Single dose of multi-clade virus-like particle vaccine protects chickens against clade 2.3.2.1 and clade 2.3.4.4 highly pathogenic avian influenza viruses

Virus-like particles (VLPs) are recognized as an alternative vaccine platform that provide effective protection against various highly pathogenic avian influenza viruses (HPAIVs). Here, we developed multi-clade VLPs expressing two HAs (a chimera of clade 2.3.2.1c and clade 2.3.4.4c HA) within a single vector. We then compared its protective efficacy with that of a monovalent VLP and evaluated its potency against each homologous strain. Chickens vaccinated with the multi-clade VLP shed less virus and were better protected against challenge than birds receiving monovalent vaccines. Single vaccination with a multi-clade VLP resulted in 100% survival, with no clinical symptoms and high levels of pre-challenge protective immunity (7.6–8.5 log2). Moreover, the multi-clade VLP showed high productivity (128–256 HAU) both in the laboratory and on a large scale, making it cheaper than whole inactivated vaccines produced in eggs. However, the PD50 (protective dose 50%) of the multi-clade VLP against clades 2.3.2.1c and 2.3.4.4c was < 50 PD50 (28 and 42 PD50, respectively), and effective antibody response was maintained for 2–3 months. This multi-clade VLP protects against both clades of HPAI viruses and can be produced in high amounts at low cost. Thus, the vaccine has potential as a pandemic preparedness vaccine.

Highly pathogenic avian influenza (HPAI) has been circulating in wild birds and poultry worldwide since the first H5N1 avian influenza virus, A/Goose/Guangdong/1/96, was identified in China in 1996 1,2 . Novel HPAI H5Nx viruses, including various NA, have emerged continuously due to extensive genetic reassortment activity; indeed, various clades have emerged due to genetic evolution and antigenic drift [3][4][5] . As a result, persistent HPAI outbreaks on poultry farms in many countries have been caused by mutated viruses, resulting in serious economic losses 1 . Moreover, various subtypes of HPAI have emerged simultaneously in countries such as China (H5N6, H5N1), Vietnam (H5N6, H5N1), and Taiwan (H5N2, H5N5), making it difficult to eradicate the virus 6 .
Since the emergence of H5N1 on a poultry farm in 2003, H5 HPAI outbreaks have occurred continuously, and new influenza viruses and genotypes have been introduced into Korea 7,8 . Various clades of H5Nx, including H5N1, H5N6, and H5N8, have been identified [9][10][11][12][13] . In particular, outbreaks of HPAI subtypes H5N6 and H5N8 occurred simultaneously in 2017 14 . During the unprecedented outbreaks of HPAI in 2016/2017, there has been increased demand for AIV vaccination by poultry producers and animal welfare organizations. Potential vaccines in the future should be considered with introduction of two or more viruses simultaneously around Korea.
Vaccination policy may be a supportable measure for preventing HPAI when implemented properly and in conjunction with accurate epidemiologic investigation and control measures 7,15 . Until recently, most AI vaccines used or registered in the field were whole inactivated AI vaccines 16  www.nature.com/scientificreports/ respond effectively to a wide range of infectious viruses 17,18 . A previous study examined the efficacy of VLP vaccines against multi-clade H5N1 2 , whereas another showed that VLP displaying H5, H7, H9, and N1 protect chicken from infection by heterologous virus 19 . However, preparation of a multivalent vaccine against various HPAI viruses by mixing monovalent whole inactivated vaccines raises biosecurity concerns and is economically unfeasible 20 . Therefore, a vaccine based on VLP requires stronger product development through further studies examining multi-subunit, chimeric, and other types of VLPs; such studies should focus on delivery of sufficient amounts of VLP antigen because these vaccines will not be competitive in the market unless they are both productive and cost-effective 21 .
Here, we constructed a single vector containing cassettes encoding multi-clade VLPs and then manufactured multi-clade VLPs as a vaccine capable of protecting against two separate clades of HPAI virus. We then compared its protective efficacy with that of a monovalent VLP and evaluated its potency against each homologous strain in specific pathogen free (SPF) chicken.

