Prof Ed Rybicki
Prof Ed Rybicki
Department of Molecular & Cell Biology, University of Cape Town
Email
Project Title
Production of HPV L1/L2 chimaeric VLP vaccines in plants and insect cells for broad protection against cervical cancer.
Project Description
Papillomaviruses (PVs) are small double-stranded DNA viruses that infect many different species such as humans, dogs, cattle, horses, rabbits, non-human primates, mice, sheep and birds. They are extremely species specific. Human papillomaviruses (HPV) are known to cause warts, and are associated with certain cancers in humans. They are divided into high and low-risk HPV types, where the high-risk types such as HPV 16, 18, 33 and 58 infect the genital epithelium and can produce lesions which progress to invasive cervical cancer. Specific high-risk types of HPV are causally associated with cervical cancer and cancer of the cervix is the most common cancer in South African women and the second most prevalent cancer in women worldwide. HPV 16 is the most prevalent high-risk HPV type found to be associated with cervical cancer.
Recently virus-like particle (VLP) vaccines against HPV have been developed, which consist of the major capsid protein L1. These vaccines are produced by Glaxo Smith Kline and Merck, by use of recombinant baculoviruses and yeast, respectively. The VLPs produced are almost indistinguishable from native virions in morphology, and induce effectively identical immune responses. In animal and human studies, VLP vaccines have been well tolerated and have induced high titres of neutralising antibodies as well as protecting against papillomaviral infection and especially disease. However, these vaccines are very expensive and are moreover very type-specific: a mixture of several types is needed to protect against the majority of circulating high-risk types alone. An ideal HPV vaccine would be affordable, safe, stable and would protect against the major oncogenic HPV types. Human and animal studies have shown that many of these criteria can be met with VLPs. However, affordability and vaccine stability will be a problem in developing countries, given the requirement for a cold chain and the predicted expense of current vaccine candidates. Further development and testing of alternative vaccines is therefore warranted as is the investigation of alternative expression systems which would reduce the cost of the vaccines.
Plants have successfully been used as production vehicles to produce a host of different proteins. It is thought that production of vaccines in plants should result in a cheap, effective alternative to conventional methods. One problem encountered so far is the low yield of proteins in plants, which in turn increases cost of production.
The two VLP vaccines on the market protect against the majority of high-risk HPV infections, such as HPV-16 and -18. However especially in Africa these vaccines might not be as effective as the infection is often caused by HPV types that do not cross-react with antibodies or T-cell receptors specific for the types contained in the vaccines. It is possible that these viruses will in time fill the niche hitherto dominated by HPV-16 and -18 and related viruses: The only way to combat this will be to expand the range of HPV types included in the VLP vaccines or to increase the breadth of immunogenic response elicited by the vaccines, by engineering the L1 protein(s) to include more cross-reactive antibody-binding epitopes. The latter approach has led to the development of HPV chimaeras: these are L1 molecules that include a specific epitopes that will be recognised by the immune system and elicit an immune response against another HPV type. This approach has the advantage that fewer antigens may need to be mixed to obtain wide coverage by the generation of many different type-specific neutralising antibodies.
Our group has developed and expressed various L1::L2 chimaeras over the years and expressed them in insect cells and plants and we could show that they neutralised HPV16 and showed some cross-neutralisation against other types. In this previous work we focused on one insertion site which resulted in the formation of capsomers instead of VLPs. Even though capsomers are also good candidate vaccines they elicit a much weaker immune response than VLPs. In the present project we aim to develop chimaeric VLP candidate vaccines that will cross- protect against more than one type of HPV. Additionally we wish to determine if plant-produced VLPs are as efficient as vaccines produced by the conventional method in insect cells by comparing the immune response elicited in mice when they are vaccinated with these VLPs.
Brown, D. R., Fife, K. H., Wheeler, C. M. & other authors (2004).Early assessment of the efficacy of a human papillomavirus type 16 L1 virus-like particle vaccine. Vaccine 22, 2936-2942.
