FAIR-CT96-3046 Production of recombinant immune complexes in transgenic plants for systemic and mucosal vaccines

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

	Contract No:	FAIR-CT96-3046
	Date Prepared:	January 2000
	Source:	Second Annual Progress Report

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Second Annual Progress Report Abstract

Objectives

The general objective of this proposal is to establish a system for molecular farming of vaccines in plants using genetically linked immune complexes, which could be suitable for both systemic and mucosal delivery. The specific objectives are:

• To engineer genetic constructs encoding antigen-antibody fusion proteins. Three model antigens will be used, tetanus toxoid, human immunodefeciency virus gp120 and bovine respiratory syncytial virus (BRSV) F-protein, with their respective specific monoclonal antibodies. For each antigen, 3 constructs will be made, in which the antigen will be fused to either the C- terminus of a MAb full length heavy chain, or to truncated heavy chains consisting of I or 2 constant region domains.
• To generate transgenic plants expressing 3 molecular forms of immune complex for each antigen.
• To determine which molecular form of immune complex is assembled correctly and expressed at the highest levels and to extract and purify recombinant material.
• To raise a panel of tetanus toxoid and HIV gp120 specific human T-cell clones from immune and non-immune donors.
• To characterise the plant derived immune complexes - by analysis of assembled molecular forms, recognition by and affinity for a panel of antibodies, analysis of glycosylation and ability to activate complement.
• To perform in vitro analysis of antigenicity of selected plant derived immune complexes in comparison to antigen administered either alone or as conventional immune complexes.
• To perform systemic and mucosal immunisation studies in mice to compare the plant derived immune complexes with antigen administered with adjuvant, as conventional immune complexes or as immune stimulating complexes (ISCOMs).

Activities

By using three different antigens, it will be possible to determine the efficacy of using transgenic plants to produce recombinant immune complex based molecules for a standard systemic bacterial immunogen, the envelope protein of an important human virus and a transmembrane fusion protein of a cattle virus that causes severe mucosal infections. The plant derived compounds will be compared with vaccine candidates that already exist, using an in vitro technique that will examine the fine specificity of antigen processing and presentation to a panel of T cells clones. The use of plant immune complexes to induce systemic as well as mucosal immune responses in vivo will be investigated.

Progress

The project is running close to schedule.

Year 1:-Despite some difficulties with obtaining relevant genes and cloning of these genes, 9 of the 12 proposed genetic constructs have been engineered and introduced into transgenic plants. The 3 remaining constructs are nearing completion and will be introduced into plants in the next 3 months. Over 500 potential transgenic plants have been produced in culture or are growing in the green house. Screening procedures have been initiated in order to determine which plants are expressing correct recombinant proteins. Preparation for analysis for the recombinant plant proteins has progressed smoothly and panels of human T-cell clones to tetanus toxoid and HIV gp 120 have been completed. In addition an early start towards optimising procedures for the immunisation studies in mice to be carried out towards the end of the project has been made. Success has also been achieved in preparing some conventional immune complexes against which the recombinant plant products will be compared.

Year 2:- Genetic engineering of DNA constructs has been completed, and all initial constructs have been inserted into transgenic plants. Preliminary characterisation of all gp 120 constructs have been carried out, the results of which show an increasing level of expression from plants expressing gp I 2O alone, gp 120-KDEL, CH2-gp 120, CH2-CH3-gp 120 and Heavy chain-gp 120. The expression of the fusion protein comprising gpl20 and murine/human immunoglobulin domains has been confirmed and optimal strategies for extraction and purification and begun preparation of material for immunisation studies have been determined. The constructs based on tetanus toxin and BRSV-F protein have also been prepared, but based on the gpl2O results, it has been decided to focus efforts on the CH2-CH3-antigen and heavy chain-antigen constructs. New T cell lines and clones obtained from human donors have been developed. In particular CD4 lymphocytes specific for antigens of HIV we have selected in vitro and expanded. In addition to T cell lines specific for envelope gp 1. 20 and for reverse transcriptase p66, CD4 T cell lines from naive donors have been generated by using a panel of different gp 120 proteins derived from different viral strains. The identification of the immunodominant epitopes is currently in progress using a panel of overlapping peptides. Finally, antigens and immune complexes have been prepared by conventional techniques in readiness for the comparative immunisation studies in mice. Furthermore procedures for systemic and mucosal immunisation, analysis of immune response have been established and preliminary dose response studies using gp 120 have been carried out in mice.

Achievements

Year 1:

• DNA sequence confirmation of 9 out of 12 clones genetic constructs for HIV gp 120, tetanus toxoid and BRSV protein-F.
• Production of panels of human T-cell clones to HIV gp 120 and tetanus toxoid.
• Preparation of conventional immune complexes of HIV gp 120 and tetanus toxoid.

Year 2:

• Completion of genetic engineering
• Generation of transgenic plants expressing all forms of gp 120 and 4/6 constructs for tetanus toxoid and BRSV-F protein.
• Initial characterisation of recombinant gp 120 constructs expressed in transgenic plants.
• Determination and optimisation of purification procedures.
• Preparation of conventional antigens
• Expansion of range of human T-cell clones and lines to HIV gp 120.
• Establishment of procedures for murine immunisation studies.

Future activities

Of immediate urgency is the reckoning of the heavy chain-tetanus toxin genetic construct and reinsertion into plants. The development of the BRSV-F-protein plants will continue, while scale up purification of gpl2O constructs for antigenicity and immunisation studies will be initiated. The project will also focus on the systemic and mucosal murine immunisation studies planned for the third year.