Results
Expression and preparation of monovalent and multi-clade VLPs. Monovalent VLP_ES2, VLP_ KA435, and VLP_KA435chi, and multi-clade VLP_ES2/KA435chi vaccines, were produced and secreted successfully from Sf9 cells. The HA titer of VLP_ES2 in the culture supernatant ranged from 128 to 256 HAU. However, the HA titer of VLP_KA435 was as low as 32-64 HAU. The titer of VLP _KA435chi ranged from 128 to 256 HAU. The titer of the multi-clade VLP_ES2/KA435chi was 128-256 HAU. After large-scale production, the titer of the multi-clade VLP was also 128-256 HAU per 25 L of medium (the same as lab-scale production) ( Table 1). The multi-clade VLP was purified from the culture supernatant and examined by transmission electron microscopy and western blot (Fig. 1). Table 1. Expression and proliferation of VLPs. HAU hemagglutination units, VLP virus-like particle. 1 VLPs were purified from Sf9 or the Tni culture supernatant. 2 Volume of the cell culture supernatant. 3 Hemagglutination activity was determined using 1% chicken red blood cells.  (Table 3).
Serology. All vaccinated groups showed detectable antibody titers against ES2 and KA435 antigens pre-challenge and post-challenge; these titers increased over time (Fig. 3). In vaccinated SPF chickens (one dose or one-tenth dose), all those receiving the multi-clade VLP vaccine seroconverted before challenge, with a mean tier of 7.2-7.8 and 3.9-4.7 log 2 after one dose and one-tenth dose, respectively. After challenge, the antibody titer against ES2 and KA435 antigens increased to 7.9-8.5 log 2 and 6.5-7.6 log 2 in the one dose and one-tenth dose groups, respectively. Unlike the one dose-and one-tenth dose-vaccinated groups, the one-hundredth dosevaccinated groups showed a weaker antibody response (1.0 log 2 ) prior to challenge. Following challenge, the HI titer of surviving chickens challenged with ES2 had more various antibody responses, with HI titers around 6.0 Table 2. Results among single and multivalent vaccinations against homologous and heterologous HPAI. HA hemagglutination activity, NT not tested, dpi days post-infection, OP oropharyngeal, CL cloacal, HI hemagglutination inhibition. *p value < 0.05. 1 No. serology positive/total survived in group (mean HI titer). 2 No. serology positive/total survived in groups (mean SN titer). 3 Homologous: same as vaccine strain; heterologous: different from vaccine strain. 4 MDT = mean death time (days). 5 No. virus positive/total in group (mean shed-virus titer).  www.nature.com/scientificreports/ log 2 (Fig. 3B). None of the sham-vaccinated chickens had detectable HI antibodies before challenge (data not shown).
Virus shedding. As shown in Fig. 4, little virus shedding was observed from 3 to 7 dpi in SPF chickens vaccinated with one dose of the multi-clade VLP. However, virus shedding was detected from 3 to 7 dpi in SPF chickens vaccinated with one-tenth dose of the multi-clade VLP and challenged with either KA435/2.3.2.1c or ES2/2.3.4.4c. In SPF chickens vaccinated with one-hundredth dose, virus shedding was detected in surviving birds, with a viral titer of 10 1.7 -10 2.8 TCID 50 /0.1 mL in OP swab samples from 3-5 dpi and 10 2.4 -10 4.0 TCID 50 /0.1 mL in CL swab samples from 3 to 5 dpi. Most birds in the sham-vaccinated groups were not tested because they died prior to sampling (Fig. 4).
Antibody persistence. We measured the mean HI titers against ES2 and KA435 antigens in SPF chickens and layer chickens inoculated with one dose of the multi-clade VLP vaccine. In SPF chickens vaccinated with the multi-clade VLP, HI titers against the KA435 antigen were 9.3 log 2 and those against the ES2 antigen were 7.8 log 2 at 3 wpv. The mean HI titers peaked between 3 wpv, and remained above 7 log 2 to provide reduction in challenge virus replication and shedding 23 by 12 wpv (KA435) and 16 wpv (ES2) (Fig. 5A). In layer chickens vaccinated with the multi-clade VLP, HI titers against the KA435 antigen were 7.4 log 2 , and those against the ES2  www.nature.com/scientificreports/ antigen were 8.6 log 2 at 3 wpv. The mean HI titers peaked at 3 wpv, and remained above 7 log 2 by 8 wpv (KA435) and 3 wpv (ES2). None of the chickens showed HI titers above 7 log 2 at 24 wpv (Fig. 5B).

