H7 avian influenza vaccine strain which differentiates infected from vaccinated animals, preparation method therefor, and application

Abstract

An H7 avian influenza vaccine strain which differentiates infected from vaccinated animals, a preparation method therefor, and an application. The highly pathogenic H7 avian influenza not only brings about huge economic losses to the livestock industry, but also seriously threatens public health safety. Conventional H7 avian influenza whole virus inactivated vaccines do have advantages such as being reliable in terms of effect, low in terms of cost and wide in terms of application range, but cannot serologically differentiate infected from vaccinated animals. The present invention uses NA of influenza B as a label to establish a method for constructing an H7 avian influenza vaccine strain which differentiates infection from vaccination, and may be used for the prevention, control and decontamination of the H7 avian influenza.

Claims

1. An H7 avian influenza vaccine comprising an H7 avian influenza virus with a label gene sequence selected from the group consisting of: the label gene sequence containing a DNA sequence for coding an influenza B virus Neuraminidase (NA) protein extracellular region amino acid sequence, or containing a DNA sequence for coding an amino acid sequence having at least 90% identity, or at least 92% identity, or at least 95% identity, or at least 98% identity with the extracellular region amino acid sequence; alternatively, the label gene sequence containing a DNA sequence for coding the extracellular region amino acid sequence in influenza B virus NA gene, or containing a sequence having at least 90% identity, or at least 92% identity, or at least 95% identity, or at least 98% identity with the DNA sequence; alternatively, the label gene sequence is a DNA sequence for coding influenza B virus NA protein, or a DNA sequence for coding an amino acid sequence having at least 90% identity, or at least 92% identity, or at least 95% identity, or at least 98% identity with the NA protein amino acid sequence; and alternatively, the label gene sequence is a DNA sequence of influenza B virus NA gene, or a sequence having at least 90% identity, or at least 92% identity, or at least 95% identity, or at least 98% identity with the DNA sequence; wherein the H7 avian influenza vaccine strain further contains an H7 subtype hemagglutinin (HA) gene encoding VPKGKRTARGLF (SEQ ID NO: 8) or a mutated H7 subtype HA gene; wherein the mutated H7 subtype HA gene has been mutated to encode VPSSRSRGLF (SEQ ID NO: 9) or VPKGRGLF (SEQ ID NO: 10) in place of VPKGKRTARGLF (SEQ ID NO: 8).

2. The H7 avian influenza virus of claim 1, wherein the influenza B virus NA is from influenza B viruses of Victoria group or Yamagata group.

3. The H7 avian influenza virus of claim 2, wherein the influenza B viruses are strains B/Massachusetts/2/2012, B/Brisbane/60/2008, B/Yamagata/16/1988 or B/Malaysia/2506/04.

4. The H7 avian influenza virus of claim 1, wherein the label gene sequence further contains packaging signal sequences at its both ends, the packaging signal is a packaging signal of H1 subtype NA, or a packaging signal sequence having at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity with the packaging signal of H1 subtype NA.

5. The H7 avian influenza virus of claim 1, wherein the label gene sequence further contains packaging signal sequences at its both ends, wherein the 5′-end packaging signal sequence comprises the noncoding region sequence, the intracellular region sequence, and the transmembrane region sequence of H1 subtype NA.

6. The H7 avian influenza virus of claim 5, wherein the intracellular region sequence encodes 5 to 7 amino acids.

7. The H7 avian influenza virus of claim 5, wherein the transmembrane region sequence encodes 24 to 32 amino acids.

8. The H7 avian influenza virus of claim 5, wherein the 5′-end packaging signal sequence of the label gene sequence is SEQ ID NO:3, or a sequence having at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity with SEQ ID NO:3.

