The Development of Porcine Reproductive and Respiratory Syndrome Virus Vaccine
Date Issued
2011
Date
2011
Author(s)
Chia, Min-Yuan
Abstract
chapter I
Porcine reproductive and respiratory syndrome virus (PRRSV) has devastated the swine industry causing tremendous economic losses throughout the world since late 90’s. Currently, there are only a limited number of inactivated or modified-live PRRSV vaccines are available on the market. Several approaches have been used to develop subunit vaccines based on plasmid DNA, baculovirus, adenovirus, pseudorabies virus, and vaccinia virus systems. However, the complexity of the host immune response to PRRSV and the ability of the virus to escape or modulate the host’s immune system make it difficult to develop a vaccine that could eradicate this disease. The glycoprotein 5 (GP5) and membrane protein (M) of PRRSV are essential in inducing host protective immunity. It has been demonstrated that DNA vaccines expressing GP5 or M alone induced relatively weak NA responses when compared to that co-expressing GP5 and M as a fusion protein. Moreover, recombinant vectored vaccinia virus vaccine co-expressing GP5 and M proteins of PRRSV with two different promoters displayed more immunogenicity than that co-expressing GP5 and M as a fusion protein; and it was speculated due to altered natural conformation of the fusion protein. A flexible linker such as the twelve-base-pair oligonucleotide sequence-encoded flexible glycine-proline-glycine-proline (GPGP) linker may preserve the intact neutralizing epitopes of these two proteins and present them to antigen-presenting cells. Thus, the first objective of the present study was to evaluate whether the immunogenicity of DNA constructs co-expressing GP5 and M proteins linked by GPGP could be enhanced in mice and pigs (Chapters II and III). Three different DNA constructs, one expressing GP5/M without GPGP linker (pcDNA-56) and two expressing GP5/M conjugated by GPGP linker (pcDNA-5L6 and pcDNA-6L5) but with different tandem orientation, were established. These constructs were then inserted into an eukaryotic expression vector, pcDNA3.1/V5-His TOPO®, as DNA vaccines to immunize mice intramuscularly for four times at a 2-week interval (Chapter II) and to immunize pigs intramuscularly for three times at a 2-week interval followed by challenge with 5 × 105 TCID50 PRRSV at three weeks after final immunization (Chapter III). The results showed that pcDNA-5L6 and pcDNA-6L5 induced higher PRRSV-specific IgG and neutralizing antibody (NA) responses, greater PRRSV-specific lymphocyte proliferation responses, and lower viremia and tissue viral load than did pcDNA-56. The results suggest that the GPGP linker may indeed preserve the natural conformation and immunogenicity of both GP5 and M proteins. It is known that PRRSV enters the host via the mucosa of gastrointestinal, respiratory, and reproductive tracts. Thus, it may be possible to activate the common mucosal immunity by using subunit oral vaccine to prevent PRRSV infection at the first line of defense. In recent years, with the development of genetic molecular biology and plant biotechnology, the genetic engineering subunit vaccine is taking on a prosperous evolvement. Expressing subunit vaccine candidates in plants opens a new avenue for producing oral/edible vaccines. Transgenic plant vaccines have advantages such as low cost, easiness in storage, and convenience in inoculation. The rigid plant cell wall can also protect the antigenic proteins from gastric acidic environment, allowing the antigens to reach gut-associated lymphoid tissue intactly. Escherichia coli heat-labile enterotoxin B subunit (LTB) is a well-characterized bacterial protein having a strong potential as a mucosal adjuvant. The antigen can be delivered across the mucosa epithelium to the underlying mucosa-associated lymphoid tissue if the antigen is genetically fused with LTB to form a pentamer. Thus, the second objective of this study was to evaluate the feasibility of co-expressing of LTB and PRRSV GP5 in transgenic tobacco plant and its immunogenicity and protective efficacy in pigs (Chapters IV and V). Two different transgenic tobacco plants, one expressing PRRSV GP5 (GP5-T) and the other co-expressing LTB and PRRSV GP5 as a fusion protein (LTB-GP5-T), were constructed. Pigs were given orally three consecutive doses of equal concentration of recombinant GP5 protein expressed in leaves of LTB-GP5-T or GP5-T at a 2-week interval and challenged with PRRSV at three weeks after final oral feeding. Pigs receiving LTB-GP5-T or GP5-T developed significantly higher PRRSV-specific antibody- (AMI) and cell- (CMI) mediated immunity and showed significantly lower viremia, tissue viral load, and milder lung lesions than wild type tobacco plant (W-T). The LTB-GP5-T-treated group had relatively higher immune responses than the GP5-T-treated group, although the differences were not statistically significant. In summary, the DNA constructs and transgenic tobacco plants indeed developed specific AMI and CMI responses in pigs against PRRSV infection. However, neither the DNA constructs nor the transgenic tobacco plants could completely eliminate the persistent infection and shedding of PRRSV in the respiratory tract and lymphoid tissue after challenge. Effects are required to further effectively improve the immunogenicity in both AMI and CMI responses of either DNA constructs or transgenic plants.
Subjects
PRRSV
vaccine
SDGs
Type
thesis
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