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Journal of Virology, December 2005, p. 14595-14605, Vol. 79, No. 23
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.23.14595-14605.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Frequency, Proliferation, and Activation of Human Memory T Cells Induced by a Nonhuman Adenovirus

Matthieu Perreau and Eric J. Kremer*

Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, IFR 122, Montpellier, France

Received 26 May 2005/ Accepted 4 September 2005


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ABSTRACT
 
Multiple human adenovirus (HAd) infections during childhood generate a memory T-cell (TM) response, which is the primary defense against HAd-induced morbidity. This cellular memory creates a conundrum for the potential clinical use of HAd-derived vectors: vector-mediated gene transfer is efficient in immunologically naïve mammals but will be compromised by memory immunity when using vectors derived from ubiquitous human pathogens. The potential lack of cellular and humoral memory is one reason we developed vectors from canine adenovirus serotype 2 (CAV-2). Here, we assayed human peripheral blood mononuclear cells for a TM response that could be stimulated by CAV-2 virion and individual capsid proteins. We found that less than half of the donors harbored a proliferating TM response directed against the CAV-2 virion (versus >85% against HAd5) in spite of a conserved antigenic Adenoviridae epitope in the CAV-2 hexon. When CAV-2 induced proliferation, it was 2.3- to >10-fold lower than HAd5 depending on the assay. The primary proliferating cells appeared to be memory (CD45RO+) CD4+ lymphocytes, differentiated into Th1 gamma interferon-producing cells, with a frequency that was up to 66-fold lower than that obtained for HAd5. We also compared CAV-2 to prototype HAd from five of the six human species and found that CAV-2-induced cellular proliferation was similar to that found with rare HAd serotypes. Individual CAV-2 capsid proteins also induced less proliferation than their HAd5 homologues. Our data suggest that CAV-2 vectors may be safer (i.e., less immunogenic) for gene transfer but are not without a theoretical risk in a subset of potential patients.


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INTRODUCTION
 
One of the more salient characteristics of adaptive immunity is the generation of immunological memory. This phenomenon enhances protection from previously encountered pathogens and is the basis for global vaccination strategies of numerous infectious diseases. T-cell memory (TM) is an evolving, dynamic process that maintains a minimum level of antigen-specific T cells that rely on a renewable population in the absence of stimuli (20, 21, 34, 75). The extraordinary flexibility for TM antigen recognition (62) is due to the ability of major histocompatibility complex (MHC) class I and II alleles to accommodate sequence divergence. There are two major subsets of TM, CD4+ and CD8+, although the former are considered more as helpers for CD8+ TM-mediated lysis and CD4+ TM can be cytotoxic (50).

Although the first line of memory defense may be humoral, TM is the most effective protection against many pathogens, including influenza A (41), cytomegalovirus (51), and Adenoviridae (23). Many of the >50 human adenovirus (HAd) serotypes can be found in most populations and can generate persistent subclinical infections that last for years (25). Nonetheless, HAds are a significant cause of morbidity and mortality in severely immunodeficient patients (23) and can be lethal in newborns and immunocompetent adults (52). The severity of lymphocytopenia is a reliable indicator of risk to HAd disease: patients with functional B cells, but lacking functional Tcells, are most susceptible to HAd-induce morbidity (32, 33). In cardiac allograft recipients, HAd was the pathogen most often isolated and was associated with reduced graft survival and acute rejection (67). Following allogeneic hematopoietic transplants, the most severely immunocompromised patients had the highest risk of HAd-induced morbidity and mortality (13, 14). HAd-specific cellular immunity is generated during childhood and may be dominated by TM with effector phenotype. HAd-specific TM declines with age and following infection with other pathogens (61, 65). Similar to cytomegalovirus and Epstein-Barr virus (EBV) (53), the potential to rapidly amplify ex vivo a HAd-specific cytotoxic T-lymphocyte (CTL) response (CD8+ or CD4+) to fight HAd-induced morbidity in immunodeficient patients is an exciting possibility (22, 36, 68).

