recorded daily for 14 days. According to IACUC guidelines, a weight loss of 25% or more was established as an end-point.
Enzyme-Linked Immunosorbant Assays (ELISAs)
Individual wells in 96-well plates were coated with purified rNA proteins (100 μl at 2 μg/ml) and held overnight at 4° C., washed 3 times with PBST (0.05% Tween-20 in PBS), and blocked with blocking buffer (1% BSA in PBS) for at least 1 h. Next, 100 μl of two-fold serially diluted sera samples were added and held at RT for 1 h, followed by 3 additional washes with PBST. HRP-conjugated goat anti-mouse IgG antibodies (Bethyl Laboratories, Inc.) were added to each well, incubated for 1 h, and washed 3 times with PBST. Anti-NA IgG titers were determined by adding TMB substrate (Biolegend), holding for 15 min at RT, and stopping reactions with 2N H2SO4. End-point titers were determined as the reciprocal of most diluted sera concentrations giving a mean optical density (OD) of 450 nm above 0.2.
Neuraminidase-Inhibiting (NI) Assays
NA-inhibiting (NI) antibodies were measured using a previously described fetuin-based assay procedure. Briefly, 96-well plates were coated with 50 μg/mL fetuin (Sigma) and held overnight at 4° C. before being washed 3 times with PBST and blocked with blocking buffer for 2 h. Two-fold serially diluted sera samples in blocking buffer were incubated with equal volumes of 1 μg VLPs (H5N1 or H7N9) or 105 p.f.u. viruses (pH1N1 or H3N2) for 1 h at 37° C., added to the fetuin-coated plates and held for another 1 h at 37° C., and then washed 3 times with PBST. Peroxidase-labeled peanut agglutinin (100 μl at 2.5 μg/mL) (Sigma) was added to each well, incubated for 1 h at RT, and washed 3 times with PBST. The NA activity levels of viruses (pH1N1 or H3N2) and VLPs (H5N1 or H7N9) were determined by adding TMB substrate (Biolegend), holding for 15 min at RT, and stopping reactions with 2N H2SO4. Plates were read with an ELISA reader (Tecan) at an OD of 450 nm. Corresponding IC50 values were defined as the reciprocal serum dilution inhibiting 50% of viral NA enzyme activity.
NA-Specific Antibody-Secreting B Cells
Splenocytes were collected from each group of rNA-immunized or PBS-immunized mice 3 weeks following the second inoculations. Multiscreen 96-well filtration plates (Millipore) were coated with rNA proteins (1 μg per well) and incubated overnight at 4° C. Plates were blocked with 200 μl/well of complete RPMI-1640 (10% FBS, 1×P/S, 1× sodium pyruvate, 1×NEAA and 100 μM β-ME) and held for 1 h at RT. Splenocytes (2×105) diluted in complete RPMI-1640 were added to individual plates and incubated for 48 h at 37° C. After 3 washes with PBST, HRP-conjugated anti-mouse IgG antibodies were added to each well and held for 2 h at RT. After 3 PBST and 2 PBS washes, AEC substrate was added to each plate and held at RT for 10 ̃60 min before stopping reactions with ddH2O. Immunospots for each immunized group were determined using an ELISPOT Plate Reader (CTL, Inc.).
Production of Chimeric pH1N1/PR8 Influenza Viruses
A mixture of 293T (3.5×105) and MDCK cells (5×105) were seeded into 2 mL OPTI-MEM media at each well of 6-well plates and incubated overnight in a CO2 incubator. The present invention used the eight distinct component plasmids of influenza virus to produce the chimeric pH1N1/PR8 viruses, inclusive of the WT HA from A/Texas/05/2009 (pH1N1) or PR8 strain, the WT or mutant (I365T&S366N) NA components from the A/Texas/05/2009 viral strain and other six components all from the PR8 viral strain. These eight DNA plasmids were simultaneously co-transfected into 293T/MDCK cell mixtures using a TransIT-LT1 transfection reagent (Mirus Bio LLC). TransIT-LT1 reagent/DNA complex mixtures were incubated overnight at 37° C. Medium was replaced with 3 mL fresh OPTI-MEM containing 0.5 μg/mL TPCK-trypsin and incubated for another 2 ̃3 days. Supernatants were collected, titrated, and held at −80° C. until used in the experiments.
Plaque Assays for Chimeric pH1N1/PR8 Influenza Viruses
MDCK cells (6×105/well) were held overnight at 37° C. Monolayer MDCK cells were infected with serially diluted PR8, PR8 x Texas HA (THA) x Texas NA (TNA) or PR8 x THA x TNA (I365T/S366N) RG viruses for 1 h at 37° C. Supernatants were removed and washed twice with PBS. Infected MDCK cells were overlaid with MEM-α plus 0.5% agarose. After another 48 hours of incubation at 37° C., infected cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet solution. Plaque numbers were observed and calculated.
Statistical Analysis
All results were analyzed using Student's t tests. Asterisks in each Fig. indicate statistical significance (*, p<0.05; **, p<0.01; ***, p<0.001).
H5N1-rNA and pH1N1-rNA Proteins Expressed and Purified Using a Baculovirus-Insect Cell Expression System
The construction diagram of soluble H5N1-rNA and pH1N1-rNA proteins presented as FIG. 1A was produced from the culture supernatants of Sf9 cells infected by recombinant baculoviruses and purified using Ni-chelated affinity chromatography. Soluble H5N1-rNA and pH1NA-rNA yields were approximately 0.5 mg/L and 0.25 mg/L, respectively, with 80-90% purity (FIG. 1B). According to results from SDS-PAGE gels with Coomassie blue staining (FIG. 1B) and Western blotting (FIG. 1C), purified H5N1-rNA and pH1N1-rNA proteins had molecular weights of 53 kDa each. Based on Eadie-Hofstee measurements, H5N1-rNA and pH1N1-rNA Km values were 2.80 and 1.90, respectively (FIG. 1D), indicating enzyme activity in insect cell-expressed H5N1-rNA and pH1N1-rNA.
