A short hepatitis C virus NS5A peptide expression by AAV vector modulates human T cell activation and reduces vector immunogenicity

Viral vector mediated gene therapies have the potential to treat many human diseases; however, host immune responses against the vector and/or the transgene pose a safety risk to the patients and can negatively impact product efficacy. Thus, novel strategies to reduce vector immunogenicity are critical for advancement of these therapies. T cell activation (TCA) is required for the development of immune responses during gene therapy. We hypothesized that modulation of TCA by incorporating a novel viral immunomodulatory factor into a viral vector may reduce unwanted TCA and immune responses during gene therapy. To test this hypothesis, we identified an immunomodulatory domain of the hepatitis C virus (HCV) NS5A protein and studied the effect of viral vectors expressing NS5A peptide on TCA. Lentiviral vector mediated expression of a short 20-mer peptide derived from the NS5A protein in human T cells was sufficient to inhibit TCA. Synthetic 20-mer NS5A peptide also inhibited TCA in primary human T cells. Mechanistically, the NS5A protein interacted with Lck and inhibited proximal TCR signaling. Importantly, NS5A peptide expression did not cause global T cell signaling dysfunction as distal T cell signaling was not inhibited. Finally, recombinant adeno-associated virus (AAV) vector expressing the 20-mer NS5A peptide reduced both the recall antigen and the TCR-mediated activation of human T cells and did not cause global T cell signaling dysfunction. Together, these data suggest that expression of a 20-mer NS5A peptide by an AAV vector may reduce unwanted TCA and may contribute to lower vector immunogenicity during gene therapy.


INTRODUCTION
Recombinant adeno-associated virus (AAV) vectors are commonly used as gene therapy delivery systems.Currently, there are two U.S. FDA-approved gene therapies that use AAV vector in vivo 1 .Although viral vector mediated in vivo gene therapies (VVIGT) are showing potential to treat various human diseases [2][3][4] , several challenges limit their wide-spread use 5 .Pre-existing immunity and de novo immune responses against the viral vector and transgene product can limit successful clinical translation of VVIGT 6 .Many preclinical and clinical studies have demonstrated activation of both the innate and the adaptive immune responses during gene therapy with common viral vectors derived from AAV, adenovirus, and lentivirus [7][8][9][10] .The host immune response against these viral vectors poses not only a safety risk to subjects receiving these therapies but can also reduce treatment efficacy and limit re-treatment options.While numerous strategies have been proposed to reduce host immune response during VVIGT 11 ; to date, the safety and efficacy of most of these strategies in humans remain unknown.
Currently, immunosuppressive (IS) drugs are commonly used to dampen host immune responses during gene therapy 12,13 .These IS drugs have systemic effect on the immune system and typically cause unwanted effects [14][15][16] .Furthermore, IS drugs have a heterogeneous response in human population 17 , and there are concerns over potential risks associated with the use of IS drugs in certain patient sub-population such as immunocompromised people who may benefit from gene therapy.In addition, vectorspecific and transgene-specific immune responses may arise after cessation of IS drug treatment 13,18,19 .These challenges impede development of gene therapies for many diseases affecting diverse human populations.Thus, developing novel strategies to reduce host immune response against viral vector during gene therapy is essential to overcome these limitations and help advance these therapies.
T cell receptor (TCR)-mediated T cell activation (TCA) is an essential step in generating both the cellular and the humoral immune response 20 .Many viruses have evolved to interfere with TCR signaling and inhibit TCA to evade immune responses and establish persistent infection 21 .Among human RNA viruses, hepatitis C virus (HCV) has a remarkable ability to cause persistent infection.HCV has evolved to inhibit both the innate and the adaptive immune responses by various mechanisms 22,23 .The HCV core protein and the envelope protein (E2) inhibits TCA and the non-structural (NS) proteins 3/4A and 5A inhibits the innate immune response [24][25][26][27][28][29][30] .
The HCV NS5A protein is a multifunctional phosphoprotein and plays an important role in viral replication 25 .Although previous studies have identified a role of NS5A in modulating innate immune response 25,31,32 , its effect on human TCA has not been studied.Here we studied the effect of HCV NS5A protein on human TCR signaling pathways and TCA.We identified a short 20 amino acid domain within the HCV NS5A protein that inhibits TCA.Furthermore, we identified the mechanism by which HCV NS5A inhibits TCA.Finally, we assessed the effect of recombinant AAV vector expressing the 20-mer NS5A peptide on human TCR signaling pathways and TCA.

