Field assessment of recombinant Schistosoma japonicum 26 kDa glutathione S-transferase in Chinese water buffaloes.

We have shown previously that anti-fecundity immunity can be induced experimentally against recombinant 26 kDa glutathione S-transferase (reSjc26GST) in Chinese water buffaloes (Bos buffelus), important reservoir hosts for Schistosoma japonicum in China. In the field study described here, we immunized buffaloes with reSjc26GST to induce protective immunity against S. japonicum and to evaluate its effectiveness in controlling schistosomiasis japonica. We selected two villages as test and control groups in inside-embankment areas endemic for schistosomiasis japonica. The buffaloes in the test village were vaccinated with reSjc26GST, whereas those in the control village were not. The indicators of the effect of the vaccine included the generation of specific IgG antibodies in the vaccinated buffaloes, changes in the prevalence and infection intensity in buffaloes and village children, changes in the density of infected snails, and changes in the infectivity of water bodies (assessed by sentinel mice) in transmission areas adjacent to both villages. Twenty months after vaccination, the infection rate of buffaloes in the test village was decreased by 60.4% (from an initial prevalence of 13.5% to 5.4%), and 67.9% when compared with that in the control village (initial prevalence of 16.7%). However, the infection rate in village children remained unchanged. The density of infected snails decreased by 71.4%, from 0.0049/0.11 m2 to 0.0014/0.11m2 in the high transmission area outside the embankment in the test village. There was no change in the infectivity of the water body transmission areas between the test and control villages. The levels of specific antibodies to reSjc26GST showed a continuous increase after vaccination. These results indicate that protective immunity was induced and maintained in buffaloes after vaccination with reSjc26GST. The vaccine could thus play a significant role in reducing S. japonicum transmission caused by water buffaloes in the Lake region of China.

[Screening and cloning of genes encoding Schistosoma japonicum antigens related to the serum antibodies in Mirotus fortis].

OBJECTIVE: To understand and identify the molecules related to the natural resistance to Schistosoma japonicum infection in Mirotus fortis. METHODS: Sera from Mirotus fortis without schistosome infection were collected. The S. japonicum adult worm cDNA library was immunologically screened with the sera. The positive recombinants were identified, cloned, sequenced and analysed with software and internet. RESULTS: Seven genes encoding antigens relevant to sera antibodies in Mirotus fortis were cloned and sequenced. These antigens included glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine protease inhibitors (SERPIN), 70 kDa heat shock protein (HSP70), 22.6 kDa membrane-associated antigen, paramyosin (Sj97), cytochrome C and cathepsin B. CONCLUSION: Many protein molecules might have been involved in natural resistance to S. japonicum infection in Mirotus fortis. The above 7 kinds of molecules may be identified as new candidates of vaccine against S. japonicum infection.

[A study on the recombinant 26 kDa glutathione-S-transferase as a vaccine candidate: dynamics of antibodies in immunized buffaloes and protection against Schistosoma japonicum infections].

OBJECTIVE: To observe the dynamics of antibodies and protection against Schistosoma japonicum infections in buffaloes after immunized with recombinant 26 kDa glutathione-S-transferase (reSjc26GST). METHODS: Buffaloes in 2 villages endemic for schistosomiasis japonica were selected as test and control groups, respectively. In test group initially 96 buffaloes were vaccinated with reSjc26GST, and 90 buffaloes in the control group did not experience vaccination. The indicators included levels of antibodies to reSjc26GST in buffaloes before and after infection with S. japonicum and changes in infection rate. RESULTS: Specific antibodies, which showed a trend of trapezoid increase, were induced in buffaloes after immunized with reSjc26GST. Twenty months after immunization, the infection rate of the test group was decreased by 62.2% when compared with that before vaccination, and by 67.7% when compared with that of the control in the corresponding period. CONCLUSION: Specific antibodies and a certain extent of protection were induced in buffaloes after immunized with reSjc26GST, which played an significant role in ameliorating morbidity.

Anti-fecundity immunity induced in pigs vaccinated with recombinant Schistosoma japonicum 26kDa glutathione-S-transferase.

We have recently reported (Liu et al. 1995) that immunization of mice with recombinant 26kDa GST (reSjc26GST) induces a pronounced anti-fecundity effect after experimental infection with Chinese Schistosoma japonicum. A similar vaccination trial was thus carried out on pigs, important reservoirs for schistosomiasis japonica, using purified, reSjc26GST and reSjp26GST from Schistosoma japonicum with alum as adjuvant; in general, similar results were obtained with the two sources of recombinant 26kDa GST. Some protection in terms of worm reduction, significant with males, against challenge infection was observed in vaccinated pigs. Moreover, prior to challenge, levels of specific anti-re26GST antibodies in the vaccinated pigs were significantly higher than in non-vaccinated pigs as determined by GST-ELISA. The most striking feature of the vaccine trial was the significant reduction in the number of eggs, especially mature eggs, in the livers of vaccinated animals. The results indicate that immunization with recombinant Sj26GST can provide some reduction in worm burden following exposure of pigs to reinfection with S. japonicum. In addition, reSj26GST can induce an anti-fecundity effect, thereby reducing pathology, coupled with a delay or interruption of the development of immature to mature eggs in the liver. As a consequence, vaccination with Sj26GST would also prove useful in affecting the transmission of schistosomiasis japonica.

