H5 avian influenza vaccine strain which differentiates infected from vaccinated animals, preparation method therefor, and application

Abstract

Provided are an H5 avian influenza vaccine strain which differentiates infected from vaccinated animals, a preparation method therefor, and an application. The vaccine strain uses an NA protein of influenza B as a label, and has application value and public health significance for the prevention, control and decontamination of H5 avian influenza.

Claims

1. An H5 avian influenza vaccine strain, comprising a label gene sequence and an H5 subtype HA gene or a mutated H5 subtype HA gene, wherein the label gene sequence comprising a DNA sequence coding an influenza B virus NA protein extracellular region amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 6, or comprising a DNA sequence coding an amino acid sequence having at least 90% homology, at least 92% homology, at least 95% homology, or at least 98% homology with the extracellular region amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 6; alternatively, the label gene sequence comprising a DNA sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 7 coding the extracellular region amino acid sequence in influenza B virus NA gene, or comprising a sequence having at least 90% homology, at least 92% homology, at least 95% homology, or at least 98% homology with the DNA sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 7; and wherein the mutated H5 subtype HA gene has been mutated to encode RETRGLF (SEQ ID NO: 9) in place of RERRRKRGLF (SEQ ID NO: 8).

2. The vaccine strain of claim 1, wherein the influenza B virus comprises influenza B viruses of Victoria group and Yamagata group.

3. The vaccine strain of claim 2, wherein the influenza B virus comprises virus strains B/Massachusetts/2/2012, B/Brisbane/60/2008, B/Yamagata/16/1988, or B/Malaysia/2506/04.

4. The vaccine strain of claim 1, wherein the label gene sequence further comprises a packaging signal sequence at both ends, wherein the packaging signal is a packaging signal of H1 subtype NA set forth in SEQ ID NO: 3 or SEQ ID NO: 4, or a packaging signal sequence having at least 80% homology, at least 85% homology, at least 90% homology, or at least 95% homology with the packaging signal of H1 subtype NA.

5. The vaccine strain of claim 1, wherein the label gene sequence further comprises packaging signal sequences at both ends, wherein the 5′-end packaging signal sequence comprises a noncoding region sequence, an intracellular region sequence, and a transmembrane region sequence.

6. The vaccine strain of claim 5, wherein the intracellular region sequence encodes 5˜7 amino acids.

7. The vaccine strain of claim 5, wherein the transmembrane region sequence encodes 24˜32 amino acids.

8. The vaccine strain of claim 5, wherein the 5′-end packaging signal sequence of the label gene sequence comprises SEQ ID NO:3, or a sequence having at least 80% homology, at least 85% homology, at least 90% homology, or at least 95% homology with SEQ ID NO:3.

9. The vaccine strain of claim 1, wherein the label gene sequence further comprises packaging signal sequences at both ends, wherein the 3′-end packaging signal sequence comprises SEQ ID NO:4, or a sequence having at least 80% homology, at least 85% homology, at least 90% homology, or at least 95% homology with SEQ ID NO:4.

10. A preparation method of an H5 avian influenza vaccine strain, comprising: contacting a label gene sequence with an HA gene or a mutated H5 subtype HA gene of H5 avian influenza virus over a reverse genetic system; and obtaining a recombinant H5 avian influenza vaccine strain; wherein: the mutated H5 subtype HA gene has been mutated to encode RETRGLF (SEQ ID NO: 9) In place of RERRRKRGLF (SEQ ID NO: 8); the label gene sequence comprising a DNA sequence for coding an influenza B virus NA protein extracellular region amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 6, or comprising a DNA sequence for coding an amino acid sequence comprising at least 90% homology, at least 92% homology, at least 95% homology, or at least 98% homology with the extracellular region amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 6; alternatively, the label gene sequence comprising a DNA sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 7 for coding an extracellular region amino acid sequence in influenza B virus NA gene, or comprising a sequence comprising at least 90% homology, at least 92% homology, at least 95% homology, or at least 98% homology with the DNA sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 7.