HCV NS5A expression inhibits TCR-mediated activation of human T cells
To assess the effect of HCV NS5A on T cell activation (TCA), a human CD4+ T cell line (Jurkat) was transduced with a control lentiviral vector (LV) or LV encoding the NS5A protein from two different HCV genotypes (GT), GT-1 or GT-2.Expression of the NS5A protein from both HCV genotypes was detected in Jurkat cells by immunoblot analysis (Fig. 1a).Following T cell receptor (TCR) engagement with anti-CD3/CD28, TCA was measured by assessing IL-2 release.Interestingly, expression of NS5A protein from GT-1 but not GT-2 resulted in inhibition of TCR-mediated TCA compared to Jurkat cells transduced with control LV expressing GFP alone (Fig. 1b).There are seven genotypes of HCV 33 and NS5A protein has considerable differences in amino acid sequence among HCV genotypes 34 .The observation that the NS5A protein from GT-1 but not GT-2 inhibited TCA prompted us to further assess both proteins to better understand mechanism by which NS5A (GT-1) inhibits TCA.
Engagement of the TCR with peptide-bound MHC complex present on the surface of antigen-presenting cells (APCs) results in activation of intracellular T cell signaling events resulting in TCA.One of the earliest events following TCR engagement is activation of lymphocyte-specific tyrosine kinase (Lck) 35 .In resting T cells, Lck is phosphorylated at the carboxy-terminal tyrosine residue (Y505) by the C-terminal Src kinase (Csk) [36][37][38] .Lck phosphorylated at the carboxy-terminal tyrosine maintains a closed conformation and is enzymatically inactive [38][39][40] .Upon TCR engagement Lck is dephosphorylated resulting in a conformational change that allows trans-autophosphorylation of the tyrosine residue (Y394) in the kinase domain 36,[38][39][40] .Lck phosphorylated at the Y394 maintains an open conformation, is enzymatically active and mediates downstream TCR signaling 36,38,[41][42][43][44] .
Since expression of the NS5A protein from HCV (GT-1) inhibited TCR-mediated TCA, we next assessed the effect of NS5A expression on Lck activation.Following anti-CD3 mediated TCR engagement, activation of Lck was assessed by immunoblots in Jurkat cells transduced with either control LV or LV-NS5A encoding HCV (GT-1) NS5A protein.Activation of Lck, as measured by phosphorylation at Y394, was significantly reduced in Jurkat cells transduced with the LV-NS5A compared to the control LV (Figs. 1c and  1d).Phosphorylation observed in T cells transduced with control LV was considered as a positive control.Active Lck phosphorylates and activates ZAP-70 tyrosine kinase, which then phosphorylates adaptor protein LAT, linker of activated T cells 36,45 .Phosphorylated LAT protein plays a key role in proximal TCR signaling by forming a multiprotein complex that activates downstream signaling factors 45 .We found that HCV (GT-1) NS5A expression reduced phosphorylation of both ZAP-70 (Figs. 1c and 1e) and LAT (Figs. 1c and 1f) following TCR engagement.Together, these data suggest that HCV (GT-1) NS5A protein inhibits proximal (upstream) TCR signaling by reducing Lck activation.

HCV (GT-1) NS5A interacts with Lck in human T cells
HCV NS5A is known to interact with numerous cellular factors 25 .A previous study found that HCV (GT-1) NS5A interacts with various members of the Src-family kinases, including Lck in non-human epithelial cell line (COS-7) via the poly-proline-rich-motif (PPRM) located at the C-terminus of the NS5A protein (amino acids, aa 340-359) 46 (Fig. 2a).The PPRM motif contains a minimal consensus sequence, proline-X-X-proline (PXXP, aa 350-353) (Fig. 2a, underlined), that interacts with proteins containing the Src-homology 3 domain (SH3 domain) such as Lck 47,48 .In a previous study, the PPRM motif of HCV (GT-1) NS5A was found to be essential for interaction with the SH3 domain of Lck, and mutation of the proline residues at position 350, 353 and 354 to alanine within the PXXP motif prevented NS5A and Lck interactions 46 .A sequence analysis revealed that the NS5A protein from HCV (GT-2) contained an alanine (A) at position 350 instead of a proline, resulting in formation of AXXP instead of PXXP (Fig 2a , underlined).Since we found that the NS5A from HCV GT-1 but not GT-2 inhibited TCA (Fig. 1b) and the NS5A (GT-2) contained an alanine at position 350 instead of a proline, we hypothesized that NS5A from GT-1 but not GT-2 interacts with Lck in human cells due to this mutation.To test this hypothesis, NS5A protein from both HCV genotypes were expressed in Jurkat T cells and HEK 293 cells.Following immunoprecipitation of NS5A, its interaction with Lck was assessed by immunoblotting.We found that NS5A from HCV (GT-1) interacted with Lck in both Jurkat T cells (Fig. 2b) and HEK 293 cells (Fig. 2c); however, in both cell lines, NS5A from HCV (GT-2) did not interact with Lck.Together, these data suggest that the NS5A protein from HCV GT-1 but not GT-2 interacts with Lck.

