Plant-derived vaccines

Abstract

Several types of human papillomavirus (HPV) are causatively associated with cervical cancer, which is the second most common cancer in women worldwide. HPV-16 and 18 are among the high risk types and responsible for HPV infection in more than 70% of the cases. The majority of cervical cancer cases occur in developing countries. Currently available HPV vaccines are expensive and probably unaffordable for most women in low and middle income countries. Therefore, there is a need to develop cost-effective vaccines for these countries. Due to many advantages, plants offer an attractive platform for the development of affordable vaccines. These include low cost of production, scalability, low health risks and the potential ability to be used as unprocessed or partially processed material. Among several techniques, chloroplast transformation is of eminent interest for the production of vaccines because of high yield of foreign protein and lack of transgene transmission through pollen. In this commentary, we focus on the most relevant aspects of plant-derived vaccines that are decisive for the future development of cost-effective HPV vaccines.

Keywords: :

• L1
• LTB
• adjuvant
• biosafety
• chloroplast transformation
• cost-effective vaccines
• human papillomavirus
• plant-derived vaccines

Background

Cervical cancer is the second most common cancer in women and the seventh overall in the world. Every year, approximately half a million new cases occur across the globe.¹ In 2008, an estimated 529,000 new cases and 275,000 deaths were reported, about 88% of which occurred in developing countries: 159,800 in Asia, 53,000 in Africa and 31,400 in Latin America and the Caribbean.² The number of deaths from cervical cancer is expected to increase to approximately 410,000 by 2030.^2,3 Overall, in developing countries, cervical cancer accounts for more than 85% of the total global burden and is the leading cause of cancer-related deaths among women.⁴ Those regions that are at high risk include South-Central Asia, South America and whole Africa with exception of North. In Eastern Africa, South-Central Asia and Melanesia, cervical cancer remains the most common cancer in women.⁴ During the last three decades, the number of cervical cancer cases has been increasing for all regions except in high income countries, while for east and South Asia, Eastern Europe, and Southern Latin America, the number of new cases has been constant.⁵

Almost all cases of cervical cancer are caused by one of 15 types of oncogenic human papillomavirus (HPV). Among these, HPV-16 and 18 are responsible for approximately 70% of invasive cervical cancer cases while HPV-16 alone contributes to 54% of the total cases.⁶ Other high-risk HPV types include 31, 33, 35, 45, 52 and 58.⁷ HPVs are non-enveloped icosahedral viruses containing double stranded circular DNA. The DNA genome encodes for two classes of proteins: the early and late. The early gene products are E1, E2, E4, E5, E6 and E7 and the late proteins are L1 and L2. The later proteins are the structural components of the viral capsid (for review see ref. 8).

Vaccines can either be prophylactic, which generate neutralizing antibodies to block HPV infection or the therapeutic vaccines, which eliminate infection by inducing a virus-specific T cell-mediated response. To date, no therapeutic vaccine is available for the treatment of already existing HPV infections. Current strategies for the development of safe and effective prophylactic vaccines are based on the induction of neutralizing antibodies against the major (L1) and minor (L2) capsid proteins of HPV.⁹ To improve the existing strategies for broad protection, a conserved and cross-protective antigen such as L2 can be included in vaccine formulations. However, to accelerate the control of cervical cancer and treat currently infected patients, it is important to develop HPV vaccines that are therapeutic. For this purpose, research is focused on early proteins encoded by the HPV genome. A recent report shows promising results for the development of such therapeutic vaccine (Pentarix) directed at the E7 proteins from five of the most prevalent high-risk genotypes of HPV (HPV16, 18, 31, 45 and 52).¹⁰ In the above mentioned report, upon administration, mice elicited strong, multi-genotype specific CD8T cell immunity. Moreover, large and established E7-expressing TC-1 tumors showed rapid and complete regression after therapeutic vaccination of mice with Pentarix.¹⁰

Are Currently Available HPV Vaccines Affordable for Women in Developing Countries?

Currently, two prophylactic vaccines, Gardasil and Cervarix manufactured by Merck and GlaxoSmithKline (GSK), respectively, are commercially available for the prevention of cervical cancer. Both vaccines contain the HPV L1 capsid protein in the form of virus-like particles (VLPs). Gardasil targets HPV types 6, 11, 16 and 18, while Cervarix targets types 16 and 18.^11-13 In the USA, the drug company’s recent prices for Gardasil and Cervarix are almost similar, i.e., approximately 130$ per dose and 390$ for three shots for private health providers.¹⁴ This price does not include the cost of receiving the injection. World Bank data estimates that the number of people living on less than $2 a day is over 2.5 billion worldwide (World Bank 2008). Due to the low resources in most developing countries, government health care programs cannot provide HPV vaccination. As a result, people need to acquire the vaccine through private health care providers, which is not affordable for a large percentage of people in low-income countries. In addition, the price of HPV vaccine from both companies is high for private use. Although, both companies have introduced price tiering systems for their respective HPV vaccines, so that they can offer vaccine to lower income countries at lower prices, the costs associated with the vaccination are still not affordable for most women in developing countries. Therefore, it is necessary to develop low-cost, stable and effective preventive vaccines that are suitable for developing countries.