In addition to HAd, there are at least 50 nonhuman Adenoviridae identified (17). Although Adenoviridae (nonenveloped double-stranded DNA viruses) are relatively species specific, they have a common origin going back millions of years to an ancestor of Tectiviridae (1, 6, 7, 17). The HAd capsid proteins are the major target antigens of the TM (18). Castelli et al. and Tang et al. identified a highly conserved Adenoviridae hexon epitope (Hx910-924) that can be restricted by a class II allele present in 75% of the population (12, 77). Olive et al. suggested that Hx910-924 is responsible for one-third of all the HAd-induced TM proliferation (45). These data suggested that the cellular anti-HAd response should cross-react with inter- and intra-Ad species (36, 69, 77). Prior to obtaining these data, we (and others) (10, 47) hypothesized that vectors derived from nonhuman Adenoviridae would be more clinically relevant than HAd vectors because of the potential lack of memory immunity. Several years ago, we began developing vectors derived from canine adenovirus type 2 (CAV-2) (28, 29, 31, 73, 74). CAV-2 vectors efficiently transduce respiratory epithelia (31), preferentially transduce neurons in the central nervous system (CNS), and lead to an efficient level of axoplasmic transport (73). Helper-dependent CAV-2 (HDCAV) vectors also lead to >1 year of in vivo transgene expression in immunocompetent rats without immunosuppression (74). These data, and those from other studies using helper-dependent HAd (HDAd) vectors (3), suggest that HDCAV vectors could be used for the treatment of some global neurodegenerative disorders (30, 44). However, one can cause more immediate harm to a patient via acute and/or chronic vector-induced cellular infiltration in the CNS than by the normal progression of most neurodegenerative disorders. We predict that an acute/chronic TM infiltration that is triggered by a vector-induced innate immune response (42, 43, 80) and may be poorly blunted by common (e.g., cyclosporine A or FK506) immunosuppression regimens (24) will lead to deleterious side effects in some patients (29).

It is in this context that we continue to assess the clinical potential of CAV-2 vectors. Some nonhuman Adenoviridae, like CAV-2, contain the Hx910-924 epitope (77), which may be conserved because complex structural constraints do not tolerate modifications of the primary sequence. Here, we describe our results analyzing the proliferation and activation of human peripheral blood mononuclear cells that potentially recognize the CAV-2 capsid proteins. We found that CAV-2 was poorly immunoreactive, based on the percent of donors responding, as well as the level in responders, which was similar to that generated by rare HAd serotypes. In the responders, the CAV-2 capsid induced primarily a Th1 type CD4+ TM proliferative response. Modest induction of T-cell activation markers and proliferation were also found following incubation of CAV-2, but again, significantly less than HAd5. We extended our study to include the TM proliferative response induced by three capsid proteins (hexon, penton base, and fiber) and the Hx910-924 peptide. Our data suggest that CAV-2 vectors may be safer than common human virus-derived vectors (e.g., HAd, adeno-associated virus [AAV], and herpes simplex virus) and could be used in the treatment of neurodegenerative disorders, but are not without a theoretical risk (in some patients) due to the presence of a detectable TM.


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MATERIALS AND METHODS
 
Vectors and viruses. Prototype HAd serotypes 4, 11, 12, and 17 were gifts from Göran Wadell (Umeå University, Sweden) and were propagated and purified as previously described (31, 72). The vectors CAVGFP and AdGFP have been described previously (31). AdGFP is a {Delta}E1/E3 vector derived from HAd serotype 5 (HAd5, from species C) and contains an enhanced green fluorescent protein (GFP) expression cassette. CAVGFP is a {Delta}E1 CAV-2 vector containing the same expression cassette. CAVGFP had a physical particle (pp)-to-infectious-unit (IU) ratio of 3:1, while AdGFP had a pp/IU ratio of 10:1 (31). AdGFP, CAVGFP, and wild-type HAd serotypes were UV/psoralen inactivated postpurification as previously described (58, 78). The nomenclature "CAV-2" and "HAd5" are used throughout the text instead of CAVGFP and AdGFP to denote that the assays were transgene independent.

Donor samples and culture conditions. Adult peripheral blood mononuclear cells (PBMC) were obtained from healthy donors from the Etablissement Français du Sang (convention #753303/00; Montpellier, France), collected in EDTA-coated tubes, and isolated by Ficoll-Histopaque separation (Sigma-Aldrich, St. Quentin Fallavier, France). For experiments requiring large amount of cells, PBMC were collected from buffy coats from healthy donors. Mononuclear cells from umbilical cord blood (UCB) were obtained following delivery of full-term infants (Clinic Saint Roch, Montpellier, France) and collected in heparinized tubes. PBMC were cultured in complete medium: RPMI 1640 (Invitrogen, Auckland, New Zealand), 10% (vol/vol) fetal calf serum (Sigma-Aldrich), and 2 mM glutamine (Merck, France).