Expression of a short HCV (GT-1) NS5A peptide inhibits TCR-mediated activation of human T cells
As noted above, HCV (GT-1) NS5A contains the poly-proline-rich-motif (PPRM) at the Cterminus, which forms a minimal consensus sequence, proline-X-X-proline (PXXP) required for its interaction with Lck 46 .To assess if expression of the PPRM motif is sufficient to inhibit TCR-mediated TCA, a lentiviral vector (LV) encoding a short 20 amino acids peptide domain of the HCV (GT-1) NS5A protein (aa 340-359) that contains the PPRM motif was generated (Fig. 3a).Furthermore, LVs encoding either the full-length (FL) HCV (GT-2) NS5A or the 20-mer peptide containing an alanine to proline mutation at position 350 (A350P), to restore the PXXP motif, were also generated to assess if restoring PXXP motif is sufficient for interactions between NS5A (GT-2) and Lck (Fig. 3a, underlined).Jurkat cells were transduced with all lentiviral vectors and transduction was assessed by analyzing GFP produced by the internal ribosomal entry site (IRES) present within the vector.All cell lines expressed GFP as measured by flow cytometry (Supplemental Fig. 1).Interestingly, restoration of the PXXP motif with A350P mutation in the NS5A (GT-2) protein did not rescue its interaction with Lck in human T cells (Fig. 3b).This suggests that mutation of alanine at position 350 to proline is not sufficient to restore interactions between HCV (GT-2) NS5A and Lck, and that other residues outside the PXXP motif are necessary for this interaction.
Next, we assessed the effect of 20-mer NS5A peptide expression on TCR-mediated TCA.Following TCR engagement with anti-CD3/CD28, TCA was measured by assessing IL-2 release.Expression of the 20-mer HCV (GT-1) NS5A peptide significantly inhibited TCA suggesting that the 20-mer peptide derived from NS5A (GT-1) was sufficient to inhibit TCA (Fig. 3c).Similar to full-length NS5A protein expression, 20-mer NS5A peptide expression also inhibited proximal TCR signaling pathways as measured by phosphorylation of Lck, ZAP-70 and LAT (Supplemental Fig. 2).These results suggest that both the fulllength NS5A protein and the 20-mer NS5A peptide inhibit TCA by inhibiting activation of proximal TCR signaling events.
Neither the full-length NS5A protein nor the 20-mer peptide from HCV (GT-2) containing the A350P mutation inhibited TCR-mediated TCA compared to control LV transduced cells (Fig. 3c).This further demonstrates that restoration of the PXXP motif in the NS5A protein from HCV (GT-2) is not sufficient for inhibition of T cell activation.
To ensure that expression of the 20-mer NS5A peptide from HCV (GT-1) did not cause global T cell signaling dysfunction, TCA was assessed following treatment with phorbol 12myristate 13-acetate (PMA) and ionomycin.Activation with PMA and ionomycin bypasses activation of the proximal TCR signaling events and activates distal (downstream) T cell signaling pathways through activation of protein kinase C (PKC) and calcineurin 49 .The 20-mer NS5A peptide from HCV GT-1 did not inhibit PMA/ionomycin induced TCA (Fig. 3d), suggesting that NS5A peptide expression does not cause global T cell signaling dysfunction.

Synthetic HCV (GT-1) NS5A peptide inhibits TCR-mediated activation of primary human T cells
Although the data obtained from Jurkat T cells strongly suggest that expression of the 20-mer NS5A peptide from HCV (GT-1) results in inhibition of TCR-mediated TCA, we sought to confirm this observation by assessing the effect of synthetic NS5A peptide on TCR-mediated activation of primary human T cells.Synthetic NS5A peptide (HGCPLPPTKAPPIPPPRRKR) representing amino acids 340-359 of the HCV (GT-1) NS5A was generated with an N-terminal HIV TAT protein transduction domain (TAT; GGGGGRKKRRQRRR) to enhance cellular uptake (TAT-GT-1).A control peptide containing TAT domain alone was also synthesized (TAT alone).Peptides were also labeled with N-terminal FITC tag to monitor cellular uptake.Primary human T cells were treated with FITC-labelled synthetic peptides and following 18 hours of incubation cellular uptake was assessed by flow cytometry.Compared to untreated cells, FITC-peptide treated cells were FITC-positive confirming cellular uptake (Fig. 4a).Following anti-CD3 mediated TCR engagement, T cell activation was measured by assessing IL-2 and interferon-gamma (IFN-γ) release.Treatment of primary human T cells with synthetic NS5A (GT-1) peptide significantly reduced TCR-induced IL-2 and IFN-γ release compared to the control (TAT alone) peptide (Fig. 4b).This data confirms that the 20-mer peptide (amino acids 340-359) derived from the HCV (GT-1) NS5A protein is sufficient to inhibit TCR-mediated T cell activation of primary human T cells.

