Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge.

OBJECTIVE: LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study. METHODS: A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography. RESULTS: Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 106 CFU of Y. pestis virulent strain 141. CONCLUSION: The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

[Evaluation of immunization protection efficacy of plague subunit vaccine].

OBJECTIVE: To evaluate the protective efficacy of plague subunit vaccine, BALB/c mice, guinea pigs and rabbits were used in this study. METHODS: Groups of mice (10 per group), guinea pigs (14 per group) and rabbits (6 per group) were immunized with F1 + rV270 vaccine, EV76 vaccine and alum adjuvant by intramuscular route, respectively. Serum antibody titres of mice, guinea pigs and rabbits were determined by ELISA and the immunized animals were challenged with 10(6) CFU of Y. pestis strain 141 at the 8th week after the primary immunization. RESULTS: The immunized mice, guinea pigs or rabbits with subunit vaccine developed anti-F1 IgG titre of 41 587.3 +/- 2.1, 11 543.7 +/- 2.1 or 522.4 +/- 22.4 and elicited statistical anti-F1 IgG titre difference among them (F = 17.58, P < 0.01). The immunized mice, guinea pigs or rabbits with subunit vaccine had anti-rV270 IgG titre of 15 748.7 +/- 1.6, 12.6 +/- 1.4 or 1648.0 +/- 5.0 and induced statistical anti-rV270 IgG titre difference among them (F value was 16.34, P < 0.01). There was significant anti-F1 IgG titre difference among mice, guinea pigs and rabbits immunized with EV76 vaccine that developed anti-F1 IgG titre of 913.4 +/- 4.5, 937.0 +/- 2.0 or 342.0 +/- 12.0 (F = 23.67, P < 0.01), whereas the immunized mice, guinea pigs and rabbits with EV76 vaccine developed anti-rV270 IgG titre of 12.0 +/- 1.0, 447.0 +/- 10.0, 40.0 +/- 11.0 and there was no anti-rV270 IgG titre difference between them (F = 2.20, P = 0.1314). The immunized mice with subunit vaccine developed significantly higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 30.57 and 19.04, respectively, P < 0.01), and there were no anti-F1 IgG titre differences between the immunized guinea pigs and rabbits (q = 0.04, P = 0.8485). The immunized mice with subunit vaccine developed significantly higher anti-rV270 IgG titres than immunized guinea pigs and rabbits (q value was 27.10 and 19.49, respectively, P < 0.01), and there were no anti-rV270 IgG titre differences between the immunized guinea pigs and rabbits with the subunit vaccine (q = 0.25, P = 0.6187). The immunized mice with EV76 elicited higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 40.67 and 29.10, respectively, P < 0.01), whereas there was no difference of F1 IgG titer between immunized guinea pigs and rabbits (q = 0.06, P = 0.8098). The immunized mice, guinea pigs and rabbits with subunit vaccine provided 100% (10/10), 86% (12/14) and 100% (5/5) protection against 10(6) CFU Y. pestis of challenge, respectively. The immunized mice, guinea pigs and rabbits with EV76 vaccine gave 100% (6/6), 93% (13/14) and 100% (6/6) protection against 10(6) CFU Y. pestis of challenge respectively. CONCLUSION: BALB/c mice is the best small animal model for valuation of protective efficacy of plague subunit vaccine. The guinea pigs showed a high individual variation for this purpose. The rabbits can be used as an alternative model for evaluating plague subunit vaccine.

Genetic variations in the pgm locus among natural isolates of Yersinia pestis.

