Smallpox subunit vaccine produced in planta confers protection in mice

Abstract

We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain (pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was expressed by using efficient transient and constitutive plant expression systems and tested by various immunization routes in two animal models. Whereas oral administration in mice or the minipig with collard-derived insoluble pB5 did not generate an anti-B5 immune response, intranasal administration of soluble pB5 led to a rise of B5-specific immunoglobulins, and parenteral immunization led to a strong anti-B5 immune response in both mice and the minipig. Mice immunized i.m. with pB5 generated an antibody response that reduced virus spread in vitro and conferred protection from challenge with a lethal dose of vaccinia virus. These results indicate the feasibility of producing safe and inexpensive subunit vaccines by using plant production systems.plant biotechnology ͉ transgenic plants ͉ B5 glycoprotein ͉ recombinant antigen P lants have emerged as an excellent alternative to other expression systems for the production of complex pharmaceutical proteins, including recombinant subunit vaccines (1-6). It was shown that some plant-derived antigens can induce systemic and mucosal immune responses and, in some cases, confer protection against challenge (1-3). Plants provide the additional advantage of direct delivery through oral or other mucosal routes (1, 6). Despite some difficulties with the expression of certain recombinant proteins, especially those of viral origin, plant biotechnology holds the promise of producing medicinal proteins to be used in vaccine formulations.Interest in a safe smallpox vaccine has been reawakened by the threat of bioterrorism (7, 8) and continuous outbreaks of orthopoxvirus diseases (9, 10). A live vaccinia virus (VV)-based vaccine has been used to eventually eradicate smallpox disease (11, 12), but does display side effects (13). Although one approach for developing a safer vaccine is to use a highly attenuated live virus, recombinant protein-based vaccines are likely to be safer. For orthopoxviruses, there are several candidate antigens that can protect mice and nonhuman primates from lethal challenge (14-20). The extracellular virus (EV)-specific membrane glycoprotein encoded by the B5R gene (21-24) is the main target of EV-neutralizing antibodies present in human-derived vaccinia gamma globulin used to treat complications arising from smallpox vaccination (25).In this study, we demonstrate that the VV B5 protein can be produced in two plant expression systems. The use of the magnifection transient expression system (26-28) enabled rapid high-yield production of soluble B5 as well as selection of optimal subcellular targeting signals to use in stable plant transformation. Preparations of purified plant-derived B5 antigen (pB5) induced a strong immune response when administered parenterally and intranasally, and mice vaccinated i.m. with pB5 were protected from VV challenge. ResultsExpression Ca...

Immunological assessment of plant-derived avian flu H5/HA1 variants

“…The Germany) were used to expedite expression [6]. To generate HA1-Fc fusions (HA1-Fc), cDNA clones encoding the heavy chain of human anti-rabies monoclonal antibody SO57 [7] and the heavy chain of murine antibody CO17-1A [8] were used as PCR templates.…”

“…HA-based polypeptide variants were isolated from plant tissues and purified by a 2-step procedure using His-and c-myc tags exactly as described [6]. HA-Fc fusion proteins were purified in a single-step protocol using protein A beads as described [7].…”

“…HA-Fc fusion proteins were purified in a single-step protocol using protein A beads as described [7]. In vitro characterization and quantification of protein expression were done by ELISA and Western blot analysis as described [6,7]. Full-size baculovirus-expressed H5 protein from BEI Resources (Manassas, VA) was used as positive control.…”

“…Six-to 8-week-old female BALB/c mice (5 per group) were immunized intramuscularly with recombinant peptides 2 times (two weeks interval) at a dose of 10 µg per immunization using alum-CpG adjuvant essentially as described [6]. Ten days after the last immunization, mice were sacrificed and sera from individual mice were analyzed by ELISA and Western blotting as described [6], followed by virus micro-neutralization and hemagglutination inhibition assays [10].…”

“…Ten days after the last immunization, mice were sacrificed and sera from individual mice were analyzed by ELISA and Western blotting as described [6], followed by virus micro-neutralization and hemagglutination inhibition assays [10]. Results are expressed as the mean ±SD for groups of five mice.…”