PURPOSE: Protein cages are promising nanoplatform candidates for efficient delivery systems due to their homogenous size and structure with high biocompatibility and biodegradability. In this study, we investigate the potential of lumazine synthase protein cage as an antigen delivery system to dendritic cells (DCs), which induce antigen-specific T cell proliferation. MATERIALS AND METHODS: Ovalbumin (OVA) peptides OT-1 (SIINFEKL) and OT-2 (ISQAVHAAHAEINEAGR) were genetically inserted to lumazine synthase and each protein cage was over-expressed in Escherichia coli as a soluble protein. The efficiency of antigen delivery and the resulting antigen-specific T cell proliferation by DCs was examined in vitro as well as in vivo. RESULTS: We successfully generated and characterized OVA peptides carrying lumazine synthase protein cages. The OT-1 and OT-2 peptides carried by lumazine synthases were efficiently delivered and processed by DCs in vitro as well as in vivo, and induced proliferation of OT-1-specific CD8+T cells and OT-2-specific CD4+T cells. CONCLUSION: Our data demonstrate the potential of lumazine synthase protein cage being used as a novel antigen delivery system for DC-based vaccine development in future clinical applications.
Antigen Presentation ; Cell Proliferation ; Dendritic Cells ; Escherichia coli ; Nanoparticles* ; Ovalbumin ; Ovum ; Peptides ; Vaccines

Vaccination is one of the most successful strategies to prevent diseases caused by pathogens. Although various expression systems including Escherichia coli, yeast, insect, and mammalian cells are currently used for producing many of vaccines, these conventional platforms have the limitation of post-translational modification, high cost, and expensive scalability. In this respect, the plant-based expression system has been considered as an attractive platform to produce recombinant vaccines due to fast, cost-effective and scalable production as well as safety. This review discusses the development of plant-derived vaccines and the current stage of plant-based expression system.
Antibodies ; Efficiency ; Escherichia coli ; Humans ; Insects ; Plants ; Plants, Genetically Modified ; Protein Processing, Post-Translational ; Vaccination ; Vaccines ; Vaccines, Synthetic ; Yeasts

In order to amplify pilA gene and ompC gene of avian pathogenic Escherichia coli (APEC) strain, two pairs of primers were designed according to the GenBank sequences, and a 549 bp pilA gene and a 1104 bp ompC gene were obtained by PCR separately. Sequence analysis indicated that the homology of the nucleotide sequence of AEPC strain to those other reference strains was 98.18% of the pilA gene and 97.28% of the ompC gene. Two expression plasmids pETpilA and pETompC were constructed by inserting pilA gene and ompC gene into the prokaryotic expression vector pET-28a. The two plasmids were transformated into E. coli BL21 separately and two recombinant strains BL21 (pETpilA) and BL21 (pETompC) were obtained. The type 1 fimbraie and the out membrane protein were highly expressed when the recombinant strain BL21 (pETpilA) and BL21 (pETompC) were induced by IPTG Two specific proteins were detected by SDS-PAGE and immunogenicity of the expressed protein was confirmed by Western blotting and ELISA. The expressed fimbraie and OmpC were transformed into vaccine. The protective immune response was proved after the mice were immunized with the two vaccines. The results showed that the recombinant strain BL21 (pETpilA) and BL21 (pETompC) could be as candidate vaccine to provide protective immune response against AEPC infection.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; immunology ; metabolism ; Escherichia coli Proteins ; genetics ; immunology ; metabolism ; Escherichia coli Vaccines ; immunology ; Fimbriae Proteins ; genetics ; immunology ; metabolism ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; Mice ; Porins ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

