An Exploratory Study Of Spore Vaccine Vehicle For Bacillus Anthracis

Bacillus anthracis is a Gram-positive spore-forming, rod-shaped bacterium. It can cause fetal anthrax, a zoonotic disease (a disease shared between animals and humans). Untreated anthrax is often fatal, especially the respiratory type of anthrax infection. Anthrax is probably the most widely studied biological warfare and bioterrorism agent. Anthrax vaccine, which is the hotspot of biosecurity research, also is the focus of pathogens and relevant vaccines. B.anthracis AP422, deriving from Chinese vaccine strain A16R, is an attenuated nontoxinogenic and nonencapsulated mutant strain. Although both of the plasmids pXO1 and pXO2 were cured in AP422, this attenuated strain can sporulate as well as A16R. For these characteristics, AP422 could be used as a delivery vehicle to design new type spore vaccine for anthrax.In section one, a procedure for high-e?ciency gene inactivation in B. anthracis has been developed. Cre-LoxP homologous recombinant system was used to disrupt eag gene in B. anthracis AP422. Some very important conditions were optimized, such as temperature-sensitive plasmid vector, length of homologous regions, high-e?ciency electroporation protocol, antibiotic concentration, and the method for the identification of positive recombinant. Using this method, the eag gene which encoded S-layer protein EA1 was knocked out. The markerless mutant strains were detected by genome PCR, RT-PCR, total proteins SDS-PAGE and western blot analysis. All the results demonstrated that the eag gene was deleted successfully and the protocol was feasible. At the same time, we found that some proteins showed very different expression levels in the AP422 and eag deleted mutant. Western blot analysis, with anti-SLH domain serum as primary antibody, indicated that many of them were potential S-layer protein. We speculated on that a compensatory mechanism exist in the synthesis process of S-layer. When the eag gene was deleted, the expression levels of some other S-layer proteins would be upgraded for retaining the integrity of S-layer.In section two, a thymidine auxotroph of B. anthracis was constructed using the gene knockout method established in initial work. A comprehensive phenotypic analysis of the mutants was executed subsequently. Specifically, we obtained the key enzymes responsible for the synthesis of thymidine in B. anthracis through bioinformatic analysis. Both thymidylate synthase (TS, encoded by thyA) and ?avin-dependent thymidylate synthase (FDTS, encoded by thyX) can catalyze the conversion of dUMP to dTMP in the presence of (CH2)2THF. So thyA and thyX were selected as the target in our research. According to homologous recombination, both thyA and thyX were deleted successfully. And combined with Cre-LoxP system, the markerless mutant was gained, as named AP422ΔthyAΔthyX. Phenotypic analysis showed that this strain was only able to grow on LB agar when extracellular thymidine was provided. At the same time, experiment showed that the the auxotroph was easy to sporulation. These results indicated that it may be possible to develop a series of vaccines based on chromosome-plasmid balanced lethal system in this attenuated B.anthracis auxotroph.And phenotypic analysis of the AP422ΔthyA strain and AP422ΔthyX strain revealed that there are two possible different thymidine synthesis pathways existing in B.anthracis, TS/DHFR biocycle and FDTS biocycle. The two pathways may play role independently in the life cycle of B.anthracis. Furthermore, Functional analysis indicated both thyA and thyX could complement the thymidine auxotroph of E. coli BW25113ΔthyA::spc. In addition, our research also showed that thyX product, FDTS, played a role in natural trimethoprim (dihydrofolate reductase inhibitors) resistance of B. anthracis.In the third part of this work, using gene knockin approach, three recombinant of B. anthracis strains were constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and other proteins. And three antibiotic marker free mutants were achieved successfully. In particularly, conservative SLH domain of EA1 protein was used as anchoring domain for the surface display of PA20, PAⅣand LFb domain. These protein domains were expressed under the control of the promoter of the S-layer component gene. SDS-PAGE and molecular weight analysis showed that these proteins were in the form of fusion protein. Immunofluorescence experiments showed these hybrid proteins were stably expressed on the cell surface of the bacterium. Sporalution assay confirmed that the recombinant strains still have the ability to form spores.Guinea pigs were immunized with vegetative cell or spore of the corresponding strains.The result showed that the hybrid proteins elicited humoral response to different antigens. In detail, nasal feeding immunization of guinea pigs with bacillus cell of the three recombinant strains, AP422::PA20, AP422::PAⅣand AP422::LFb, were sufficient to elicit systemic IgG antibody responses immune response. Serum IgG titers were more than 1:1000. When different doses of AP422::PAⅣspores were administered guinea pigs by oral route, sIgA responses in mucosal tissues as well as systemic IgG antibody responses to the PAⅣcould be detected. At the same time, this administration could develop strong humoral responses against spore proteins, which was very important to protective effect of anthrax vaccine. In addition, these recombinant strains did not carry any resistance markers, tally with the current requirements of the attenuated live vaccine vehicle. Collectively, these results have shown that the AP422 can be used as a delivery vehicle of B. anthracis spore vaccine candidate strain.In the last part of this work, recombinant bacterium which spore protein BclA fused LF bind domain was used as original strain to construct a new strain with PAⅣdisplaying on the cell surface, using conservative SLH domain of EA1 protein as anchoring domain. Different doses of spores of this recombinant strain were administered guinea pigs by oral route, and the animals developed strong humoral responses against both PAⅣand LFb. The anti-PAⅣsIgA and anti-LFb sIgA in mucosal tissues also could be detected. More importantly, the two antigens could play key roles in different phases and worked as two valents vaccine.All in all, B. anthracis AP422 can be developed as a very promising spore vaccine delivery vehicle for anthrax.