Recombinant AAV vector expressing the 20-mer NS5A peptide inhibits TCA
Recombinant AAV vector has limited packaging capacity.Thus, incorporation of large immunomodulatory factor(s) to reduce vector immunogenicity can limit transgene packaging capacity of the vector.Since we identified a short 20-mer NS5A peptide that can inhibit the proximal TCR signaling and activation of primary human T cells, we sought to test the ability of a recombinant AAV vector (serotype 6) encoding this short peptide to modulate TCA ex vivo.Primary human T cells from healthy donors were incubated either with the control AAV vector encoding GFP (AAV) or encoding NS5A peptide and GFP (AAV-NS5A).After 48 hours, T cells were activated using anti-CD3 and TCA was assessed by measuring IL-2 and IFN-γ released in the culture supernatant.In the absence of anti-CD3 stimulation (unstimulated), IL-2 was detected in T cells from 2 out of 6 donors incubated with the control AAV vector but was not detected in cells from the same donors incubated with AAV-NS5A.In absence of CD3 stimulation, IL-2 release is likely due to activation of pre-existing memory T cells in these donors.This suggests that AAV vector expressing NS5A peptide may also inhibit recall antigen-mediated T cell response (Fig. 5a).Following anti-CD3 mediated TCR engagement, IL-2 and IFN-γ were detected in T cells obtained from all donors that were transduced with control AAV vector (Fig. 5a).However, TCR-stimulated IL-2 and IFN-γ were significantly reduced in T cells from same donors that were transduced with AAV-NS5A vector.This is consistent with previous findings (Figs. 3  and 4) and demonstrates that expression of a short NS5A peptide of HCV (GT-1) either via a lentiviral vector or an AAV vector inhibits TCA in human T cells.
To ensure that AAV-NS5A does not cause global dysfunction of T cell signaling pathways, control AAV or AAV-NS5A transduced T cells were also stimulated with PMA and Ionomycin (PMA+Iono).Consistent with previous observation (Fig. 3d), AAV-NS5A did not inhibit PMA+Iono mediated TCA as measured by IL-2 and IFN-γ release, suggesting that the NS5A peptide expression does not cause global T cell signaling dysfunction (Fig. 5b), but rather acts in a targeted manner affecting proximal/upstream signaling events.