Depletion of CD4+ and CD8+ T cells from PBMC. PBMC were incubated with anti-CD4 or anti-CD8 (Pharmingen, France) monoclonal antibodies (MAbs), followed by the addition of mouse anti-immunoglobulin G (IgG)-conjugated magnetic beads (Dynal-Biotech, Oslo, Norway). Cells not bound to beads, the CD4– or CD8– population, were collected (Dynal-Biotech). The purity of each cell isolation step was monitored by flow cytometry after staining with appropriate fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated MAbs.

MoDC, CD4+/CD45RA–, and CD8+/CD45RA– T-cell isolation. PBMC were isolated from buffy coats from three donors. CD14+ cells were isolated using the Miltenyi Biotec Macs isolation system (Paris, France). Monocytes were seeded at 106 cells/ml in complete medium containing 4 ng/ml human interleukin-4 (IL-4), 50 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Sigma-Aldrich) for 7 days in six-well plates to differentiate them into monocyte-derived dendritic cell (MoDC). At days 3 and 5, we replaced half of the original medium with fresh medium containing IL-4 and GM-CSF. CD4+ and CD8+ TM were purified from buffy coats. CD45RA+ cells were depleted using the Miltenyi Biotec Macs isolation system. Then CD4+ and CD8+ cells were positively isolated. CD4+CD45RA– and CD8+CD45RA– cells were resuspended at 106 cells/ml in complete medium.

Cellular proliferation. Freshly isolated PBMC were seeded at 105 cells/well in flat-bottom 96-well plates (Greiner Bio-One, France) and incubated with 1 µg phytohemagglutinin (PHA) and 100 U/ml of human IL-2, HAd5 (103 or 102pp/cell), or CAV-2 (103 pp/cell). Control cells (nonstimulated) were cultured in complete medium. Three, 5, 7, and 10 days poststimulation, the cells were pulsed for 18 h with 1 µCi of [3H]thymidine/well (Amersham, France). Cell proliferation was recorded using a microplate harvester.

Surface markers and gamma interferon (IFN-{gamma}) expression. CD25, CD69, and HLA-DR expression was assayed by incubating cells for 20 min on ice with FITC or PE-conjugated MAb (clones B1.49.9, TP1.55.3, and immu-357, respectively; Immunotech, France), at days 3, 5, and 7. Background fluorescence was measured using an Ig isotype control Ab. PBMC (106 cells/ml) were stimulated with medium alone, PHA/IL-2, 103 pp/cell of HAd5, or CAV-2. On day 6, cells were pretreated for 24 h with 20 µg/ml Brefeldin A (Sigma) 6 h prior to labeling with 50 ng/ml phorbol myristate acetate/and 1 µg/ml ionomycin. The cells were then stained with an anti-CD4 MAb conjugated with FITC (clone 13B8.2; Pharmingen), fixed using 4% paraformaldehyde-PBS, and stained with an anti-IFN-{gamma} MAb conjugated with PE in a buffer containing 0.05% saponin (Sigma). The specificity of the anti-IFN-{gamma} staining was verified by the use of an isotype control MAb. Cells were washed with PBS (GIBCO, Invitrogen) and analyzed on a FACSCalibur (BD Biosciences, San Jose, CA) instrument, and data analysis was performed using CellQuest (BD Biosciences).

Enzyme-linked immunospot (ELISPOT) assay. PBMC were seeded at 105 or 2.5 x 105/well in quadruplicate on 96-well plates (Pharmingen, France) precoated with either anti-IFN-{gamma} (5 µg/ml) or anti-IL-4 MAb (5 µg/ml) (Pharmingen, France). Cytokine secretion was assayed 6, 12, 18, 24, 48, and 72 h postincubation with either 1 µg PHA and 100 U/ml IL-2 (positive control), 103pp/cell of HAd5 or CAV-2, or 3 µg/ml full-length tetanus toxin (Sigma-Aldrich). Cells were rinsed with 0.05% Tween 20-phosphate-buffered saline (PBS). Secondary biotinylated anti-IFN-{gamma} MAb (2 µg/ml) or anti-IL-4 MAb (2 µg/ml) was added and incubated overnight at 4°C. Plates were washed again, and cytokine expression was revealed with avidin-peroxidase and 3-amino-9-ethylcarbazole substrate (Pharmingen, France). Spots were quantified using an Immuno-Spot series I analyzer.

