material, the grid configuration of the filter wall, or even the electrical and physi-

cochemical properties of the filter, VLPs can be retained or continuously harvested.

Selecting a filter that separates unassembled free Gag monomers and allows a

continuous harvest of VLPs is the optimal configuration to facilitate further

downstream processing steps.

10.5

EXAMPLES OF GAG-BASED VLPs

Different examples using the Gag polyprotein as scaffold for pseudotyped VLPs are

highlighted in this section to illustrate the potential of Gag-based VLPs.

In the first one, a stable cell line expressing the main influenza viral antigens

hemagglutinin (HA) and neuraminidase (NA) (subtypeH1N1) under the regulation

of a cumate inducible promoter was developed (293HA-NA cells). The Gag gene

was transiently transfected to the cell to produce VLPs. Interestingly, it was found

that the production yield when Gag was used as scaffold was 10 times higher than

the yield obtained when the influenza core protein M1 was used. Then, the pro-

duction process was transferred to a 3L bioreactor scale. The produced VLPs were

purified by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential

flow filtration. The VLPs obtained were characterized using electron microscope,

where VLPs of 150–200 nm were observed, HA was quantified by single radial

immunodiffusion, hemagglutination assay and Dot-Blot and NA was quantified

using Dot-Blot. More importantly, the produced influenza VLPs were used to im-

munize mice intranasally. This immunization induced strong antigen-specific mu-

cosal and systemic antibody responses and provided full protection against a lethal

intranasal challenge with the homologous virus strain.

In a second example, Gag-based VLPs were produced by transient transfection

to develop a vaccine for the foot-and-mouth disease virus (FMDV) [27]. The

Gag-GFP polyprotein was cotransfected with a novel fusion rabies glycoprotein

(RVG), which carries in its N-term the FMDV main antigen: the G-H loop.

Observing the cells under confocal microscopy it is confirmed that the G-H loop

colocalize at the cell membrane. The Gag polyprotein accumulates at the vicinity

of the cell membrane and takes part of the cell membrane and the G-H loop

during the budding process resulting on VLPs that display the GH-RVG on its

surface. This was confirmed by gold immunolabeling. The expected size of Gag-

VLPs of around 130–140 nm was observed by TEM, CryoTEM, and NTA.

Immunostainings with a FMDV hyperimmune serum showed that the hetero-

logous antigenic site, genetically fused to RVG, is recognized by specific G-H

loop antibodies. Additionally, the cVLPs produced expose the G-H loop to the

liquid surrounding (analyzed by specific ELISA). Finally, these FMD cVLPs can

induce a specific humoral immune response, based on antibodies directed to the

G-H loop in experimental animals.

10.6

SCALABLE DSP FOR HIV-1 GAG VLPS

The first step in VLP purification is clarification. In the case of HIV-1 Gag VLPs,

they benefit from being an extracellular product; thus, the cell lysis step is not

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Bioprocessing of Viral Vaccines