One of the authors, Karen Logan, is now employed at a commercial company Emergent BioSolutions, but her contributions to the work described in this manuscript were made while an employee of Imperial College. The company has never had or has any interests commercial or otherwise in this manuscript. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: AB FL SS LK GD SP. Performed the experiments: AB AM SH TP TA. Analyzed the data: AB AM KL LK GD SP. Contributed reagents/materials/analysis tools: AM TP TA SS FL. Wrote the paper: AB AM SH TP TA VB CH KL LK GD SP.
Current address: Emergent BioSolutions, Emergent Product Development UK Ltd., Wokingham, United Kingdom
¶ Members of a Collaboration for AIDS Vaccine Discovery (CAVD) consortium.
High mutation rates of human immunodeficiency virus (HIV) allows escape from T cell recognition preventing development of effective T cell vaccines. Vaccines that induce diverse T cell immune responses would help overcome this problem. Using SIV gag as a model vaccine, we investigated two approaches to increase the breadth of the CD8 T cell response. Namely, fusion of vaccine genes to ubiquitin to target the proteasome and increase levels of MHC class I peptide complexes and gene fragmentation to overcome competition between epitopes for presentation and recognition.
Three vaccines were compared: full-length unmodified SIV-mac239 gag, full-length gag fused at the N-terminus to ubiquitin and 7 gag fragments of equal size spanning the whole of gag with ubiquitin-fused to the N-terminus of each fragment. Genes were cloned into a replication defective adenovirus vector and immunogenicity assessed in an
Fragmentation but not fusion with ubiquitin increases the breadth of the CD8 T vaccine response against SIV-mac239 gag. Thus gene fragmentation of HIV vaccines may maximise responses.
For human immunodeficiency virus (HIV) and its simian counterpart (SIV), the role of cytotoxic T lymphocyte (CTL) responses in controlling viral replication and disease progression has been highlighted in numerous studies (reviewed in
One reason for the failure of CD8 T cell inducing HIV/SIV vaccines to induce protective immunity may reflect the limited breadth of the response. Primary CD8 T cell responses to pathogens, including HIV and SIV, and vaccines tend to be focused on very few epitopes, with most of the response directed against a single dominant epitope and lesser responses against one or two subdominant epitopes
One strategy to overcome immune escape and enable virus control is to develop vaccines that induce CD8 T cells against multiple epitopes. Several vaccine strategies have been described to overcome immunodominance and stimulate a broader CD8 T cell response. For example, a polyvalent mosaic immunogen approach
Although both ubiquitination and gene fragmentation have been described previously in the context of vaccination in mice
SIV-mac251 codon optimised gag gene was used as a model gene to study whether ubiquitination and gene fragmentation diversify the T cell response. An
Recombinant Ad5 vectors were generated by insertion of mRNA sequence optimised Ub(G76V)-fused HA-tagged SIV gag genes (GeneArt, Germany) into Ad5 shuttle vectors (pShuttle(−) from Capital Biosciences USA) and ligation with Ad5 vector backbones, followed by large-scale production in packaging cell lines and virus purification (Vector Biolabs, USA). A schematic representation of the different vector constructs used in this study is shown in
Blood leukocyte cones, products of leukapheresis, were purchased from the North London Blood Transfusion Service (London, UK). Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Lymphoprep™ 1077 (PAA Laboratories GmbH, UK) and then separated into a low-density fraction enriched for monocytes and a high-density fraction enriched in T lymphocytes by further centrifugation over a 50% Percoll gradient (Sigma-Aldrich, UK). Both fractions were aliquoted, frozen, and stored in liquid nitrogen until further use.
Human monocyte-derived DC (moDC) were generated as previously described by Sallusto and Lanzavechia
Between 105 and 106 DC were transduced with the different Ad5 vectors separately. For each sample donor, DC were divided into 9 fractions of 100 µl each of serum-free RPMI-1640. They were transduced at 25 pfu of Ad5 vector/cell and incubated for 1 hour at 37°C. For some experiments, replication defective Ad5 with no insert (Ad5-empty) was used at 2500 vp/cell (kindly donated by Dan Barouch, MIT and Harvard, Boston, USA). Cells were then harvested and resuspended at a final concentration of 105 cells/ml in culture medium (RPMI-1640 supplemented with 5% pooled human AB serum [Sigma Aldrich, UK], 1% penicillin, 1% streptomycin and 1% L-glutamine [PAA Laboratories, UK]). Cells were incubated for 24 hours then matured overnight with bacterial lipoplysacharide (LPS, 1 µg/ml, Sigma Aldrich, UK) and IFN-γ (1000 U/ml, Miltenyi Biotec, Germany). To assess targeting of vaccine genes to the proteasome, DC were transduced with the different Ad5 constructs, cultured in the presence or absence of LPS and IFN-γ for 24 h and then cultured for a further 24 h in the presence or absence of 10 µM of the proteasomal inhibitor MG132 (Sigma Aldrich, UK). The cellular level of transgene protein was inferred by measuring HA expression. DC were stained with anti-HA tag-PE antibody (Abcam, UK) in the presence of 0.5% Saponin (Sigma Aldrich, UK) in PBS. Cells were then fixed with 4% paraformaldehyde (Sigma Aldrich, UK) in PBS and analysed by flow cytometry.