CFSE labeling. PBMC were washed and resuspended in PBS at 2 x 106cells/ml for labeling with the fluorochrome carboxy-fluorescein diacetate-succinimidyl ester (CFSE, Molecular Probes, Eugene, OR) at a final concentration of 2.5 µM for 3 min at room temperature. Labeling was terminated by the addition of fetal calf serum (30% of total volume); cells were washed twice and then cultured as follows. PBMC (106 cells) were incubated with complete medium, PHA/IL-2, or 103 pp/cell of HAd5 or CAV-2. To detect T-cell divisions, 105 PBMC were incubated for 20 min on ice with anti-CD3-labeled Cy5-conjugated MAb (clone UCHT1; Immunotech). Division was analyzed on a FACSCalibur instrument.

Proteins and peptide. The CAV-2 hexon protein was purified from CAV-2 virion. Briefly, 4 mg of CsCl-purified CAV-2 was dialyzed in 20 mM Tris-Cl (pH 8.8), 1 mM dithiothreitol (DTT), and heat disrupted at 56°C for 2 min. Viral DNA was removed by benzonase digestion for 30 min at 37°C, and the solution was adjusted to 10% pyridine (Sigma) and incubated for 30 min at room temperature to further dissociate the virions. The viral suspension was centrifuged 10min at 20,000 x g, and supernatant proteins were separated using two-step fast-performance liquid chromatography (FPLC). Initially, the supernatant was passed through a MonoQ column (Pharmacia) and hexon was eluted using a 0 to 0.5 M NaCl gradient in 20 mM Tris (pH 8.8)-1 mM DTT. We purified the hexon fraction using a preequilibrated Superpose-12 ion exchange column. His-tagged CAV-2 penton base and fiber were produced using the pFastBac baculovirus protein production system (Invitrogen, France) and purified using His affinity columns by following standard protocols (cloning details available on request). Hx910-924 was synthesized according to the Fmoc-tert-butyl strategy using an AMS 422 multiple-peptide synthesizer (Abimed, Langenfeld, Germany). Purification was performed using reverse-phase high-pressure liquid chromatography (Water-prepLC40; Waters).


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RESULTS
 
CAV-2-induced proliferation. Flomenberg et al. demonstrated that PBMC from 29 of 30 donors harbored a subset of cells that proliferated following incubation with a species C HAd (18). These data highlighted the risk of adverse side effects following gene transfer with vectors derived from ubiquitous human pathogens (e.g., Ad, AAV, or HSV) and their nonhuman counterparts (10) (e.g., canine [29], bovine [5], ovine [9], and chimpanzee [81] Ad vectors and simian AAV [19]). To address this caveat in the context of a nonhuman Adenoviridae vector, we assayed human PBMC for proliferation and activation induced by CAV-2. Initially, we determined the kinetics of the induced cellular proliferative response and antigen dose in PBMC (Fig. 1a). These data demonstrated that the proliferative response in our assay was dose and time dependent and that CAV-2- and HAd5-induced proliferation peaked at day 7. We subsequently assayed proliferation on day7 and with 102 and 103 pp/cell of HAd5 and 103 pp/cell ofCAV-2.



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  		FIG. 1. Kinetic and dose effect of the proliferative responses and comparison of HAd5- versus CAV-2-specific proliferation in PBMC. (a) PBMC from three donors were stimulated with 102 or 103 pp/cell of HAd5 or CAV-2 for 3, 5, 7 or 10 days, or mock treated (medium alone) (not shown). The proliferation levels (one of three donors shown) were measured by [3H]thymidine incorporation 18 h poststimulation. The lower multiplicity of infection of CAV-2 (102 pp/cell) led to proliferation near background (an SI of >3) (22) in all donors. At day 5, both multiplicities of infections of HAd5 gave signals above background. At day 7, CAV-2- and HAd5-induced proliferation peaked: 103 pp/cell of HAd5 induced ~1.5-fold higher proliferation than 102 pp/cell. Error bars denote standard errors of the mean. Results are expressed in SI (cpmmean from sample cells) (cpmmean from control cells)–1. (b) Mononuclear cells isolated from UCB (n = 6) and donors (n = 71) were stimulated with 102 or 103 pp/cell of HAd5 or 103 pp/cell of CAV-2 for 7 days. Proliferation response is shown as the SI. Mock-treated PBMC, UCB (naïve T cells), or PBMC incubated with PHA/IL-2 were used as controls. The SImean of 103 pp/cell of CAV-2 was ~4-fold lower than 103 pp/cell of HAd5 (P < 0.01). There was no significant difference (P < 0.13) between 103 pp/cell of CAV-2 versus 102 pp/cell of HAd5. The horizontal bar denotes SImean. All stimulations were performed in triplicate. *, P < 0.05. All statistical analyses were performed using Student's t test unless otherwise noted.