9. The H7 avian influenza virus of claim 1, wherein the label gene sequence further contains packaging signal sequences at its both ends, wherein the 3′-end packaging signal sequence is SEQ ID NO:4, or a sequence having at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity with SEQ ID NO:4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the structure schematic diagram of artificially synthesized A/B chimeric NA gene;

(2) FIG. 2 is the pFLu vector map and the clone schematic diagram of influenza virus gene segments;

(3) FIG. 3 is detecting the reactivity of anti-Re-Mu2H7-DIVA-ΔNS serum with influenza ANA by immunofluorescence.

DESCRIPTION OF THE EMBODIMENTS

(4) The present invention will be illustrated in detail in conjunction with the following specific examples and the accompanying figures, and the embodiments of the invention are not limited to this. For unnoted conventional experimental methods, see “Guideline for Molecular Cloning”, the 3rd edition (Sambrook, ed., Science press, 2002).

Example 1 A Preparation Method of Avian Influenza Vaccine Strain Re-MuH7-DIVA-ΔNS Virus

(5) (1) Construction of Low Pathogenic HA Mutant Gene

(6) The pFlu vector is a kind of bidirectional transcription vector, which may transcribe viral RNA by the human poll promoter, and also transcribe viral mRNA by CMV promoter, thus synthesizing the viral proteins (Hoffmann et al., PNAS, USA 97, 6108-6113, 2000).

(7) HA gene (KY855526) in the artificially synthesized wild type H7 avian influenza, of which the (KRTA) sequence in the highly pathogenic characteristic sequence (VPKGKRTARGLF) in this wild type HA amino acid sequence is deleted through site-directed mutagenesis to obtain the corresponding low pathogenic Mu1HA gene sequence; or the highly pathogenic characteristic sequence (VPKGKRTARGLF) is mutated into (VPSSRSRGLF) to obtain the corresponding low pathogenic Mu2HA gene sequence; the mutated Mu1HA, Mu2HA genes are cloned into the pFlu vector through a site to obtain the recombinant plasmid pFlu-Mu1HA and pFlu-Mu2HA, with the construction schematic diagram shown in FIG. 2.

(8) (2) Construction of Low Pathogenic A/B Chimeric NA Gene

(9) Constructing the artificially synthesized A/B chimeric NA gene as shown in FIG. 1, which contains a DNA sequence (SEQ ID NO: 2) for coding an extracellular region amino acid sequence (SEQ ID NO: 1) in influenza B virus NA as the label gene sequence, the sequence containing type B NA extracellular region as shown in SEQ ID NO: 2 deriving from B/Massachusetts/2/2012 in the influenza B virus Yamagata group (Ping J et al, PNAS, 2016, 113(51):E8296-E8305), the label gene sequence further contains packaging signal sequences at its both ends, wherein the 5′-end packaging signal sequence (SEQ ID NO:3) includes the noncoding region sequence, the intracellular region sequence and the transmembrane region sequence, the 3′-end packaging signal sequence is SEQ ID NO:4. The chimeric NA is inserted into the pFlu vector through the BsmBI site to obtain a recombinant plasmid pFlu-PR8-BNA.

(10) (3) Acquisition of Re-MuH7-DIVA-ΔNS Vaccine Strain

(11) For ensuring the safety property of the vaccine strain, the wild type virus NS1 gene is modified, the nucleotide sequence of the modified mutant gene ΔNS is as shown in SEQ ID NO:5. The virus containing the mutant gene ΔNS has lost the function of antagonizing interferons, thus only can grow and propagate in interferon-deficient cells or chick embryos with underdeveloped interferon systems, therefore having good safety property.

