Development of transgenic alfalfa plants containing the foot and mouth disease virus structural polyprotein gene P1 and its utilization as an experimental immunogen
Introduction
The use of transgenic plants for the expression of recombinant antigens has been increasingly used for the production of experimental immunogens [1], [2], [3], [4], [5], [6], [7], [8], [9], [10] since they are, potentially, an inexpensive source of antigens to be parenterally administrated or, more interestingly, to be used as edible vaccines [1], [2], [3], [10]. Although a very promising technology, most of the antigens expressed to date have been epitopes or single proteins containing linear epitopes [8], [10] or adopting simple tridimensional structures [3], [4], [9]). However, the practical use of transgenic plants as a source of antigen for vaccine production would undoubtedly require, in most cases, the expression of more complex antigenic structures.
Foot and mouth disease virus (FMDV) is the causative agent of a very significant economic disease affecting meat and milk producing domestic animals [11]. Current vaccines are based on the utilization of inactivated virus and, although they have proved to be effective tools for the prevention of the disease, their production is both costly and risky because of manipulation of massive amounts of virulent virus could result in virus dissemination [11]. Thus, it is important to develop new approaches, which could provide practical alternatives to the current methodology of vaccine production.
With that scope, we have focused on the development of alternative methods of FMDV vaccine production using transgenic plants as bioreactors. We have successfully demonstrated that the FMDV VP1 protein, which carries critical epitopes responsible for the induction of protective neutralizing antibodies, could be expressed in plants of Arabidopsis thaliana, alfalfa and potato [12], [13], [14], and was able to elicit a virus-specific antibody response and protection against the virulent challenge after parenteral or oral administration.
Here, we report the production of transgenic alfalfa plants expressing the FMDV VP1 polyprotein and show that the expressed products are immunogenic in a mouse experimental model by inducing a strong FMDV-specific antibody response against complete virus particles and viral subunits as well as a complete protection against the experimental challenge with the virulent virus. These results demonstrate, for the first time, the possibility of using transgenic plants to express complex antigenic structures and their utilization as effective experimental immunogens.
Section snippets
Production of transgenic plants of alfalfa
A 3327 bp DNA fragment (spanning the P1-3C protein coding region) was introduced in the genome of transgenic plants. P1-3C encodes for the FMDV O1C genomic regions P1 (1A, 1B, 1C, 1D), 2A, the first 61 amino acid residues of the N terminus of 2B, the complete sequence of 3B1, 3B2, 3B3, 3C and the first 16 amino acid residues of the N terminus of 3D. The construction also contains a start and a stop codon at positions 1 of 1A and following the 16th codon of 3D, respectively.
P1-3C was obtained by
Production and genetic analysis of transformed plants
Nine lines of transgenic plants containing the P1-3C gene were initially produced. The presence of the P1-3C genes was detected in all the pRokP1-3C transformed plants by amplification of a PCR product of the expected size (655 bp) that was regularly absent in the non-transformed plants (Fig. 1B). Additionally, the complete sequence of 3C was detected in the transgenic plants (data not shown).
RT-PCR of pRokP1-3C transformed plants consistently resulted in products corresponding to the
Discussion
The use of transgenic plants for antigen production has received further experimental support from several groups who have reported the expression of different antigens using this methodology [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. Nevertheless, most antigens expressed in transgenic plants constitute unique proteins or simple structures. Hepatitis B surface antigen [3], Norwalk virus capsid protein [4] and human papilloma virus capsid protein [9] are the only antigenic structures
Acknowledgements
Authors wish to thank the technical assistance of Mrs. Cristina Gomez and the personnel of Laboratory Bayer in Argentina where the challenge experiments were conducted. This work was supported by Grant PICT 08-08718 from SECYT, Argentina.
References (17)
- et al.
Passive immunization of mice pups through oral immunization of dams with a plant-derived vaccine
Immunol Lett
(2003) Vaccination against foot and mouth disease virus
Vaccine
(1992)- et al.
Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunized with alfalfa transgenic plants expressing the viral structural protein VP1
Virology
(1999) - et al.
A novel methodology to develop a FMDV peptide based vaccine in transgenic plants
Vaccine
(2002) - et al.
Efficacy of a food plant based oral cholera toxin B subunit vaccine
Nat Biotechnol
(1998) - et al.
Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene
Vaccine
(1998) - et al.
Production of hepatitis B surface antigen in transgenic plants for oral immunization
Nat Biotechnol
(2000) - et al.
Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes
J Infect Dis
(2000)
Cited by (74)
Immunogenic profiling and designing of a novel vaccine from capsid proteins of FMDV serotype Asia-1 through reverse vaccinology
2021, Infection, Genetics and EvolutionCitation Excerpt :Similarly, Xiao et al. (2016) established 11.75 PD50 per dose for cattle by expressing Asia-1 vp0, vp3, and vp1 in the E. coli expression system. Plant-based expression of FMDV P1 polyprotein conducted by Wang et al. (2012) and Santos et al. (2005) indicated the potency of the empty capsid encoding region against viral infection in the experimental mouse model. Taking these results into account, we selected the P1 + 3C coding region for the commercial synthesis of genes to be expressed in plants.
Minimally processed crude leaf extracts of Nicotiana benthamiana containing recombinant foot and mouth disease virus-like particles are immunogenic in mice
2018, Biotechnology ReportsCitation Excerpt :Until 2018, only two approaches to produce plant-based FMD VLPs (both using transgenic plants) had been reported. Although these approaches were not able to conclusively demonstrate that the recombinant capsid proteins assembled into VLPs, immunization with foliar extracts from transgenic alfalfa [14] and tomato plants [15] proved to evoke a strong antibody response and protection in mice and guinea pigs, respectively. During the last few years, there has been an increasing trend towards the use of transient expression of recombinant proteins in plants, mostly because this approach is fast and flexible, allowing the rapid expression of high concentrations of heterologous proteins in a matter of days [13].
Transient expression of heat- and acid-resistant foot-and-mouth disease virus P1-2A mutants in Nicotiana benthamiana
2018, Virus ResearchCitation Excerpt :The latter is proteolytically cleaved by 3C to yield capsid proteins VP0, VP1 and VP3, after which VP0 is cleaved into VP2 and VP4 upon virus maturation. Recombinant co-expression of P1-2A with 3C in various expression hosts has been shown to result in the assembly of virus-like particles (VLPs) (Li et al., 2012; Lewis et al., 1991; Xiao et al., 2016; Cao et al., 2009; Roosien et al., 1990; Abrams et al., 1995; Mignaqui et al., 2013; Dus Santos et al., 2005; Dus Santos and Wigdorovitz, 2005; Porta et al., 2013a). In some cases, these VLPs are immunogenic in animals (Li et al., 2012; Cao et al., 2009; Mignaqui et al., 2013; Dus Santos et al., 2005; Porta et al., 2013a) making them ideal vaccine candidates.
Immunogenicity of T7 bacteriophage nanoparticles displaying G-H loop of foot-and-mouth disease virus (FMDV)
2017, Veterinary MicrobiologyMucosal Vaccines from Plant Biotechnology
2015, Mucosal Immunology: Fourth Edition