proteins. However, the limited capacity of their N-glycosylation
pathway to produce only high-mannose oligosaccharides remains
an important drawback when expressing proteins that are structurally or functionally dependent on N-linked glycan processing to
complex type structures [2, 3]. In this case, the mammalian cell is
the host of choice as it provides all posttranslational modifications
of the native protein.
The Hepatitis B Virus (HBV) small (S) surface antigen
(HBV-S) is a multispanning transmembrane protein that forms
disulfide bridge-stabilized dimers within the endoplasmic reticulum
(ER) of the host cells [4]. The dimers spontaneously associate into
20 nm-diameter subviral particles (SVPs) that do not incorporate
the viral capsid and genetic material and are secreted from cells,
independent of virions (Fig.1) [5]. These virus-like particles (VLPs)
are highly immunogenic, non-infectious and can be produced in
large amounts in heterologous expression systems in the absence of
any other viral components, which has led to their development
into efficient and safe vaccines against HBV [6]. These remarkable
properties of the HBV-S protein have been exploited to generate
chimeric SVPs carrying foreign and HBV-derived epitopes either
fused or co-expressed with HBV-S [7–11]. Several insertion sites
have been tested for their ability to accommodate epitopes of
different lengths within the luminal domain of the S proteins,
containing the major B-cell epitopes (the “a” determinant), and
two have been shown to be compatible with SVP production and
secretion. Chimeric HBV-S proteins bearing foreign peptide
sequences allowed for VLP formation and secretion and triggered
specific humoral and cellular immune responses against the native
protein [7, 8].
Here we describe a method for mammalian cell production and
purification of chimeric HBV particles that combines relevant virus
neutralization epitopes of the large (L) and S envelope proteins for
further use in immunological investigations. Our previous studies
have indicated that the HBV S/preS1
21–47 chimera obtained by
insertion of the 21–47 amino acids sequence of the preS1 domain
of the L protein between residues 126 and 127 of the “a” determinant of S (genotype D) preserves the SVPs properties and is secretion competent. Moreover, the chimeric protein is a more efficient
immunogen than the HBV-S protein, the major component of the
current vaccine [10, 11]. Our approach employs transient transfection of adherent HEK293T cells with plasmids encoding for HBV
S/preS1
21–47
and HBV-S, followed by purification of
corresponding SVPs from cell supernatant by ultracentrifugation
on sucrose cushion and step gradients. This method is simple and
scalable and may be applied to similar chimeric HBV particles
displaying relevant immunogenic peptides derived from other
pathogens of medical interest.
84
Mihaela-Olivia Dobrica et al.
pathway to produce only high-mannose oligosaccharides remains
an important drawback when expressing proteins that are structurally or functionally dependent on N-linked glycan processing to
complex type structures [2, 3]. In this case, the mammalian cell is
the host of choice as it provides all posttranslational modifications
of the native protein.
The Hepatitis B Virus (HBV) small (S) surface antigen
(HBV-S) is a multispanning transmembrane protein that forms
disulfide bridge-stabilized dimers within the endoplasmic reticulum
(ER) of the host cells [4]. The dimers spontaneously associate into
20 nm-diameter subviral particles (SVPs) that do not incorporate
the viral capsid and genetic material and are secreted from cells,
independent of virions (Fig.1) [5]. These virus-like particles (VLPs)
are highly immunogenic, non-infectious and can be produced in
large amounts in heterologous expression systems in the absence of
any other viral components, which has led to their development
into efficient and safe vaccines against HBV [6]. These remarkable
properties of the HBV-S protein have been exploited to generate
chimeric SVPs carrying foreign and HBV-derived epitopes either
fused or co-expressed with HBV-S [7–11]. Several insertion sites
have been tested for their ability to accommodate epitopes of
different lengths within the luminal domain of the S proteins,
containing the major B-cell epitopes (the “a” determinant), and
two have been shown to be compatible with SVP production and
secretion. Chimeric HBV-S proteins bearing foreign peptide
sequences allowed for VLP formation and secretion and triggered
specific humoral and cellular immune responses against the native
protein [7, 8].
Here we describe a method for mammalian cell production and
purification of chimeric HBV particles that combines relevant virus
neutralization epitopes of the large (L) and S envelope proteins for
further use in immunological investigations. Our previous studies
have indicated that the HBV S/preS1
21–47 chimera obtained by
insertion of the 21–47 amino acids sequence of the preS1 domain
of the L protein between residues 126 and 127 of the “a” determinant of S (genotype D) preserves the SVPs properties and is secretion competent. Moreover, the chimeric protein is a more efficient
immunogen than the HBV-S protein, the major component of the
current vaccine [10, 11]. Our approach employs transient transfection of adherent HEK293T cells with plasmids encoding for HBV
S/preS1
21–47
and HBV-S, followed by purification of
corresponding SVPs from cell supernatant by ultracentrifugation
on sucrose cushion and step gradients. This method is simple and
scalable and may be applied to similar chimeric HBV particles
displaying relevant immunogenic peptides derived from other
pathogens of medical interest.
84
Mihaela-Olivia Dobrica et al.