In order to obtain an attenuated vaccine candidate for enteropathogenic Escherichia coli (EPEC) O45, a ler deletion mutant of pig enteropathogenic E. coli (PEPEC) O45 was constructed by using the suicide vector pCVD442, termed as PEPEC O45(deltaler). The culture supernatant of PEPEC O45(deltaler) deletion mutant was inoculated in vero cell culture. PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the PEPEC O45(deltaler) deletion mutant and the virulent strain O45 respectively. Mice were observed daily for clinical signs and weight changes. Test group of mice inoculated with PEPEC O45(deltaler) gained weight normally and experienced no clinical signs. In contrast, control group of mice inoculated with virulent strain O45 exhibited weight loss and all died in four days. In another experiment, pregnant mice and pig were orally vaccinated by PEPEC O45(deltaler) twice at interval of 14 days respectively. Subsequently, the suckling mice and pig were orally challenged with O45 at 7 days of age respectively. The results showed that 80% of the sucking mice born by vaccinated mice and 75% of the sucking pig born by vaccinated pig were survival; 15% of the sucking mice born by non-vaccinated mice and 10% of the sucking pig born by non-vaccinated pig were survival. This study demonstrated that PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell and to be safety to mice and pig. Oral immunization can induce specific immune responses in mice and pig, and this mutant strain could be used as an attenuated vaccine candidate against PEPEC O45.
Animals ; Enteropathogenic Escherichia coli ; genetics ; immunology ; Escherichia coli Infections ; microbiology ; prevention & control ; Escherichia coli Proteins ; genetics ; Escherichia coli Vaccines ; biosynthesis ; genetics ; immunology ; Gene Deletion ; Mice ; Mutagenesis, Site-Directed ; Swine ; microbiology ; Swine Diseases ; microbiology ; prevention & control ; Trans-Activators ; genetics ; Vaccines, Attenuated ; biosynthesis ; genetics ; immunology

OBJECTIVETo express a fusion protein of Neisseria gonorrhoeae with a mucosal adjuvant.

METHODSThe gene coding Loop VI-VIII(PL678) of porin, an out-membrane protein of Neisseria gonorrhoeae, was obtained by PCR. It was inserted into a plasmid fused with subunit B of heat labile enterotoxin. The recombinant was transformed in E. coli. The expression of fusion protein was analysed by ELISA, SDS-PAGE and Western-blot.

RESULTFusion protein with LTB was successfully expressed, and displayed both the ability of binding GM1 and the reactogenicity with polyclonal antibodies against Neisseria gonorrhoeae.

CONCLUSIONThe expression of fusion protein laid a foundation for the study of the intramolecular vaccine against Neisseria gonorrhoeae.

Bacterial Outer Membrane Proteins ; biosynthesis ; Bacterial Toxins ; biosynthesis ; Bacterial Vaccines ; immunology ; Enterotoxins ; biosynthesis ; Escherichia coli ; genetics ; Escherichia coli Proteins ; Neisseria gonorrhoeae ; chemistry ; immunology ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; Vaccines, Synthetic ; immunology

The study evaluated whether a transgenic carrot vaccine could induce a K88-specific immune response in sows and whether the resultant maternal antibody could protect piglets against enterotoxigenic Escherichia coli (ETEC) K88ac infection. Sows (n = 15) selected randomly from a farm in Korea were assigned to three groups (n = 5 per group: control [untreated]), group A (orally inoculated with a non-transgenic and transgenic carrot vaccines at 2 and 4 weeks ante partum, respectively), and group B (conventionally vaccinated according to the manufacturer's instructions). After 7 days of lactation, 5 piglets selected randomly from each group were challenged with 1 x 1010 colony forming units/mL ETEC K88ac. Group C had the lowest mean fecal consistency score on post-challenge days 1 and 7. Histiologically, On post-challenge day 7, group C showed an increased duodenum and ileum villus:crypt ratio, compared to group A in the duodenum, with group B displaying the highest ratio. Groups B and C had more increased villus width than group A in the jejunum. Group C displayed the greatest increase in villus width in the ileum. The colostrums and serum from groups B and C displayed higher concentrations of IgA and IgG against ETEC K88, compared to group A. Based on the results, it was concluded that the transgenic carrot vaccine in sow per oral may have an effect on preventing piglet diarrhea as good as commercial recombinant vaccine.
Ants ; Colostrum ; Daucus carota ; Diarrhea ; Duodenum ; Enterotoxigenic Escherichia coli ; Female ; Ileum ; Immunoglobulin A ; Immunoglobulin G ; Jejunum ; Korea ; Lactation ; Vaccines

