FAIR-CT95-0720 The Plant as a Factory for the Production of Oral Vaccines and Diagnostics

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Biotechnology : FAIR Area 3 - Generic Science and Advanced Technologies for Nutritious Foods : Pharmaceuticals/Cosmetics : Plant Genetics

	Contract No:	FAIR-CT95-0720
	Date Prepared:	November 1998
	Source:	Final Report Abstract

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Final Report Abstract

Note: The project coordinator Axis Genetics has ceased trading

Introduction

For many years the pharmaceutical industry has been extremely interested in the production of oral vaccines. To date the only successful oral vaccines are based on live, attenuated pathogens (e.g. polio virus and Salmonella), which have either the potential to revert to the virulent form or are associated with concerns with respect to environmental safety. This has led to attempts to develop inert vaccines but these have not been successful as very large amounts of a vaccine are required per dose due to dilution and degradation in the harsh conditions of the alimentary tract. Current methods for producing large amounts of a vaccine are economically prohibitive. The goal of this project was to overcome these obstacles using plants for the production of oral vaccines based on Axis Genetics' EPICOAT® technology. This technology utilises cowpea mosaic virus (CPMV) and molecular biology techniques to produce plant chimaeric virus particles (CVPs) that express epitopes from viral pathogens on its. surface. These CVPs can be easily obtained in large quantities and it is possible to envisage the production in plants of many vaccines and other pharmaceuticals at a very low cost.

The objectives of this project were essentially to identify epitopes that could be used with EPICOAT, to develop and evaluate the feasibility of edible oral vaccines based on CVPs and to develop and evaluate the feasibility of developing diagnostic reagents based on CVPs.

The initial disease targets for evaluation for both vaccine and diagnostic development included canine parvovirus (CPV), porcine parvovirus (PPV), rabbit haemorrhagic disease (RHDV), African horsesickness virus (AHSV) and Aleutian disease virus (ADV) of minks. Work was carried out on all of these viruses but it was determined that PPV and ADV were not suitable for further evaluation.

PEPSCAN™ analysis resulted in the identification of nine antigenic sites in the PPV capsid protein, but only one elicited a low level neutralising antibody titre when used to immunise pigs as peptide- KLH conjugates. It is now known that ADV gives rise to an immune complex disease in which vaccination can make animals worse. Although it was shown that the immunogenicity of the ADV VP2 terminal peptide could be enhanced, the protective effect was found to be transient. Although the ADV non-structural proteins may be useful as a source of antigenic determinants, no suitable epitopes were defined. Thus after preliminary work on these two viruses, it was decided not pursue them further.

The EPICOAT technology harnessed plant viruses as a peptide display system. Plant viruses are genetically modified to display multiple copies of an immunogenic peptide on their surface. The resultant chimaeric virus particles (CVPs) are multiplied within the plant and extracted for use as vaccines, delivered nasally, orally or by injection.

The technology utilises cowpea mosaic virus (CPMV), a plant virus that infects the cowpea plant, Vigna unguiculata, otherwise known as the "black-eyed bean" (UK) or "black-eyed pea" (USA). CPMV is the type member of the comovirus group of plant viruses whose genome consists of two positive sense polyadenylated RNA strands, RNA-1 and RNA-2. RNA-1 codes for the proteins involved in replication and processing of the virus whereas RNA-2 codes for the two virus capsid proteins, S (small) and L (large), together with a protein that facilitates virus movement through the plant.

Objectives

The project aimed at using genetically engineered plant viruses to develop oral vaccines and diagnostic reagents. The diseases to be targeted in this project were canine parvovirus, porcine parvovirus (PPV), rabbit haemorrhagic disease (RHDV), African horsesickness (AHSV), and Aleutian disease (ADV). The initial goal of the project was to identify immunogenic peptides from these viruses that when administered to an animal would protect the animal from disease. Once immunogenic peptides were identified, a plant virus (cowpea mosaic virus, CPMV) would be engineered so that it expressed multiple copies of the peptide on its surface. The genetically engineered plant virus is referred to as a chimaeric virus particle (CVP) and it acts a multi-epitope particulate antigen. The CVPs were then evaluated as vaccines and as diagnostic reagents.

Activities

From the results obtained in the early part of the project, it was decided to focus efforts on canine parvovirus, RHDV and AHSV. The canine parvovirus work focused on optimisation of the presentation of a parvovirus epitope on plant virus particles and evaluation of the lead CVP as a vaccine delivered by parenteral or oral administration. A large number of variant CVPs containing the previously defined canine parvovinis epitope were made with the aim of optimising the presentation of the epitope on the surface of the plant virus particle. A challenge study in dogs was been carried out and immunisation studies involving the oral administration of CVPs to a number of different animals have also been conducted. With both RHDV and AHSV, work has concentrated on the identification of viral epitopes using antibodies (monoclonal and polyclonal), developed during the first part of the programme, in conjunction with PEPSCAN analysis and the expression of recombinant protein fragments.

