Influenza viral vectors expressing two kinds of HA proteins for bivalent vaccines against clade 2.3.4.4 and clade 2.3.2.1 H5 HPAIVs

The H5 highly pathogenic avian influenza viruses (HPAIVs) in China pose a serious challenge to public health and the poultry industry. In this study, we constructed a replication-competent recombinant influenza A virus of clade 2.3.4.4 Н5N1 expressing the clade 2.3.2.1 H5 HA1 protein from a tricistronic NS segment. We used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. H5 HA1 and nuclear export information were fused in frame with a truncated NS1 open reading frame, separated by 2A self-processing sites. The resulting PR8-H5-NS1(73)H5 stably expressed clade 2.3.4.4 H5 HA and clade 2.3.2.1 H5 HA1 proteins and exhibited similar in vitro growth kinetics as the parental PR8-2344H5 virus. PR8-H5-NS1(73)H5 induced specific hemagglutination-inhibition (HI) antibody against clade 2.3.4.4 H5 that was comparable to that of the combination vaccine of PR8-2344H5 and PR8-2321H5. HI antibody titers were significantly lower against clade 2.3.2.1 H5 virus than with the combination vaccine. PR8-H5-NS1(73)H5 completely protected chickens from both clade 2.3.4.4 and clade 2.3.2.1 H5 HPAIVs challenge. Our results suggested that PR8-H5-NS1(73)H5 was highly immunogenic and efficacious against both clade 2.3.4.4 and clade 2.3.2.1 H5 HPAIVs in chickens.


Viruses and cells. HPAIVs
were isolated from chickens that died in outbreaks and propagated in 10-day-oldspecific-pathogen-free (SPF) embryonated chicken eggs (ECEs). Their intravenous pathogenicity indexes were 3.00 and 2.86 using ten 6-week-old SPF chickens intravenously inoculated with 0.1 ml 1/10 dilution of fresh infectious allantoic fluid from FJ/5 and JS/7, according to the World Organization for Animal Health manual. The 293 T human embryonic kidney cells (HEK 293 T) were maintained in Dulbecco's modified Eagle medium supplemented with 10% FCS and kept at 37 °C in 5% CO 2 .
Construction of plasmid pHW-NS1(73)-H5HA1-NEP. The coding sequence of Dmd/FMDV-2A was generated synthetically (Sangon, Shanghai, China) and cloned into the pcDNA3 vector using the restriction sites NotI and XbaI. The sequence for the first 73 amino acids of NS1 was amplified by PCR from plasmid pHW198-NS 8 and cloned 5′ to and in frame with Dmd/FMDV-2A using BamHI and EspEI. NS(73)-Dmd/ FMDV-2A was cloned into the pHW2000 plasmid using BamHI and MunI restriction sites.
The mature HA1 coding sequence derived from JS/7 was cloned 3′ of and in frame with the FMDV-2A cleavage site using BglII and EcoRI. The PTV-1 2A cleavage site was fused to the NEP coding sequence by fusion PCR and cloned 3′ of and in frame with the mature HA1 coding sequence using EcoRI and BstEII restriction sites.
Generation of recombinant viruses. All viruses were generated by a standard reverse genetics method using 8 bidirectional plasmids pHW2000 9 . HEK 293 T cells were co-transfected with 0.8 μg of each of the six pHW-plasmids (pHW191-PB2, pHW192-PB1, pHW193-PA, pHW195-NP, pHW197-M, and pHW-NS1(73)-H5 HA1-NEP), as well as the HA and NA genes of the FJ/5 virus using Lipofectamine 3000 transfection reagent (Life Technologies, Carlsbad, CA, USA). The pHW-plasmids expressing six internal genes were all originated from A/PR/8/34 (H1N1) (PR8) virus. The HA sequence of FJ/5 virus was attenuated by removing the multibasic amino acid motif from RERRRKRG to RETRG in the HA cleavage site. After 24 h, TPCK-treated trypsin (Sigma-Aldrich Corporation, St. Louis, MO, USA) was added to a final concentration of 2 μg/ml. After 72 h, supernatants of transfected cells were collected and used to inoculate10-day-old SPF ECEs incubated at 37 °С for 72 h. Vaccine batches were produced in SPF ECEs after five egg passages of viral constructs.
Determination of the 50% embryo infectious dose (EID 50 ) of the viruses. Infectious titers of viruses were determined by standard methods using 10-day old SPF ECEs. Viral suspensions (10 −1 to 10 −9 dilutions) were prepared in PBS (pH 7.2) and allantoic cavities of five ECEs were infected with 0.1 ml dilution. ECEs were incubated at 37 °C at relative humidity 60 for 72 h. Viral titers were determined by hemagglutination assays. Viral titers were calculated and expressed as log10 EID 50 /ml. SPF  Genetic stability of PR8-H5-NS1(73)H5 virus. Ten consecutive passages of PR8-H5-NS1(73)H5 virus were conducted in 10-day-old ECEs for genetic stability testing. The allantoic cavity of ECEs was infected using10 4 EID 50 . Genetic stability of viral constructs was confirmed by reverse transcription polymerase chain reaction (RT-PCR) using NS segment-specific primers (sense 5′-GTA GAT TGC TTT CTT TGG-3′ and antisense5′-CTA AAT AAG CTG AAA CGA-3′). At passages 1, 3, 5 and 10, the size of the NS amplicon was compared to the pHW plasmid encoding the corresponding gene. NS1-fusion proteins encoding genes of viral constructs were sequenced at passages 1, 3, 5 and 10 using the Sanger method with commercial kit Prism BigDye ™ Terminator v3.1 (Applied Biosystems, Foster City, CA, USA) on an automatic sequencer Genetic Analyser 3730XL.

