In a previous study we demonstrated immunologic memory for the peptide expressed by r-GV was elicited, however the assessments of r-GV in that study involved only the expression of peptide sequences from a single SIV gag gene . The ELISA data presented here tested quite different peptide sequences incorporated into the r-GvpC protein and the results indicate that in this context, these too are stimulatory and immune visible. 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. Here we tested peptides encoded by three different SIVsm genes. The Tat protein can function indirectly to promote virus replication via receptor mediated signal transduction, while intracellularly Tat normally is transported into the host cell nucleus. The protein is essential for viral replication and is currently a candidate vaccine component. Rev functions as a nucleocytoplasmic shuttling protein normally found primarily in host cell nuclei/nucleoli. One of its functions is to "escort" unspliced viral mRNA out of the nucleus. In contrast, Nef is a peripheral membrane protein, and appears to have a linker function, among others. Thus Nef mediates a variety of protein-protein interactions via its SH3 binding surface and down regulates MHC class I, and CD4 expression, perhaps through its capacity as a linker. Nef therefore can exert significant effects on the host cell functions [21–27]. From the data presented here, clearly the DNA segments tested, representing sequences from proteins with significant inherent functional differences, were stably incorporated into the gvpC gene. The data also shows the insert peptide is retained in the recombinant GvpC protein, and the chimeric proteins produced are effectively incorporated to produce recombinant gas vesicles. For all three test peptide sequences the functionality of the r-GvpC was shown by the generation of gas vesicles that float, and r-GvpC release from isolated gas vesicles during sample preparation for SDS-PAGE, verifies r-GvpC were incorporation as part of the organelles themselves. Likewise, immunogenicity was demonstrated through the anti-peptide responses elicited. These demonstrated r-GV effectiveness as a delivery vehicle and support the conclusion that irrespective of the gene source, the displayed peptides are immune system visible and immuno-stimulatory. The known structure of GV microparticles and their resistance to degradation, support the suggestion that these functional characteristics of this unusual display system are keys to their effectiveness as an adjuvant.
The basis of this self adjuvanting effect remains to be clarified but characteristics of certain halobacterial proteins may be relevant. Molecular chaperones are essential components of organisms that function as part of the normal physiology, and in response to stress, and pathology. A molecular chaperone system also is present in Halobacterium sp. NRC-1 and represented by the proteins DnaK, DnaJ, GrpE, and Hsps1–4 (small heat shock proteins). The peptide-binding function of Hsp's (heat shock proteins) allows chaperon proteins such as Hsp70 (heat-shock protein 70) to acquire antigenic proteins within cells, even when administered outside the cell and thereby induce priming of CD8+T lymphocytes in vivo. In addition to peptide binding and delivery, Hsp70 also can assist as a carrier of peptides or proteins that are effective immunogens for B cells, CD4+, and CD8+T cells, in the absence of an adjuvant. Thus it has been shown that the C-terminal portion of Hsp70 (amino acids 359–610) induces human monocytes to produce CC chemokines (chemotactic cytokines), IL-12 (interleukin-12), TNF a (tumour necrosis factor alpha), as well as stimulating the maturation of dendritic cells . Additionally there is evidence that for microbial Hsp, this C-terminal portion of Hsp70 is involved as an adjuvant in the induction of the innate and adaptive elements of the immune response .
The slow degradation of wt- and r-GV could readily initiate Hsps up-regulation in the host cell. Therefore, the self-adjuvanting characteristics of gas vesicles could be analogous to the chaperon activities of Hsp 70, produced by the host cells in response to perturbation by the gas vesicle presence, and in fact there is a notable similarity between the GvpC protein and the DnaK and DnaJ proteins. In addition, or alternatively, the geometric display on gas vesicle surfaces may be analogous to the display achieved when bacterial S-layers are used. Such displays are known to enhance immune responses .