Immunisation studies using a number of different CVP particles expressing the previously defined canine parvovirus epitope indicated that the original CVP, CPMV-PARVO1 was probably the best in terms of eliciting an immune response. This CVP was used in second, more comprehensive, challenge study involving the parenteral immunisation of dogs and subsequent challenge with canine parvovirus. The CVP was treated by UV-irradiation prior to administration in order to destroy its ability to replicate in plants. The irradiated CVP was shown to be at least as efficacious as a vaccine as immunisation with the peptide-KLH conjugate when given as equivalent doses with respect to the amount of peptide.

In this project, the use CVPs as oral immunogens was investigated by testing their capacity to induce humoral immune responses in mice, guinea pigs, rabbits, and mink. We have shown that although the plant virus carrier is highly immunogenic in mice when delivered through parenteral, oral and intranasal routes, antibody responses to the displayed peptide were elicited in a consistent manner only after parenteral or nasal immunisation. A number of different additives and formulations were evaluated for their effect on the oral immunogenicity of the CVPs, but no significant improvements were achieved. Also, the antibody responses after oral immunisation of mink were much lower than those achieved in mice, and protection against disease was at best, partial.

The antigenic and structural topography of the RHDV capsid was dissected using MAbs generated to rRHDV particles and VP60-truncated fragments expressed in E. coli. A combination of MAb and PEPSCAN analysis was then used to identify four potential epitopes in RHDV VP60. Four CVPs were constructed by inserting the selected sequences into the betaB-betaC site in the CPMV S protein. The four CVPs were used to immunise rabbits that were subsequently challenged with RHDV.

The commercial Cylap vaccine was used as a positive control. In summary, none of the treatments, with the exception of the Cylap vaccine, showed any degree of protection in rabbits upon challenge with RHDV. It was concluded that, despite the promising results generated by PEPSCAN analysis, the whole of the VP60 protein is required in order to elicit neutralising antibodies in vivo.

A similar antigenic analysis of AHSV was also conducted. In conclusion, an immunodominant region, comprised between residues 54-269, and eight different antigenic sites were mapped in the AHSV-4 VP5. The N terminus of VP5 seems to be the most immunodominant region and may be the surface area in VP5 that is most exposed and accessible to antibodies. In contrast to the VP5 proteins from other orbiviruses, AHSV VP5 alone is able to induce neutralizing antibodies albeit at lower levels than VP2. Since the defined epitopes are bound by neutralising MAbs and one of them is conserved among different orbiviruses, the amino terminal region of VP5 could be of special interest for inclusion in a new generation of vaccines for AHSV and diagnostic tests for orbiviruses.

CVPs comprising recombinant cowpea mosaic virus displaying peptides derived from canine parvovirus were evaluated as diagnostic reagents for canine parvovirus. Two CVPs were evaluated; CPMV-PARVO1 displayed a peptide corresponding to amino acids 3-19 of the VP2 capsid protein of canine parvovirus, and CPMV-PARVO2 displayed the same peptide in the reverse orientation. Both CVPs were evaluated in an ELISA format for detection of canine parvovirus-specific antibodies in dog sera, and for detection of canine parvovirus-specific antibodies in sera from rabbits immunised with either of the two CVPs or with the canine parvovirus VP2 peptide coupled to keyhole limpet haemocyanin (KLH). In summary, the studies indicate that although the parvovirus epitopes are exposed on the surface of the modified cowpea mosaic virus, in their present configuration they are not a good alternative for canine parvovirus diagnosis.

Future work

Further studies with more immunodominant epitopes need to be carried out to ascertain the real potential of this system for diagnostic applications.

Conclusions

In conclusion, the experiments showed that CPMV-displayed peptides are highly immunogenic when given parenterally or intranasally and are capable of inducing both serum and mucosal immune responses. This ability makes CPMV a promising epitope presentation system. Continued studies on Es oral delivery should focus on stabilising the epitope towards proteolysis in the digestive tract, and on elucidating the uptake mechanism leading to the unusual immunogenicity of these particles. Such an understanding could in term be used for further improvement in immunisation schedule, dosage, and in development of more efficient formulations of this new type of epitope carrier system.

Potentially useful epitopes have now been identified for RHDV, for use in the development of a CVP-based vaccine, and AHSV, for use in the development of a CVP-based diagnostic. Confirmation of the potential of a parvovirus CVP-based vaccine was achieved through a successful dog challenge study. Studies involving the oral administration of purified CVPs have demonstrated the strong immunogenic response of the carrier, although it is likely that the CVPs will have to be formulated correctly in order to protect the particles from loss of the epitope during passage through the stomach.