Abstract
BackgroundPrevious studies indicated that recombinant gas vesicles (r-GV) from a mutant strain of Halobacterium sp. NRC-1 could express a cassette containing test sequences of SIVmac gag derived DNA, and function as an antigen display/delivery system. Tests using mice indicated that the humoral immune response to the gag encoded sequences evoked immunologic memory in the absence of an exogenous adjuvant.ResultsThe goal of this research was to extend this demonstration to diverse gene sequences by testing recombinant gas vesicles displaying peptides encoded by different SIV genes (SIVtat, rev or nef). Verification that different peptides can be successfully incorporated into the GvpC surface protein of gas vesicle would support a more general biotechnology application of this potential display/delivery system. Selected SIVsm-GvpC fusion peptides were generated by creating and expressing fusion genes, then assessing the resulting recombinant gas vesicles for SIV peptide specific antigenic and immunogenic capabilities. Results from these analyses support three conclusions: (i) Different recombinant gvpC-SIV genes will support the biosynthesis of chimeric, GvpC fusion proteins which are incorporated into the gas vesicles and generate functional organelles. (ii) Monkey antibody elicited by in vivo infection with SHIV recognizes these expressed SIV sequences in the fusion proteins encoded by the gvpC-SIV fusion genes as SIV peptides. (iii) Test of antiserum elicited by immunizing mice with recombinant gas vesicles demonstrated notable and long term antibody titers. The observed level of humoral responses, and the maintenance of elevated responses to, Tat, Rev and Nef1 encoded peptides carried by the respective r-GV, are consistent with the suggestion that in vivo there may be a natural and slow release of epitope over time.ConclusionThe findings therefore suggest that in addition to providing information about these specific inserts, r-GV displaying peptide inserts from other relevant pathogens could have significant biotechnological potential for display and delivery, or serve as a cost effective initial screen of pathogen derived peptides naturally expressed during infections in vivo.
Highlights
Previous studies indicated that recombinant gas vesicles (r-GV) from a mutant strain of Halobacterium sp
Our earlier studies to generate recombinant gas vesicles provided evidence that when inserted into an appropriate site in the gvpC gene, this halobacterial gene would accept Simian Immunodeficiency Virus (SIV) gag gene DNA segments of different lengths and the resultant sub microscopic organelles would express this recombinant protein at the gas vesicle surface [19]
The results presented above extend and strengthen the initial hypothesis that r-GV can serve as a flexible cassette based epitope display/delivery system that supports expression of genes or gene segments encoding peptides from proteins representing diverse functions in their native context
Summary
Previous studies indicated that recombinant gas vesicles (r-GV) from a mutant strain of Halobacterium sp. NRC-1 genome is completely sequenced [4] and the genomic data supports a wide range of studies that enhance its potential for commercial uses, as well as basic biochemical analyses of adaptation to extreme conditions. Both an halobacterium transformation system [5,6] and E. coli/Halobacterium sp. The cell envelope of Halobacterium salinarum consists of a single lipid bilayer membrane surrounded by an S-layer assembled from the cell-surface glycoprotein This fact is important in terms of simple release of gas vesicles, or cytosolic proteins, from the organism. Fourteen gvp genes have been identified as encoding components involved in the genesis of organelles, and the regulation of gvp gene expression has been shown to occur at the transcriptional and translational levels [7,8,9,10]
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