We assayed PBMC from >70 donors for their proliferative response to CAV-2 virion (Fig. 1b). Similar to the studies by Flomenberg and colleagues (18), we used 103 pp/cell of HAd. The vectors used in our study were replication defective and unlikely to express significant amounts of de novo virus proteins in transduced cells. Furthermore, we detected no significant level (<1%) of gene transfer (i.e., GFP expression) following incubation of PBMC or T cells with AdGFP or CAVGFP. Control experiments using a {Delta}E1 CAV-2 vector containing a lacZ expression cassette (31) gave results equivalent to CAVGFP (not shown), demonstrating that we were detecting HAd5- or CAV-2-specific antigens and not those due to the transgene. Unlike the case with EBV (18, 37, 76), no HAd-specific cellular proliferation could be detected in UCB.

Initially, we found that a 10-fold difference in the HAd5 pp/cell ratio led to a 2.4-fold variation in the mean stimulation index (SImean) (Table 1). Although the absolute frequency showed interdonor variations, the frequency within a specific donor was stable (not shown). Secondly, unlike our previous data demonstrating the lack of significant levels of anti-CAV-2 neutralizing antibodies (NAbs) in 98% of a random cohort (31), we found a detectable, albeit low and reproducible, level of CAV-2-induced cellular proliferation. However, the CAV-2-induced SImean was >4-fold lower than an equivalent dose ofHAd5. More notably, 55% of the donors were "nonresponders" for CAV-2 while 16% (103 pp/cell) and 35% (102pp/cell) were nonresponders for HAd5 (Table 1). Our "percent responders" and SImean range were similar to those found by Heemskerk et al. (22) (76% responders and SI range from 4.5 to 234). Given this criterion, the responder SImean for CAV-2 was 2.5-fold lower than an equivalent dose of HAd5 and significantly (P < 0.05) lower than 10-fold fewer (102) HAd5 pp/cell.


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  		TABLE 1. Summary of induced SI by HAd5 and CAV-2a

CD4+ proliferation: quantification and characterization. Ficoll-purified PBMC contain ~33% CD4+ and ~16% CD8+ lymphocytes. To identify the CAV-2-induced proliferating cells, we depleted CD8+ or CD4+ cells from the PBMC and assayed for HAd5- or CAV-2-induced proliferation. The results from one of three donors are shown (Fig. 2a). Depletion of CD8+ cells increased the SI versus total PBMC, while depletion of CD4+ cells reduced the signal to levels equal to UCB (not shown). The higher proliferation in the CD8-depleted population is likely due to the increase in the percent of CD4+ cells/well (from ~33 to ~50%). Our data suggested that CD4+ cells were the primary HAd5- or CAV-2-induced proliferating population. Our data are in agreement with that of others studies (18, 65). We also asked if our proliferation assay was detecting a population of cells that was dividing multiple times or a larger population dividing less frequently. To address this question, CFSE-labeled PBMC from donor 2b were incubated with CAV-2 or HAd5 (Fig. 2b). CFSE labeling leads to random uniform fluorescent labels on secondary NH2 groups of cellular proteins and allows one to identify and quantify the number of times antigen-reactive cells divided. In PHA/IL-2-treated cells, >80% of the T cells proliferated more than once. In contrast to HAd5, CAV-2 induced 17-fold fewer cells to proliferate, demonstrating that fewer T cells recognize the CAV-2 virion.



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  		FIG. 2. Role of CD4+ T lymphocytes in the proliferative response. (a) Total, CD8-depleted (CD4+), and CD4-depleted (CD8+) PBMC, isolated from three buffy coats, were stimulated with 103 pp/cell of either HAd5 or CAV-2 for 7 days and labeled with [3H]thymidine. Results are shown as SI, and error bars denote the standard error of the mean. The three donors generated similar patterns, although they varied in intensity. Results from one donor are shown. (b) CFSE-labeled PBMC were incubated with an anti-CD3 antibody conjugated with Cy3, and the percentage of HAd5- and CAV-2-specific (103 pp/cell for each) proliferating CD3+ T lymphocytes was monitored using flow cytometry. PHA-induced proliferation was used as a control. All stimulations were done in triplicate. The purity of the selected populations was >95% (not shown). We used the negative selection strategy to avoid activation/stimulation of T cells. Donor 2b had an SI of 43 using 103 pp/cell of HAd5 and an SI of 12 using 103 pp/cell of CAV-2.