Preparation of vaccines.
Vaccine samples were prepared from viral constructs PR8-2344H5, PR8-2321H5, and PR8-H5-NS1(73)H5, accumulated in 10-day-old ECEs at 37 °C for 72 h. Formalin (final concentration, 0.1%) was added to inactivate allantoic suspensions with viral constructs before incubating at 4 °C for 72 h. Inactivated viruses were concentrated, purified through 10-50% sucrose density gradients, and resuspended in phosphate-buffered saline (PBS). Allantoic suspensions of PR8-2344H5 and PR8-2321H5 were combined into single pools at a 1:1 ratio to obtain the combination vaccine formulation. Oil-adjuvant whole-virus inactivated vaccine was prepared with viral constructs. Inactivated virus was mixed with mineral oil adjuvant at 1:2 (vol/vol) and emulsified and the HA protein content in the final vaccine preparation is about 9.24 μg/ml, which was quantified as previous described 10 . Vaccination and challenge test. Eighty three-week-old white Leghorn SPF chickens were randomly divided into four groups with 20 chickens in each group. Three groups were injected intramuscularly (i.m.) with 0.3 ml formalin-inactivated PR8-H5-NS1(73)H5 vaccine, combination vaccine of PR8-2344H5 and PR8-2321H5, or PR8-2344H5 vaccine, respectively. One group was injected i.m. with 0.3 ml PBS as a control. Three weeks after vaccination, serum samples were taken from all chickens for antibody detection, and birds were challenged intranasally with 10 6 EID 50 of lethal H5 virus in a volume of 0.2 ml. Oropharyngeal and cloacal swabs were collected on day 3 and day 5 postchallenge (p.c.) for virus isolation and titration in eggs. All birds were observed for signs of disease or death for 10 days after challenge.
Antibody detection using the HI assay. Specific antibodies in the chicken sera were detected using the HI assay as described previously 11 . Briefly, the sera were inactivated by incubation at 56 °C for 30 min. Then, the sera were 2-fold serially diluted with PBS, and incubated with four hemagglutination units of the target influenza virus for 30 min. This was followed by adding equal volumes of fresh 1.0% (v/v) chicken red blood cells and further incubation of 30 min. The sample HI titer was defined as the reciprocal of the highest dilution that completely inhibits the agglutination.
Virus isolation and titration. Oropharyngeal 12 in 2.0 ml microcentrifuge tubes.The samples were centrifuged at 10000 g for 5 min at 4 °C, and then the supernatants were applied in ten-fold dilution series and the EID 50 was determined as above.

