Molecular characterization and humoral immunity of bovine rotavirus VP4, VP6, and VP7
Date
1992-01
Authors
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Publisher
ORCID
Type
Degree Level
Masters
Abstract
The studies described in this thesis include the production and
characterization of three bovine rotavirus structural proteins, VP4, VP6
and VP7, which are very important in eliciting protective immune
responses in convalescent animals. To understand these proteins at a
molecular level, and the role of individual proteins in immunity, rotavirus
VP4, VP6 and VP7 proteins were expressed by recombinant vaccinia virus
and baculovirus. The evaluation of the immunogenicity of the recombinant
proteins may aid in the development of more effective subunit vaccines to
prevent and control rotavirus infection.
The recombinant forms of VP4, VP6 and VP7 expressed by
recombinant vaccinia virus and baculovirus were analyzed by
immunoprecipitations and western blotting. These in vitro characterizations
revealed that the different recombinant forms of VP4 are similar in
size and antigenicity to authentic BRV VP4. I have shown that: (1) the
recombinant VP4 protein produced in mammalian cells or insect cells had
the same molecular weight as the authentic protein; (2) the trypsin
cleavage patterns of recombinant VP4 were similar to the pattern produced
by trypsin treatment of authentic VP4, and; (3) recombinant VP4 was stable
in BSC-1 and Sf9 cells, while recombinant VP4 expressed by baculovirus
was not stable during post-translational processing. The reason for this
instability has been found. There is a proteolytic agent in infected Sf9 cells
which cleaves the VP4 protein.
The characterization of recombinant VP6 demonstrated that its
configuration closely mimicked the authentic VP6 protein. It was shown
that: (1) the recombinant VP6 protein produced in mammalian cells or insect cells had the same molecular weight and antigenicity as authentic
BRV VP6; (2) recombinant VP6 is not expressed on the cell surface but in
the cytoplasm of infected cells, and; (3) recombinant VP6 protein could be
assembled into single shelled particles in vitro in a fashion identical to
authentic VP6 protein.
Characterization of recombinant VP7 produced by vaccinia virus
revealed that it was similar to authentic VP7. However, VP7 expressed by
recombinant baculovirus was similar to the native form of the primary
translation product but differed in its post-translational modification. The
differences are: (1) the molecular weight of VP7 expressed by recombinant
baculovirus was lower than the molecular weight of authentic VP7 or VP7
expressed by recombinant vaccinia virus, and; (2) VP7 expressed by
recombinant baculovirus was secreted into the medium, which is different
from VP7 expressed by rotavirus and recombinant vaccinia virus.
However. when tunicamycin was used to inhibit glycosylation, the VP7
expressed by recombinant baculovirus was of the same molecular weight as
VP7 expressed by rotavirus or recombinant vaccinia virus and none were
secreted into the medium. This also suggested that VP7 expressed by
recombinant vaccinia virus and baculovirus were decorated with N-linked
oligosaccharides, as is authentic VP7.
The antigenicity of recombinant VP4, VP6 and VP7 and their ability
to induce protective immune responses were analyzed by immunizing mice
with live recombinant vaccinia virus or recombinant proteins produced by
baculovirus mixed with adjuvant. ELISA results indicate that antibodies
raised in mice which recognize rotavirus after two immunizations.
Antibodies in the milk were also analyzed by ELISA. The results indicate
that vaccination elicits high lacteal Ab titers. We also examined whether antibodies raised in vaccinated mice reacted with individual rotavirus
proteins. The ELISA and immunoprecipitation results indicate that
antibody induced by the recombinant viruses were specific to the BRV
protein expressed by the specific vaccine viruses.
Protection of neonates from rotavirus infection was tested by
challenging mice suckling from vaccinated dams. The results indicate
that protection from rotavirus diarrhea occurred in neonates nursed by
dams immunized with recombinant vaccinia virus expressing VP7, a
mixture of recombinant vaccinia viruses expressing VP4, VP6 and VP7,
recombinant baculovirus expressing VP7 and a mixture of recombinant
baculoviruses expressing VP4, VP6 and VP7, while protection was not
conferred to offspring suckling from dams immunized with recombinant
baculovirus expressing VP6, wild type vaccinia virus, placebo and sentinel.
In the vaccinia virus system, two promoters were used, one was a
vaccinia virus 7.5 K gene promoter, the other was a vaccinia virus
consensus late gene promoter. The results indicate that the consensus late
gene promoter expresses significantly higher levels of protein than the 7.5
K promoter. However, the increase in the level of protein expression is
dependent on the gene being expressed. For example, the consensus late
gene promoter produced > 16 times the quantity of VP6 protein when
compared to that produced by the 7.5 K promoter, while gene 4 (VP4) and 8
(VP7) expressed only 4-8 times the quantity of protein. Thus, both the gene
and the promoter appear to influence the level of expression.
In conclusion, these studies characterized recombinant BRV VP4,
VP6 and VP7 proteins expressed by vaccinia virus and baculovirus and
defined the immunogenicity of these proteins in dam vaccination studies. These studies may lead to the development of more effective vaccines to
deliver rotavirus antigens and to prevent rotavirus infection.
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Citation
Degree
Master of Science (M.Sc.)
Department
Veterinary Microbiology
Program
Veterinary Microbiology