Inherent features of Halobacterium sp. NRC-1 and their biosynthesis of gas vesicles are highly relevant to biotechnological utilities and these are evident here, in the steps to generate and test the recombinant organelles. The plasmid pFM101d allows simple insertion of exogenous DNA cassettes and subsequent transfer into the Halobacterium strain SD109 transformation vector. A simple restriction digest cleavage of the rightward operon is followed by ligation into pMS104d, the large (18.9 kb) plasmid used to transfer the 14 genes needed for gas vesicle biogenesis. As application of simple agarose gel analysis showed, successful SD109 transformation can be readily assessed via EtBr (Ethidium Bromide) staining to verify amplification products with the appropriate bp sizes. The actual transformation of plasmid recipient is readily achieved by a one step treatment of the Vac- SD109 strain with spheroplasting solution. A cell wall, such as is characteristic of gram positive or gram negative bacteria is not present in these archaeal organisms. This fact simplifies transformation as well as the subsequent gas vesicle isolation processes. Further, spheroplast formation itself can be verified by simple microscopic examination of the treated halobacterial samples. The SD109 cells undergo a shape change and exhibit a spherical rather than the normal rod morphology (Figure 3). Such spheroplasts were readily transformed without additional treatments by simple incubation with isolated pMS104D::tat ::rev:: or :: nef1 DNA. Similarly, successful transformation and appropriate function of the genes transferred by the transforming plasmid is also easily determined. For liquid media, the formation of "creamy-white" halobacterial cultures vs. the rust brown of Vac- SD109 cultures is diagnostic for gas vesicle production and readily evident by simple visual inspection as seen in Figure 4.
The specific verifications provided here are keys in the context of validating this system and the peptides tested. First, as clearly shown, recombinant GvpC expressed by the vesicles demonstrate an expected decrease in electrophoretic mobility vis-à-vis GvpC from gas vesicles produced by wild type Halobacterium sp. NRC-1. Second, in the present study, the gene segments inserted into the gvpC encode peptides from proteins exemplifying a variety of functions, and as Figures 5, 6, 7 showed, when displayed by r-GvpC, each one was recognized not only by anti-GvpC antibody (Figure 5), validating retention of the r-GvpC protein, but also by antibody elicited in vivo during infection of monkeys with SHIV virus (Figures 6, 7). Since the anti-SHIV antibody did not recognize wild type GV protein (Figure 6B), the incorporated SIV peptides were being specifically identified when displayed as part of the r-GvpC protein which is present at sites all over the gas vesicle surface. Thus the insert into the GvpC protein is both immune system visible and accessible. Equally important, display of the SIV peptides in this manner did not abrogate their recognition by appropriate antisera. Finally, it is also highly relevant that as the titration of sera collected 43 weeks post re-immunization shows, titers remained for all three peptides although at 1:160, the anti-Rev titer was the lowest of the three. However, the titer for Rev likely reflects the fact that only one animal in this group was extant. At this very late time point the titers of the animals immunized with Nef1 and Tat r-GV (~1:640 and 1,280) were very notably higher.
From the perspective of general utility and ease of recombinant production, the characteristic inherent in the system used here and the effective incorporation of exogenous pathogen DNA sequences, both were demonstrated in the course of generating the recombinant gas vesicles. The features critical to their unusual utility include characteristics inherent to the gas vesicle organelle itself as well as the Halobacteria sp. in which they form. They are relevant to r-GV use in biotechnology applications such as a pathogen peptide display and/or delivery system. Thus, despite the unusual intracellular environment that halophiles maintain, and the unusual nature of their native proteins [31, 32], the pathogen gene sequences were retained and also translated with fidelity in the halobacteria SD109 expression system as demonstrated by SHIV antibody recognition. The recombinant GvpC proteins themselves are incorporated into the gas vesicle structures as demonstrated by r-GvpC release from isolated organelles and recognition by both anti-GvpC and anti-SHIV antibody. Inherently, the GvpC protein, as the "molecular glue" for the organelle, is present at the surface of recombinant gas vesicles . In addition, as previously published electron micrograph show, this surface intrinsically exhibits a highly organized, patterned surface [7, 18]. Finally, gas vesicle formation itself is quality controlled by the gvp gene cluster carried by the transformed SD109 strain and release of the organelles themselves is easily achieved by simply suspending the halobacteria in water with 1.0 mM MgSO4.