Antibody responses in mice stimulated by various doses of the potato-derived major surface antigen of hepatitis B virus.

The ability of potato-derived major surface antigen of hepatitis B virus (P-HBsAg) to elicit antibody responses to different dosages of P-HBsAg ranging from 0.02 to 30 µg administered orally in mice was examined. All immunized groups produced specific serum IgG and fecal IgA antibodies against P-HBsAg, even at low levels (<5 µg), after administration of a 0.5-µg yeast-derived HBsAg (Y-HBsAg; LG Life Sciences, Republic of Korea) booster.

Oral transgenic plant-based vaccine for hepatitis B.

In addition to improving vaccine formulations in order to elicit robust and long-lasting immune responses, there is also an increasing need for improving the manner in which these vaccines are delivered. As most current vaccines are administered by injection by a health care giver, there is the ever-present danger of needlestick injuries. Therefore, needle-free vaccinations are a viable option toward limiting needle-associated injuries and additionally increasing compliance with vaccination schedules, as both children and adults have an aversion to injections. Noninvasive methods of vaccination will also facilitate speed of vaccine delivery and likely also reduce cost, both important factors for health care in developing countries. One alternative to current injectable immunizations is orally delivered vaccines. A specific approach that we and others are evaluating is the use of transgenic plant tissue that expresses vaccine antigens for oral immunization. Herein, we review the development of an oral HBV vaccine expressed in transgenic potato tubers and the immune responses generated in human subjects given this novel vaccine.

Transgenic lettuce seedlings carrying hepatitis B virus antigen HBsAg

The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L.) using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety "Vitória de Verão" genetically modified.

[Construction of a plant effective expression vector containing the gene of hepatitis B virus surface antigen].

OBJECTIVE: To construct a plant effective expression vector driven by a fruit specific promoter for the expression of hepatitis B virus surface antigen (HBsAg), to further improve the expression of exogenous gene in plant, and to prepare for the development of an effective anti-hepatitis vaccine. METHODS: Tomato fruit-specific promoters' gene 2A12 and E8 were respectively introduced to pBPFOmega7 to form pB2A12 and pBE8. The DNA fragment containing HBsAg-s gene from plasmid YEP-HBs was inserted respectively into pB2A12 and pBE8 to form pB2A12-HBs and pBE8-HBs. The fragment containing "p35S+2A12+Omega+HBsAg-s+Tnos" of the pB2A12-HBs was sub-cloned into plasmid pCAMBIA1301 to yield the reconstructed plant binary expression plasmid pCAM2A12-HBs, and the fragment containing "p35S+E8+Omega+HBsAg-s+Tnos" of the pBE8-HBs was sub-cloned into plasmid pCAMBIA1301 to yield the plasmid pCAME8-HBs. The inserted gene HBsAg and fruit-specific promoters in the reconstructed plant binary expression vectors were confirmed by sequencing. Then, pCAM2A12-HBs and pCAME8-HBs were directly introduced into Agrobacterium tumefaciens strain EHA105. RESULTS: Digestion with restriction enzymes proved that all recombinant vectors had the inserts with expected length of the target fragments, and the sequencing results were confirmed correct. CONCLUSION: In this study, plant expression vector containing HBsAg gene driven by fruit specific promoter and CaMV35s promoter was successfully constructed.

Immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis B and human immunodeficiency viruses.

A synthetic chimeric gene, TBI-HBS, encoding the immunogenic ENV and GAG epitopes of human immunodeficiency virus (HIV-1) and the surface protein antigen (HBsAg) of hepatitis B virus (HBV), was expressed in tomato plants. Tomato fruits containing the TBI-HBS antigen were fed to experimental mice and, on days 14 and 28 post-feeding, high levels of HIV- and HBV-specific antibodies were present in the serum and feces of the test animals. Intraperitoneal injection of a DNA vaccine directing synthesis of the same TBI-HBsAg antigen boosted the antibody response to HIV in the blood serum; however, it had no effect on the high level of antibodies produced to HBV.

Mucosal immunogenicity of the hepatitis B core antigen.

The hepatitis B virus (HBV) core antigen (HBcAg) is a potent immunogen in animal models and humans and has been used as a carrier for several antigens, however, the mucosal immunogenicity of HBcAg or chimeric HBcAg proteins has been poorly studied and only using the truncated variant of the HBcAg. In this study we explored the mucosal immunogenicity in mice of the recombinant complete nucleocapside of HBcAg. The antigen was administered by different mucosal and parenteral routes. The antibody response in sera was evaluated after each immunization and mucosal lavages were tested with the final extraction. To characterize the immune response, the serum IgG antibody response was tested during six months and also the ratio IgG2a to IgG1 was determined. The results obtained evidenced that the mucosal immunogenicity of HBcAg depended on the administration route, being the intranasal (i.n.) route the one that generated the higher IgG responses in sera, similar in intensity and duration to parenteral administrations. The IgA response in mucosal washes was superior for nasally immunized mice compared to the rest of mucosal and parenteral groups. The nasal route also induced the higher IgG2a to IgG1 ratio, evidencing a Th1-like Ab subclass pattern. In addition to the high Ab responses, preliminary results of the cellular response induced by nasal administration evidenced the induction of strong lymphoproliferative responses in spleen cells.

Enhanced type I immune response to a hepatitis B DNA vaccine by formulation with calcium- or aluminum phosphate.

DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. When compared with conventional vaccines, however, DNA vaccines often induce lower antibody titers. We have now found that formulation of a DNA vaccine encoding hepatitis B surface antigen with calcium- or aluminum phosphate adjuvants can increase antibody titers by 10-100-fold and decrease the immunogenic dose of DNA by 10-fold. Furthermore, boosting an HBs protein-primed response with the adjuvanted DNA vaccine resulted in a dramatic increase in the HBs-specific IgG2a response reflecting a shift towards a TH1 response. The mechanism by which aluminum phosphate exerts its adjuvant effect is not through increased expression of HBsAg in vivo; rather, the adjuvant appears to increase the number and affinity of HBs peptide antigen-specific IFN-gamma and IL-2 secreting T cells.