Development and evaluation of bivalent dna vaccine against avian infectious bronchitis
Infectious bronchitis (IB) constitutes a major challenge to the poultry industry worldwide. The widely used live attenuated IB vaccines suffer from numerous limitations, including but not limited to, reversion to virulence, recombination and mutations. Whereas killed IB vaccines induce poor im...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2015
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/70897/1/FPV%202015%201%20-%20IR.pdf |
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| Summary: | Infectious bronchitis (IB) constitutes a major challenge to the poultry industry
worldwide. The widely used live attenuated IB vaccines suffer from numerous
limitations, including but not limited to, reversion to virulence, recombination and
mutations. Whereas killed IB vaccines induce poor immune response and require
repeated dosing. DNA vaccines on the other hands, have the potentials to induce both
humoral and cell mediated immune responses against infectious pathogens with
minimal unwanted effects. In this study, the S1 glycoprotein of M41 and CR88 IBV
strains were selected for the development of bivalent DNA vaccine against IB.
Bioinformatic analysis revealed structural and phylogenetic differences between the
two virus strains. However, several conserved B-cells and T-cells epitopes were
predicted to occur among these strains. To develop a broad-based DNA vaccine against
the two strains, the full-length S1 glycoprotein genes of M41 and CR88 IBV strains
were cloned individually as well as in combination to obtain a pBudCR88-S1,
pBudM41-S1 and pBudCR88-S1/M41-S1 plasmids. In vitro expressions of the
constructed IB-DNA plasmids were confirmed by immunofluorescence and RT-PCR
assays 42 hrs post transfection in chicken embryo fibroblast cells. In order to improve
the delivery potential of the candidate DNA vaccine plasmid and protect it from
endonuclease degradation, a chitosan - saponin (CS-SP) nanoparticle was synthesized,
characterized and used for the encapsulation of the bivalent IB DNA plasmids.
Characterization of the CS-SP nanoparticle revealed a spherically-shaped nanoparticle
with a size below 100 nm and a zeta potential of 38.9 mV. The encapsulated bivalent
IB-DNA plasmid was found to be thermostable, less toxic and protected against in vitro
enzymatic degradation. Subsequently, the efficacy of the candidate DNA vaccine was
evaluated by immunizing 3 weeks old SPF chickens. Six groups of chickens
comprising of vaccinated and control chicken received 100 μg of plasmid as follows: -
PBS-control (A) empty-plasmid control, pBudCE (B); pBudM41-S1 (C); pBudCR88-
S1 (D); pBudCR88-S1/M41-S1+nano (E) and pBudCR88-S1/M41-S1-without nano
(F). All chickens except those in group E received two booster vaccination at two week
intervals. To assess the effect of CS-SP, chickens in group E only received the first
booster vaccine. To evaluate the vaccine’s protective capacity, the initial six chicken
groups (A, B, C, D, E, F) were divided each into 2 parts as M41-challenged (n=6) and
CR88-challenge (n=6) subgroups and then challenged accordingly with 105 EID50 of
M41 and CR88 IBV strains two weeks after the last booster vaccination. All chickens were monitored before terminating the experiment two weeks after challenge.
Evaluation of immune response was carried out by measuring the IBV specific
antibodies (weekly); percentage CD3+, CD4+ and CD3+, CD8+ T-cells (two weeks
after the last booster); clinical signs (after the challenge); oropharyngeal and cloacal
virus shedding (day 3, day 5, day 10 and day 15) and histopathological lesion scores
(two weeks after challenge). Analysis of humoral and cell mediated immune (CMI)
response revealed a significant increase in anti-IBV antibody as well as CD3+, CD4+
and CD3+, CD8+ T cells responses in chickens vaccinated with bivalent IB-DNA
vaccines as determined by ELISA and flow-cytometry respectively. Vaccinated
chickens exhibited milder clinical signs following virus challenge as compared to the
control groups. Furthermore, vaccination with a bivalent DNA plasmid especially the
nanoencapsulated plasmid (pBudCR88-S1/M41-S1+nanoparticles) protected chickens
against heterologous virus challenge as revealed by a significant reduction in the
(p<0.05) in orophyrengeal and cloacal virus shedding following M41 and CR88-IBV
strains. However, monovalent IB-DNA vaccines could only protect against
homologous virus challenge (P<0.05). Histopathologically, lesion scores tend to be
higher in the trachea and kidney of control-unvaccinated chickens (A and B) compared
to the vaccinated chicken groups (C-F), although this difference was not statistically
significant (p>0.05). In conclusion, this study demonstrated that bivalent DNA vaccine
expressing S1 glycoproteins from M41 and CR88 IBV strains is able to induce
significant humoral and CMI responses that is able to protect vaccinated chickens
against challenge with both M41 and CR88 IBV serotypes. These findings highlight the
potential applications of bivalent DNA vaccine as an alternative strategy for the
control of infectious bronchitis in poultry. |
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