Experiment Study On A Recombinant Pertussis DNA Vaccine

Pertussis or whooping cough is a highly contagious disease of the humanrespiratory tract, which is a major cause of infant death throughout the world. Theincidence of whooping cough has been greatly reduced since the advent of pertussisvaccines in the 1940s. However, whole-cell vaccines have been associated withserious side effects, such as local and systemic reactions. In these 30 years, acellularpertussis vaccines were used to substitute with whole-cell vaccines for pertussisprovention in Japan and other developed countries. Now whole-cell vaccines weremost used in many developing countries. Both whole-cell vaccines and acellularvaccines were used in China.Form 1990s the incidence of pertussis disease is increasing in both developingcountries and developed countries. There are approximately 45 million cases, 90% ofwhich are in developing countries, and 300 000 fatalities are estimated to occurworldwide each year. So it's valuably to study on a new pertussis with highperformance and low side effects.Being an extracellular pathogen, B. pertussis was thought to be a humoralpathogen only, whereas evidence suggests that cellular and humoral immunity mayact in concert to eliminate B. pertussis infection. Furthermore, cellular immunitymemory lasts longer than humoral immunity, and may play a role in protectiveimmunity after infection with B. pertussis. Now a problem in pertussis vaccines is thelong-term protection, and it's one of the reason of the increasing pertussis cases.DNA vaccine is considered as the third generation vaccine following classicalvaccine and genetically engineering vaccine. DNA vaccine mimic the process ofnatural infection, and can induce cellular and humoral immunity. So, DNA vaccinemay being the candidate vaccine for pertussis prevention.In this study, we combine the second and third generation vaccine techniques, construct a recombinant pertussis DNA vaccine and discuss its immunogenicity.Firstly, we select the protective antigen fragments PTS1, PRN and FHA, then mutatePTS1 at amino acid 9 and amino acid 129, recombine them together with plasmidpVAX1 as a recombinant plasmid pVAX1/PPF. The three fragments were recombinedwith pVAX1 respectively and were mixed as mix plasmid group. After the miceimmunized with these recombinant plasmids and mix plasmids, anti-PTS1, anti-PRN,anti-FHA, cytokine IL-10, IL-4, IFN-γand TNF-αwere induced. So the combinantpertussis DNA vaccine can induce humoral and cellular immune response in mice.Then, we try to improve the protective immunity of the pertussis vaccine throughthese three methods: 1, co-inject with cytokine GM-CSF plasmid as prime; 2,co-inject with cytokine GM-CSF plasmid as prime and use homologous recombinantprotein as boost; 3, vaccine on AdEasy adenoviral vector system.It's the first study using GM-CSF plasmids in the recombinant pertussis DNAvaccine. As the result, compared with the mice immunized with pVAX1/PPF only,more anti-PTS1, IL-4 and TNF-αwere produced in the mice coinjected withpGM-CSF. It means that coinjected with pGM-CSF can improve the cellular andhumoral immune responses. Both of the pVAX1/PPF and pVAX1/PPF coinjectedwith pGM-CSF induced protection effects against a lethal dose of B. pertussis.There's no distinguished difference between the two groups.A new strategy in DNA vaccine studies was introduced in this study. That is,coinjected with pGM-CSF as prime and homologous protein as boost. As the result,compared to mice immunized with pVAX1/PPF only, mice boosted with protein wereinduced more antibodies whether coinjected pGM-CSF or not. It means that miceboost with protein were induced a much more enhanced humoral immune response.Mice with GM-CSF prime and protein boost were induced more anti-PTS1, anti-PRN,anti-FHA, IL-10, IL-4, IFN-γ, TNF-αand a better splenocyte proliferation than thoseinjected with pVAX1/PPF only. Compared to the strategy coinjected with pGM-CSFor DNA prime and protein boost, the new strategy combined this two strategies. Micecoinjected with pGM-CSF as prime and homologous protein as boost were induced anenhanced humoral and cellular immune responses. It's a hopeful strategy to improve the immune responses produced by DNA vaccine.In this study, we try to reconstruct the pertussis recombinant fragment PPF to anadenovirus vector. Adenovirus vector can induce an enhanced protein expression andan enhanced immune response. We try to reconstruct the vector throuth a shuttleplasmid pShuttleCMV, then construct the virus in AD293 cells and infect mice toinduce the immune response.Pertussis vaccine were used about almost a century. But there are still health andeconomic problems originated by pertussis. DNA vaccine can induce cellular andhumoral immune response against many pathogens in many animal models. In thisstudy, we proved that DNA vaccine could be a candidate vaccine for pertussisprevention.Now the difficulties in DNA vaccine studies are the limited immune responseand protection induced in big animals. A hot spot is looking for good strategies toimprove the immune response induced by DNA vaccine. The strategies introduced inthis study could be a helpful essay.