(12) The recombinant vaccine strain Re-MuH7-DIVA-ΔNS is rescued with the classical “6+2” influenza reverse genetic system. Each 0.5 ug of 6PR8 internal genes pFlu-PR8-PB2, pFlu-PR8-PB1, pFlu-PR8-PA, pFlu-PR8-NP, pFlu-PR8-M, pFlu-PR8-ΔNS and 2 external genes pFlu-Mu1-HA/pFlu-Mu2-HA, pFlu-PR8-BNA are co-transfected into 293T cells (Lipofectamine 3000). 24 h after transfection, a culture medium containing TPCK-Trypsin at a final concentration of 0.5 ug/ml is exchanged, and 48 h after transfection, the cell supernatant is collected, which is inoculated into 8-day-old SPF chick embryos at 0.2 ml per embryo by allantoic cavity inoculation. After inoculation, chick embryos are cultured in an incubator at 37° C. for 48 h. The chick embryo allantoic fluid (F0 generation) is collected to obtain the vaccine strains Re-Mu1H7-DIVA-ΔNS and Re-Mu2H7-DIVA-ΔNS respectively, and it is determined whether they have hemagglutination titers. If they have no hemagglutination titers, the obtained viruses are blind passaged for one generation, and then determined whether they have hemagglutination titers.

Example 2 Growth Properties of Vaccine Strains Containing Different Low Pathogenic Modified Mutant Genes Mu1HA, Mu2HA on Chick Embryos

(13) The vaccine strains Re-Mu1H7-DIVA-ΔNS and Re-Mu2H7-DIVA-ΔNS obtained in Example 1 are serially passaged on 8-day-old SPF chick embryos (F0-F3) respectively. 48 hours after vaccination, each generation of viruses are harvested and determined their hemagglutination titers (HA titers).

(14) The detection results are shown in Table 1, from which it can be seen that the growth properties of Re-Mu2H7-DIVA-ΔNS are obviously superior to those of Re-Mu1H7-DIVA-ΔNS. As the genetic backgrounds of Re-Mu1H7-DIVA-ΔNS and Re-Mu2H7-DIVA-ΔNS reassortant viruses are almost the same, only different in the modifications of the highly pathogenic wild type HA, therefore, the modification mode on Mu2-HA is more favorable for the growth of H7 avian influenza on chick embryos, reaching 5 log 2˜6 log 2. No chick embryo deaths are observed during the passages, indicating that the recombinant viruses exhibit low pathogenic or no pathogenic on chick embryos, with good safety property. Taking F0 and F3-generation viruses of which the artificially synthesized A/B chimeric NA gene is amplified by RT-PCR, it is demonstrated by sequencing that chimeric NA gene can be stably passed to progeny viruses.

(15) In conclusion, the rescued Re-Mu2H7-DIVA-ΔNS strains become ones with low pathogenicity or without pathogenicity, which only can grow and propagate in interferon-deficient cells or low-age chick embryos with underdeveloped interferon systems, therefore having good safety property. After incubation on 8-day-old SPF chick embryos for 48 hours, their HA titers may reach 6 log 2. Due to NS1 partial deletion of Re-Mu2H7-DIVA-ΔNS strain, its growth titer on chick embryos is lower than that of normal non-deleted viruses, but better than non-deleted wild type viruses in terms of safety.

(16) TABLE-US-00001 TABLE 1 Growth properties of vaccine strains Re-Mu1H7-DIVA- ΔNS, Re-Mu2H7-DIVA-ΔNS with different low pathogenic modifications on chick embryos Passage HA Titers (log2) Number Re-Mu1H7-DIVA-ΔNS Re-Mu2H7-DIVA-ΔNS F0 0 5 F1 2 6 F2 3 6 F3 3 6

(17) The applicants have preserved the inventive vaccine strain Re-Mu2H7-DIVA-ΔNS in China Center for Type Culture Collection, the address of which is Wuhan University, China. The Collection Center received the vaccine strain provided by the applicants on Oct. 19, 2017. The preservation number of the culture issued by the Collection Center is CCTCC NO: V201742, the proposed classification name is H7 avian influenza vaccine candidate strain Re-Mu2H7-DIVA-ΔNS, the preserved vaccine strain has been identified as viable on Oct. 28, 2017.

