Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.

Abstract

(1) and expression of the capsid protein in recombinant baculovirus-infected insect cells (4) have facilitated the study of the virus and the development of a candidate vaccine (5). Expression of the Norwalk virus capsid protein (NVCP) in insect cells yields a protein with an apparent Mr of 58,000 that self-assembles into insect cell-derived Norwalk virus-like particles (i-rNVs) lacking viral RNA, which are reactive with sera from Norwalk virus-infected humans (4). Electron cryomicroscopy of i-rNV shows that the 38-nm empty capsid is composed of 90 dimers of NVCP that form arch-like capsomeres (6). The particles are morphologically and antigenically similar to authentic virus particles, stable on storage at 4°C, stable after lyophilization, and resistant to pH 3.0 treatment (4, 7). These qualities make i-rNV attractive for use as a potential vaccine against Norwalk virus. Recent studies showed that oral immunization of mice with as little as 50 ,ug of i-rNV per dose resulted in the production of serum and mucosal antibodies against NVCP (5). This result is striking in view of the fact that i-rNV is a nonreplicating vaccine and no cholera toxin (CT) adjuvant is needed to achieve immunization.We have experimented with the use of plants as an economical alternative for expression and delivery of recombinant vaccines (8-10). Hepatitis B surface antigen (HBsAg) expressed in tobacco leaves forms subviral particles (8) that are similar to the recombinant yeast-derived antigen, which is licensed for parenteral immunization (11). The plant-derived HBsAg retains both B-and T-cell epitopes when studied in a mouse model (10). Furthermore, the Escherichia coli heatlabile enterotoxin B-subunit expressed in potato tubers and fed to mice without preparation (other than slicing) stimulates serum and gut mucosal antibodies against E. coli labile enterotoxin B-subunit (9). These studies provide proof that recombinant antigens can be produced in transgenic plants, and, at least in some cases, these antigens are orally immunogenic.We report here the expression of recombinant NVCP in transgenic tobacco leaves and potato tubers. The NVCP from tobacco leaves self-assembles into tobacco-derived virus-like particles (t-rNVs) that are morphologically and physically similar to i-rNVs. Further, we show that either partially purified t-rNV given orally or potato tubers expressing NVCP fed directly to mice stimulate the production of antibodies against NVCP. We conclude that a plant-derived edible vaccine for Norwalk virus is feasible. Together with our previous studies, these findings bolster the concept of using transgenic plants for a novel, safe vaccine production and delivery system for developing countries. MATERIALS AND METHODSConstruction of Plant Expression Vectors. A 2.4-kbp DNA fragment containing the gene encoding NVCP was obtained by partial EcoRI digestion of pUCNV4145 (1) and subcloned into pBluescript-KS (Stratagene) at the EcoRI site. One clone (pKSNV2.4) was digested with SmaI and SstI; the resulting 1.9-kbp fragmen...

Arabidopsis thaliana plants expressing Rift Valley fever virus antigens: Mice exhibit systemic immune responses as the result of oral administration of the transgenic plants

“…Viral capsid proteins have previously been shown to be easily expressed in plants [12,13,34] and here we show that this is the case also for the RVFV N protein, as is evident from its expression at high concentrations. To the best of our knowledge, this is the first time RVFV antigen expression has been demonstrated in plants.…”

“…It is therefore likely that the non-responders had lower intake, or none at all, compared with the responders. Others have previously shown that plant extracts delivered by gastric intubation [13] or pelleted plant material [15] are feasible alternatives to free feeding. These are delivery modes that would help to ensure that every mouse would consume equal amounts of plant material.…”

“…The antigen dose has previously been shown to be a factor influencing the inducible immune response after oral administration [13]. Since only half of the animals responded in the first experiment when 21 g of plant material was administered per four animals, the amount of plant material in the second experiment was slightly increased to 26 g per four animals ( Table 2).…”

“…Various viral proteins have successfully been expressed in plants and have shown antigenic properties in animal experiments [12][13][14][15][16]17]. Whereas some of these proteins were delivered invasively [12,16], others, such as antigens for Crimean-Congo Hemorrhagic fever virus, Human-immunodeficiency virus, and Norwalk virus, were able to elicit systemic immune responses when administered orally, either as pellets, plant extracts or fresh plant material [13][14][15]. Plants are ideal as oral delivery systems since the natural bioencapsulation of vaccine antigens allows for safe passage through the acidic environment in the stomach of non-ruminants, such as humans or laboratory animals, into the duodenum and the small intestine [18].…”

A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate

“…Edible plant vaccines against enterotoxic Escherichia coli (ETEC; Refs. 14 & 15), cholera toxin [17,18], and norovirus [19,20] have already passed pre-clinical trials and preliminary human clinical trials show very promising results -transgenic plants can stimulate a two-way immune response, both systemically and mucosally. Improvement of administration protocols and the use of adjuvants during oral vaccination could then be important ways of further increasing efficacy of edible vaccines.…”