Expression of antigens aimed at veterinary vaccine development.
Parasitic diseases fecally transmitted, such taeniasis/cysticercosis Taenia solium binomial, represent a health problem whose incidence continues due to the prevalence of inadequate sanitary conditions, particularly in developing countries. When the larval stage of the parasite is established in the central nervous system causes neurocysticercosis a disease than can severely affect human health. It can also affect pigs causing cysticercosis causing economic losses. Since pigs are obligatory intermediate hosts, they have been considered as the targets for vaccination to interrupt the transmission of the parasitosis and eventually reduce the disease. Progress has been made in the development of vaccines for the prevention of porcine cysticercosis. In our research group, three peptides have been identified that, expressed synthetically (S3Pvac) or recombinantly (S3Pvac-phage), reduced the amount of cysticerci by 98.7% and 87%, respectively, in pigs exposed to natural conditions of infection. Considering that cysticercosis is orally acquired, it seems feasible to develop an edible vaccine, which could be administered by the pig farmers, simplifying the logistical difficulties of its application, reducing costs, and facilitating the implementation of vaccination programs. This chapter describes the most important advances towards the development of an oral vaccine against porcine cysticercosis.
- T. solium
- oral vaccine
- transgenic plant
- Carica papaya
Between control measures it has been explored the improvement of health education, sanitary conditions, standards of meat inspection and the rearing of pigs. It has also been explored the impact of massive or individual treatment of taeniasis and the treatment and/or vaccination of pigs, all of them with promising results [2, 3, 4]. Vaccination of pigs would imply an unlikely immediate and potent effect upon the number of tapeworm-carriers in rural communities, interrupting the parasite’s life cycle and eventually reduce human neurocysticercosis. Developing an effective vaccine against
In our group, an anti-cysticercosis vaccine named S3Pvac based on three peptides expressed was developed. The vaccine synthetically (S3Pvac) or recombinant (S3Pvac-phage) produced, reduced the number of cysticerci by 98.7% and 87% [7, 8] in randomized field trials, respectively. The recombinant vaccine was subsequently used in a control program applied in the State of Guerrero, confirming its usefulness. Indeed, S3Pvac-phage significantly reduce the prevalence of porcine cysticercosis from 7 to 0.5% and 3.6 to 0.3% estimated by tongue examination or ultrasound, respectively . In the course of this control program, we were able to evaluate the difficulties involved in using an injectable vaccine. Pigs are produced free rurally reared, thus the application of an injectable vaccine requires their capture and subjection, a laborious procedure that increases the costs of vaccination and limits its massive application. Considering that cysticercosis is orally acquired, it seems feasible to develop an oral vaccine , which could be administered by the pig breeder, simplifying the logistical difficulties of its administration, reducing costs and facilitating the implementation of vaccination control programs.
For the design of an oral vaccine the use of plants is increasingly recognized as valuable platform. Plants offer the production of antigens at low costs, circumventing costly purification procedures. Plants also offer a natural way of antigen encapsulation preventing its degradation by the detrimental environment to which an oral intake vaccine is exposed such us antigen degradation by low pH, mucosal enzymes [10, 11] and the use of cell cultures will avoid non-desirable environmental effects due to the release of transgenic plants into the environment.
Moreover, plants also frequently include components with adjuvant properties like saponins that may increase the immunogenicity of the vaccine . Considering this, the recombinant peptides KETc7, KETc1.6His, and KETc12.6His were expressed in transgenic clones of papaya embryogenic calli . The three clones together constitute the oral S3Pvac-papaya vaccine candidate. Papaya was selected because the high efficiency of transformation and its own antiparasitic properties .
This third version of the vaccine has been shown to be immunogenic in mice and pigs and is being produced in suspended culture systems to massively produced this oral version of the vaccine that must be evaluated on the field against pig cysticercosis.
2. Parasite and oral immunity
Oral vaccination is an interest route to prevent infections caused by orally acquired pathogens overcoming the limitations of current injection-based vaccines in providing front-line protection against pathogen invasion and dissemination . It offers a painless, safe and low-cost route that does not require trained personnel. Moreover, this route can elicit mucosal and systemic immunity. Vaccine antigen can be recognized and translocated by M cells, which act as sentinels and enter directly into the Peyer’s patches. Then antigens can be transported to the intestinal mesenteric lymph nodes, stimulating the host’s systemic and mucosal immune response resulting in the production of IgA and IgG antibodies with the ability to neutralize of invading pathogens before they are able to cause a widespread infection. Oral vaccination can also trigger an effective cellular immunity. However, the development of oral vaccines is a major challenge due to an inefficient transport to reach M cells and the possibility to induce local and systemic immune tolerance. Considering that plant-based vaccines usually expressed low content of antigen it seems feasible to avoid oral tolerance using the proper dose and vaccine schedule. It remains to be elucidated if plants-derived vaccines could overcome mucosal tolerance when administered to human beings.