NA-Specific IgG Antibodies were Induced by H5N1-rNA and pH1N1-rNA Immunizations
Groups of five female BALB/c mice were i.m. immunized with two doses of H5N1-rNA or pH1N1-rNA proteins (2 or 20 μg per dose) over a three-week interval. Antisera were collected 2 weeks following the second immunizations. Data for NA-specific IgG titers against the same immunogens (H5N1-rNAs or pH1N1-rNA proteins) in each group are presented in FIG. 2A. The present invention used an anti-mouse N1NA-specific antibody as a positive control and PBS-immunized sera as a negative control (103.5-4 and undetectable NA-specific IgG titers, respectively). As shown in FIG. 2A, mice immunized with 20 μg of H5N1-rNA or pH1N1-rNA exhibited slightly higher NA-specific total IgG titers compared to mice immunized with 2 μg of H5N1-rNA or pH1N1-rNA. The present invention also performed ELISA assays with H5N1 VLPs (FIG. 2B) and the pH1N1 (A/California/04/2009) virus (FIG. 2C). Our data indicate that at either 2 or 20 μg, both H5N1-rNA and pH1N1-rNA cross-reacted with H5N1 VLPs and the pH1N1 virus to induce similar amounts of total IgG titers (FIG. 2B-2C). Similar cross-reactive results were also found for the H3N2 virus (A/Udorn/307/1972) and H7N9 VLPs (FIGS. 2D-2E). No significant differences were noted for either IgG1 or IgG2a subclass titers in the same immunization groups. The single exception was significantly greater IgG2a for mice immunized with 20 μg H5N1-rNA (data not shown).
NI Antibodies are Elicited by H5N1-rNA and pH1N1-rNA Immunizations
To measure NI antibody titers, two-fold serially diluted serum samples from each immunization group were mixed with 1 μg of H5N1-VLP, 105 p.f.u. of the pH1N1 (A/California/04/2009) virus, 105 p.f.u. of the H3N2 (A/Udorn/307/1972) virus, or 1 μg of H7N9-VLP, and then examined using fetuin-based assays. NA-inhibiting percentages and NI titers against the same group two viruses, H5N1 and pH1N1, in each immunization group are shown in FIGS. 3A-3B and 3E-3F. Those results indicate dose-dependency, with NI curves from all rNA immunization groups significantly higher than the curves for the PBS immunization group. NA-inhibiting percentages and NI titers were also demonstrated in the H3N2 virus and H7N9 VLPs, with similar results but lower NI titers (FIGS. 3C-3D and 3G-3H). Regarding IC50 values, similar NI titer ranges were observed against the homologous viruses: 3.7-3.8 for H5N1-rNA immunization against H5N1 and 4.3-4.4 for pH1N1-rNA immunization against pH1N1 (FIG. 4). However, significant differences were noted among the six immunization groups in terms of NI titers against the H5N1, pH1N1, H3N2 and H7N9 heterologous viruses. Both H5N1-rNA immunization groups (2 and 20 μg) expressed higher heterosubtypic NI titers against pH1N1 compared to titers elicited by the two pH1N1-rNA immunization groups against H5N1 viruses. In contrast, H5N1-rNA and pH1N1-rNA elicited lower NI titers, with IC50 values less than 2.32 for all groups immunized against the H3N2 and H7N9 viruses (FIG. 31). Our data indicate that the effect of H5N1-rNA immunization was limited to more potent heterosubtypic NI antibodies against pH1N1 viruses.
Antibody-Secreting B Cells Detected in Splenocytes
To measure anti-NA IgG-secreting B cells elicited by H5N1-rNA or pH1N1-rNA, splenocytes were collected from immunized mice 3 weeks after their second immunizations, reacted with 1 μg H5N1-rNA or pH1N1-rNA protein per well, and examined using ELISPOT assays. The results shown in FIG. 5 indicate that the numbers of spots against homologous viruses due to H5N1-rNA immunization were slightly higher than those resulting from pH1N1-rNA immunization. Significantly higher numbers of spots against H5N1 and pH1N1 heterologous viruses were only noted in the 20 μg H5N1-rNA immunization group, which also exhibited higher quantities of ASCs in splenocytes against homologous and heterologous H5N1 and pH1N1 viruses.
Protective Immunity Against H5N1, pH1N1 and H7N9 Virus Challenges
To assess protective immunity triggered by rNA immunizations, mice immunized with 2 or 20 μg of H5N1-rNA or pH1N1-rNA proteins were challenged with 10 MLD50 of H5N1 (RG-14), pH1N1 (CA/09) or H7N9 (TW/13) viruses 3 weeks following their second immunizations. According to the results shown in FIGS. 6A-6B with the exceptions of an 80% survival rate for mice immunized with 2 μg of pH1N1-rNA and a 0% rate for the PBS control mice, all immunization groups had 100% survival rates against homologous H5N1 or pH1N1 viral challenges. Significantly lower body weight losses were observed for mice in the 20 μg H5N1-rNA and 20 μg pH1N1-rNA immunization groups challenged with homologous H5N1 or pH1N1 compared to those in the 2 μg H5N1-rNA and 2 μg pH1N1-rNA immunization groups (FIGS. 7A-7B). In terms of cross-protection levels, mice receiving either 2 or 20 μg H5N1-rNA inoculations exhibited complete protection against pH1N1 viral challenges (FIG. 6C), with significantly lower body weight losses noted in the 20 μg group (FIG. 7C). A 60% survival rate (FIG. 6D) and faster weight loss recovery following challenges with the heterologous H5N1 virus were observed in the 20 μg pH1N1-rNA immunization group (FIG. 7D). Zero protection against a H7N9 virus challenge was observed in the 20 μg H5N1-rNA and 20 μg pH1N1-rNA immunization groups (FIGS. 6E and 7E).