Example 3 A Preparation Method of an H7 Avian Influenza Vaccine Strain Re-MuH7-DIVA-ΔNS which Differentiates Influenza a Virus Infection from Vaccination

(18) The preparation method of Example 3 is the same as that of Example 1, except that in constructing the artificially synthesized A/B chimeric NA gene as shown in FIG. 1, the DNA sequence for coding the extracellular region protein amino acid sequence in influenza B virus NA is different from that in Example 1, the remaining are all the same as Example 1.

(19) In this Example, the DNA sequence for coding the extracellular region protein amino acid sequence (SEQ ID NO: 6) in influenza B virus NA is shown in SEQ ID NO: 7, which is used as the label gene sequence, the sequence shown in SEQ ID NO: 7 deriving from B/Brisbane/60/2008 of influenza B virus Victoria group (Ping J et al, PNAS, 2016, 113(51):E8296-E8305).

(20) The Re-MuH7-DIVA-ΔNS vaccine strain prepared in the present invention will be further detected for its effects below.

(21) Process: Re-Mu2H7-DIVA-ΔNS vaccine strain prepared in Example 1 (NA extracellular region gene is derived from B/Massachusetts/2/2012 of Yamagata group), Re-MuH7-DIVA-ΔNS vaccine strain prepared in Example 3 (NA extracellular region gene is derived from B/Brisbane/60/2008 of Victoria group), PR8-ΔNS virus (NS-deficient PR8 virus) of the control group 1, PR8-WT virus (wild type PR8 virus) of the control group 2 are respectively inoculated into the allantoic cavities of 8-day-old SPF chick embryos at 0.2 ml per embryo. The inoculated chick embryos are cultured in an incubator at 37° C. for 48 h. The chick embryo allantoic fluid (F0-generation) is collected for determining its hemagglutinin titer. F0-generation viruses are diluted and inoculated into 10 SPF chick embryos, cultured for 48 h to obtain viruses which are defined as F1-generation. With the same process, F1-generation viruses are serially passaged to F3-generation.

(22) Results: the detection results are shown in Table 2, from which it can be seen that, for demonstrating whether type B NA gene of different branches can match with H7 subtype HA(H7-BNA) well, NA genes of representative strains from different groups: B/Brisbane/60/2008(Victoria group) and Massachusetts/2/2012(Yamagata group) are selected for study, it is found from the results that type B NA genes of different branches (Victoria group and Yamagata group) both exhibit good matching with H7, the Re-PR8-MuH7-ΔNS vaccine strain obtained from Examples 1 and 3 can approach its upper limit (5 log 2˜6 log 2) without the need of passage adaptation on chick embryos. It also can be seen from Table 2 that the growth titers of vaccine strains containing mutant ΔNS are lower than that of wild type by 2 log 2˜3 log 2, however, the vaccine strains containing mutant ΔNS are better in terms of safety.

(23) TABLE-US-00002 TABLE 2 Growth properties of different chimeric recombinant H7 avian influenza viruses on chick embryos Virus HA Titers (log2) Passage Example 1 Example 3 Control Group 1 Control Group 2 Number Re-Mu1H7-DIVA-ΔNS Re-Mu2H7-DIVA-ΔNS PR8-ΔNS PR8-WT F0 5 4 6.5 9 F1 6 5 7 10 F2 6 5.5 7 9 F3 6 5 7 10

(24) For representative influenza B virus strains from different groups: B/Brisbane/60/2008 (Victoria group) and Massachusetts/2/2012 (Yamagata group), the homology between the two NA whole gene nucleotide sequences is 94.9%, the homology of the amino acid sequences is 94.9%; the homology between the two DNA sequences for coding NA protein extracellular region is 95.1%, the homology of the NA protein extracellular region amino acid sequences is 94.6%. Because influenza B is only classified into Victoria group and Yamagata group, it is demonstrated in the invention that representative NA strains from the two groups (Example 1 and Example 3) both have good compatibilities with H7 HA, showing that influenza B virus NA gene may all be used in preparing an H7 avian influenza vaccine strain which differentiates influenza A virus infection from vaccination.

