| ObjectiveTo establish the mouse model of tuberculosis or multi-drug resistant tuberculosis (MDR-TB) in mice, and to evaluate the therapeutic effects of chimeric Ag85A/ESAT6 DNA or Ag85A alone or in combination with chemotherapy in mouse models of Mycobacterium tuberculosis infection to establish new therapeutic agents or regimens to treat tuberculosis, especially MDR-TB.Methods1. The drug resistance of the M. tb isolates was determined by conventional drug susceptibility testing using the absolute concentration method on Lowenstein–Jensen medium. The rpoB and katG gene from the isolates mentioned above were amplified by PCR and were then sequenced by Canada Sangon Ltd. The sequencing data were compared with those registered in the Gene Bank database by BLAST analysis.2. The mice with tuberculosis were treated with M. tb chimeric Ag85A/ESAT6 DNA alone or combined with RFP. Eighty female BALB/c mice were infected intravenously via the tail vein with 2500000 CFU of M. tb clinical isolate HB240-1, then randomly divided into 8 groups based on the treatment recipe as follow: (1) saline, (2) plasmid vector pVAX1, (3) RFP, (4) ESAT6/Ag85A DNA combined with RFP, (5) RFP and INH, (6) ESAT6/Ag85A DNA combined with RFP and INH, (7) ESAT6-Ag85A DNA, (9) HSP65 DNA. The mice infected with HB240-1 were treated on the third day after infection. Ag85A/ESAT6 DNA vaccines were injected intramuscularly 5 times at 10 days intervals. The lungs and spleens from the mice were quantitated the viable mycobacteria, estimated the organ weight index (WI) and observed the gross pathological changes at 2 weeks after terminative treatment.3. M. tb chimeric Ag85A/ESAT6 DNA or Ag85A DNA vaccines alone or combined with RFP were used to treat the mice with MDR-TB. Ninety female BALB/c mice were infected intravenously via the tail vein with 220000 CFU of M. tb clinical isolate HB361 which was resistant to RFP and INH, then randomly divided into 9 groups based on the treatment recipe as the following: (1) saline, (2) plasmid vector pVAX1, (3) RFP, (4) vaccae, (5) ESAT6/Ag85A DNA combined with RFP, (6) ESAT6/Ag85A DNA, (7) Ag85A DNA combined with RFP, (8) Ag85A DNA, (9) HSP65 DNA. The mice infected with HB361 were treated on the third day after infection. DNA vaccines were injected intramuscularly 5 times at 15 days intervals. The lung and spleen tissues were harvested to quantitate the viable mycobacteria in the organs, to estimate the organ WI and to observe the gross pathological and tissue histopathological changes at 4 weeks after terminative treatment.4. M. tb DNA or Ag85A DNA vaccines alone or combined with RFP or PZA were used against mice MDR-TB. 70 female BALB/C mice were infected intravenously via the tail vein with 220000 CFU of clinical isolate M. tb HB361 which was resistant to RFP and INH, then randomly divided into 7 groups based on the treatment recipe as the following: (1) plasmid vector pVAX1, (2) RFP, (3) PZA, (4) vaccae vaccine, (5) Ag85A DNA, (6) Ag85A DNA combined with RFP, (7) Ag85A DNA combined with PZA. The mice infected with HB361 were treated on the second day after infection. DNA vaccines were injected intramuscularly 5 times at 15 days intervals. The lung and spleen tissues were harvested to quantitate the viable mycobacteria in the organs, to estimate the organ WI and to observe the tissue histopathological changes at 4 weeks after terminative treatment.Results1. M. tb clinical isolates HB361, HB240 and HB401 were resistant to INH and RFP. 2. Pulmonary and splenic bacterial loads in each therapeutic group significantly decreased compared to saline group. RFP group, chimeric Ag85A/ESAT6 DNA combined with RFP group, INH and RFP group, Ag85A/ESAT6 DNA combined with INH and RFP group reduced the pulmonary bacterial loads by 1.82, 0.86, 1.87, 1.88 logs, and reduced splenic bacterial loads by 1.75, 1.38, 1.84 and 1.54 logs (p<0.01), respectively. Their weight indexes of the lungs and spleens were significantly lower than that in saline group (P<0.05). The Pathological changes of lungs in RFP group, Ag85A/ESAT6 DNA combined with RFP group, INH and RFP group, Ag85A/ESAT6 DNA in combination with INH and RFP group were slight and limited.3. Compared with sailine group and RFP group, vaccae group, ESAT6/Ag85A DNA combined with RFP group, ESAT6/Ag85A DNA group, Ag85A DNA combined with RFP group, Ag85A DNA group and HSP65 DNA group reduced the pulmonary bacterial loads by 0.32, 0.28, 0.34, 0.66, 0.50, 0.26 logs, and reduced the splenic bacterial loads by 0.29, 0.31, 0.37, 0.68, 0.46, 0.28 logs(p<0.05,p<0.01), respectively. The pathological changes of lungs in vaccine groups were slight and limited, the profile of the alveoli was relatively clear compared with control groups. 4. The death rates of mice in vector group, RFP group, and vaccae group were 10%. Compared with vector group and RFP group, PZA group, vaccae group, Ag85A DNA group, Ag85A DNA combined with RFP group, Ag85A DNA group combined with PZA reduced the pulmonary bacterial loads by 0.91, 0.93, 1.32, 1.25, 0.87 logs, and reduced the splenic bacterial loads by 0.67, 0.62, 0.97, 1.21 and 0.75 logs (p<0.01), respectively. The pathological changes of lungs in therapeutic groups were slight and limited, the profile of the alveoli was relatively clear compared with control groups.Conclusions1. BALB/c mice were infected intravenously with 2500000 CFU or 220000 CFU of M. tb clinical isolate HB240-1 which was sensitive to drugs or HB361 which was resistant to RFP and INH, and the mouse models of TB or MDR-TB were successfully established. 2. The therapeutic efficacy of Ag85A/ESAT6 DNA vaccine combined with chemotherapy was significantly stronger than that of Ag85A/ESAT6 DNA vaccine alone in the mouse model of tuberculosis.3. The therapeutic effects of Ag85A DNA alone or Ag85A DNA combined with RFP was significantly stronger than that of chimeric Ag85A/ESAT6 DNA,HSP65 DNA vaccine and vaccae in the mouse model of MDR-TB.4. The therapeutic effects of Ag85A DNA alone or combined with RFP were strong than that of vaccae, PZA and Ag85A DNA combined with PZA in the mouse model of MDR-TB.The therapeutic Ag85A DNA vaccine and the combination with anti-TB drugs are the promising and affordable strategies for the treatment of MDR-TB disease in developing countries.
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