Abstract
In the development of genetically inactivated bacterial vaccines, plasmid retention often requires the antibiotic resistance gene markers, the presence of which can cause the potential biosafety hazards such as the horizontal spread of resistance genes. The new lysis plasmid was constructed by utilizing the approach of balanced-lethal systems based on auxotrophic gene Aspartate semialdehyde dehydrogenase (asd). The PhiX174 lysis gene E and λPR37-cI857 temperature-sensitive regulatory system was cloned in the asd gene positive plasmid and this novel approach allowed the production of antibiotic resistance marker free Salmonella Enteritidis (S. Enteritidis) ghost. The immunogenic potential of the biosafety enhanced antibiotic resistance gene free S. Enteritidis ghost was evaluated in chickens by employing the prime-boost vaccination strategy using a combination of oral and intramuscular routes. A total of 75 two-week-old chickens were equally divided into five groups: group A (non-immunized control), group B (intramuscularly primed and boosted), group C (primed intramuscularly and boosted orally), group D (primed and boosted orally), and group E (primed orally and boosted intramuscularly). Chickens from all immunized groups demonstrated significant increases in plasma IgG, intestinal secretory IgA levels, and antigen-specific lymphocyte proliferative response. After a virulent S. Enteritidis challenge, all immunized groups showed fewer gross lesions and decreased bacterial recovery from organs in comparison with the non-immunized control group. Among the immunized chickens, groups B and D chickens showed optimized protection, indicating that the prime-booster immunization with the ghost via intramuscular or oral route is efficient. Taken together, our results demonstrate that an antibiotic resistance gene free lysis plasmid was successfully constructed and utilized for production of safety enhanced S. Enteritidis ghost, which can be used as a safe and effective vaccine against virulent S. Enteritidis infections.
Highlights
The development of genetic engineering technology has allowed the construction of genetically modified organisms and inactivation systems, opening up new horizons in the production of highly immunogenic inactivated vaccines against infectious diseases [1,2,3,4,5]
The ghost cassette contained the PhiX174 lysis gene E, which was transcriptionally controlled by the mutated rightward lambda promoter λPR and the gene for the corresponding temperaturesensitive cI857 repressor
Enteritidis ghost The asd gene containing the lysis plasmid pJHL101 was transformed into JOL1254, and bacterial ghosts were produced by protein E-mediated lysis of bacterial cells
Summary
The development of genetic engineering technology has allowed the construction of genetically modified organisms and inactivation systems, opening up new horizons in the production of highly immunogenic inactivated vaccines against infectious diseases [1,2,3,4,5]. When the approach of vaccine production through genetic inactivation of bacteria is used, the gene encoding lysis protein E is expressed from the multi-copy plasmids in the host bacterial cell [8]. The production of plasmid-based inactivated vaccines usually relies on the presence of an antibiotic resistance gene marker, which ensures the plasmid stability during the culture of the bacteria in the presence of antibiotic [4,9,10,11,12,13,14]. The constitutive expression of antibiotic resistance genes puts the host bacterial strain in additional metabolic stress, which slows down the growth rate of bacteria resulting in low cell mass production [16]. The addition of antibiotics in the culture increases the cost of vaccine production, and there is the possibility of leaving antibiotic residues in the final product [9,17]
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