| Summary: | The aim of this project was to assess the expression, purification and immunogenicity in mice of two important HIV antigens, the core protein p24 and the regulatory protein Nef, produced in transplastomic <i>Nicotiana tabacum </i>plants. A comparison of p24-expressing tobacco Petite Havana transplastomic plants, containing gene constructs with different 5’ untranslated regions (UTRs), demonstrated the importance of regulatory and stabilizing elements. Plants containing a p24 construct with a bacteriophage T7 gene 10 (<i>T7g10</i>) 5’ UTR and generating a protein with 5 extra amino acids at the N-terminus, accumulated almost twice as much protein as plants containing other constructs. Analysis of p24 accumulation in leaves of the high biomass tobacco variety Maryland Mammoth revealed a dramatic difference between a transplastomic line containing a codon-optimized construct encoding five extra N-terminal amino acids, which accumulated p24 in all leaves of the plant, and a transplastomic line containing a native p24 construct which accumulated p24 only in young leaves. To assess the subcutaneous and nasal immunogenicity of plant-derived p24, the protein was purified to ~95% homogeneity from Maryland Mammoth transplastomic plants. After immunization of experimental mice, ELISAs for p24-specific IgG and T-cell proliferation assays showed that chloroplast p24 could elicit a strong humoral response following subcutaneous administration and a cellular response following intranasal administration. Oral administration of partially purified tobacco p24-Nef as a booster, after subcutaneous priming with either p24 or Nef, elicited strong humoral and cellular responses, with both IgG1 and IgG2 subtypes found in sera. In addition, tobacco BiP was found to be as good an adjuvant as <i>Vibrio cholerae </i>toxin B and <i>Mycobacterium tuberculosis </i>Hsp70 for enhancing the immune response to p24.
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