Discussion
Here, we constructed a vector containing cassettes for multi-clade VLP into which two kinds of HA can be inserted. We then manufactured a multi-clade VLP vaccine by inserting the HA sequence of clade 2.3.2.1c(chimera) and 2.3.4.4c vaccine strains from the Korean AI national antigen bank. Finally, we compared the efficacy of monovalent and multi-clade VLP vaccines in SPF chickens challenged with homologous strains. Chickens vaccinated with the multi-clade VLP shed less virus and were better protected against challenge than chickens vaccinated with the monovalent vaccine. This is likely because multivalent vaccines trigger stronger cellular and humoral responses, thereby inhibiting viral replication more effectively than the monovalent VLP vaccines 24,25 . Moreover, one chicken vaccinated with the monovalent VLP_KA435chi died, despite showing a higher HI titer (9.0 log 2 ) ( Table 2); this may be because the HA2 of KA435 was substituted with the HA2 of Buan2, coupled with challenge with the more virulent 2.3.2.1c strain as reported previously 26 , and induction of lower SN titer which more closely related to the capability of antibodies controlling the replication of virus comparing HI titer 27 .
For use in an antigen bank, an emergency vaccine should show a minimum 50 PD 50 per dose, or antibody persistence above and HI of 1/128 HI for 6 months post-vaccination; in Korea, the guidelines are a minimum 80% protection from mortality and an HI titer of greater than 1/128 14 . In this study, the PD 50 and antibody persistence of the multi-clade VLP did not meet these criteria or were lower than those of whole inactivated vaccinated chickens in comparison with the results of a previous study 26 . Whole inactivated vaccines induce a   www.nature.com/scientificreports/ stronger immune response than subunit vaccines such as VLP vaccines 28 . Therefore, we believe that our results are due to the fact that the VLP vaccine is based on part (M and HA) of the virus. Indeed, other studies show that whole inactivated vaccines are more effective in potency tests than split, subunit vaccines and virosomes; also, whole inactivated vaccines have a significantly higher probability of inducing seroconversion than subunit vaccines 29,30 . Further study is needed on how multi-clade VLP increases protective efficacy comparing to whole inactivated vaccines. Our multi-subtype vector includes a cassette into which two full HAs are co-localized within one VLP structure 2 ; we then ensured high protein expression (128-256 HAU) by producing a chimeric VLP under optimized cell culture conditions. Indeed, productivity in cell culture was equal to that after large-scale (25 L) production (Table 2), and higher than that after large-scale production reported by other studies (e.g., 16-128 HAU in Sf9 cells) 2,31 . This process enables cost savings of approximately 0.02 dollars per dose when compared with mixing two types (0.068 dollars per dose) of whole inactivated vaccine using the egg product system of the Korean AI national bank (data not shown). This means that the multi-clade VLP we developed in this study may be economically more viable than vaccines cultured in eggs. In addition, multi-clade VLPs effectively induce Th1 type immune responses, and plasma and memory B cells 32 . Only single vaccination of multi-clade VLP with a dose of 512 HAU showed immunogenicity, survival rate and reduction of virus shedding in similar to results boosting other VLP vaccination 2,19,33 .
In conclusion, we constructed a single vector containing cassettes encoding multi-clade VLPs and produced a chimeric vaccine capable of protecting chickens against simultaneous challenge with clade 2.3.2.1c and 2.3.4.4c HPAIVs. Large amounts of vaccine were produced in cell culture, making the vaccine potentially cost-effective. However, the PD 50 and antibody persistence were below national standards and lower than those of whole inactivated vaccines. Taken together, these results suggest that the multi-clade VLP vaccine is an economically effective vaccine that protects against infection by multiple HPAIVs.
The constructed pFastBac™ dual vector plasmids pHA_ES2, pHA_KA435, pHA_KA435chi, and pHA_ES2/ KA435chi were transformed into DH10Bac™ competent cells (Invitrogen) to generate recombinant bacmids. Recombinant bacmid DNA was transfected into Sf9 cells seeded in 6-well plates (8 × 10 5 cells/well) using Cell-fectin® Reagent (Invitrogen), resulting in the release of recombinant baculovirus (rBV) into the culture medium. At 72 h post-transfection, culture medium was harvested and inoculated into Sf9 cells to generate a high titer rBV stock. Titration of the baculovirus stock was performed in a plaque assay on Sf9 cells. Transmission electron microscopy of VLPs. The VLP suspension was placed on formvar-coated (copper 300 mash) grids, negatively stained with 1% uranyl acetate, and dried by aspiration. The VLP particles were then examined under a transmission electron microscope (Hitachi7100FA, Tokyo, Japan).

Efficacy of the monovalent and multi-clade VLP vaccines.
To compare the efficacy of the monovalent vaccines with that of the multi-clade VLP vaccine, 60 5-week-old SPF chickens were divided into six groups (10 chickens per group). The four groups used for vaccination comprised two groups of chickens receiving monovalent VLP vaccines (VLP_ES2 or VLP_KA435chi) and two groups receiving the multi-clade VLP (VLP_ES2/KA435chi) vaccine. All VLP vaccines were emulsified in Montanide ISA VG70 adjuvant and injected via the intramuscular route; each chicken received 0.5 mL. Another 20 SPF chickens were split into two sham groups and injected with an emulsified solution of PBS plus ISA VG70 in the same ratio as the VLP vaccines. Serum samples were collected from all chickens at weekly intervals post-vaccination and then at 14 days post infection (dpi). To determine the immunogenicity of the VLP vaccines, all sera were tested by HI test against homologous and heterologous, and Serum neutralizing antibody test (SN test) in Dermal Fibroblast 1 (DF1) cells, as described previously 36 . Three weeks after vaccination, two groups of vaccinated chickens were challenged with 0.1 mL ES2/2.3.4.4c and KA435/2.3.2.1c (10 6.0 EID 50 /0.1 mL). All birds were observed daily for 14 dpi to check mortality, clinical signs, and viral shedding. To determine viral shedding, oropharyngeal (OP) and cloacal (CL) swab samples were collected at 1, 3, 5, 7, 10, and 14 dpi. Each OP or CL sample was suspended in 1 mL of maintenance medium containing an antibiotic-antimycotic mixture (Invitrogen). Samples were used to inoculate cultures of Dermal Fibroblast 1 (DF1) cells, and virus growth was determined by detection of cytopathic effects and measurement of HA activity. Virus titers were calculated as described elsewhere 22 . The limit of virus detection was < 1 log 10 TCID 50 /0.1 mL. All experiments with live H5 virus were performed in biosafety level 3 facilities and in accordance with guidelines approved by the Animal Ethics Committee of the Animal and