DISCUSSION
Viral vector-mediated in vivo gene therapy is rapidly evolving, and many new therapies are currently being developed.Many of these therapies likely will reach clinical studies with the promise of curing previously incurable human diseases.One of the major challenges with viral vectors is induction of host immune responses against the vector and the transgene product.Currently, immunosuppressive (IS) drugs are commonly used to mitigate host immune responses during gene therapy, but IS drugs have numerous unwanted systemic effects and can have deleterious side effects both for the patient and the therapeutic vector 50 .These drugs also tend to have temporal effects and the immune response particularly to transgene may arise after drug cessation.Thus, novel strategies to favorably modulate the host immune responses are warranted.
In this study, we characterized the effect of hepatitis C virus (HCV) non-structural protein 5A (NS5A) on human T cell activation (TCA) signaling pathways with the goal to identify novel, viral-based strategies to modulate host T cell responses during gene therapy.As previous studies had identified immunomodulatory functions of the HCV NS5A protein 31,46 , we sought to further characterize these immunomodulatory effects on human TCA, with the potential to apply this to gene therapy vectors.We found that lentiviral vector expressing the full-length HCV NS5A protein from GT-1 but not from GT-2, inhibited proximal TCR-mediated activation of human T cells (Fig. 1).Lck, as well as ZAP-70 and LAT are proximal T cell signaling molecules that are phosphorylated and activated following TCR engagement 51 , it is also known that Lck is targeted by numerous viruses to inhibit TCA 52 .We found that the NS5A (GT-1) protein expression resulted in inhibition of proximal TCR signaling pathways in part due to inhibition of Lck activation and its downstream targets ZAP-70 and LAT.The inhibition of proximal TCR signaling pathways resulted in inhibition of IL-2 release from Jurkat cells expressing NS5A (GT-1) (Fig. 1b).Although, we did not measure IL-2 transcripts in these cells, IL-2 is transcribed only after T cell activation, which is translated and released from the cells.As we found that GT-1 NS5A protein inhibits T cell activation, IL-2 transcripts are likely also inhibited, as our previous work demonstrated that IL-2 transcripts are reduced along with IL-2 protein levels following inhibition of TCA pathways 53 .
To better understand the mechanism for Lck inactivation by NS5A (GT-1), we also assessed interactions between NS5A and Lck protein.A previous study found that a poly-proline rich motif (PPRM) of the NS5A from HCV GT-1 binds to the Src-homology 3 (SH3) domain containing proteins including Lck 46 .We found that NS5A from GT-1 but not GT-2 interacted with Lck suggesting that the HCV GT-1 NS5A binds to Lck and inhibits its activation following TCR engagement.Sequence analysis revealed that the PPRM of the NS5A GT-1 has a consensus SH3-domain binding motif (PXXP); however, instead of a proline at position 350, the NS5A protein of HCV GT-2 has an alanine resulting in AXXP, a non-consensus sequence.To test if the alanine residue at position 350 is responsible for the inability of NS5A (GT-2) to interact with Lck, alanine was mutated to proline (A350P) to restore the PXXP motif in the NS5A from GT-2.However, neither the interactions with Lck nor the reduction in TCA was observed with the mutant NS5A (GT-2) protein containing A350P.This suggests that other residues outside the PPRM or other differences between GT-1 and GT-2 may be responsible for interactions with Lck.Lack of interactions between NS5A GT-2 and Lck may explain why NS5A GT-2 does not inhibit T cell activation; however, further studies are necessary to better understand the relationship between NS5A protein from different HCV genotypes and its effect on TCA.
Our results indicate that expression of a short 20 amino acids (20-mer) peptide of HCV NS5A (GT-1) is sufficient to inhibit TCA in primary human T cells (Figs. 3 and 4).We also found that recombinant adeno-associated virus (AAV) vector expressing the 20-mer peptide inhibits primary human T cell activation ex vivo (Fig. 5) suggesting that incorporation of short 20-mer HCV peptide may help reduce unwanted T cell activation during gene therapy.In this study, recombinant AAV vector containing capsid derived from AAV serotype 6 (AAV6), which efficiently transduces T cells, was used to assess the ability of a short NS5A peptide to modulate T cell function.For AAV vectors that do not transduce T cells or if the target cells of AAV vectors are non-T cells, NS5A peptide expression in the target cells alone may not be sufficient to modulate vector immunogenicity.In those cases, strategies such as engineering AAV vector to secrete NS5A peptide, so that secreted peptide will be taken up by bystander T cells, would be necessary to modulate T cell responses.
One of the limitations of AAV vector is its packaging capacity, thus, incorporation of any additional factor can limit transgene packaging capacity.Furthermore, incorporation of larger proteins or peptides may also inherently induce an immune response.Thus, the use of a smaller peptide, such as the 20-mer peptide identified in our study has advantage over expression of larger and potentially antigenic proteins or peptides identified in other studies to modulate host immune response against AAV vector 54 .
In conclusion, immunogenicity of gene therapy products is a significant challenge that needs to be addressed by developing novel immunomodulatory approaches.Our study has identified a novel strategy to dampen the host T cell response during AAV mediated gene therapy.Current studies are underway to assess the potential of the 20-mer NS5A peptide to modulate immune response against AAV vector in vivo.

Peripheral blood mononuclear cells (PBMCs):
Whole blood was obtained from healthy donors at the National Institutes of Health (NIH) Blood Bank.All the donors provided written consent for their blood products to be used in research projects.All the samples provided to the investigators were de-identified.This study was exempted by the FDA's IRB, Research Involving Human Subject Committee (RIHSC).PBMCs were isolated from whole blood using Ficoll-Hypaque gradient centrifugation.Isolated PBMCs were washed in cold PBS and resuspended in complete RPMI.To obtain primary T cells, PBMCs were incubated overnight at 37°C with 5% CO 2. The following day, T cells in suspension were harvested and the adherent cells were discarded.Up to 70% of cells in the final culture were CD3+ T cells.

Cells:
Jurkat T cell line (clone E6.1) was obtained from ATCC and 293T cells from Cell Genesys, Foster City, CA.Jurkat cells and primary T cells were maintained in RPMI 1640 and 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM).Media were supplemented with 10% heat-inactivated FCS, 2mM Glutamax, 100 IU/ml penicillin, and 100 μg/ml streptomycin.Cells were grown at 37°C in 5% CO 2 .
Cell viability was determined using a trypan blue exclusion method and counted using the Countess II FL automated cell counter (Invitrogen).293F suspension cells, obtained from Thermo Fisher Scientific, were maintained in shaker flasks at 120 rpm, under 8% CO 2 at 37°C and propagated in its respective FreeStyle media (serum free/defined).