Table 1 shows various plants that have been used to express antigens from different pathogens to be evaluated as edible vaccines. Tobacco has been used as an experimental model of transformation and expression. However, the use of other species such as tomatoes, lettuce, potatoes, corn, soybeans, alfalfa, Arabidopsis, papaya and carrots has been expanded [11, 28, 29, 30, 31, 32, 33]. In some of these plants, the expressed recombinant antigen has shown efficacy when evaluated in experimental models or directly in the naturally affected host. Recombinant antigens have been reported to induce an immune response with the production of IgG, IgM or IgA antibodies, regardless of the route of administration .
|Foot and mouth virus||Arabidopsis||VP1||Ip||Mice|||
|Foot and mouth virus||Potato||VP1||Ip||Mice|||
|Foot and mouth virus||Alfalfa||Peptide VP1|
|Gastroenteritis virus||Arabidopsis||GP- S||Im||Mice|||
|Gastroenteritis virus||Potato||GP- S||Oral||Mice|||
|Gastroenteritis virus||Tobacco||GP- S||Ip||Pig|||
|Gastroenteritis virus||Seeds of corn||GP- S||Oral||Pig*||[25, 26]|
|S3Pvac-papaya||Embryogenic Transgenic clones||KETc1, KETc12 and KETc7 peptides||Sc||Mice|||
|S3Pvac-papaya||Embryogenic Transgenic clones||KETc1, KETc12 and KETc7 peptides||Oral||Pig|||
3. Transgenic plant platform
Many different advantages of expression of recombinant proteins in transgenic plants for vaccine production can be mentioned over other commonly expression systems, such as bacteria, yeasts and baculoviruses. Plants can be constitutively or tissue-specific expressed in single or multiple transgenes, antigens can be stable in seeds without the requirement of refrigeration, no purification nor cold chain for preservation.
Transgenic plants can also be used as bioreactors to produce high amounts of the recombinant protein of interest [34, 35]. They can also be produced as
One nice study of veterinary interest is the expression of the glycoprotein S of the porcine gastroenteritis virus in corn seeds for the production of an oral vaccine, which has also the ability to induce protection, through colostrum, in piglets [25, 26].
Morphological type: Arboreal.
Climate: Equatorial tropical.
|Food fiber||Caricain||Strengthens Immunity||Spermatocide|
|Retinol (vit. A)||Class II chitinase||Analgesic|
|β-carotene||Class III chitinase||Antibiotic|
|Thiamine (vit. B1)||Serin protease inhibitor||Stimulation of pancreatic juices|
|Riboflavin (vit. B2)||Glutamyl cyclotransferase||Hypotensive|
|Niacin (vit. B3)||Beta-1,3-glucanase||Febrifuge|
|Pantothenic acid (vit. B5)||Cystatin||Anti-inflammatory|
|Folic acid (vit. B9)||Acetogenins*||Anti-helminthic|
Carica papayaas a cestode vaccine
Papaya is an alternative system for the exploration of tropical tree genomes, containing a genome of 372 megabase (Mb), of diploid inheritance with 9 pairs of chromosomes and presents the smallest gene number, 24,746 genes . Papaya exhibits some properties of possible advantages to be used as a platform to express
Among many papaya components, the papain contained in the latex has been widely evaluated in its ability to damage the cuticle of intestinal parasites by proteolytic digestion. Table 3 shows some reports on the characterization and evaluation of antiparasitic activity of papaya against
Raw latex (Sigma)
|25 mM||Disrupt the surface of the cuticle|||
|Not available||Damage to the strobile|
Reduced motility and subsequent death of the parasite
|300 mM||Reduced motility and induced death of the parasite|||
|Papaya latex supernatant||2.4 μmol||Affected worm growth|||
|Latex supernatant||240 nm for 6 days||Minimal and temporary efficacy|||
3.3 S3Pvac-papaya anticysticercosis vaccine
For the development of the S3Pvac-papaya cysticercosis vaccine, three genetic constructions were used for the expression of recombinant peptides, KETc1.6His, KETc12.6His and KETc7 . Figure 1 summarize the methodology employed for the production of S3Pvac-papaya vaccine.