Harper, D. M., Franco, E. L., Wheeler, C. & other authors (2004).Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet 364, 1757-1765.
Rybicki, E. R. (2009). Plant-produced vaccines: promise and reality. Drug Discovery Today 14, 16-24.
zur Hausen H. (1996). Papillomavirus infections–a major cause of human cancers. Biochim Biophys Acta 1288, F55-F78.
Non-scientific report
In the present project we aim to develop chimaeric VLP candidate vaccines that will cross- protect against more than one type of HPV. Additionally we wish to determine if plant-produced VLPs are as efficient as vaccines produced by the conventional method in insect cells by comparing the immune response elicited in mice when they are vaccinated with these VLPs.
We designed chimaeras which consist of the major HPV16 coat protein, L1, that have sequences from the minor capsid protein, L2, inserted at 2 sites. The three L2 amino acid sequences 17-36, 56-81 and 108-120 have previously been shown to have cross-protection abilities. That means if one is vaccinated with a HPV 16 chimaera one is potentially also protected against other HPV types such as HPV18 and HPV 52.
The conserved L2 amino acid regions were subcloned into the HPV-16 L1 surface C- and E-loops. Each C- and E-loop chimaera was cloned into four plant expression vectors: pTRAc, pTRAcTP, pRIC3 and pEAQ. pTRAc and pTRAc-cTP, target proteins to the cytoplasm and chloroplast, respectively. The pEAQ expression vector was used as it has shown high expression levels in previous studies. pRIC3.0, a self-replicating vector, was also used to determine if higher protein expression levels would be achieved. Each of these plant expression vectors provides the opportunity to make a comparison between the expression levels of the different chimaeras and if protein targeting to different cell compartments plays a role in expression.
Tobacco plants have been infiltrated with Agrobacterium each harboring the C- and E-loop chimaeras, in all four different plant expression vectors. Plant leaf discs were harvested at time intervals of 1, 3, 5 and 7 days post-infiltration to find the optimal day for extraction. Levels of protein expression were determined by comparing density levels of bands on gels. Transmission electron microscopy was used to look at the structural integrity of the chimaeras and the formation of VLPs or capsomeres was visualised. It was found that the best expression occurred at 3/5 days post infiltration at an Agrobacterium optical density of 0.5 or 1.0. The structural integrity of the VLPs produced from each chimaera was investigated by looking at crude extracts under a transmission electron microscope. Furthermore, the localization of the VLPs in the plant cells were also looked at by using confocal microscopy and sectioning of leaves.
Once the structural integrity and the localization of the VLPs were confirmed, the plant-produced VLPs were purified by ultracentrifugation. For this we had to optimize the protocol, testing three different buffers. With the optimized protocol we could purify structurally intact VLPs from the tobacco plants. We will now scale up production and purification of VLPs and use them to immunise mice. Sera form mice will be used to test if the VLPs protect against more than one type of HPV.
We have also produced HPV types 11, 31 and 58 pseudovirions that will be used in pseudovirion-based neutralization assays (PBNA). Pseudovirions are basically VLPs that harbor a reporter gene inside the capsid, and are commonly used in PBNAs to test wether candidate vaccines are able to neutralize the PSVs, visualized by the loss of reporter gene expression. The PSVs produced in this study are stored away and will be used later once mice have been immunized with our plant-produced VLPs, and will be compared to the VLPs produced in insect cells.
The same chimaeras that are produced in tobacco plants will also be expressed in insect cells via recombinant baculovirus for comparison. The insect cell produced chimaeras will be used as the standard to compare the plant produced chimaeras to. Insect cell produced HPV VLPs are deemed the gold standard as one of the human vaccines, CervarixTM (GSK) on the market presently is produced in insect cells. The chimaeras have been subcloned into the pFastbac1, a baculovirus expression vector. Insect cell lines are currently growing and transfection with the chimaeras will commence soon. Optimisation of concentration and cell harvesting (as done in plants) will be performed.