nanoparticles [23]. Kaczmarczyk et al. generated fusion Gag proteins with several

prodrugs and enzymes and demonstrated its directed delivery in vitro [24]. The

immunogenicity of VLPs has been also studied in cancer research [25].

10.3

PRODUCTION OF HIV-1 GAG VLPs

HIV-1 Gag VLP production has been achieved in several cellular platforms [17],

but most of the research is performed with animal cell cultures, as shown in

Table 10.1. Three main strategies can be distinguished in the production of viral-

based products in animal cell cultures: viral infection, transient gene expression

(TGE), and stable gene expression (SGE). From the three mentioned strategies,

infection with alphavirus, vaccinia virus, adenoviral vectors, or baculovirus has

been used for the rapid production of bioproducts [38]. Among them, the BEVS is

the most used system in the production of Gag VLPs with insect cells (Table 10.1)

[39]. BEVS has been widely used with the insect cell lines, Sf9 [40] and High Five

[41], and the new TNMS-42 [42]. BEVS is a very productive system achieving

productions of milligrams per liter [13]. However, the obtention of the recombinant

viral stock is not always straightforward and the lytic cycle caused by the infection

may affect product quality. Furthermore, its main disadvantage is the baculovirus

interference in the purification process, due to their similarities with enveloped viral

structures, as with Gag VLPs.

TGE on the other hand, uses non-viral vectors to introduce a DNA plasmid

coding for the protein of interest. In this regard, mammalian cell lines, especially

HEK 293, is the workhorse in TGE strategies for viral-based products [39]. Still,

shorter production times of 2−4 weeks are required to obtain up to grams of a

protein of interest [43]. Several physical and chemical methods could be found in

literature to this end, as reviewed in [44]. However, in industrial biotechnology, the

use of transfection reagents such as calcium phosphate and in a more extent the

cationic polymer, polyethylenimine (PEI), are the most extensively used [43].

Alternative transfection reagents, such as cationic lipids, have been also used for

DNA delivery in a highly efficient way. Nonetheless, their high cost has relegated

their use to SGE strategies [45]. HEK 293 cell cultures [26,46], but also the

mammalian cell lines HeLa [34], CHO [47], or CAP-T [48] have been used for the

production of HIV-1 Gag VLPs by means of TGE and SGE. Alternatively, the

development of SGE and TGE strategies in insect cell lines devoid of BEVS has

been proven in recent years [49–52].

10.3.1

PEI-MEDIATED TRANSIENT TRANSFECTION

The use of PEI for gene delivery in vitro and in vivo has been on the spotlight for more

than three decades [43,53]. PEI is simple to use, efficient with suspension cells,

compatible with serum free media and cost-effective. Since its first use as transfection

reagent described by Boussif et al. in 1995 [54], several polymer lengths and struc-

tures, namely linear and branched PEI, have been tested in DNA delivery approaches

[55]. Improved PEI formulations are nowadays commercially available like JetPEI,

FectoPro, PeiPro, ExGene500, or the clinical-grade PEIPro®-HQ from Polyplus

Recombinant vaccines: Gag-based VLPs

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