Lentiviral vectors expressing NS5A protein:
HCV NS5A GT-1 sequence was obtained from pCMV NS5A TAG1 plasmid (Addgene Plasmid #17646) and HCV NS5A GT-2 sequence was obtained from the J6/JFH infectious clone kindly provided by Dr. Charles Rice, Rockefeller University 55 .Full-length HCV NS5A sequence for each genotype was amplified and cloned into a modified pCIG3 vector (Addgene Plasmid #78264) containing a C-terminal hemagglutinin (HA) tag.For expression of a short NS5A peptide, synthetic oligos with the restriction sites (BamHI/ EcoRI) were obtained from Integrated DNA Technologies (Coralville, IA) and directly cloned into pCIG3-HA vector.Point mutation in the NS5A protein was created with QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) following manufacturer's recommendation using primers containing the desired mutation.DNA sequences were confirmed by sequencing at DNA core facility (CBER/FDA).Sequences of primers and oligos are provided in supplemental table 1.

Lentiviral vector production:
Lentiviral vectors were manufactured in 293T cells using PEI transfection reagent (Polysciences).Briefly, 2 × 10 6 cells were plated in 10cm dishes and incubated overnight.The next day the cells were washed with RPMI 3 times and replenished with 10ml of fresh media.The transfection reaction mix was prepared with 36μl of PEI, 6μg of pCIG3-HA encoding HCV NS5A, 2μg of VSV-G envelope encoding plasmid, and 4ug of the packaging plasmid pCD/NL-BH*DDD (a gift from Dr. Jakob Riser, FDA).Transfection complexes were gently mixed in 1ml of Opti MEM (Thermo Fisher Scientific) and incubated for 15 minutes at room temperature.The mix was added dropwise to the cells and the dishes gently rocked.The next day the media was changed, and supernatants were collected after 72 hours.Supernatants were clarified (centrifugation at 1500 rpm for 10 mins) and concentrated using Amicon 100kDa Centrifugal Filter Unit (Millipore).Final aliquots were then filter sterilized using a .45μmfilter.Concentrated vectors were stored at −80°C.Vector transduction units were determined by transducing HEK293T cells and assessing GFP by flow cytometry.

HCV NS5A synthetic peptides:
FITC labelled synthetic peptides with an N-terminal HIV TAT protein transduction domain (TAT) alone (GGGGGRKKRRQRRR), or with the HCV NS5A (GT-1) aa 340-359 (GGGGGRKKRRQRRRHGCPLPPTKAPPIPPPRRKR), or the HCV NS5A (GT-2) (GGGGGRKKRRQRRRAGCALPPPKKAPTPPPRRRR) were purchased from GenScript Biotech.Peptides were dissolved in 100% DMSO.Healthy donor T cells (1×10 6 cells/ml) were incubated with 500μg/ml peptide at 37°C overnight, followed by washing twice with media before stimulation with 100ng/ml of soluble anti-CD3.Cells were activated for 24 hours in culture media containing 500μg/ml of peptide.Peptides were used at 500μg/ml as preliminary experiments demonstrated that at this concentration synthetic NS5A peptides inhibited T cell activation without affecting cell viability.After 24 hours, supernatants were collected and analyzed for IL-2 or IFN-gamma.

Recombinant adeno-associated virus (AAV) vector production:
AAV production was performed using Thermo Fishers 293F Freestyle suspension cell line and respective media under a modified triple transfection and double Iodixanol ultracentrifugation protocol.For transfection, cells were grown at 1×10 6 cells/ml.A two plasmid AAV system utilizing pscAAV-GFP (CellBioLabs #AAV-410) and pDGM6 (Addgene #110660) was used in conjunction with PEI (Molar ratio of 2 pDGMC:1 pscAAV-GFP for plasmids, along with a 2:1 PEI to DNA ratio).For expression of a short NS5A peptide, synthetic oligos with the restriction sites were obtained from Integrated DNA Technologies (Coralville, IA) and directly cloned into pscAAV-GFP vector.DNA sequences were confirmed by sequencing at DNA core facility (CBER/FDA).Transfection complexes were carried out in OptiMEM and incubated for 15 min at room temperature, then applied to cells with fresh media.Following 72 hours post transfection, cells were harvested, washed and resuspended in AAV cell lysis/stabilization buffer (.001 P-F68, 200mM NaCl, 200mM MgCl 2 , 50mM Tris-HCL pH 7.4 in dH 2 0), then further lysed using a combination freeze/ thaw (liquid nitrogen/37°C water bath) and sonication.Sodium benzonase (final conc. of 50 U/ml), was added to the lysed cells for 1 hour at 37°C, which were then spun for 30 min at 10,000 rpm to collect cellular debris.Clarified cell lysates were then loaded onto a discontinuous Iodixanol gradient (15, 25, 40 and 60% fractions prepared in PBS+MgCl 2 and KCl).Samples were spun at 32,000 RPM (SW32ti Beckman Coulter rotor) overnight.The viral fraction was then collected and diluted 1:1 in PBS for a second spin under identical conditions with the exception of a gradient change (30, 40 and 60% fractions).Following ultracentrifugation, vector was passed through a 1-micron PES filter, then diluted in a Vector Wash Buffer (.001 P-F68200mM NaCl, 200mM MgCl 2 , in PBS), for concentration and removal of Iodixanol using an Amicon 100 kDa PES cutoff column.Concentrated vector was then reformulated in AAV Resuspension Buffer (5% glycerol, 35mM NaCl, 10mM MgCl 2 in PBS, filtered using .2μMPES, then aliquoted and stored at −80°C.Vector titering was performed through a determination of transducing titers using serial dilutions in Jurkat cells for GFP expression and were expressed as TU/ml.