A PCR-based screening method was used to study the genetic variations of the pgm locus among natural isolates of Yersinia pestis from China. Our results indicate that genetic variations in the pgm locus are well correlated with biovars of Y. pestis and plague foci, suggesting that the pgm locus plays a role in Y. pestis adaptation to its environment. The gene encoding two-component regulatory system sensor kinase became a pseudogene in all strains of biovar Orientalis due to a thymidine deletion, while it is intact in all the strains of the other biovars. Only strains from Foci H and L are the same as Yersinia pseudotuberculosis in that they have an intact transmembrane helix in the sensor kinase protein, which is lost in all the other strains because of the 18 bp in-frame deletion. The IS100 element that flanks the 39 terminus of the pgm locus was inserted into the chromosome during the within-species microevolution of Y. pestis, which is absent in strains from Foci G, H and L and also in Y. pseudotuberculosis. This fact indicates that the strains from these three foci are of an older lineage of Chinese Y. pestis. It is this IS100 element's absence that maintained high stability of the pgm locus in the Y. pestis strains from these three foci. The IS285 element insertion in the pigmentation segment and the IS100 element insertion in the downstream flanking region of the pgm locus are only present in strains from Foci H and L. The flanking region outside the 59 terminus of the upstream IS100 element is identical in the strains from these two foci, which is different in the other strains. All of these unique characteristics suggest that they are of a special lineage of Chinese Y. pestis.

[Study on the pathogen of plague in Sanjiangyuan area in Qinghai province].

OBJECTIVE: To study the biological characteristics of Yersinia pestis and to develop prevention and control program on plague in Sanjiangyuan areas, Qinghai province. METHODS: To identify the biologic types and molecular biological features of Y.pestis isolated in Sanjiangyuan area from 1954-2007. RESULTS: Among the 411 strains of Y. pestis, 12 strains belonged to the microtus type Y. pestis with denitrification (-) and donkey-hide gelatin carbohydrate (-) and glycerine (+). 399 strains belonged to classic type Y. pestis with denitrification (+) and donkey-hide gelatin carbohydrate (+) and glycerine (+). 411 Y. pestis strains had factor F I and Pst I. Among them, VW+ strains of Y. pestis accounted for 95.13% (391/411), VW-accounted for 4.87% (20/411), Pgm(+) accounted for 80.78% (332/ 411), Pgm(+/-) accounted for 9% (37/411) and Pgm(-) accounted for 10.22% (42/411) respectively. 96.82% (213/220) of the Y. pestis strains showed strong virulence to laboratory mice while 3.18% (7/220) of the strains carried medium virulence. 90.02% of the tested Y. pestis (370/411) strains had 6 x10(6), 45 x 10(6), 65 x 10(6) plasmids. 8 types of genome were found among 80 strains of Y. pestis, with 6 of them resembling ZHOU Dongsheng' s classification. Two new genome types were found. CONCLUSION: The Y. pestis in the Sanjiangyuan area had the characteristics of plague pathogen, identified in Qinghai-Tibet plateau. It is estimated that human beings are highly susceptible to the disease which spread fast, causing serious signs and symptoms with high death rate.

[A molecular epidemiological study on human plague fulminant epidemic in Qinghai, 2004].

OBJECTIVE: To study the epidemiology of genotyping Yersinia pestis isolated in the fulminant epidemics of human plague in Qinghai province in 2004. METHODS: Primer pairs targeting the twenty-three different identified regions (DFRs) were designed to detect the presence or deletion of each DFR in 13 strains of Yersinia pestis isolated from the fulminant epidemic of human plague in Qinghai province in 2004. RESULTS: There were 4 genomovars, i.e. Genomovar 8, 10, 15 and 16 in the 13 strains of Yersinia pestis identified. The genomovar of all the strains of Yersinia pestis isolated from Nangqian county was Genomovar 10. Among the two strains of Yersinia pestis isolated from Wulan county, the genomovar of one strain was Genomovar 8 and the other was Genomovar 10. The genomovars of all the strains of Yersinia pestis isolated from Qilian, Qumalai and Chengduo county belonged to Genomovar 16. CONCLUSION: It was demonstrated that the genotyping of Yersinia pestis appeared to be a powerful tool for investigating human plague epidemics.