Rotascaffold:
Nano-biotechnological recombinant vaccines
Elías Rafael Ruiz Morales, Diego Ramírez Martínez, Enrique Paz, Laura A. Palomares.
Instituto de Biotecnología, Universidad Nacional Autónoma de México.
INTRODUCTION
The rotavirus capsid is composed by four proteins: VP2, VP6, VP7 and VP4.
Rotavirus capsid structure.
James Z. Chen et al. (2009). PNAS.
Under certain conditions, some of these proteins can self-assemble, forming
complex protein structures.
The most interesting structures are those formed by the protein VP6, which
can assemble as nanospheres or nanotubes. VP6 nanotubes have wonderful
traits, highlighting their high adjuvant capacity and immunogenicity.
VP6 is a polymorphic protein, made by 397 a.a. Grouped as trimers, this
protein composes the medium layer of rotavirus, and makes contacts with the
other three capsid proteins.
Inspired in the natural interactions and properties of VP6, here we propose a
novel technology to develop as a new recombinant vaccine candidate with
important advantages over existing vaccines. Recombinant vaccines use non-
infectious organisms to produce the antigens that can trigger the immune
response. This has been used to develop new, safe and effective vaccines.
NEW VACCINATION TECHNOLOGY
In order to exploit the potential of VP6 nanotubes, we propose the creation of
a scaffold, composed by a VP6 nanotube backbone, that can carry many
antigens attached to it through linker peptides with the ability to bind naturally
to the VP6 nanotube.
Rotascaffold system.
Blue: VP6. Orange: VP6 binding site.
Green: polypeptide sequence. Red: antigen.
NANOTUBE PRODUCTION
VP6 was produced using the baculovirus-insect cell expression system.
Purification was carried out first by anion exchange chromatography in order
to separate VP6 from other proteins. Then, size exclusion chromatography
was used to discriminate the nanotubes from monomeric VP6, as was
previously described by Plascencia-Villa G., et al. (2011).
Nanotubes were visualized by transmission electron microscopy (TEM).
VP6 nanotubes. TEM.
Left: 4000x. Right: 25000x
LINKERS PRODUCTION
Linker peptides were designed with a VP6 binding site made using a fragment
of the VP4 binding domain, expecting that they will bind to the VP6 nanotube.
Gene constructions were inserted in an expression plasmid and then
transformed in Escherichia coli, strain DH5⍺. Antibiotic resistance and a RFP
bounded to the antigen site were used to isolate the desired colonies.
VP6
binding
site Polypeptide Antigen
sequence
1500 bp
1000 bp
Left: linker peptide diagram.
Right: 1% agarose gel showing the size of linker DNA sequence
A poly-histidine tag was included in the linker sequence in order to facilitate
linker purification by affinity chromatography and its identification by western
blot.
Linker peptide’s production is identifiable by measuring the fluorescence
intensity. Growth between producer and non producer bacteria is similar.
100 200 300
PB NC W NBP mM mM mM
50 kD
37 kD
25 kD
Left: Wester blot of linker peptides. Antibody: ⍺ -6x Histidine.
NC: negative control. PB: producer bacteria lysate. W: wash protein. NBP: non
binding protein. 100/200/300mM: imidazole concentration used to elute.
Middle: growth kinetics of producing bacteria.
Right: fluorescence kinetics of producing bacteria.
CONCLUSIONS
Production and purification of VP6 nanotubes and linker peptides have been
achieved. Nanostructures have been observed and characterized using
TEM.
The successfull production of Rotascaffold will be a breakthrough in
vaccinology, thanks to its advantages, being safer, with greater efficiency in
protection and with the potential of being able to protect against many
infectious agents in a single immunization, as several antigens can
simultaneously be linked to nanotubes.
PERSPECTIVES
Transmission electron microscopy is currently being performed to test the
nanotube-linker to confirm the binding of linker peptides to VP6 nanotubes.
To test Rotascaffold’s effectiveness mice will be immunized against zika virus,
loading the system with Zika proteins, then the humoral response will be
measured by ELISA and challenge studies will be performed of titers.
ACKNOWLEDGMENTS
Dr. Ricardo Castro, Dr. Octavio Tonatiuh Ramírez M.C. Ruth Pastor and to all
the GPR staff.
REFERENCES
Malm, M., et al. (2017). Rotavirus capsid VP6 tubular and spherical nanostructures
act as local adjuvants when co‐delivered with norovirus VLPs. Clinical &
Experimental Immunology. Lappalainen, S., et al. (2013). Comparative
immunogenicity in mice of rotavirus VP6 tubular structures and virus-like particles.
Human vaccines & immunotherapeutics, 9(9), 1991-2001. Boigard, H., et al. (2017).
Zika virus-like particle (VLP) based vaccine. PLoS neglected tropical diseases,
11(5), e0005608. Tagliamonte, M., et al. (2016). Virus-Like Particles. Micro-and
Nanotechnology in Vaccine Development, 205. Pumpens, P., et al. (2001). HBV core
particles as a carrier for B cell/T cell epitopes. Intervirology, 44(2-3), 98-114. Chen,
J. Z., et al. (2009). Molecular interactions in rotavirus assembly and uncoating seen
by high-resolution cryo-EM. PNAS, 106(26), 10644-10648. Pastor, A. R., et al.
(2014). The assembly conformation of rotavirus VP6 determines its protective
efficacy against rotavirus challenge in mice. Vaccine, 32(24), 2874-2877.
Plascencia-Villa, G., et al. (2011). Strategies for the purification and characterization
of protein scaffolds for the production of hybrid nanobiomaterials. Journal of
Chromatography B, 879(15), 1105-1111.