Escherichia coli (E. coli) O157:H7 is an important cause of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). LPS-based vaccines need improvement since the anti-LPS antibodies raised by the vaccines are bactericidal and release toxin that may precipitate the development of HUS. Despite huge efforts, the treatment of infection with E. coli O157 has been difficult because antibiotics do not change the course of the enteritis caused by E. coli O157 and may increase the incidence of HUS through the release of Shiga-like toxin (Stx)-I. For this aim we tried the conjugate of E. coli O157 OSP bound to the nontoxic B subunit of Stx-I B as a vaccine that can induce both serum IgG anti-LPS antibody with bactericidal activity and neutralizing antibody to Stx-I. Mice were immunized s.c. with OSP-Stx-I B conjugate. Anti-sera were analyzed for the anti-LPS antibody, anti-Stx-I B antibody, complement-dependent bactericidal activity, and Stx-I neutralization activity. Mice injected with the bivalent conjugate elicited both bactericidal antibodies to E. coli O157 and neutralization antibodies to Stx-1. We also analyzed the distributional, functional changes of T lymphocytes in vitro and in vivo. After the injection of Stx-I, splenocytes showed a decrease in proliferation when stimulated with phytohemagglutinin (PHA) or LPS, and the number of CD4+ and CD8+ T cells also decreased. Interleukin (IL)-2, IL-4 and IFN-gamma productions by CD3+ T cells were slightly increased in the Stx-I injected mice.
Animals ; Anti-Bacterial Agents ; Antibodies ; Antibodies, Neutralizing ; Colitis ; Enteritis ; Escherichia coli O157* ; Escherichia coli* ; Escherichia* ; Hemolytic-Uremic Syndrome ; Immunoglobulin G ; Immunotoxins* ; Incidence ; Interleukin-4 ; Interleukins ; Mice ; O Antigens ; T-Lymphocytes ; Vaccines*

Avian pathogenic Escherichia coli (APEC) is a causative agent for a number of extra intestinal diseases and account for significant losses to the poultry industry. Since protective immunity against APEC is largely directed to virulence antigens, we have individually expressed four different viulence antigens, papA, papG, IutA, and CS31A, using an attenuated Salmonella Typhimurium and a plasmid pBB244. Following oral immunization of mice with combination of two or four of these strains, serum IgG and mucosal IgA responses were elicited against each antigen represented in the mixture. The antigen-specific mucosal IgA responses were significantly higher in the group of mice immunized with the heat-labile Escherichia coli enterotoxin B subunit (LTB) strain than those in the group of mice immunized without the LTB strain. While, there was no significant difference between these two groups in antigen-specific serum IgG responses. The results showed that LTB could act as mucosal immune adjuvant. To assess the nature of immunity, the distribution of antigen-specific IgG isotypes was analyzed. All groups promoted Th1-type immunity as determined by the IgG2a/IgG1 ratio. Thus, our findings provided evidence that immunization with a combination of several vaccine strains is one of the strategies of developing effective vaccines against APEC.
Animals ; Enterotoxins ; Escherichia coli ; Immunity, Mucosal* ; Immunization ; Immunoglobulin A ; Immunoglobulin G ; Intestinal Diseases ; Mice* ; Plasmids ; Poultry ; Salmonella typhimurium ; Salmonella Vaccines* ; Salmonella* ; Vaccines ; Virulence

OBJECTIVESTo evaluate the efficacy of antitumour colon cancer cell vaccine modified by escherichia coli cytosine deaminase (EC-CD).

