Production of antigenic recombinant cholera toxin subunit B.

Cholera is a major public health problem confronting developing countries, where outbreaks occur in a regular seasonal pattern and are particularly associated with poverty and poor sanitation. Primer set was designed to flank binding region of cholera toxin subunit B [ctxB] gene that was amplified using PCR. PCR products were purified using gel purification technique. Cholera ctxB gene was cloned into cloning vector. Stop codons contained in the insert were deleted and then the insert was subcloned into pQE expression vector. Cloned vectors were subjected to DNA sequence analysis. Small-scale culture was done for preparative rCTB production and purification. Mice were immunized with purified rCTB to test ability of rCTB to elicit antibody production. Primer set showed the ability to detect and excise ctxb gene successfully form cholera genomic DNA by PCR. After deleting the stop codons, sequence analysis revealed that the insert was in open reading frame with start codon of the vector. Small bacterial cultures revealed the presence of specific band at approximately 12-125 Kda in induced culture. rCTB was able to elicit specific antibody production after animal immunization

Antigenic potential of whole cell-recombinant cholera toxin subunit B [WC-CTB] preparation

Colonisation of the small intestine by V. cholerae, a typical non-invasive pathogen, is an important early step in the pathogenesis of cholera. In the present study, trial to make cholera whole-cell vaccine with addition of recombinant B subunit of cholera toxin [rCTB] was-done. Cholera chB gene was cloned into pQE expression vector. Large-scale culture was done for preparative rCTB production and purification. Commercial CTB from V. cholerae was used for induction of polyclonal anti-CTB antibodies in mice. These polyclonal antibodies were used to test the antigenicity and identity of rCTB. Cholera whole-cell vaccine was prepared by resuspending equal volumes of dead Inaba and Ogawa vaccine strains in PBS. Orochol E Berna was used as control vaccine. Mice were divided into 4 groups [20 mice each]; group 1 [G1]: control unimmunised, group 2 [62]: rCTB immunized, group 3 [G3]: rCTB + killed Inaba and Ogawa immunised, and group 4 [G4]: Orochol immunized. Sera and faeces from all groups were collected and used in evaluation of anti-rCTB antibody levels. The spleens of animals were aseptically removed and used in lymphoproliferative assay. Small bacterial cultures revealed the presence of specific band at approximately 12-12.5 KDa in induced culture. Anti-commercial CTB antibodies were successfully prepared and used in Western blot analysis and verified the presence and antigenicity of rCTB. Large scale production and purification of rCTB resulted in 12.9 mg protein. Both serum and secretory antibody level in mice of G2 was significantly less than in mice of both G3 and G4. G3 was significantly higher than 62, while there was no significant difference between G3 and G4. G4 was significantly higher than G2, while there was no significant difference between G4 and G3. Stimulation index of splenic cells in G 1 was not significantly different from that of G2, while it was significantly lower than GB and G4. SI of G2 was significantly lower than G3 and G4. G3 was significantly higher than G1, G2, and G4. G4 was significantly higher than 01 and 02, but it was significantly lower than G3. The results in this study showed the ability of rCT B to induce immune responses in the presence of cholera bacteria more than if used alone. The inclusion of rCTB in addition to vibrios, increased systemic, intestinal, and cellular responses. Depending on previous studies, adding any new cholera strain which are present or will appear in future is possible. In addition, other vaccines [either killed bacteria or vaccine subunit] can be added to the formula indicated in this study

Possible antigenic cross reactivity between hepatitis c virus and schistosoma soluble worm antigens

The possible cross-reactivity between antibodies [Abs] to hepatitis-C virus [HCV] and Schistosoma mansoni was investigated. Ninety-one serum samples were collected from Egyptian adult healthy male blood donor volunteers. Sera were assayed for HCV antibody using a third generation ELISA technique, and divided into two groups. Group I [n = 61] included the anti - HCV positive sera, and group II [n = 30] anti HCV negative sera. For the two groups of sera, the seropositivity for anti - S.mansoni adult microsomal antigen [MAMA] was determined by Falcon assay screening test - ELISA [FAST-ELISA] and found to be higher in group I [78.7%] than group II [33.3%]. In contrast, screening for anti - Escherchia coli Abs by passive haemagglutination test demonstrate positive results in 44 [72.1%] of group I, and 28 [93.3%] of group II. Elimination of schistosoma Abs from HCV +ve sera did not affect detection of Hcv Abs when re-tested. It is concluded that Abs to immunogenic epitopes of Hcv and schistosoma adult worm antigens are not cross-reactive