CD69, the earliest known inducible protein to appear on activated T cells, and CD25, the {alpha} chain of the IL-2 receptor, are upregulated during cellular stress and antigen recognition. We therefore assayed CD25 and CD69 upregulation following incubation with HAd5 or CAV-2 (Fig. 3a and b). In the proliferation assay, donor 3a had an SI that was near the highest for each virus (Fig. 3 legend). HAd5-induced CD25+/CD69– and CD25+/CD69+ expression was four- and eight-fold over the background. Similar to the proliferation assay, PBMC from the 15 donors tested responded modestly to CAV-2: in donor 3a, the only notable difference versus the mock-treated sample was a threefold increase in CD25+/CD69+ cells (2.2-fold less than HAd5), demonstrating a significant difference in these donors.



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  		FIG. 3. Activation and frequency of T-cell response. PBMC from 15 donors (results from 2 are shown) were stimulated for 3, 5 (not shown), or 7 days with 103 pp/cell of HAd5, CAV-2, or PHA/IL-2 or mock treated. Peak activation was at 5 days. We assayed T-lymphocyte activation using flow cytometry. PBMC were labeled with FITC- or PE-conjugated antibodies against the activation markers CD25 and CD69. (a) Donor a was a strong responder (SI of 155 and 84 using 103 pp/cell of HAd5 and CAV-2, respectively), while the donor in panel b was near the mean (Table 1). (c) Flow cytometry analysis of HLA-DR expression on T cells (CD3+) from donor in 3b (seven donors were assayed for HLA-DR expression). We performed all stimulations in triplicate.

A second donor (3b), who scored close to the SImean for CAV-2 and HAd5, gave a greater variation between HAd5- and CAV-2-induced cell activation (Fig. 3b). Similar to donor 3a, we found an ~7-fold increase in CD25+/CD69+ cells following incubation with HAd5. However, we found little difference between CAV-2 and the mock-treated cells, demonstrating the interpatient variability and lack of a CAV-2-reactive TM in some donors.

To further characterize the cellular response, we also assayed the upregulation of HLA-DR on T cells (CD3+). We found an ~3.5-fold increase following incubation with the HAd5 assay versus the mock- or CAV-2-treated cells (Fig. 3c). Combined, these data demonstrated a notable interpatient variability between the CAV-2 and HAd5-induced activation of PBMC.

Identification and quantification of Th1 or Th2 type response. To determine if CAV-2 induced a Th1 or Th2 type response in PBMC, we assayed for IFN-{gamma} and IL-4 secretion using an ELISPOT assay (35). We found that CAV-2- and HAd5-induced IFN-{gamma} expression was time and dose dependent (Fig. 4a). At 18 h postincubation, CAV-2 induced ~2-fold fewer spot-forming cells (SFC) than HAd5. At 48 and 72 h, the number of CAV-2-induced SFC remained constant while HAd5 continued to increase. We found no CAV-2- or HAd5-induced IL-4 secretion in eight donors tested (Fig. 4b), suggesting that the response was primarily Th1, as previously reported for HAd2/5 antigens.



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  		FIG. 4. Kinetics of IFN-{gamma} and IL-4 secretion. PBMC were stimulated with 103 pp/cell of HAd5 or CAV-2 for 6, 18, 48, and 72 h, with PHA/IL-2, or mock treated (data not shown). The number of IFN-{gamma}- (a) or IL-4- (b) producing T lymphocytes were monitored using the ELISPOT assay. The results are expressed in SFC/106 cells. The results from one of eight donors are shown; the assay was performed in quadruplicate. Error bars denote the standard error of the mean. (c) Intracellular staining of IFN-{gamma} assayed by flow cytometry. PBMC were incubated with 103 pp/cell of CAV-2 or HAd5. PHA/IL-2 and mocked-treated cells were used as controls. This donor had an SI of 93 and 83 for 103 pp/cell of HAd5 and CAV-2. CAV-2 induced 120 SFC/106 PBMC at 48 and 72 h, while HAd5 increased from 150 to 180 SFC/106 PBMC at 72 h. (d) PBMC isolated from three donors (HAd5-induced SI of 293, 57, and 32; CAV-2-induced SI of 25, 0.8, and 4.3) were stimulated with 102 or 103 pp/cell of either HAd5 or CAV-2 or with tetanus toxin (3 µg/ml) for 24 h. The average SFC induced by tetanus toxin was 60 (±10)/106 PBMC, which is consistent with others (40). PHA/IL-2 and mocked-treated cells were used as controls (not shown). The number of IFN-{gamma}-producing cells was monitored using the ELISPOT assay, and the results are expressed in SFC/106 cells. All donors varied in IFN-{gamma} (*, P < 0.05) between 102 and 103 pp/cell of HAd5. The responses from donors 2 and 3 were not significant for 102 or 103 pp/cell CAV-2 (P < 0.10). Error bars denote standard errors of the means and can be hidden by points.