11. The method of claim 10, wherein the label gene sequence further comprises packaging signal sequences at both ends.

12. The method of claim 11, wherein the 5′-end packaging signal sequence of the label gene sequence comprises SEQ ID NO:3, or a sequence having at least 80% homology, at least 85% homology, at least 90% homology, or at least 95% homology with SEQ ID NO:3.

13. The method of claim 11, wherein the 3′-end packaging signal sequence of the label gene sequence comprises SEQ ID NO:4, or a sequence having at least 80% homology, at least 85% homology, at least 90% homology, or at least 95% homology with SEQ ID NO:4.

14. The method of claim 10, further comprising 6 additional PR8 internal genes, wherein the 6 additional PR8 internal genes comprise a wild type NS, a mutated ΔNS gene, PB2, PB1, PA, NP, or M; wherein ΔNS is a mutated NS gene, and the nucleotide sequence of ΔNS is as set forth in SEQ ID NO:5.

15. An H5 avian influenza vaccine strain, which is named as H5 avian influenza vaccine candidate strain Re-MuH5-DIVA-ΔNS, has been preserved in China Center for Type Culture Collection, with the preservation number of CCTCC NO: V201741.

16. An avian influenza vaccine, comprising the vaccine strain of claim 15.

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-MuH5-DIVA-ΔNS (A/B chimeric NA) serum with influenza A NA 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-MuH5-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) With the artificially synthesized wild type H5 gene (A/Duck/Hubei/49/2005), the high conservative sequence (RERRRKRGLF, SEQ ID NO: 8) in the highly pathogenic wild type HA amino acid sequence is mutated into a low pathogenic amino acid sequence (RETRGLF, SEQ ID NO: 9) through site-directed mutagenesis, to obtain the corresponding low pathogenic MuH5HA gene sequence. The modified MuH5HA gene is cloned into the pFlu vector through the BsmBI site to obtain the recombinant plasmid pFlu-MuH5HA, 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-MuH5-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-MuH5-DIVA-ΔNS is rescued with the classical “6+2” influenza reverse genetic system. Each 0.5 ug of 6 PR8 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-MuH5HA, 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 48h 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 hs. The chick-embryo allantoic fluid (F0 generation) is collected to obtain the vaccine strain Re-MuH5-DIVA-ΔNS, and it is determined whether it has a hemagglutination titer.

(13) The rescued Re-MuH5-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, there is no need for serial passage adaptation on chick-embryos, the HA titers of which may reach 7 log 2. Due to NS1 partial deletion of Re-MuH5-DIVA-ΔNS strain, its growth titer on chick-embryos is lower than that of non-deleted viruses, but better than non-deleted viruses in terms of safety. The obtained virus containing allantoic fluid is inactivated with formalin and further formulated into inactivated vaccines.

(14) The applicants have preserved the inventive vaccine strain Re-MuH5-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: V201741, the proposed classification name is H5 avian influenza vaccine candidate strain Re-MuH5-DIVA-ΔNS, the preserved vaccine strain has been identified as viable on Oct. 28, 2017.

Example 2 a Preparation Method of Avian Influenza Vaccine Strain Re-MuH5-DIVA-ΔNS Virus

(15) The preparation method of Example 2 is the same as that of Example 1, except that in constructing the artificially synthesized AB 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.

(16) 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).

(17) The Re-MuH5-DIVA-ΔNS vaccine strain prepared in the present invention will be further detected for its effects below.

(18) Process: Re-MuH5-DIVA-ΔNS vaccine strains obtained from Examples 1 and 2, PR8-ΔNS (NS-deficient PR8 virus) of the control group 1, PR8-WT (PR8 wild type virus) of the control group 2 are respectively inoculated into 8-day-old chick-embryos at 0.2 ml per embryo for serial passages, the inoculated chick-embryos are cultured in an incubator at 37° C. for 48 hs. 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 48hs to obtain viruses which are defined as F1-generation. With the same process, F1-generation viruses are serially passaged to F3-generation.