Autologous naïve T cells were isolated by negative selection using magnetic beads from the high density Percoll fraction. First, total T cells were enriched using the Pan T cell isolation kit II (Miltenyi Biotec, Germany). CD45RO positive memory T cells were subsequently depleted using CD45RO microbeads (Miltenyi Biotec, Germany) according to the manufacturer's guidelines. The purity of isolated cells was found to be greater than 90% as assessed by double staining with antibodies against CCR7 (R&D systems, UK) and CD45RA (BD Biosciences, UK) by flow cytometry.
Antigen-specific naïve T cells were primed and expanded as previously described in
After 3–4 weeks of T cell expansion, cells were harvested, washed twice and rested in cytokine-free culture medium for 48 hours. For some experiments CD4 T cells were depleted using CD4 Dynabeads (Invitrogen, UK) according to the manufacturer's guidelines. Cells were recounted and mixed with autologous PBMC at a 1∶1 ratio in culture medium. 96-well Polyvinylidene fluoride (PVDF) membrane plates (MSIPS4510, Millipore, UK) were activated with 70% ethanol for 4 minutes. Plates were washed 3 times with sterile PBS and coated overnight with mouse anti-human IFN-γ monoclonal antibody (10 µg/ml, clone 1-D1K, Mabtech AB, Sweden) in sterile PBS. Plates were washed then blocked for at least 2 hours with RMPI-1640 containing 10% human AB serum. After further washing cells were added at a concentration of 105 cells/well. One hundred and twenty five individual SIV-mac239 gag overlapping peptides (15-mers overlapping by 11aa, obtained from the NIH AIDS Reference Reagent Program. Sequences of peptides 1–125 are given at
DC were transduced with the different Ad5 vectors as previously indicated. Cells were matured overnight with LPS and IFN-γ followed by co-culture with autologous expanded T cells and after 3 h brefeldin A was added and culture continued for a further 15 h. Cells were harvested and surface stained for 20 mins at 4°C with anti-CCR7-PE (R&D Systems, UK), CD3-PE-Cy5, CD45RA-APC, CD8-APC-H7, CD4-HorizonV500 (all from BD Biosciences, UK). Cells were washed and fixed with BD Stabilising fixative (BD Biosciences, UK) for 10 mins, washed and resuspended in 0.5% Saponin (Sigma Aldrich, UK) in PBS (PAA Laboratories, UK). They were then stained for 30 mins at room temperature with anti-IFN-γ-PE-Cy7, IL-2-HorizonV450, and TNF-α-FITC antibodies. Finally, cells were washed and fixed with BD stabilising fixative (BD Biosciences, UK). Cells were acquired using a 3-laser configuration LSRII flow cytometer (BD Biosciences, USA). Cytokine secretion by antigen specific T cells were analysed by FlowJo (Tree Star Inc, USA). The gating strategy for the Identification of multifunctional T cells was performed as previously published in
Confluent A549 cells were infected for 24 h with 10 pfu/cell of each Ad5 construct. Cells were then harvested or actinomycin D (8 µg/ml) added and cells incubated at 37°C for a further 3 h before harvesting. mRNA was extracted using the Miltenyi Biotec mRNA isolation kit and treated with DNase prior to reverse transcription to synthesise cDNA employing AMV reverse transcriptase and oligo-dT. Quantitative real time PCR was performed with a Roche 4 LightCycler 480 and the LightCycler 480 Syber Green master kit (Roche, UK) with the primers shown in
Data depicted in the figures are shown as means ± standard deviation using Graphpad Prism 5 (GraphPad Software, San Diego, CA, USA). Statistical evaluations were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). A one-way ANOVA (repeated-measures) was employed to assess differences between groups. When significance was obtained (p values below 0.05), a Fisher LSD post-hoc test was used to examine pairwise comparisons.