Plants as a Platform for the Production of Cost-effective Vaccines

The majority of the currently available vaccines are expensive because of their complex manufacturing processes through various cell culture systems, requirement of fermenters and purification by complex technologies. Moreover, additional expenses are associated with adjuvant, transportation, cold storage and sterile delivery.¹⁵ In contrast, plants offer a cost-effective platform for the production of low-cost vaccines. Production of vaccines from plants has many advantages: low cost, scalability, low health risks and the potential ability to be administered as unprocessed or partially processed material (for review see refs. 16–18). Plant-derived vaccines can be produced either stably by nuclear and chloroplast transformation or transiently by tobacco mosaic virus (TMV) based expression. Each technique has its own advantages and disadvantages. However, regarding vaccine production in plants, chloroplast transformation suits well, because of high yield of recombinant protein.¹⁹ Moreover, chloroplast transformation is safe for the environment as transgenes cannot spread via pollen in most plant species²⁰ Various vaccine antigens against different human diseases have been successfully expressed in chloroplast.¹⁹ However, none of the chloroplast-derived vaccine candidate has yet entered into clinical trials. So far, few plant-derived vaccines against human diseases, expressed either by nuclear transformation or TMV based expression, have shown promising results in phase I and II clinical trials upon oral delivery (for review see refs. 21–22).

HPV-16 L1 antigen has been expressed in plants by nuclear and chloroplast transformation, either as VLPs or capsomeres,^23-29 and the plastid-derived VLPs were shown to be highly immunogenic in mice.²⁷ Recently, our group expressed HPV-16 L1 protein along with Escherichia coli heat labile enterotoxin subunit B (LTB), as a fusion protein.³⁰ LTB can act as an adjuvant and by this facilitate the transport of the antigen from the gut lumen to the gut associated lymphoid tissues. In addition to L1, many studies have been performed regarding the expression of E6 and E7 in plants (for review see ref. 31). Currently, research is underway for the development of a therapeutic vaccine against HPV from plants, by a renowned research group in South Africa.³²

There are several advantages of plant/plastid transformation that can be exploited for cost-effective production of HPV vaccines. These are:

• Very high yields of recombinant proteins can be achieved by chloroplast transformation. In two recent reports,^33,34 approximately 70% and 72% of total soluble protein (TSP) has been obtained by plastid transformation, respectively. Since stimulation of the mucosal immune system generally requires much higher doses of the antigen than injection, plastid transformation can be optimized to express L1 or other antigenic proteins in large amounts to meet the prerequisite for the initiation of proper immune responses. In addition, with the increase in the yield of the recombinant protein, the downstream processing costs can be decreased.³⁵

• Scalability: growth of plants can be scaled up according to the required amount of protein.

• Biosafety issues can be covered by the application of chloroplast transformation and/or growing the plants in contained facilities. Furthermore, an inducible system can be used to control the transgene expression when required.^19,36

• Transgenic plants can be grown at the site where the vaccine is needed. This advantage can save the costs related to transportation and cold storage.

• Plant-derived vaccines have the potential to be used as oral vaccine, thus evading the costs related to sterile needles and trained medical staff.

• Stability: plants-derived vaccines are likely to be more stable. A recent report³⁷ shows that a chloroplast-derived vaccine candidate was stable at room temperature for 20 months. Moreover, mice immunized with the vaccine stored at room temperature showed similar IgA/IgG levels as those of mice immunized with the vaccine stored at 4°C.³⁶ This characteristic is very important for the development of a vaccine for developing countries where cold chains are difficult to maintain in remote areas.

• Due to the prokaryotic nature of the gene expression system of the plastid, multiple genes can be linked in operons and co-expressed simultaneously. This advantage of multigene expression in plastids can be adopted for the production of vaccines with improved immunogenicity by coupling the antigens with specific adjuvants. By this, costs related to separate production and administration of adjuvants can be saved. Moreover, this feature of plastid transformation can be used to co-express L1 antigens from different HPV types and/or combine L2 for broad protection against different types.

In conclusion, plants have a great potential to produce cost-effective vaccines against HPV. There are many advantages of plant-derived vaccines that can be used to produce low-cost vaccines for low and middle income countries. Exceptionally high yields obtained through plastid transformation make this system very promising for vaccine production. Moreover, the issue of biosafety can be covered by using chloroplast as an expression system. However, for plant-derived vaccines, the major hurdles are the costs related to clinical trials. Limited resources in research and lack of interest/investment from companies are also further reasons for the slow advancement of plant-based vaccines. Nevertheless, a couple of plant-derived pharmaceuticals has entered the clinical trials^38,39 and these successful developments markedly increase the chances that further plant-derived vaccines will soon be available in the market to serve patients in low and middle income countries.

Acknowledgments

The research work reported in this commentary by our group was supported by Higher Education Commission (HEC), Pakistan in co-operation with ÖAD, Austria, and partially funded by the GoF foundation, Germany. We also acknowledge the collaboration with the Norwegian Research Council (NRC) on plant derived vaccines.