3.4 Protective properties of S3Pvac papaya against cysticercosis
The S3Pvac vaccine expressed in embryogenic papaya clones has demonstrated high protective capacity against experimental murine and
|Experimental model||Immunized with:||Mean ± SD (% Protection)†||Immune response||Reference|
|Saline||27.2 ± 14.2|
|S3Pvac papaya saline (μg/dose)|
|0.1||12.3 ± 3.4* (55)||CD4, CD8 proliferation|
|1||10.8 ± 2.5* (60)||CD4, CD8 proliferation|
|10||9.2 ± 1.0*|
|Specific IgG Abs; CD4 and CD8 proliferation|
|100||41.4 ± 62|
|Saline||18.6 ± 17.3* (86.8)|
|Corn starch§||29.8 ± 31.4 (41.4)|||
|Soy oil||10.3 ± 2.2* (75)|
|Canola oil||11.5 ± 8.6* (84)|
|Rabbit experimental ||Saline||4.33 ± 4.01|||
|S3Pvac papaya£||0.25 ± 0.62*|
We previously reported non-specific protection that was induced by the wild type soluble extract [52, 54] has been attributed to the antiparasitic properties described to papaya itself mentioned above.
3.5 Biotechnological approach for the production of papaya anti-cysticercosis vaccine
Plant biotechnology is a rapidly evolving area with major impact in the production of molecules with high pharmaceutical value.
Plant biotechnology involves relevant procedures in the manufacture of oral vaccines enabling the production of higher amounts of active biomasses from transgenic plants, by means of massive propagation of cells, tissues and organs [12, 55]. Among others, these procedures include the growth of callus (aggregates of undifferentiated cells growing in solid media), suspension cultures (individualized undifferentiated cells growing in liquid media); as well as embryo cultures that could be grown in solid or in liquid nutrient media.
The three callus lines expressing KETc1, KETc7 and KETc12 peptides were generated, and further efforts to optimize the massive growth of the corresponding callus and suspension cultures, were conducted. These
3.5.1 Callus cultures
In the establishment and optimization of callus cell lines,
3.5.2 Cell suspension cultures
Ten grams fresh weight (FW) of the friable callus line KETc7 were inoculated in 250 ml Erlenmeyer baffled flasks containing 100 ml nutrient medium without phytagel, and placed for 30 days on a rotary shaker at 100 rpm, 25°C and dark conditions. To disaggregate cell clusters and increase oxygen transfer, baffled flasks were used (Figure 3).
The cultures were sub-cultured in fresh medium every 15 days, and the best results were observed when using B5 nutrient medium, cultivated in darkness, and producing uniform suspended cultures without phenolization (Figure 4).
Once the friable uniform cell suspension cell line was established, it became possible to evaluate the growth kinetics of the culture during 45 days by collecting samples every 3 days, and determining the following growth parameters: fresh weight, dry weight, cell viability, pH and carbohydrate consumption .
The results showed that the KETc7 suspension cell line grew very well, reaching a doubling time of 6.9 days and a specific growth rate (μ) of 0.10 d−1. The maximum biomass value was 14.36 gPS L−1 obtained at 15 days in culture.
3.5.3 Embryo suspension cultures
The KETc7 embryos callus cell line generated in solid B5 medium without phytoregulators was used to establish embryo suspension cultures. An inoculum of 10% was added into 250 ml Erlenmeyer flasks containing 100 ml of liquid B5 medium (Figure 6). The flasks were kept on an orbital shaker at a stirring speed of 115 rpm, under constant light conditions (24 μmol.m−2. S−1) and 25°C. After 15 days, the biomass was sub cultivated in the same conditions described above, and the culture was propagated.
3.5.4 Cell suspension cultures in bioreactors
To scale-up the
3.5.5 Embryo suspension cultures in bioreactors
This review addresses oral vaccination as a feasible approach to prevent parasitic diseases. Since most anti-parasitic vaccines currently available are parenterally administered, their use involves high production and logistic costs and become inaccessible for underdeveloped countries. To cope with this issue, the use of papaya transgenic clones is herein proposed to develop an anti-cysticercosis oral vaccine and to assay its effectiveness against other parasitic infections of veterinary and/or public health interest. The use of biotechnological tools by escalation of suspension cultures would allow us to produce a vaccine in sufficient, controlled amounts for its direct application, reducing the use of antibiotics, and therefore the risk of bacterial resistance.
Thanks to Georgina Díaz, B.Sc., and Gonzalo Acero, B.Sc., for their technical support. Special thanks to Juan Francisco Rodríguez, B.Sc., for his help in preparing the original manuscript. This work was supported by the Programa de Investigación para el Desarrollo y la Optimización de Vacunas, Inmunomoduladores y Métodos Diagnósticos, Institute of Biomedical Research, UNAM.
Conflict of interest
The authors declare no conflict of interest.