In order to detect expression of SIV-gag gene fragments within DC, all genes used in this study were tagged at the 3′ prime end with a hemaglutinin (HA) sequence (
DC were transduced with Ad5 constructs for 24 hours and cultured in the presence or absence of LPS and IFN-γ for an additional 24 hours. Untransduced DC were used as controls. MG132, a proteosomal inhibitor, was added to half of the samples in the last 16 hours of culture. Expression of HA intracellularily was monitored by flow cytometry. Representative dot plot profiles are shown in
To ensure responses were not due to major differences in expression or stability of mRNA of different constructs, A549 cells, an epithelial cell line, were transfected with equal infectious units of each construct and after 24 h mRNA was extracted or transcription inhibited by addition of actinmycin D for 3 h prior to mRNA extraction. The levels of mRNA expressed relative to that of β actin were determined for each construct and the relative amount of mRNA for each modified gag construct compared with that for full length unmodified gag. In two independent experiments there were no marked differences in transgene mRNA expression, apart from a somewhat higher expression of MF2, between the different modified gag constructs and that of mRNA for full length gag (
Isolated naïve T cells were stained for expression of CD4, CD8, CD45RA and CCR7 receptors prior to co-culture with Ad5-transduced autologous DC. Expanded T cells were also analysed for expression of these markers on a weekly basis. As shown in
Purified naїve T cells were primed and boosted with Ad5-trasnduced DC expressing non-ubiquitinated full-length SIV-gag (0xUb), ubiquitinated full-length SIV-gag (1xUb), or ubiquitinated SIV-gag mini genes (MF1–MF7). A) The percentages of expanded CD3+ T lymphocytes expressing CD4 (open bars) or CD8 (Gray bars) are shown on day 0, day 7, day 14 and day 21 post initial DC-T cell priming. B) the proportions of CD3+ CD8+ T cell subsets out of total CD8 T cells that were CCR7+ CD45RA+ (Naїve T cells, gray bars), CCR7− CD45RA+ (Terminal effector cells [TEMRA], white bars), CCR7−CD45RA− (Effector memory [EM], hatched bars), and CCR7+ CD45RA− (Central Memory [CM], closed bars) are shown for days 0, 7, 14 and 21 post initial DC-T cell co-cultures. C) same as B except that data show memory differentiation of cytokine (IFN-γ, IL-2, or TNF-α+) CD3+ CD8+ cells on day 7, 14, and 21 in response to overnight stimulation with transduced mature DC. Bars show mean values out of four samples ± standard deviations.
Fusion with ubiquitin would be expected to preferentially target the antigen to the MHC class I pathway, possibly at the expense of the class II pathway, and since CD4 T cells are required for development and maintenance of CD8 T cell memory we asked whether fusion to ubiquitin or fragmentation would influence the CD8 T cell memory phenotype. Expanded CD8 T cells were stained with antibodies against CCR7 and CD45RA to identify the different differentiation stages of T cell memory development. CD8 T cells that were CCR7+ CD45RA+, CCR7− CD45RA+, CCR7−CD45RA−, and CCR7+CD45RA− were defined as naïve, terminal effector (TEMRA), effector memory (EM), and central memory (CM) T cells respectively
For the total naïve CD8 T cell population co-culture with Ad5-SIV-gag-transduced DC resulted in their differentiation into terminal effector and effector memory cells in similar proportions after 7 days (
We also addressed the question of whether ubiquitination or gene fragmentation would alter the cytokine profiles of memory CD4 and CD8 T cells. T cells were primed and expanded as previously indicated using the different Ad5 vectors. IFN-γ, IL-2 and TNF-α production by CD4 and CD8 T cells was measured by intracellular cytokine staining on a weekly basis in response to overnight culture with DC expressing the respective SIV gag genes. As illustrated in
Purified naive T cells were primed and boosted weekly with Ad5-trasnduced DC expressing non-ubiquitinated full-length SIV-gag (0xUb), ubiquitinated full-length SIV-gag (1xUb), or ubiquitinated SIV-gag mini genes (MF1–MF7). T Cells were restimulated overnight with Ad5-trasnduced mature DC expressing the respective genes on days 6, 13, and 20 post initial DC-T cell priming. The percentages of IFN-γ (open bars), IL-2 (Gray bars), and TNF-α (hatched bars) producing CD8 (left panels) and CD4 T cells (right panels) are shown in A). The proportions of CD8 (left panels) and CD4 T cells (right panels) that were producing a single cytokine (open bars), two cytokines (gray bars), or all three cytokines (hatched bars) out of the total population of cytokine producing cells are shown in B). For both A and B, bars represent mean values out of four samples whilst error bars represent standard deviations.
