Data presented in this study addresses two different strategies aimed at broadening T cell responses to codon optimised SIV gag, namely fragmentation and fusion with ubiquitin. A marked increase in the number of epitopes recognised in ubiquitin-fused mini genes compared to ubiquitin-fused full-length gag was observed. Thus gene fragmentation is a potential strategy for improving the breadth of HIV vaccines. Since each gag fragment was fused to ubiquitin the appropriate comparison for assessing the response to fragmentation is with full length gag fused to ubiquitin. However, since fusion of ubiquitin to full length gag greatly reduced responses compared to unmodified full length gag the response to gag fragments that have not been fused to ubiquitin needs to be investigated. To ensure that DC were not transfected with more than one construct, thus potentially increasing competition which fragmentation aimed to reduce, separate cultures transfected with single constructs were performed. For vaccination it would be desirable to deliver different gene fragments to different sites to avoid DC being transduced with more than one fragment. Although the results of the fragmentation experiments were encouraging it is possible that in vitro results may not mirror responses in vivo and vaccination studies are needed to confirm that fragmentation of gag is advantageous. In contrast we found that fusion of gag to ubiquitin reduced the breadth of the T cell response. Fusion of ubiquitin to a number of different pathogen genes including influenza NP 
, LCMV NP 
, HIV nef 
and some but not other malarial antigens 
has been reported to enhance immunogenicity. We found that fusion of ubiquitin to SIV gag resulted in a lower T cell responses both in magnitude and breadth. Interestingly, Fluet and colleagues demonstrated in a murine study that vaccination with the matrix capsid portion of SIV-gag fused at the N-terminus to ubiquitin resulted in a 2–3 fold reduction in the magnitude of T cell responses against SIV-peptide pools compared to unmodified SIV-gag 
. Similarly, an earlier study by Wong et al 
did not show enhanced responses in mice vaccinated with vectors containing unstable gag constructs.
We anticipated that increased degradation of protein via the ubiquitin-proteasome (UPS) pathway (reviewed in 
) would preferentially direct antigen through the MHC class I pathway at the expence of the class II pathway leading to higher proliferation of CD8 T cell over CD4 T cells, a hypothesis similar to that proposed by Wong et al 
. This however was not the case in our system, where the percentages of expanded CD4 and CD8 T cells over time were comparable. However, it is possible that the addition of IL-2, IL-7 and IL-15 throughout the in vitro
culture period may have influenced the differentiation of naïve T cells towards effector and central memory cells (
and references therein). Although there was higher targeting of SIV-gag to the proteasome using ubiquitinated constructs, which would be expected to increase peptide density on DC surface 
, neither ubiquitination nor fragmentation altered the memory differentiation profiles of expanded CD8 T cells. Notably, responses generated against Ad5-empty resulted in a similar memory profile over time suggesting that the experimental setup of this in vitro assay may be the main driving factor for CD8 T cell differentiation.
Our homologous prime/boost strategy was found to induce T cell responses against the Ad5 vector, responses to the inserts were also induced as indicated by IFN-γ ELISPOT against SIV-gag peptides. With the exception of sample A (), which represents antigen specific CD8 T cell responses, insufficient numbers of cells were generated to deplete CD4 T cells from the expanded cells prior to conduction of the ELISPOT assays. Nonetheless, the observation that ubiquitination of SIV-gag resulted in markedly fewer responses compared to its non-ubiquitinated counterpart was consistent throughout the samples. Fragmentation dramatically increased the number of peptides recognised in all samples tested indicating that total T cell responses, and specifically CD8 T cell responses in sample A, were broadened. Though, these responses remained similar or lower than those observed using non-ubiquitinated full length gag.
As fusion with ubiquitin to full-length gag severely reduced the breadth of the response, it is important that the effect of fragmentation is assessed by comparison with ubiquitin-fused full-length gag rather than with unmodified gag. If fragments without the ubiquitin sequence were available and tested, an even broader response may have been observed. Of particular interest was the observation that fragmentation induced many responses against peptides that were not induced by priming with unmodified full-length gag. This may suggest differential antigenic processing induced by fragmentation. A key question is whether these new epitopes whose recognition is stimulated by vectors containing fragmented SIV gag would be expressed by SIV-infected cells in vivo
. Of relevance here are the requirements for inducing a primary T cell response compared to a secondary response. The requirements for a primary response are more stringent than for restimulation of memory cells where it has been proposed that a memory CD8 T cell could be activated by a single MHC class I peptide complex 
. Thus if these new epitopes are expressed at too low a concentration in natural infection to induce a primary response there may be a sufficient amount to activate killing by memory CD8 T cells.
Numerous reports in the literature have shown that ubiquitination of genes induces improved antigen degradation which is translated into increased T cell activation, either in clonal size or avidity of response depending on the conditions of the assays 
. The observation that despite proteasomal targeting ubiquitination of SIV gag genes in our study resulted in loss or narrowing of T cell responses compared to wild type SIV gag is puzzling. Although we cannot rule out the possibility that this reduction may reflect our in vitro system, in vivo murine studies have shown similar results 
. Interestingly we have also found that responses to HIV gag fused to ubiquitin are also reduced (Herath et al unpublished data). Notably, T cells were stimulated on a weekly basis and it remains plausible that shorter stimulation intervals may influence the outcome of ubiquitination. The specificity of T cell responses were identified in this study on the basis of secretion of IFN-γ alone () and it is likely that these may not be fully representative of all T cell responses as indicated by intracellular cytokine staining for TNF-α and IL-2 (). In addition, since we were unable to quantify the number of expanded CTL by tetramer staining or other means, it is difficult to rule out the possibility that ubiquitination may have in fact resulted in expansion of SIV-gag-specific CD8 T cells that do not secrete IFN-γ, IL-2, or TNF-α and are maybe only positive for perforin and granzymes or perhaps neither of these. Nonetheless, CD8 T cells generated in this study were found to produce mainly IFN-γ by day 21 which is when the ELISPOT assays were carried out. In addition, most clinical trials rely on IFN-γ ELISPOTS to determine the immunogenicity of vaccine candidates in the case of HIV and SIV studies 
Genetic fragmentation has previously been suggested as a strategy to overcome antigenic competition and widen the breadth of the T cell response. Employing a similar gag fragmentation strategy to that described here to vaccinate mice, broader immune responses have been induced 
. Here we show that fragmentation is effective at broadening the immune response in a human system. In both studies of Liu et al 
and Singh and Barry 
the fragmented genes were administered as a mixture in a single injection site. Although physically separated, the vectors containing different gene fragments may have been picked up and presented to T cells by the same DC, which may therefore hinder the effects of fragmentation. Thus future in vivo
studies should consider vaccination regimens where minigenes are administered at different anatomical sites to maximise the effects of fragmentation.