Transduction of cells:
Jurkat cells were transduced with either control or HCV NS5A expressing lentiviral vector at MOI of 10.Following 24 hours of transduction, cells were washed and GFP was analyzed at 72-96 hours.GFP expressing cells were bulk sorted using BD FACSAria (CBER Flow Cytometry Core) and expanded in culture for stimulation.Human T cells were transduced with AAV vector at MOI of 2.5.Following 48 hours of transduction, cells were stimulated for 24 hours before analysis.

ELISA:
IL-2 or IFN-gamma cytokines released in cell culture supernatant were quantified using human IL-2 or human IFN-gamma ELISA kits (BD Biosciences) according to the manufacturer's instructions.

Immunoprecipitation:
Cells were washed in PBS and lysed in lysis buffer (Pierce IP Lysis Buffer).Following cell lysis, lysates were clarified by centrifugation at 5000rpm for 5 mins.Pellet was discarded, and clarified lysate was mixed with respective antibodies and protein G magnetic beads (Dynabeads, Invitrogen) and incubated for 3 hours at room temperature in a tube rotator.Beads were washed three times with PBS, and bound proteins were eluted by resuspending the beads and protein complex in Laemmli sample buffer and heated for 10 mins at 95° C. Beads were then separated using a magnet, and proteins in the sample buffer was subjected to immunoblot analysis.HCV GT-1 NS5A protein was immunoprecipitated using anti-NS5A (GT-1) antibodies (Abcam) and HCV GT-2 NS5A was immunoprecipitated using anti-HA antibodies that cross-reacted to the C-terminus HA-tag present in the NS5A (GT-2) protein.NS5A proteins were detected in immunoblots using two separate antibodies that were used in the immunoprecipitation.

Immunoblot Analysis:
Cellular lysates were mixed with Laemmli sample buffer, heated at 95°C for 5 minutes and separated on NuPAGE Bis-Tris gels by electrophoresis and transferred to nitrocellulose membranes using the iBlot transfer system (Thermo Scientific).Membranes were incubated in previously heated 3% fat-free dry milk for 1 hour at room temperature followed by overnight incubation with primary antibodies.Proteins were detected with Super Signal West Dura (Thermo Scientific #34075) using a Bio-Rad ChemiDoc MP imaging system.Immunoblots were quantified using ImageJ (NIH) by stripping and reprobing the same membrane for the indicated loading controls for normalization.Integrated Density (IntDen, the product of area and mean gray value) was calculated for each condition.In each experiment, the IntDen value was obtained for each condition, and was normalized to the value obtained in the control stimulated LV condition using the following formula: (IntDen value/ IntDen value at stimulated LV).Primary antibodies used were: pLck (Y394, R&D Systems), pZAP-70 (Y319), total Lck, total ZAP-70, total LAT and anti-HA from Cell Signaling, pLAT(Y226) from Biolegend, beta-Actin and GAPDH from Sigma.