The antigenicity of GST antigen extracted from Chinese strain of Schistosoma japonicum.

The GST antigen, similar to Sj26 (Philippine strain), which plays an important role in inducing protective immunity against Schistosoma japonicum, can be extracted and purified from adult worms of the Chinese strain of S. japonicum. There are two bands at 26 kDa and 28 kDa of GST antigen called the 26-28 kDa GST antigen as identified by SDS-PAGE, and these have GST activities. Mice were immunized with the 26-28 kDa antigen and the specific antibody response in serum was assayed by ELISA, IFA and western blot. The antigenicity of the 26-28 kDa GST antigen in mice was significant. For example, the antigen could stimulate mice to increase the level of serum IgM and IgGl; the antibodies in serum of immunized mice could be localized in the antigenic determinants of tegument or body of the worms; specific antibodies against the antigens increased markedly after immunization as measured by ELISA or IFA; the antibody from mice immunized with the 26-28 kDa GST antigen can recognize 26-28 kDa antigenic molecules, identified by immunoblot assay.

The temperature--dependent expression of GST of Schistosoma japonicum (Philippine strain).

Obtained from pSj5, the cDNA gene encoding GST antigen of Schistosoma japonicum (Philippine strain) was ligated with efficient temperature-dependent PBV220 vector which was constructed in CAPM, and then introduced into host bacterium-DH5 alpha (E. coli) by transformation. Transformants were selected by ampicillin and recombinant clones were identified by restriction mapping. The result showed that recombinant clone 43 was the one carrying recombinant plasmid PBV 220 with the correct insertion of the gene fragment. The GST expression ability of clone 43 was investigated by GST enzymic activity assay and SDS-PAGE. A relatively high level of GST enzymic activity was expressed by this clone under the temperature-dependent condition, that is, cultured at 30 degrees C and expressed at 42 degrees C. A more strongly stained 26 kDa protein band was identified by SDS-PAGE. The result indicated that GST of S. japonicum (Philippine strain) could be expressed not only by IPTG induction, but also by the temperature-dependent method.

Comparative study on antigenicity and immunogenicity of 26-28 kDa antigen and recombinant Sj26 (RSj26) of Schistosoma japonicum.

This paper reports a comparison of the recombinant Sj26 (rSj26) antigen derived from the Philippine strain and the 26-28 kDa antigen isolated and purified from the Chinese strain of Schistosoma japonicum with respect to their antigenicity and immunogenicity. The results showed that there were obvious cross reactions between rSj26 and 26-28 kDa antigen when rSj26 antigen was tested against specific antibodies in sera of mice infected with the Chinese strain of S. japonicum or the 26-28 kDa antigen was tested against specific anti-rSj26 antibodies by ELISA, IFA and Western blotting. Both the 26-28 kDa and the rSj26 antigen had weak cross reactions with SEA antigen. The worm reduction rate after challenging with Chinese strain cercariae in mice immunized with rSj26 was 26-32%, similar to that in mice immunized with 26-28 kDa antigen. It is suggested that rSj26 antigen can induce a certain level of specific protective immunity in the host against infection by the Chinese strain of S. japonicum cercariae.

The possibility of GST antigen from Chinese strain of Schistosoma japonicum for immunodiagnosis of schistosomiasis.

The GST antigen (called 26-28 kDa antigen) extracted and purified from Schistosoma japonicum adult worms was applied to the detection of specific antibodies in sera of infected mice and mice immunized with the above protein antigen by ELISA technique. The 26-28 kDa antigen was better than crude antigens (SEA, SWAP) when used to detect specific antibodies in sera from immunized mice. As with crude antigens (SEA and SWAP), the 26-28 kDa antigen could be used to detect specific antibodies in infected sera, with titers as high as 1:160-1:320. There were no false positive reactions and a positivity rate as high as that using SWAP occurred when the 26-28 kDa antigen was used in schistosomiasis patients and normal subjects by intradermal test. It is suggested that the 26-28 kDa antigen may be a suitable candidate for immunodiagnosis of schistosomiasis.

[Comparative study on antigenicity and immunogenicity of the 24-26 kDa antigen and the recombinant Sj26 antigen of Schistosoma japonicum].

