**3. Recombinant Platforms for Expression of H7N9 VLPs**

The recombinant platforms using either mammalian, insect, or plant cells have many potential advantages including scalability and intrinsic safety, especially for HPAI viruses such as H7N9 (Table 2). Mammalian cells, such as MDCK, Vero and PERC.C6 cell lines have been extensively studied and applied to influenza vaccine production. The use of VLPs expressed from mammalian cell platform has advantages for VLP production such as flexibility and a similar glycosylation pattern to the human virus. In some reports, VLPs expressed from HEK293T cells containing HA and NA proteins on their surface with a HA:NA ratio of 1:1, which is higher than natural influenza viruses, where the HA:NA ratio is 4:1 [57]. VLPs isolated from HEK293SF cells were also characterized by nano-LC-MS/MS analysis to identify the protein cargo. Nucleolin was the most abundant cellular protein present in VLPs, in addition to the recombinant influenza VLP proteins [58]. However, production of VLPs in mammalian cell expression system is difficult to scale up to the commercial scale, especially when using the transient transfection method. This limitation can be addressed by preparing stable cell line of corresponding genes. For example, a stable cell line expressing H1 and N1 genes under the cumate inducible promoter was generated to create 293HA-NA cells. Plasmid with either Gag or M1 gene was transiently transfected in 293HA-NA cells and VLP yield was compared with Gag or M1 protein. 293HA-NA cells produced seven times more HA and NA with Gag plasmid transfected cells than its counterpart matrix M1-encoding plasmid [59]. Gag VLPs produced by this approach elicited strong antibody response and provided complete protection against the homologous virus strain. Optimization of cell density at the time of induction in inducible stable HEK293 cell lines also increased VLP yield by five-fold [60].

Insect cells such as Sf9 can be maintained in serum-free suspension cultures and have been often used to prepare recombinant VLPs including H7N9. When influenza VLPs were compared between insect Sf9 cells and mammalian HEK293 suspension cells, Thompson et al. found that Sf9 cells produce approximately 35 times more VLP than HEK293 cells [61]. VLPs produced from Sf9 cells also showed higher HA titer and more homogenous VLPs than HEK293 cells derived VLPs [61]. As expected, the high titer of baculovirus vector was also present in VLPs prepared in Sf9 cells. However, VLPs generated in HEK293 cells contained extracellular vesicles, such as exosomes and microvesicles. VLPs produced from Sf9 insect cells may have a safety advantage over other expression system because insect cells are unlikely to contain human pathogens, and baculovirus vector cannot replicate in human cells. VLPs produced from insect cells show higher yields as compared to mammalian cell-produced VLPs, which is beneficial for commercial production. In the above study, Sf9 cells produced up to 35 times higher yield than the HEK293 cells' expression system [61]. In addition, VLPs had higher HA activity and they were more homogenous in morphology and size than mammalian cells produced VLPs [61]. Sf9 cells are usually maintained in a serum free, animal product-free medium. Some of the chemical compounds known as M2 inhibitors, such as amantadine-enhanced expression of HA protein in VLPs by seven-fold in Sf9 expression system [62]. However, the challenge for VLPs expressed using

baculovirus expression system is that it is difficult to separate VLPs from the baculovirus expression vector during vaccine manufacturing.

H7N9 VLPs have been prepared from the full-length HA, NA, and matrix M1 genes in Sf9 cells. The H7 HA was derived from A/Shanghai/2/2013 (H7N9) virus, while both NA and M1 proteins were derived from A/Puerto Rico/8/1934 (H1N1) virus, a standard donor virus that is often used for preparation of influenza reassortant viruses used in human influenza vaccine production [63]. VLPs have been isolated from Sf9 cells and their morphology resembling native H7N9 virus envelope was confirmed.

In another study, recombinant H7N9 VLP vaccine was prepared from HA and NA derived from the A/Anhui/1/2013 (H7N9) virus and the matrix M1 protein derived from the H5N1 virus (A/Indonesia/05/2005) [42]. As control vaccines, VLPs were prepared from A/chicken/Jalisco/CPA1/2012 (H7N3) and A/Indonesia/05/2005 (H5N1) viruses. The H7N9 VLPs elicited hemagglutination-inhibition (HAI) antibody response against the homologous H7N9 virus, cross-reactive HAI against the heterologous H7N3 virus, and three- to four-fold higher HAI response if VLPs were administered with ISCOMATRIX™ saponin-based adjuvant. Similarly, all doses of H7N9 VLP elicited anti-NA antibody, with three- to four-fold higher responses measured in the corresponding ISCOMATRIX subgroups. A lethal murine wild-type A/Anhui/1/2013 (H7N9) challenge demonstrated 100% survival of all animals receiving H7N9 or H7N3 VLP vaccines, versus 0% survival in A(H5N1) vaccine and placebo groups [42].