Statistics:
One-way ANOVA was used to compare results from multiple groups and two-sided Student's t test was used to compare results from two groups.P values less than 0.05 were considered statistically significant.GraphPad PRISM (GraphPad Software Inc.) was used for statistical analysis.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.(a) Panel A illustrates a peptide region of HCV NS5A protein (amino acids 340 -359) from two genotypes (GT-1 and GT-2) that contains the poly-proline rich motif (underlined).HCV NS5A protein was immunoprecipitated from (b) Jurkat T cells or (c) HEK293 cells expressing either NS5A, GT-1 or GT-2 and Lck.The precipitates were analyzed by immunoblots using anti-Lck and anti-NS5A antibodies.Two different antibodies were used to detect NS5A proteins from genotype 1 and genotype 2 as described in the methods section.Isotype matching antibodies were used as a negative control during immunoprecipitation.A representative immunoblot analysis is shown.Each experiment was independently performed at least three time with similar results.(a) Panel A illustrates a peptide region of NS5A protein (amino acids, aa 340 -359) from GT-1 and GT-2 that contains the poly-proline rich motif (underlined).HCV NS5A (GT-2) was mutated to contain a proline instead of alanine at position 350 (A350P, arrow).(b) HCV NS5A protein (GT-2) containing an alanine to proline mutation at position 350 (A350P) was immunoprecipitated from Jurkat T cells using anti-HA antibodies that react to a C-terminal HA tag present in the NS5A protein.The precipitates were analyzed by immunoblots using anti-Lck and anti-HA antibodies.Isotype matching antibody were used as a negative control during immunoprecipitation.The input cell lysate used for immunoprecipitation was also assessed for Lck expression.A representative immunoblot analysis is shown and was independently performed at least three time with similar results.(c) Following TCR engagement with anti-CD3/CD28, T cell activation was measured by assessing IL-2 release in Jurkat cells transduced with a control lentiviral vector (LV) or LV expressing a short peptide (aa 350-359) derived from NS5A protein GT-1 or GT-2 with A350P mutation or LV expressing a full-length (FL) NS5A protein of HCV (GT-2) containing A350P mutation.(d) Following PMA/ Ionomycin stimulation, T cell activation was measured in Jurkat T cells transduced with a control lentivirus vector (LV) or LV expressing NS5A (GT-1) peptide (aa 340-359).IL-2 was normalized to levels released by cells transduced with control LV.Data represent the average of three technical replicates and the standard deviation is shown.
Each experiment was repeated at least three times with similar results.*P<0.01,ns= not significant.

Figure 1 :
Figure 1: HCV NS5A expression inhibits TCR-mediated activation of human T cells.(a) Immunoblot analysis of Jurkat T cells transduced with a control lentiviral vector (LV) or a lentiviral vector expressing hepatitis C virus (HCV) NS5A protein (LV-NS5A) from genotype 1 (GT-1) or genotype 2 (GT-2).GAPDH expression was measured as a loading control.(b) Following TCR engagement with anti-CD3/CD28, T cell activation was measured by assessing IL-2 release.(c) Activation of proximal TCR signaling events was measured in LV or LV-NS5A transduced Jurkat cells.Cell were either unstimulated (−) or anti-CD3 stimulated (2 minutes, +) and phosphorylation of Lck at tyrosine 394 (pY394), ZAP-70 at pY319 and LAT at pY226 was assessed.Phosphorylation of both 56 and 61 kDa Lck isoforms were detected.Actin protein expression was measured as a loading control.Quantification of immunoblots for (d) phospho-Lck, (e) phospho-ZAP70, and (f) phospho-LAT obtained from 3 independent experiments are shown.Phospho proteins levels were normalized to actin.Data represent the average of three independent experiments, and the standard deviation is shown.*P<0.01.

Figure 3 :
Figure 3: Expression of a short HCV (GT-1) NS5A peptide inhibits TCR-mediated activation of human T cells.

Figure 4 :
Figure 4: Synthetic HCV (GT-1) NS5A peptide inhibits TCR-mediated activation of primary human T cells.(a) Flow cytometry analysis of primary human T cells following 24-hour incubation with FITC-labelled synthetic peptide.Cells were either left untreated (No peptide) or treated with peptide with an HIV TAT protein transduction domain sequence at the N-terminus (TAT peptide).Each experiment was conducted in triplicate and repeated on a separate day with consistent results.(b) Primary human T cells were treated with TAT control peptides or peptides representing HCV NS5A (GT-1) sequence (aa 340-359).Following TCR engagement with anti-CD3, T cell activation was measured by assessing IL-2 and IFN-γ release.Data represents the average of three technical replicates, and the standard deviation is shown.Each experiment was independently performed using three different donors with similar results.*P< 0.01; us = unstimulated.

Figure 5 :
Figure 5: Recombinant AAV vector expressing the 20-mer NS5A peptide inhibits TCA.Primary human T cells were either mock transduced or transduced with a control AAV vector or a vector expressing HCV NS5A (GT-1) peptide (340-359) (AAV-NS5A).Transduced cells were either unstimulated (US) or stimulated with (a) anti-CD3 or (b) PMA/ Ionomycin and T cell activation was measured by assessing IL-2 and IFN-γ release.Cytokine levels were normalized to cytokines released by mock transduced T cells following activation.Data represents the average from six donors and the standard deviation is shown.**P<0.0001;*P<0.001,ns = not significant.