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Development of an oral DNA vaccine against MG7-Ag of gastric cancer using attenuated salmonella typhimurium as carrier - PubMed

Development of an oral DNA vaccine against MG7-Ag of gastric cancer using attenuated salmonella typhimurium as carrier

Chang-Cun Guo et al. World J Gastroenterol. 2003 Jun.

Abstract

Aim: To develop an oral DNA vaccine against gastric cancer and evaluate its efficacy in mice.

Methods: The genes of the MG7-Ag mimotope and a universal Th epitope (Pan-DR epitope, PADRE) were included in the PCR primers. By PCR, the fusion gene of the two epitopes was amplified. The fusion gene was confirmed by sequencing and was then cloned into pcDNA3.1(+) plasmid. The pcDNA3.1 (+)-MG7/PADRE was used to transfect an attenuated Salmonella typhimurium. C57BL/6 mice were orally immunized with 1X10(8) cfu Salmonella transfectants. Salmonella harboring the empty pcDNA3.1(+) plasmid and phosphate buffer saline (PBS) were used as negative controls. At the 6th week, serum titer of MG7-Ag specific antibody was detected by ELISA. At the 8th week cellular immunity was detected by an unprimed proliferation test of the spleenocytes by using a ((3)H)-thymidine incorporation assay. Ehrlich ascites carcinoma cells expressing MG7-Ag were used as a model in tumor challenge assay to evaluate the protective effect of the vaccine.

Results: Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than that in control groups (0.841 vs 0.347, P<0.01; 0.841 vs 0.298, P<0.01), while in vitro unprimed proliferation assay of the spleenocytes showed no statistical difference between those three groups. Two weeks after tumor challenge, 2 in 7 immunized mice were tumor free, while all the mice in the control groups showed tumor formation.

Conclusion: Oral DNA vaccine against the MG7-Ag momitope of gastric cancer is immunogenic. It can induce significant humoral immunity against tumor in mice, and the vaccine has partially protective effects.

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Figures

Figure 1
Figure 1

Incorporation of the epitope gene into the pcDNA3.1 fragment by PCR: A product of 620bp was amplified by the first PCR (a), and a fragment of 660bp was amplified by the second PCR (b).

Figure 2
Figure 2

Hind III digestion of the recombinant plasmid after subcloning the PCR product into pcDNA3.1 (+) from the pUCm-T vector: A fragment of 660bp was released (c), which corresponded to the size of the carrier fragment.

Figure 3
Figure 3

Sequencing of PCR product (partial sequence): By PCR, the two epitopes were fused together and incorporated into a pcDNA3.1 fragment. The amino acid sequence of KPHVHTKGGGS correspondeds to the sequence of MG7-Ag mimotope. AKFVAAWTLKAAZ corresponds to the sequence of universal Th epitope PADRE.

Figure 4
Figure 4

PCR identification of the pcDNA3.1 (+)-MG7/PADRE plasmid harbored by the Salmonella typhimurium SL3261: By PCR, a fragment of 800bp was amplified (d), suggesting the existence of epitope genes and removal of carrier fragment.

Figure 5
Figure 5

Immunohistochemical staining of the Ehrlich ascites carcinoma cells (EAC): Positive signal was seen in the cytoplasm and membrane of the EAC cells (A). When stained with a negative control monoclonal antibody (anti-E-tag antibody), the EAC cells showed no positive staining (B).

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