Cross-Reactive NI Antibodies Elicited by pH1N1-rNA Mutant Proteins
To add detail to our investigation of cross-reactive NI epitopes, the present invention aligned the amino acid sequences of A/Vietnam/1203/2004 (H5N1), A/Texas/05/2009 (pH1N1), A/Udorn/307/1972 (H3N2) and A/Shanghai/02/2013 (H7N9). As shown in FIG. 8, the present invention identified 34 different amino acids (in red) in the NA ectodomains of the two sequences (94.6% identical). Previous reports have identified influenza NA enzyme catalytic sites at residues 118-119, 151-152, 198, 224, 227, 243, 274, 276-277, 292, 330, 350 or 425, therefore the present invention targeted residues 149, 344, 365 and 366—all located close to NA enzyme-active sites and contributing to NI antibody elicitation. Site-directed mutagenesis at these four residues produced three mutant pH1N1-rNA proteins: I149V, N344Y and I365T/S366N (not illustrated). Results from an analysis of anti-sera from the wild type and three mutant pH1N1-rNAs show that the I149V and I365T/S366N mutant proteins elicited more NI antibodies against the homologous pH1N1 strain (FIG. 10A), with the I365T/S366N protein eliciting more potent cross-reactive NI antibodies against the H5N1 (FIG. 10B), H3N2 (FIG. 10C) and H7N9 viruses (FIG. 10D). Corresponding IC50 values calculated from NI response curves indicate that the I149V and I365T/S366N mutant proteins resulted in increased NI titers against the homologous pH1N1 viruses, and that all three mutant proteins resulted in increased NI titers against the heterosubtypic H3N2 and H7N9 viruses (FIG. 11). The I365T/S366N mutation of pH1N1-rNA induced the highest quantities of NI antibody titers against the homologous and heterosubtypic strains. The present invention also constructed and expressed the other mutant pH1N1-rNA proteins changing from pH1N1 to H3N2 or H7N9, as FIG. 12 described. Results from an analysis of anti-sera from the wild type and three mutant pH1N1-rNAs show that the pH1N1-N344H (N2) and pH1N1-I365T/S366A (N9) mutant proteins elicited more NI antibodies against the homologous pH1N1 strain (FIG. 13A) and heterologous H7N9 strain (FIG. 13D), with similar cross-reactive NI antibodies against the H5N1 (FIG. 13B) and H3N2 (FIG. 13C) viruses. Corresponding IC50 values calculated from NI response curves indicate that the pH1N1-N344H (N2) and pH1N1-I365T/S366A (N9) mutant proteins resulted in increased NI titers against the homologous pH1N1 viruses, and that pH1N1-N344H (N2) mutant protein resulted in increased NI titers against the heterosubtypic H7N9 viruses (FIG. 14).
Construction of the I365T/S366N Mutant of Chimeric pH1N1/PR8 Viruses
The present invention used a PR8 (A/Puerto Rico/8/1934 (H1N1))-based reverse genetic system with replacement HA and NA genes of A/Texas/05/2009 (pH1N1) to construct chimeric pH1N1/PR8 viruses of the wild type NA gene (PR8 x THA x TNA), and a I365T/S366N mutant of the NA gene (PR8 x THA x TNA-I365T/S366N). The obtained chimeric pH1N1/PR8 viruses with the I365T/S366N mutation had similar plaque morphologies (FIG. 15A) with the titers of 6×106 PFU/ml in MDCK cells. The present invention also measured the replication kinetics of the three viruses (PR8 RG, PR8 x THA x TNA and PR8 x THA x TNA-I365T/S366N) in MDCK cells at MOIs of 0.01. No significant differences in replication kinetics were observed in the I365Y/S366N mutant viruses (FIG. 15B) or in NA enzymatic activity compared to the PR8 and chimeric pH1N1/PR8 viruses (FIG. 15C).
CONCLUSION
NA-based influenza vaccines are attractive because of the smaller number of changes in NA antigens compared to HA antigens in host immune systems. For this study, the present invention constructed and purified H5N1-rNA and pH1N1-rNA proteins from Sf9 insect cells, and found that mice immunized with H5N1-rNA and pH1N1-rNA proteins exhibited higher quantities of NA-specific total IgG, IgG1, IgG2a subclass and NI antibody titers, increased numbers of ASCs in splenocytes, and better protective immunity against live virus challenges. H5N1-rNA immunization was found to induce more potent cross-reactive NI antibodies and protective immunity against pH1N1 viruses compared to pH1N1-rNA immunization against H5N1 viruses. Cross-reactive NI epitopes were further dissected by immunization of pH1N1-rNA proteins with I149V, N344Y and I365T/S366N NA mutations. The I365T/S366N mutation of pH1N1-rNA was found to increase cross-reactive NI antibodies against the H5N1, H3N2 and H7N9 viruses.
While the invention can be subject to various modifications and alternative forms, a specific example thereof has been shown in the drawings and is herein described in detail. It should be understood, however, that the invention is not to be limited to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the appended claims.