METHODSMouse colon cancer cell vaccine CT26/CD was established by gene modification using retrovirus plasmid pLCDSN. Its tumorigenicity and effect on liver metastasis model established with wild-type colon cancer were evaluated.

RESULTSCT26/CD was established successfully and proliferated in medium containing 0.6 g/L G418 stably. EC-CD gene expression on these mutant cells was confirmed by RT-PCR. These mutant cells were more sensitive to 5-fluorocytosine (5-FC) compared with the wild-type ones (P=0.000), and presented excellent bystander effect. CT26/CD had the same tumorigenicity as its parental cells (P=0.892). CT26/CD, combined with the prodrug 5-FC, could inhibit tumor progress and live metastasis, and prolonged the survival of the liver metastasis model animals (P=0.000).

CONCLUSIONThe colon cancer cell vaccine modified by EC-CD presented anti-tumor effect in vivo, when treated with the prodrug.

Amino Acid Motifs ; Animals ; Cancer Vaccines ; therapeutic use ; Cell Line, Tumor ; Colonic Neoplasms ; therapy ; Cytosine Deaminase ; genetics ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; genetics ; Humans ; Mice ; Mice, Inbred BALB C

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important pathogen. One of the important virulence traits of EHEC O157:H7 is the capacity to produce attaching and effacing (A/E) lesions on enterocyte. This property encoded by a pathogenicity island termed the locus of enterocyte effacement (LEE). LEE contains ler (LEE-encoded regulator) gene. The product of ler is a central up-regulator of many virulence genes of the LEE. Another important virulence factor of EHEC O157: H7 is Shiga toxin (Stx), encoded by a prophage integrated into the chromosome of O157:H7. In order to obtain an attenuated vaccine candidate, a ler deletion mutant of O157: H7 was constructed by use of suicide vector pCVD442. Meanwhile, due to potential instability of the prophage carrying the stx gene, the prophage was cured with serial passages of bacteria and confirmed by PCR and DNA sequencing. A ler/stx deletion mutant of EHEC O157:H7 was constructed, termed as O157:H7(deltaler/deltastx). The cultural supernatant of O157 ler/stx deletion mutant was inoculated in vero cell culture, and the result indicating that O157 ler/stx deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the O157 ler/stx deletion mutant and the virulent strain O157:H7 EDL933, respectively. Mice were observed daily for clinical signs and weight changes. After inoculation of the deletion mutant, test group of mice (inoculated with O157:H7(deltaler/deltastx)) gained weight normally and experienced no clinical signs. In contrast, control group of mice (inoculated with O157: H7) exhibited weight loss and all died in four days. In another experiment, pregnant mice were orally vaccinated by O157:H7(deltaler/ deltastx) twice at interval of 14 days. Subsequently, the suckling mice were orally challenged with O157:H7 EDL933 at 7 days of age. The results showed that 78.34% of the sucking mice born by vaccinated mice were survival and 12.73% of the sucking mice born by non-vaccinated mice were survival. This study demonstrated that O157 ler/stx deletion mutant lost the toxigenicity to vero cell and to be safety to mice. Oral immunization can induce specific immune responses, and this mutant strain could be used as an attenuated vaccine candidate against EHEC O157.
Administration, Oral ; Animals ; Animals, Suckling ; Antibodies, Bacterial ; blood ; immunology ; Cercopithecus aethiops ; Escherichia coli Infections ; immunology ; mortality ; prevention & control ; Escherichia coli O157 ; genetics ; immunology ; pathogenicity ; Escherichia coli Proteins ; genetics ; Escherichia coli Vaccines ; administration & dosage ; genetics ; immunology ; Female ; Gene Deletion ; Genetic Vectors ; genetics ; Genomic Islands ; genetics ; Immunization ; Lactation ; immunology ; Male ; Mice ; Mutation ; Pregnancy ; Shiga Toxin ; genetics ; Survival Rate ; Trans-Activators ; genetics ; Vaccines, Attenuated ; administration & dosage ; genetics ; immunology ; Vero Cells ; Virulence ; genetics