A limitation of the ELISPOT assay is the inability to identify the IFN-{gamma}-secreting cells. To complement this assay, PBMC from three donors were incubated with CAV-2 or HAd5 and assayed for CD4 expression and intracellular IFN-{gamma} expression. Two of the donors responded with <1% IFN-{gamma}-secreting cells for each virus, and the results from the third are shown (Fig. 4c). In donor 4c, we found no difference between CAV-2 and HAd5 induction of IFN-{gamma}. These data also demonstrated that at least 80% of the IFN-{gamma}-secreting cells were CD4+.

As well as a time-dependent IFN-{gamma} expression, a dose effect is also critical to the level of the subsequent immune reaction. We therefore quantified the HAd5 and CAV-2-induced SFC in three donors (Fig. 4d). To put our data in the context of a well-characterized antigen, we also used tetanus toxin as a stimulus (tetanus vaccinations are advised once every 5 to 10years). We found a dose-dependent IFN-{gamma} induction using HAd5. Consistent with our previous data, CAV-2 induced fewer (versus HAd5) SFC in each of the three donors. Only donor 1 gave a statistically significant dose-dependent effect for CAV-2. Notably, HAd5 induced more SFC than tetanus toxin in two out of three donors, demonstrating that HAd infections are controlled by relatively high levels of TM (23). Together, these approaches generated quantitative and qualitative data, demonstrating that the HAd5- and CAV-2-induced response was primarily IFN-{gamma}-secreting CD4+ cells and that CAV-2 was less immunoreactive than HAd5. A priori, one would predict that in our Ad-induced CD4+ TM proliferative assay, these cells would be from the effector TM (TEM) population, which are found preferentially in the peripheral circulation. Consistent with this assumption, TEM are CCR7–, secrete IFN-{gamma}, or contain perforin granules (57). However, Sester et al. (65) found in their assay that Ad-induced proliferating cells were IL-4–, IFN-{gamma}+ (similar to our data, see below), and perforin negative.

CAV-2 versus HAd species A, B, C, D, and E and MoDC presentation. While HAd2 and -5 (species C) are the best characterized members of the Adenoviridae and have been used for gene transfer vectors for >20 years, they represent a fraction of the HAd serotypes. In addition, serotype prevalence is spread irregularly throughout different populations and geographical locations. To address our biased perspective of Adenoviridae-specific TM, we compared CAV-2-induced T-cell proliferation to prototypes from five of the six human species (Fig. 5a). We found that species C and D (HAd5 and HAd17, respectively) induced the highest level of proliferation. Interestingly, in two of the three donors, HAd17 induced the highest response (HAd17 seroprevalence is ~30%, yet morbidity is rare) (25, 79). CAV-2-induced proliferation was <3 SI in the three donors and was similar to HAd12, a relatively rare human serotype (25). These data demonstrated that the TM proliferative response was due to the input capsid load (psoralen-inactivated viruses were used) and not de novo protein synthesis (either enhanced GFP or viral proteins). More markedly, these data suggested that the interspecies Adenoviridae TM response poorly cross-reacted and that the antigenic epitopes (e.g., Hx910-924)—in the context of whole virion—were not equally effective at inducing proliferation.