Generation of recombinant influenza A virus expressing H5 HA1 protein.
We designed a tricistronic NS-derived gene segment with a single open reading frame comprising NS1(1-73)Dmd, H5 HA1 and NEP separated by two different 2A self-processing sites. We used different 2A peptide sequences to reduce the risk of recombination at these sites, which could lead to excision of the HA1 coding information. A foot-and-mouth disease virus (FMDV) 2A autoprocessing site was inserted between NS1(1-73)Dmd and HA1, with HA1 separated from NEP by a porcine teschovirus-1 (PTV-1) 2A cleavage site (Fig. 1A). The FMDV 2A peptide was the first 2A cleavage site to be described, and has been used for applications including the generation of recombinant influenza viruses [13][14][15][16] . The PTV-1 2A cleavage site has a high cleavage efficiency 17,18 . This artificial NS segment was used to rescue influenza viruses expressing dimeric NS1(1-73), HA1 and NEP in a PR8 virus genetic background 8 . Based on the HA and NA gene sequences of FJ/5 replacing the corresponding gene sequences of PR8 virus, this rescue was successful. We named the resulting virus PR8-H5-NS1(73)H5.

Discussion
H5N1 AIVs have become enzootic in poultry and wild birds in China 2 . The government of China undertook a mass poultry vaccination practice against HPAI in 2005 19 . Vaccine strains used in China have been updated several times since 2004 to ensure an antigenic match between the vaccines and the prevalent strains 5 . Currently, combination vaccine of H5 Re-8 and H7 Re1 has been used to control highly pathogenic H5 and H7N9 AIVs throughout the country since August 2017. However, clade 2.3.2.1 H5 virus is also a threat for poultry, with some outbreaks caused by clade 2.3.2.1 H5 virus.
In this study, we generated a replication-competent recombinant influenza A virus of subtype Н5N1 expressing another H5 HA1 protein. We demonstrated that this vaccine could be used as a new candidate for H5 subtype avian influenza vaccines against two clades. Current killed influenza virus vaccines predominantly induce anti-HA that specifically targets antigenic sites in the globular head domain of the HA1 region and block receptor binding 20,21 . Therefore, we chose mature HA1 as an antigenic determinant inserted into the NS segment between NS1 and NEP. Our strategy resembles that of De Baets et al., who reported inserting GFP into an NS1 segment of     16 . Nonetheless, the efficiency of different 2A sites must be determined empirically, as they may depend on context. To reduce the NS segment size, we used a truncated NS1 protein that generally results in in vivo virus attenuation 23 . Some attenuation can be reversed by adding a heterologous dimerization domain to the truncated NS1, which improves stability of the dimeric NS1 protein 24 . Foreign gene-expressing viruses ideally have high genetic stability. This is difficult to accomplish with engineered influenza viruses. PR8-H5-NS1(73)H5 virus has in vitro replication kinetics that are similar to parental PR8-H5 virus, although PR8-H5-NS1(73)H5 replicated slower than PR8-H5. Sequencing showed that there was no mutation in the third, fifth and tenth passage of PR8-H5-NS1(73)H5 virus stock compared to the pHW plasmid encoding the corresponding gene. This result indicated the stability of the PR8-H5-NS1(73)H5 virus. The reason for the virus grows better after several passages needs further investigation. A previous study demonstrated that NS1 tolerates foreign sequences exceeding its own length 7 . As far as we know, GFP is the longest fragment inserted, at 702 bp. We have successfully inserted 978 bp of H5 mature HA1 and obtained stable virus.
As compared with FJ/5 (clade 2.3.4.4 H5) and S/7 (clade 2.3.2.1 H5), the amino acid identity of HA was 90.5%, suggesting that these viruses are likely distinct in antigenicity. This is consistent with the results of the cross-HI tests and the vaccination-challenge tests.