A Pilot Study Of Intragastric Immunization With Hybrid Filamentous Phage Induced Systemic Cell-mediated Immune Response And Protection Against Tumor Development In Vivo

In 1985, George Smith, who pioneered the phage-display technology, proposed that foreign sequences could be inserted within the minor coat protein, pIII, of filamentous phage. As a result of powerful methodologies, a large-scale effort to measure and analyze molecular interactions is under way. Phage display has been used originally for the screening of immunogenic epitopes or mimotopes on displayed peptides. These, in turn could become lead molecules for the design of peptide-based vaccines.In recent years, phage display was considered for vaccine development. Firstly, the work in a handful cases indicated that parenteral administration of filamentous phage could induce a strong immunological responses including humoral response and systemic T cell response. The study of phage displayed the repeat regions of circumsporozoite protein of Plasmodium falciparum on pIII were both antigenic and immunogenic when injected to rabbits reported in 1988. The potential of phage display for neutralizing the virus was demonstrated in 1994. Phage displayed epitopes such as the V3 loop of gp120 from HIV-1, HBV surface antigen and the murine mastocytoma P81A, indicated that such hybrid phage could induce cell-mediated immune response in vivo and conferring protection to mice from lethal challenges with antigen-expressing tumor cells in 2000,2001 respectively. Furthermore, several papers have shown that the best immunogens are represented by mimotopes displayed in the phage rather than on other carriers.Secondly, the current studies revealed that epitope-displaying bacteriophage could induce strong systemic and mucosal antigen-specific antibody responses via the oral route. It has been shown that the response induced by oral administration is specifically cross-reactive with the original antigen in 1997. Zuercher et al. have shown that displaying phage has the ability to survive the harsh environment of the stomach in an antigenically intact form in 2000. As a universal vector, displaying phage has then provided a new horizon for vaccine development.In order to explore whether displaying phage could induce a sytemic CTLs response via oral route, the study of recombinant phage with CTL epitope peptides for protection against tumor development in vivo by oral administration is then discussed in the paper.By way of phage display technology and epitope-based vaccine design, we genetically engineered a recombinant phage, M13KEova, displaying at its surface a chimeric CTL epitope SIINFEKL from the chicken ovalbumin, which can express MHC class I restricted CTL epitope OVA257-264 peptide. The hybrid phage were then digested with restriction enzyme BamHI and performed discontinuous procedure of H. Sch?gger and G. Von Jagow 's low molecular weight SDS gel electrophoresis for identification. The results have shown that foreign sequences were cloned into the N-terminal region of the major coat protein (pVIII) of filamentous bacteriophage M13KE and OVA257-264 peptide could be expressed by this recombinant phage. This approach lead to OVA257-264 peptide display in multiple copies on the surface of the virion and avoid the shortcoming that the quantities of single-stranded DNA produced by phagemids vary from one host to another and are often low.Immunizing C57BL/6 (H-2K~b restricted) mice intragatrically with the appropriate amount of hybrid filamentous phage particles and CT/CTB adjuvants was employed the scheme of two times at 7-day intervals. Fifteen days after the last immunization, C57BL/6 mice were injected with EL4 tumor cells that transfected by electroporation with the plasmid pAc-neo-OVA (E.G7-OVA, H-2K~b restricted) and monitored for tumor development. To determine whether intragastric immunization with hybrid phage has the ability to protect against tumor growth in vivo.We have observed that recombinant phages bearing CTL epitope OVA257-264 peptides successfully inhibited the tumor growth in vivo compared with the control counterparts. It indicated that M13KEova induced H-2K~b-restricted CTL...