Protective immune response of bacterially-derived recombinant FaeG in piglets.

FaeG is the key factor in the infection process of K88ad enterotoxigenic Escherichia coli(ETEC) fimbrial adhesin. In an attempt to determine the possibility of expressing recombinant FaeG with immunogenicity for a new safe and high-production vaccine in E. coli, we constructed the recombinant strain, BL21 (DE3+K88), which harbors an expression vector with a DNA fragment of faeG, without a signal peptide. Results of 15% SDS-polyacrylamide slab gel analysis showed that FaeG can be stably over-expressed in BL21 (DE3+K88) as inclusion bodies without FaeE. Immunoglobulin G (IgG) and M (IgM) responses in pregnant pigs, with boost injections of the purified recombinant FaeG, were detected 4 weeks later in the sera and colostrum. An in vitro villius-adhesion assay verified that the elicited antibodies in the sera of vaccinated pigs were capable of preventing the adhesion of K88ad ETEC to porcine intestinal receptors. The protective effect on the mortality rates of suckling piglets born to vaccinated mothers was also observed one week after oral challenge with the virulent ETEC strain, C83907 (K88ad, CT+, ST+). The results of this study proved that the adhesin of proteinaceous bacterial fimbriae or pili could be overexpressed in engineered E. coli strains, with protective immune responses to the pathogen.

Oral immunization of mice with plant-derived fimbrial adhesin FaeG induces systemic and mucosal K88ad enterotoxigenic Escherichia coli-specific immune responses.

The importance of adhesins in pathogenicity has resulted in them being useful targets in the defense against bacterial infections. To produce edible vaccines against piglet diarrhea caused by enterotoxigenic Escherichia coli (ETEC), plants were genetically engineered to produce recombinant fimbrial adhesin FaeG. To evaluate the efficacy of the edible vaccine FaeG in mice, the soluble protein extracts were examined by about 15 microg recombinant FaeG for each oral immunization dose per mouse. After four doses of vaccination, both IgG and IgA antibodies specific to K88ad fimbriae were elicited in serum, and specific IgA antibodies were also evoked in feces of the immunized mice. Moreover, visible K88ad ETEC agglutination by the specific serum from the immunized mice was observed, implying the antibody was highly specific and effective. Results from an in vitro villous-adhesion assay further confirmed that serum antibodies of the immunized mice could inhibit K88ad ETEC from adhering to pig intestinal receptors, further demonstrating the oral immune efficacy of the plant-derived FaeG. This study provides a promising, noninvasive method for vaccinating swine by feeding supplements of transgenic plant. Moreover, the low cost and ease of delivery of this edible ETEC vaccine will facilitate its application in economically disadvantaged regions.

A modified TMV-based vector facilitates the expression of longer foreign epitopes in tobacco.

Based upon a mutant isolated from tobacco infected with a recombinant tobacco mosaic virus (TMV), a new TMV-based vector was developed in which four to six C-terminal amino acid residues were deleted from the viral coat protein (CP) subunit. The new vector was quite similar to the original TMV-based vector, which all expressed a well characterized epitope peptide F11 (P(142)-A(152)) of VP1 from foot-and-mouth disease virus (FMDV) serotype O in tobacco, in the infectivity, yield of the virus particles and more importantly protective activity of F11 in guinea pigs and swine against the FMDV. Furthermore, the capacity of the length of foreign peptide encoded by this new vector was much improved to successfully express a peptide F25 containing two fused epitopes F14 (R(200)-L(213)) and F11 of FMDV VP1, which was failed using the original vector in tobacco. Although animal assays indicated that such expressed F25 was not as efficient as F11 in the immunity, possibly due to lack of a spacer arm between the two fused epitopes, the new TMV-based vector may meet the requirement of expressing longer foreign peptides for different vaccines and other medicines.

Immunogenicity of the epitope of the foot-and-mouth disease virus fused with a hepatitis B core protein as expressed in transgenic tobacco.

A novel plant-based vaccine protecting against foot-and-mouth disease (FMD) was developed by inserting the VP21 epitope into the internal region of the hepatitis B virus core antigen gene (HBcAg). The specific sequence of the VP21 epitope is located within the VP1 capsid protein of the FMD virus (FMDV). It spans 21 amino acids located between positions 140 and 160 of the G-H loop. The fusion gene, HBCVP, was inserted into the plant binary vector pBI121 and then transformed into tobacco (Nicotiana tabacum) plants via Agrobacterium tumefaciens strain LBA 4404. The presence of HBCVP in the tobacco genome was confirmed by polymerase chain reaction (PCR); its transcription was verified by reverse transcription-PCR; and the recombinant protein expression was confirmed by Western blot analysis. The results of immunologic microscopic observation demonstrated that recombinant fusion protein HBCVP can form a virus-like particle (VLP) structure in transgenic tobacco leaves. Mice, immunized intraperitoneally with a soluble crude extract of transgenic tobacco leaves, were found to produce specific antibody responses to both HBcAg and FMDV VP1. A virus challenge demonstrated that the immunized mice were highly protected against virulent FMD. This work describes a new way to develop an FMD vaccine from plants that will aid the development of new vaccines using HBcAg fused to the conserved epitopes of other pathogenic antigens.

Expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector.

We expressed two immunogenic dominant epitopes of foot-and-mouth disease virus (FMDV) serotype O in tobacco plant using a vector based on a recombinant tobacco mosaic virus (TMV). The recombinant viruses TMVF11 and TMVF14 contained peptides of 11 and 14 amino acid residues, respectively, from FMDV VP 1 fused to the open reading frame of TMV coat protein (CP) gene between amino acid residues 154 and 155. TMVF11 and TMVF14 systemically infected tobacco plant and produced large quantities of stable progeny viral particles assembled with the modified CP subunits. Guinea pigs, mice and swine were used to test the protective effects of the recombinant viruses against FMDV infection. Most guinea pigs were protected against FMDV challenge after parenteral injection with TMVF11, TMVF14, or the mixture TMVF11/TMVF14, but not wtTMV. The TMVF11/TMVF14 mixture protected all animals when challenged with 150 guinea pig 50% infection dosage (GPID(50)) FMDV. Oral administration of the TMVF11/TMVF14 mixture (3mg total) protected 3/8 guinea pigs against the same FMDV challenge. Most of the suckling mice parenterally injected with antiserum from guinea pigs immunized with the TMVF11/TMVF14 mixture, but not with wtTMV, were also protected against FMDV challenge with 10 suckling mouse 50% lethal dosage (SMLD(50)), indicating that antibodies produced in guinea pigs immunized with the TMVF11/TMVF14 mixture specifically neutralized FMDV. Western blot analysis indicated that antiserum from those guinea pigs reacted with the FMDV VP1 protein. The protective effect of TMVF11 was also demonstrated in swine, where preliminary tests showed that nine pigs immunized with TMVF11 in three experiments were protected against FMDV challenge with 20 minimal infecting dose (MID).

Production of FaeG, the major subunit of K88 fimbriae, in transgenic tobacco plants and its immunogenicity in mice.

Transgenic tobacco plants stably expressing recombinant FaeG, which is the major subunit and adhesin of K88ad fimbriae, were obtained. Analysis of sera from immunized mice indicates that in mice, the immunogenicity induced by plant-derived FaeG protein is comparable to that generated with traditional approaches.