## Claims

1. A recombinant neuraminidase, comprising an ectodomain provided with an amino acid sequence at least 95% identical to SEQ ID NO: 1, wherein the amino acids at positions 365 and 366 are replaced from IS to TN.

2. The recombinant neuraminidase as claimed in claim 1, wherein the amino acid sequence is at least 97% identical to SEQ ID NO: 1.

3. The recombinant neuraminidase as claimed in claim 1, wherein the amino acid sequence is at least 99% identical to SEQ ID NO: 1.

4. The recombinant neuraminidase as claimed in claim 1, wherein the amino acid sequence is selected from Seq ID NO: 4.

5. A recombinant influenza virus, comprising:
a recombinant neuraminidase, comprising an ectodomain provided with an amino acid sequence at least 95% identical to SEQ ID NO: 1, wherein the amino acids at positions 365 and 366 is replaced from IS to TN.

6. The recombinant influenza virus as claimed in claim 5, wherein the amino acid sequence is at least 97% identical to SEQ ID NO: 1.

7. The recombinant influenza virus as claimed in claim 5, wherein the amino acid sequence is at least 99% identical to SEQ ID NO: 1.

8. The recombinant influenza virus as claimed in claim 5, wherein the amino acid sequence is selected from Seq ID NO: 4.

9. An influenza virus vaccine, comprising:
a recombinant neuraminidase, comprising an ectodomain provided with an amino acid sequence at least 95% identical to SEQ ID NO: 1, wherein the amino acids at positions 365 and 366 is replaced from IS to TN.

10. The influenza virus vaccine, as claimed in claim 9, wherein the amino acid sequence is at least 97% identical to SEQ ID NO: 1.

11. The influenza virus vaccine as claimed in claim 9, wherein the amino acid sequence is at least 99% identical to SEQ ID NO: 1.

12. The influenza virus vaccine as claimed in claim 9, wherein the amino acid sequence is selected from Seq ID NO: 4.

13. The influenza virus vaccine as claimed in claim 9, wherein the influenza virus vaccine is an inactivated vaccine, an attenuated influenza, a virus-like particle vaccine or a subunit vaccine.