Depending on the complexity of the final structure, there are several production

platforms available (Figure 10.1). The simpler VLP types could be produced in

prokaryotes and assembled in cell-free environments [9,10]. The bacteria

Escherichia coli or the yeast Pichia pastoris have been described as the

most productive platforms with bulk concentrations up to 4.38 mg/mL [11] and

400 mg/mL [12], respectively, although post-translational modification (PTMs)

may limit their application to the production of complex VLPs [3,7,13]. Transgenic

plants such as potato or tobacco [3,7,14] and baculovirus-vector expression system

(BEVS) with High Five or Sf9 insect cell lines are also used to produce VLPs.

Despite their different PTM characteristics [14,15], several products have been li-

censed and numerous phase III clinical trials are ongoing with VLPs produced in

these systems [5,10]. Finally, the mammalian CHO and HEK 293 cell lines, which

present better glycosylation patterns, are preferred for the expression of highly

complex VLP candidates.

Up to date, more than 100 ongoing or completed clinical trials have evaluated

VLPs as vaccine candidates [5]. From these studies, two main applications can be

highlighted: infectious viral diseases and cancer [4], influenza and HPV-cancer-

related vaccine candidates being the ones in more advanced phases. Importantly,

there are already two licenced VLP vaccines against HPV and widely distributed

(Gardasil and Cervarix). Of note, many diseases such as malaria or Chikungunya

fever are being tested in Phase I, whereas new VLP vaccine candidates against

Ebola, Zika, MERS (Middle East Respiratory Syndrome), coronavirus, or AIDS

(acquired immune deficiency syndrome) have been investigated in pre-clinical

trials, among others [5]. Also, during the COVID-19 pandemic, several candidates

tested for vaccination were based on VLPs.

10.2

HIV-1 GAG VLPs

HIV-1 contains two copies of single-stranded RNA (ssRNA) genome composed of

nine open reading frames (ORF), with three main structural genes: gag, pol, and

env, two regulatory proteins (tat and rev) and four accessory genes (nef, vif, vpu,

and vpr) (Figure 10.2). From the three main genes, gag codes for the structural

polyprotein, encompassing the nucleocapsid (NC), capsid (CA), and matrix (MA),

whereas pol and env encode for the main enzymes (PR, RT, and IN) and the re-

ceptor binding proteins (gp120 and gp41), respectively (Figure 10.2). At the end of

20th century, Göttlinger described the capacity of the Gag polyprotein to generate

non-infectious viral particles on its own [18]. Once produced in the cytoplasm of a

host cell, myristoylation of the N-terminal is produced, increasing its affinity for the

cell membrane and with the aid of the ESCRT (endosomal sorting complexes re-

quired for transport) machinery, immature particles are secreted to the extracellular

space taking part of the cell membrane through a budding process [19]. The first

application of VLPs consisted of the generation of HIV-1 vaccine candidates by

expressing the native Env glycoprotein on its surface. However, this strategy was

shown to be very inefficient and the successive approaches for HIV vaccine de-

velopment moved toward using recombinant-truncated and conserved Env frag-

ments [17–20]. Although increased knowledge has been generated on HIV-1 VLPs,

Recombinant vaccines: Gag-based VLPs

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