Construction And Evaluation Of A Novel MntA Mutant Vaccine Candidate Of Bacillus Anthracis

Anthrax is an acute infectious disease of livestock and humans caused by Bacillus anthracis,and they are usually vaccinated by injection,once for a year.This way of vaccination is inconvenient and easily lead to stress response.Besides,A16 R is still relatively virulent,therefore it is necessary to develop a novel oral attenuated vaccine for anthrax.The protein Mnt A,coded by the gene mnt A of Bacillus anthracis,the solute-binding component of manganese ion ATP-binding cassette transporter,can be expressed both in vitro and during infection.Furthermore,expression of Mnt A is independent of the virulence plasmids p XO1 and p XO2.Mnt A is a novel Bacillus anthracis virulent factor and is essential for its virulence.Some research suggested that deletion of mnt A could lead to further attenuated.Our research is to develop a more attenuated novel vaccine by deleting the gene mnt A of Bacillus anthracis attenuated strains AP422 and AP429,and studied biological characteristics of the candidate vaccine strains,then evaluate the safety and immunogenicity of the vaccine.We knocked out the gene mnt A to make the strains of AP422 and AP429 more attenuated by Cre-Lox P system and homologous recombination.The upstream and downstream homologous arms of mnt A gene were amplified by PCR and were linked to the thermo sensitive plasmid PMAD::spc to construct the targeting vector,which was then transformed to the attenuated Bacillus anthracis AP422 and AP429.Under the pressure of antibiotic and temperature,mnt A genes of AP422 and AP429 were knocked out by homologous recombination,and then the resistance selection marker was deleted using Cre-Lox P system.By PCR verification and sequencing,the gene mnt A was completely deleted,and only one Lox P site was left.Furthermore,the Mnt A protein could not be detected by Western blotting.The results suggested that the gene mnt A of AP422 and AP429 was successfully knocked out and the resistance selection marker was also deleted.The strains AP422?mnt A and AP430 knocked out the gene mnt A were obtained.The experiment on biological characteristics of the candidate vaccine strains suggested that Compared with the pre-knocked strains,their OD600 per hour are the same,that was the knocked out strains had a similar speed,so they had a basically similar ability of growth and spore formation.When cultured in LB medium under the condition of 37? and 250 rpm,cultured for 96 h,the spores of knocked out strains formed well and the concentration could reach to 109CFU/m L,which could be used for animal experiments.When cultured together to explore the different growing ability of the pre-knocked and knocked strains,only the pre-knocked strain grew,no knocked strains grew,so the pre-knocked strains had a more significant growth advantage,which suggested that the knocked out strains had a poor survival ability.To evaluate the safety of the novel vaccine strain,C57 mice of 8 weeks old were immunized by injecting on the left leg with the prepared spores,the control groups were injected with A16 R spores and the experimental groups were AP430 spores.When immunized at a dose of 2.5×108 CFU A16 R spores per mouse,we found edema on the left leg and abdomen,and 4/7 mice of the group died on the second day,2/7 mice of the group died on the third day;When immunized at another dose of 2.5×107CFU A16 R spores per mouse,we also found edema on the left leg and abdomen,and 4/7 mice of this group died on the third day.On the contrast,the physical and mental condition of the mice C57 injected by AP430 were very well without edema and death.The results suggested that the virulence of AP430 decreased significantly and might be a promising vaccine strain.To evaluate the immunogenicity of the novel vaccine strain,female guinea pigs weighting 250 g were vaccinated orally with the prepared spores of A16 R and AP430 at the dose of 4.0×108CFU/m L and 4.0×107CFU/m L respectively.Then we measured the antibody titers by ELISA.The serum from the guinea pigs injected by 4.0×108CFU/m L AP430 spores was diluted 100 times to measure anti PA Ig G and its OD450 was 0.8.The OD450 of the other group of 4.0×107CFU/m L AP430 spores was 0.7 while the two control groups were 0.70,0.55 respectively.The intestinal fluid from guinea pigs injected by 4.0×108CFU/m L AP430 spores was not diluted to measure anti PA Ig G and their OD450 was 0.90.The OD450 of the other group of 4.0×107CFU/m L AP430 spores was 0.80 while the control groups were 0.75,0.68 respectively.It is suggested that the mnt A mutant Bacillus anthracis strains are more attenuated and are promising as live attenuated vaccine candidate.