Example 4 Preparation of Re-MuH7-DIVA-ΔNS Inactivated Vaccine

(25) 50 ml of F0, F1, F2 or F3-generation allantoic fluids from Re-MuH7-DIVA-ΔNS vaccine strains prepared in the above examples are harvested, and inactivated with a formalin solution at a final concentration of 0.25% at 37° C. for 24 h. The inactivated allantoic fluids are added into 2% of Tween-80, dissolved sufficiently and then emulsified with white oil containing 3% of Span 80 at a proportion of 1:3, at a shear emulsification rate of 12000 rpm for 3 min. Upon a dosage form test, a sizing test, a viscosity test, and a stability test, it is determined that the inactivated vaccine is an off-white water-in-oil emulsion with low viscosity, uniform particle sizes, good stability and suitable for injection.

Example 5 Detection of Effects of Re-MuH7-DIVA-ΔNS Inactivated Vaccine on Vaccinating Animals

(26) Process: 10 3-week-old SPF chickens are vaccinated with Re-Mu2H7-DIVA-ΔNS vaccine prepared above at 0.3 ml per chick by subcutaneous injection at the neck, blood is sampled 21 days after vaccination, serum is isolated and HI antibodies are determined.

(27) Results: it is demonstrated from experiments that Re-Mu2H7-DIVA-ΔNS stimulates the organism to generate high level of HI antibodies, the average HI titer (log 2) for week 3 after vaccination is 9.3±0.95. For HA and HI tests, reference to GBT 18936-2003 (diagnosis technology of highly pathogenic avian influenza).

Example 6 Serological Experiments

(28) N1, N2, N6, and N9 genes of the existing influenza A are cloned into pCAGGS eukaryotic expression plasmid through KpnI and NheI sites, which are named as pCAGGS-N1, pCAGGS-N2, pCAGGS-N6, pCAGGS-N9. Each 1 μg of pCAGGS-N1, pCAGGS-N2, pCAGGS-N6, pCAGGS-N9 plasmid is transfected to 293T cells pre-coated on 24-hole cell culture plates. 30 h after transfection, the reactivities of the following 7 groups of chicken serum with N1, N2, N6, N9 are detected by immunofluorescence.

(29) The profiles of the 7 groups of chicken serum are as below:

(30) Anti-Re-Mu2H7-DIVA-ΔNS chicken serum: chicken serum which is only vaccinated with the inventive Re-Mu2H7-DIVA-ΔNS inactivated vaccine;

(31) Anti-H7N9 standard chicken serum: H7N9 standard serum, purchased from Harbin Veterinary Research Institute.

(32) Anti-H5+H7 serum: clinical serum of vaccinated H5N1 Re-8 strain+H7N9 Re-1 strain whole virus inactivated vaccines.

(33) Anti-N1 chicken serum: one-week-old SPF chicken are vaccinated with 100 μg pCAGGS-N1 (by intramuscular injection) respectively, the whole blood is harvested 4 weeks after vaccination to prepare the serum.

(34) Anti-N2 chicken serum: one-week-old SPF chicken are vaccinated with 100 μg pCAGGS-N2 (by intramuscular injection) respectively, the whole blood is harvested 4 weeks after vaccination to prepare the serum.

(35) Anti-N6 chicken serum: one-week-old SPF chicken are vaccinated with 100 μg pCAGGS-N6 (by intramuscular injection) respectively, the whole blood is harvested 4 weeks after vaccination to prepare the serum.

(36) Anti-N9 chicken serum: one-week-old SPF chicken are vaccinated with 100 μg pCAGGS-N9 (by intramuscular injection) respectively, the whole blood is harvested 4 weeks after vaccination to prepare the serum.