Another study reported a VLP from Sf9 cells that consisted of HA, NA and M1 proteins derived from the human isolate A/Taiwan/S02076/2013(H7N9) as a potential vaccine. In animal experiments, BALB/c mice and specific-pathogen-free chickens receiving the VLP immunization developed HAI serum titer and antibodies against NA and M1 proteins [45].

The immunogenicity and protective efficacy of a H7N9 VLP vaccine were evaluated in the ferret challenge model considered the most appropriate animal model for influenza [31]. Purified recombinant H7N9 VLPs morphologically resembled influenza virions and elicited high-titer serum HAI and neutralizing antibodies to A/Anhui/1/2013 (H7N9) virus. H7N9 VLP-immunized ferrets were challenged with homologous virus. After challenge, VLP-vaccinated animals displayed reductions in fever, weight loss, and virus shedding as compared to the same parameters in the unimmunized control ferrets. H7N9 VLP was also effective in protecting against lung and tracheal infection. The addition of either ISCOMATRIX or Matrix-M1 adjuvant further improved immunogenicity and protection of the VLP vaccine against H7N9 virus [31].

Another recombinant influenza vaccine was developed by expressing H7 from H7N9 (A/Shanghai/2/2013) on the surface of recombinant baculovirus. Although this approach would not result in a typical influenza VLP morphology, the spatial conformation of H7 in the resulting vaccine is expected to be similar to that in an influenza virion or in a cognate VLP. Mice were immunized twice, either intranasally or subcutaneously, with the vaccine. The immunogenicity and cross-protective efficacy of the vaccine were evaluated against H7N9 or H7N7 subtype challenges. The authors concluded that intranasal administration of H7 protein expressed on the baculovirus envelope can be an alternative way to prime the immune system against influenza infection during a pandemic situation [64]. Recently, a baculovirus vaccine expressing the HA of H7N9 strain A/Chicken/Jiaxing/148/2014 was prepared [65]. The recombinant baculovirus-HA generated in this study showed favorable growth characteristics in insect cells, good safety profile, and induced high-level hemagglutination inhibition antibody titer. Moreover, this vaccine demonstrated better efficacy than inactivated whole-virus vaccine JX148, provided complete protection of chickens against challenge with HPAI H7N9 virus, and effectively inhibited viral shedding.

Plant-based VLP vaccines can be produced from *Agrobacterium*-mediated transient expression of influenza HA proteins in *Nicotiana benthamiana*. Plant-made H1 or H5 VLPs mimicked the structure of influenza virions to some extent, they are immunogenic and elicit both humoral and cellular responses [66,67]. Each HA-only particle contains 30–50 homo-trimer HA incorporated into lipid bilayer envelope of plant cell origin [68]. Plant-based recombinant VLP vaccines elicited humoral and cellular responses and protected against challenges. For example, a plant-derived VLP vaccine based on the HA of influenza H7N9 A/Hangzhou/1/2013 was prepared, with no other influenza proteins included in the vaccine. The immunogenicity of such H7-only VLP vaccine was assessed in mice and ferrets after one or two intramuscular dose(s) with, and without, adjuvant (alum or GLA-SE™). In ferrets, H7-specific cell-mediated immunity was also evaluated. The mice and ferrets were challenged with H7N9 A/Anhui/1/2013 influenza virus. A single immunization with the adjuvanted vaccine elicited a strong humoral response and protected mice against an otherwise lethal challenge. Two doses of unadjuvanted vaccine significantly increased humoral response and resulted in 100% protection with significant reduction in clinical signs, leading to nearly asymptomatic infections. In ferrets, a single immunization with the alum-adjuvanted H7 VLP vaccine induced strong humoral and CMI responses with antigen-specific activation of CD3(+) T cells. This plant-made H7 vaccine therefore induced protective responses after either one adjuvanted or two unadjuvanted doses [39]. Potentially, the plant-based transient expression system can allow the production of VLP structures, which contain HA only, without other influenza proteins.

The recombinant HA-only H7 vaccine against H7N9 virus has been also prepared in insect cells [30]. The study has shown that purified HA formed rosette-shaped nanoparticles of approximately 30–50 nm in diameter [69]. Similarly, subviral particles containing the full-length H7 rHA (A/Anhui/1/2013 (H7N9) were isolated from Sf9 cells treated with mild detergent [38]. Similarly to a previous study [30], purified rHA formed particulate structures; the particles were approximately 20 nm in diameter, exhibited hemagglutination activity, and consisted of approximately 3–4 trimers of the full-length HA molecules.