We also addressed the polyfunctionality of these responses weekly. The majority of antigen-specific CD4 and CD8 T cells produced a single cytokine (around 80% of the response,
Although no major differences were observed between the different constructs utilised in terms of T cell memory differentiation or cytokine production, it is probable that these responses include Ad5-specific T cells which may obscure SIV-specific T cell responses. To address this issue, we repeated T cell priming and expansion using DC that were transduced with either Ad5 with no insert (Ad5-empty) or Ad5 expressing 0xUb-gag using cells from four donors. We monitored T cell memory differentiation and cytokine production over a four week period (Figures S3 and S4). No differences were observed in CD4/CD8 T cell ratios between cultures primed with Ad5-empty or Ad5-0xUb gag (
The specificity of T cells that were primed and expanded against Ad5-empty or Ad5-0xUb-gag were assessed on a weekly basis by intracellular cytokine staining (
Since we were unable to distinguish SIV-specific responses from those generated against the Ad5 vector using intracellular cytokine staining, we assessed the specificity and breadth of responses against SIV transgenes from the individual vectors by performing ELISPOT assays using individual peptides (15 mers overlapping by 11 aa) spanning the entirety of SIV-mac239 gag were used to stimulate the expanded T cells in IFN-γ ELISPOT assays (
Purified naive T cells were primed and boosted weekly with Ad5-transduced DC expressing non-ubiquitinated full-length SIV-gag (0xUb), ubiquitinated full-length SIV-gag (1xUb), or ubiquitinated SIV-gag mini genes (MF1–MF7) separately. SIV-specific T cell responses against 125 overlapping SIV-gag peptides were measured using IFN-γ ELISPOT assays on day 21 post initial DC-T cell priming. Spot forming cells (SFC) per one million T cells in cultures that were initially stimulated with 0xUb (closed bars), 1xUb (open bars), or SIV-gag mini fragments (gray bars) are shown for a representative sample in the left panel, where each graph from top to bottom shows responses against individual SIV peptides spanning the mini-fragment regions MF1 to MF7. The data from 4 independent samples are summarised in the right hand side tables where strong responses (>600 SFC/106 cells) are shown in black and medium responses (>400 SFC/106 cells) are highlighted in gray. All data were corrected for background IFN-γ production in response to mock stimulations. Note that sample A represents CD8 specific T cell responses whereas samples B, C, D represent responses using a mixture of CD4 and CD8 T cells. Sequences of all 125 peptides numbered as in these experiments may be found at
Overall, there was a mean frequency of 16.5 peptides (range 2–29 peptides) recognised by cells expanded using vectors expressing non-ubiquitinated full-length SIV-gag (
Number of peptides recognised (>400 SFC/10E6 cells) | Number of Strong positive responses (>600 SFC /10E6 cells) | |||||||||||
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0xUb | 1xUb | MF | 0xUb | 1xUb | MF | ||||||
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22 | 4 | 15 | 10 | 3 | 8 | ||||||
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13 | 3 | 18 | 3 | 1 | 9 | ||||||
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29 | 7 | 14 | 7 | 0 | 4 | ||||||
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2 | 0 | 2 | 0 | 0 | 0 | ||||||
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Indicates statistically significant differences
Percentage Similarity (%) |
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Construct A | 0xUb | 1xUb | MF | |||
Compared with Construct B | 1xUb | MF | 0xUb | MF | 0xUb | 1xUb |
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13.64 | 22.73 | 75 | 50 | 33.33 | 13.33 |
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7.69 | 7.69 | 33.33 | 0 | 5.56 | 0 |
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20.69 | 10.34 | 85.71 | 14.29 | 21.43 | 7.14 |
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0 | 0 | n.a. | n.a. | 0 | 0 |
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The percentage similarity amongst the constructs used in this study was calculated as follow: the number of common peptides that are recognised by both construct A and B divided by the total number of peptides recognised by construct A multiplied by 100. n.a. not applicable.
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
We anticipated that increased degradation of protein via the ubiquitin-proteasome (UPS) pathway (reviewed in
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 (
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
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
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
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