**8. Challenges for Recombinant VLPs as Influenza Vaccines**

Heterogenous composition is one of the challenges associated with the use of influenza VLPs as a vaccine candidate. VLPs produced by using baculovirus expression system contain live baculovirus vector as impurity. Moreover, considerable fraction of HA protein is bound to viral envelope of baculovirus [107]. Therefore, it is difficult to distinguish whether the HA activity is associated with influenza VLP or with baculovirus-displayed HA in the unpurified samples during vaccine manufacturing, which also causes difficulty for HA quantification. VLPs produced from mammalian expression system do not contain baculovirus contamination but they contain extracellular particles and produce low yields, with VLPs needing to be concentrated up to 200 times to reach measurable titers [72]. The current methods that are used for quantification are mostly applicable to concentrated and purified VLPs; however, better methods need to be developed to characterize the materials at the different stage of bioprocessing during the manufacturing process. Other potential challenges include the presence of endogenous rhabdovirus in standard Sf9 cell lines [108]. Although insect rhabdovirus is not associated with any human disease, the development of rhabdovirus-free cell line can resolve this potential safety concern [108,109].

To overcome manufacturing challenges, technological improvements to production of H7N9 VLPs in baculovirus-Sf9 insect cell cultures have been reported [110–112]. The yield and quality of influenza VLPs produced in insect cells could be improved by inhibiting cytopathic effects of the protein M2 [62]. The ion channel activity of M2 induces significant cytopathic effects in Sf9 insect cells. These effects include altered Sf9 cell morphology and reduced baculovirus replication, resulting in impaired influenza protein expression and thus VLP production. The use of the M2 inhibitor amantadine indeed improved Sf9 cellular expression not only of M2 (∼three-fold), but also of HA (∼seven-fold) and of matrix protein M1 (∼three-fold) when co-expressed to produce influenza VLPs. This increased cellular expression of all three influenza proteins further led to ∼two-fold greater VLP yield. The quality of the resulting influenza VLPs was significantly improved, as demonstrated by the ∼two-fold, ∼50-fold, and ∼two-fold increase in the antigen density to approximately 53 HA, 48 M1, and 156 M2 per influenza VLP, respectively [62].

For purification of influenza VLPs, a nitrocellulose membrane-based filtration system [113] and a cascade of ultrafiltration and diafiltration steps, followed by a sterile filtration step [109,114] were used successfully. The bioprocessing of influenza VLPs was recently reviewed [115]. Recently, a fast chromatography step purification method was developed for chimeric HIV-1 Gag influenza-HA VLPs produced in *Tnms*42 insect cells using the baculovirus insect cell expression vector system [116]. The absence of the baculovirus capsid protein p39 in the product fraction was confirmed by HPLC-MS. The process is simple and includes only a few handling steps, which has promising characteristics to become a platform for purification of these types of VLPs [116].

Regarding the detection and quality assessment of influenza vaccines, several methodologies are being used. These include development of standards for potency determination [117]. A high-resolution LC-MS method allows the quantitation of both HA and NA protein concentrations in influenza VLP vaccine candidates [118].