(19) Results: the detection results aere shown in Table 1. For demonstrating whether type B NA gene of different branches can match with H5 subtype HA (H5-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 H5. Wherein, the titer of Re-MuH5-DIVA-ΔNS vaccine strain of Example 1 possessing Massachusetts/2/2012 (Yamagata group) NA gene on chick-embryo is 7 log 2 HA titer; while the titer of Re-MuH5-DIVA-ΔNS vaccine strain of Example 2 possessing B/Brisbane/60/2008 NA gene on chick-embryos may reach 5.5 log 2 HA titer. Taking F0 and F3 viruses, through amplification of chimeric NA gene by RT-PCR, it is demonstrated by sequencing that chimeric NA gene can be stably passed to progeny viruses.

(20) As also can be seen from Table 1, 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.

(21) TABLE-US-00001 TABLE 1 Growth properties of different chimeric recombinant H5 avian influenza viruses on chick-embryos HA Titers (log2) Example 1 Example 2 Control Control Passage Re-MuH5- Re-MuH5- Group 1 Group 2 Number DIVA-ΔNS DIVA-ΔNS PR8-ΔNS PR8-WT F0 7 5 6.5 9 F1 7 5.5 7 10 F2 7.5 5 7 9 F3 7 5 7 10

(22) 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 2) both have good compatibilities with H5 HA, showing that influenza B virus NA gene may all be used in preparing an H5 avian influenza vaccine strain which differentiates influenza A virus infection from vaccination.

Example 3 Preparation of Re-MuH5-DIVA-ΔNS Inactivated Vaccine

(23) 50 ml of F0, F1, F2 or F3-generation allantoic fluids from Re-MuH5-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 hs. 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 mins. 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 4 Detection of Effects of Re-MuH5-DIVA-ΔNS Inactivated Vaccine on Vaccinating Animals

(24) Process: 10 3-week-old SPF chicken are vaccinated with Re-MuH5-DIVA-ΔNS vaccine prepared in the present invention 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.

(25) Results: it is demonstrated from experiments that Re-MuH5-DIVA-ΔNS stimulates the organism to generate high level of HI antibodies, the average HI titer (log 2) for week 3 is 9.5±0.85. For HA and HI tests, reference to GBT 18936-2003 (diagnosis technology of highly pathogenic avian influenza).

Example 5 Serological Experiments

(26) 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 hs after transfection, the reactivities of the following 7 groups of chicken serum with N1, N2, N6, N9 are detected by immunofluorescence.

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

(28) Anti-Re-MuH5-DIVA-ΔNS chicken serum: chicken serum which is only vaccinated with the inventive Re-MuH5-DIVA-ΔNS inactivated vaccine;

(29) Anti-H5N1 standard: H5N1 standard serum, purchased from Harbin Veterinary Research Institute.

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

(31) 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.

(32) 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.

(33) 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.

(34) 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.

(35) The immunofluorescence process is as below:

(36) 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.

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

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

(39) 4) Primary antibodies are diluted with PBS containing 1% BSA by corresponding factors (anti-Re-MuH5-DIVA-ΔNS, anti-H5N1 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.

(40) 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.

(41) 6) Observing with a fluorescence microscope.

(42) 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-MuH5-DIVA-ΔNS. It is found that the anti-Re-MuH5-DIVA-ΔNS serum does not cross react with N1, N2, N6 and N9 proteins (e.g., as shown in Table 2 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-H5N1 standard serum can strongly react with N1 protein. It is demonstrated from this experiment that vaccination with the Re-MuH5-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 H5 subtype whole virus vaccine is unable to differentiate infected from vaccinated animals.

(43) TABLE-US-00002 TABLE 2 Comparison of reactivities between chicken sera vaccinated with different antigens and various NA subtypes Antigens Antibodies N1 N2 N6 N9 Anti-Re- HI: 91og2 No No No No MuH5- reactivity reactivity reactivity reactivity DIVA- ANS Anti-H5N1 HI: 81og2 Reactivity ND ND ND standard Anti- HI: 81og2 Reactivity ND ND ND H5 + H7 (H5) 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

Example 6 a Preparation Method of an H5 Avian Influenza Vaccine Strain Re-MuH5-DIVA-ΔNS which Differentiates Influenza a Virus Infection from Vaccination

(44) The preparation method of Example 6 is the same as that of Example 1, except that in constructing the artificially synthesized AB 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).

(45) 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.