This paper reports on the comparison of the recombinant Sj26 (rSj26) antigen originated from the Philippine strain and 24-26 kDa antigen isolated and purified from Chinese mainland strain of S. japonicum for their antigenicity and immunogenicity. The results showed that there were obvious cross reactions between rSj26 and 24-26 kDa antigen when rSj26 antigen was tested against specific antibodies in sera from mice infected with the mainland strain of S. japonicum or 24-26 kDa antigen was tested against specific anti-rSj26 antibodies by ELISA, IFA and Western blotting etc. Both of 24-26 kDa and rSj26 antigen had weak cross reaction with SEA antigen. The worm reduction rate after challenging with mainland strain cercariae in mice immunized with rSj26 was 26-32%, similar to that in mice immunized with 24-26 kDa antigen. It is suggested that rSj26 antigen can induce certain level of specific protective immunity to protect the host against infection by Chinese strain of S. japonicum cercaciae.

[Anti-antibody responses of rabbit to mouse monoclonal antibody against schistosome "target antigen"].

The supernatant of protective McAb (isotype IgG2a) secreted by 1E2 hybridoma line was purified by protein A-sepharose CL-4B column and rabbits were immunized with purified 1E2 McAb. Dot-ELISA, immunodiffusion assay, Western blotting and ELISA were used to observe the presence of anti-McAb and anti-anti-McAb. The main results are: 1. anti-McAb antibody could be detected as early as 20 days post immunization in the immune sera and would be decreased gradually without boosting; otherwise, it would be increased quickly and the titers of anti-McAb were increased within 5 days post-boosting and could be maintained at a plateau for 30-50 days, and then decreased gradually to the normal; 2. there were different anti-anti-McAbs in the immune sera. Anti-anti-McAb, like anti-anti-idiotype antibody, can recognize different schistosome antigens and 90, 68 or 45 kDa antigenic determinants. The level of anti-anti-McAbs varied from titers 1:100 to 1:1600 at different time post-immunization with 1E2. The results provide evidence to understand the appearance of anti-McAb antibody and the way to develop anti-idiotype and anti-anti-idiotype McAb for research purpose.

[Study on McAbs against protein "target antigen" in Schistosoma japonicum].

In the studies presented here, we demonstrated the feasibility of producing large number of hybridoma cell lines secreting McAbs against S. japonicum including 16 cell lines secreting IgG McAbs (7 for IgG1, 5 for IgG2a, 1 for IgG2b, 3 for IgG3) and 13 McAbs for IgM, on the basis of successfully extracting 24-26kD and 90kD "target antigen" proteins known as important antigens for inducing schistosome protective immunity. All the above cell lines were characterized for localization of the McAbs, mediating in vitro ADCC against schistosomula, epitope recognized by the McAbs on different kinds of schistosome antigens. Studies have shown the evidences that the McAbs can be used to identify the "target antigen" on the surface of schistosome, to isolate and purify "target antigen" of S. japonicum by chromatography column bearing McAbs, as well as to immunize animals as antigens for accumulation of data in the development of vaccine against S. japonicum via anti-idiotype antibodies.

[Further study on the immunity induced by mutagenic NTG attenuated cercariae of Schistosoma japonicum].

In order to understand the immunity induced by NTG attenuated cercariae of S. japonicum, investigations were carried out on the survival rate of mice, worm reduction rate, liver and spleen granulation etc. at different intervals between immunization and challenge (4, 6, 8, 10 and 12 wk) in mice exposed percutaneously to NTG attenuated cercariae (30 micrograms/ml NTG for 15 min). Scanning electron microscopy on tegumental surface of schistosomes from immunized mice was also performed. Results showed that better protection was achieved at a challenge time of 8 wk after initial immunizing infection as demonstrated by the higher worm reduction rate and the longer survival rate with a lower degree of liver granulation. It is suggested that acquired immunity did develop against a challenge in mice exposed to NTG-attenuated cercariae, but there were different levels of immunity at different times post-immunization. Thus week 8 appeared to be the optimal time for acquired immunity developed against a challenge infection. The possibility of induction of different levels of immunity was discussed.

[Humoral and cellular immunity in mice immunized with mutagenic NTG-attenuated cercariae of Schistosoma japonicum].

Observation was made on humoral and cellular immunity in mice immunized with NTG-attenuated cercariae. Results showed that the specific antibody in serum of immunized mice increased gradually. The titer by ELISA and IFA were more than 1:1,280 respectively at week 7 post-immunization with NTG-attenuated cercariae (15 min. pc or 60 min. ip). SDS-PAGE electrophoresis showed that there was an extra-band of IgG with MW, between 50,000 and 40,000 in the serum of immunized mice and there was no obvious difference in IgM bands. It suggested that there were differences in not only the increase of antibody titre, but also the composition of antibody post-immunization. The results also indicated that there were differences in levels of lymphocyte proliferative response to schistosome antigens at different weeks post-immunization. The proliferative response to SEA was greater in immunized mice than in infected mice, especially in lymph node lymphocytes. It suggested that NTG-attenuated cercariae did stimulate mice to produce immunity. The dynamic characteristics of humoral and cellular immunity may play roles in the induction of protective immunity against S. japonicum.