Plastids: The Green Frontiers for Vaccine Production.

Infectious diseases pose an increasing risk to health, especially in developing countries. Vaccines are available to either cure or prevent many of these diseases. However, there are certain limitations related to these vaccines, mainly the costs, which make these vaccines mostly unaffordable for people in resource poor countries. These costs are mainly related to production and purification of the products manufactured from fermenter-based systems. Plastid biotechnology has become an attractive platform to produce biopharmaceuticals in large amounts and cost-effectively. This is mainly due to high copy number of plastids DNA in mature chloroplasts, a characteristic particularly important for vaccine production in large amounts. An additional advantage lies in the maternal inheritance of plastids in most plant species, which addresses the regulatory concerns related to transgenic plants. These and many other aspects of plastids will be discussed in the present review, especially those that particularly make these green biofactories an attractive platform for vaccine production. A summary of recent vaccine antigens against different human diseases expressed in plastids will also be presented.

Chloroplast-derived vaccines against human diseases: achievements, challenges and scopes.

Infectious diseases represent a continuously growing menace that has severe impact on health of the people worldwide, particularly in the developing countries. Therefore, novel prevention and treatment strategies are urgently needed to reduce the rate of these diseases in humans. For this reason, different options can be considered for the production of affordable vaccines. Plants have been proved as an alternative expression system for various compounds of biological importance. Particularly, plastid genetic engineering can be potentially used as a tool for cost-effective vaccine production. Antigenic proteins from different viruses and bacteria have been expressed in plastids. Initial immunological studies of chloroplast-derived vaccines have yielded promising results in animal models. However, because of certain limitations, these vaccines face many challenges on production and application level. Adaptations to the novel approaches are needed, which comprise codon usage and choice of proven expression cassettes for the optimal yield of expressed proteins, use of inducible systems, marker gene removal, selection of specific antigens with high immunogenicity and development of tissue culture systems for edible crops to prove the concept of low-cost edible vaccines. As various aspects of plant-based vaccines have been discussed in recent reviews, here we will focus on certain aspects of chloroplast transformation related to vaccine production against human diseases.

Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes.

Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus-like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource-poor countries, development of an improved mucosal and cost-effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat-labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co-administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV-16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB-L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1-ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild-type plants. Taken together, these results pave the way for the possible development of a low-cost adjuvant-coupled vaccine with potentially improved immunogenicity against cervical cancer.