(37) The immunofluorescence process is as below:

(38) 1) Into each cell is added 0.5 ml of 4% paraformaldehyde for immobilization for 20 minutes, and then washed with PBS for three times.

(39) 2) It is permeated with 0.2% Triton X 100 for 10 minutes, and then washed with PBS for three times.

(40) 3) It is blocked with 5% BSA for 1 hour, and then washed with PBS for three times.

(41) 4) Primary antibodies are diluted with PBS containing 1% BSA by corresponding factors (anti-Re-Mu2H7-DIVA-ΔNS, anti-H7N9 standard, anti-H5+H7, for 100-fold; anti-N1/N2/N6/N9, for 20-fold), and added into each hole at 0.5 ml, incubated in a wet box at 37° C. for 1 hour, and then washed with PBS for three times.

(42) 5) Anti-Chicken secondary antibodies (Alexa Fluor 594 Donkey Anti-Chicken IgY) are diluted with PBS containing 1% BSA for 200-fold, added into each hole at 0.5 ml, incubated at room temperature for 0.5 hours, and then washed with PBS for three times.

(43) 6) Observing with a fluorescence microscope.

(44) Results: Influenza N1, N2, N6 and N9 neuraminidases are respectively expressed in 293T cells, the immunofluorescence process is used to detect whether serum has reacted with N1, N2, N6 and N9 3 weeks after vaccination with Re-Mu2H7-DIVA-ΔNS. It is found that the anti-Re-Mu2H7-DIVA-ΔNS serum does not cross react with N1, N2, N6 and N9 proteins (e.g., as shown in Table 3 and FIG. 3), both clinical serum vaccinated with the existing whole type A virus vaccines (H5N1 Re-8 strain+H7N9 Re-1 strain) and anti-H7N9 standard serum can strongly react with N9 protein. It is demonstrated from this experiment that vaccination with the Re-Mu2H7-DIVA-ΔNS vaccine can not only induce high level of HI antibodies, but also can differentiate infected from vaccinated animals, which overcomes the disadvantage that the existing H7 subtype whole virus vaccine is unable to differentiate infected from vaccinated animals.

(45) TABLE-US-00003 TABLE 3 The reactivity profiles between chicken sera vaccinated with different antigens and each NA subtype Antigens Antibodies N1 N2 N6 N9 Anti-Re-Mu2H7-DIVA-ΔNS HI: 9 log2 No No No No reactivity reactivity reactivity reactivity Anti-H7N9 standard HI: 8 log2 ND ND ND Reactivity Anti-H5 + H7 HI: 9 1og2 ND ND ND Reactivity (H7) Anti-N1 HI: N/A Reactivity ND ND ND Anti-N2 HI: N/A ND Reactivity ND ND Anti-N6 HI: N/A ND ND Reactivity ND Anti-N9 HI: N/A ND ND ND Reactivity Note: N/A: not applicable; ND: not detected.

Example 7 A Preparation Method of an H7 Avian Influenza Vaccine Strain Re-MuH7-DIVA-ΔNS which Differentiates Influenza A Virus Infection from Vaccination

(46) The preparation method of Example 7 is the same as that of Example 1, except that in constructing the artificially synthesized A/B chimeric NA gene as shown in FIG. 1, the influenza B virus NA sequence used is the DNA sequence for coding NA whole protein sequence, the remaining are all the same as Example 1, wherein, the DNA sequence of NA derived from the NA whole gene sequence of B/Massachusetts/2/2012 in the Yamagata group of influenza B virus (Ping J et al, PNAS, 2016, 113(51): E8296-E8305).

(47) The above examples are the preferable embodiments of the invention, however, the detailed description of the invention is not limited to the examples described above, any other changes, modifications, substitutions, combinations, simplifications made without deviating from the spirit and principle of the invention should all be considered as equivalent replacements, which are all within the scope of the present invention.