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  		FIG. 5. T-cell-induced proliferation: CAV-2 versus a prototype from each of the five HAd species and MoDC antigen presentation. (a) PBMC from three donors (same donors as in Fig. 4d) were stimulated for 7 days with 103 pp/cell of psoralen-inactivated CAV-2, HAd5, HAd4, HAd11, HAd12, or HAd17 (102 pp/cell). Monocytes and CD4+/CD45RA– and CD8+/CD45RA– T lymphocytes were isolated from three donors (buffy coats). Monocytes were differentiated into MoDC using IL-4/GM-CSF for 7 days. CD45RA–/CD4+ (b) and CD45RA–/CD8+ (c) T lymphocytes were cocultured with MoDC (ratio, 1:20) and stimulated for 7 days with 103 pp/cell of psoralen-inactivated CAV-2, AdGFP, Ad4, Ad11, Ad12, or Ad17. Results are shown as SI. UV/psoralen inactivation reduced the infectious titer >10,000-fold (not shown). Notably, we did not detect a difference between CAV-2 and UV/psoralen-inactivated CAV-2 or between HAd5 and UV/psoralen-inactivated HAd5 (not shown). The purity ofthe MoDCs, CD8+/CD45RA–, and CD4+/CD45RA– was >95% (notshown).

Leen et al. (36) and Smith et al. (69) demonstrated an improved efficacy of de novo CTL production via MoDC antigen presentation. Although their approach was to generate HAd-specific CTL from naïve T cells, we asked if MoDC could increase the sensitivity of our in vitro proliferation assay via improved antigen presentation and retested the proliferation capacity of CD8+ TM cells (Fig. 2). To eliminate the signal from proliferating naïve T cells, we incubated the viruses with MoDC and then cocultured them with purified CD4+ or CD8+ TM. Confirming our previous results, we detected only CD4+ TM proliferation following incubation with capsid antigens (Fig. 5b and c). Similar to Fig. 5a, the proliferation was highest for HAd5 and HAd17 and lowest for CAV-2 and HAd12. Together, these data demonstrated the preferential proliferation of CD4+ TM, and the low level of CAV-2-induced TM proliferation.

To determine if CAV-2 capsid proteins activated CD8+ TM cells, we repeated the above conditions (purified CD8+ TM alone or cocultured MoDC) and assayed for IFN-{gamma} secretion using an ELISPOT assay. In the five donors tested, we were unable to detect activation (data not shown). With the inherent limitations of the in vitro assays used here, our data suggested that CD8+ TM were unable to respond to Adenoviridae antigens. Furthermore, we found that MoDC antigen presentation (versus PBMC) did not notably increase the sensitivity of our TM proliferation assay (not shown).

Which CAV-2 capsid proteins induce the greatest proliferation. As mentioned previously, up to 30% of the anti-HAd TMproliferation may be due to the Hx910-924 epitope (910-DEPTLLYVLFEVFDV-924) in the Adenoviridae hexon. This motif (underlined) can be restricted by a class II allele present in the majority of the population (12, 77) and may be primordial for the inter- and intraspecies anti-Ad TM response. The CAV-2 hexon also harbors this epitope and, a priori, one would predict that if donors responded to HAd5, then CAV-2 (as well as the other human serotypes) would generate at least a partial response. As shown above (Fig. 2 and 5), we did not find that to be the case.

To examine this inconsistency, we incubated PBMC with CAV-2, HAd5, the CAV-2 fiber, penton base, and hexon, the HAd5 fiber, penton base, and hexon, or Hx910-924 and assayed for the proliferative response (Fig. 6 and Table 2). We performed dose-response assays and found that the highest concentrations presented here (e.g., 25 nM hexon) were near saturating (not shown). Notably, the doses we used were comparable to those used by Olive et al. (45). We found a dose-dependent proliferative response to each stimulus in all responders (for examples, see Fig. 6a, data from a single donor). Consistent with our previous data, we also found an interpatient variability in the relative response to each antigen (Fig. 6b). Combined data from our cohort showed that under saturating conditions, the SImean was greatest for HAd5 hexon, followed by the penton base. In five of the eight donors, hexon induced the greatest proliferation, while for the remaining donors, it was the penton base. There was no significant difference between the SImean of the whole capsid, the fiber, or the peptide. A similar profile, although less intense, was seen with CAV-2 virion and proteins. Saturating concentrations of Hx910-924 generated an SImean of ~30% of the total HAd5-induced TM proliferation (Fig. 6c). Importantly, the SImean for HAd5 and CAV-2 in these donors was similar to that found with the total cohort (Tables 1 and 2), suggesting that this small cohort was a close representative of the total cohort.



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  		FIG. 6. T-cell proliferation induced by CAV-2, HAd5, individual capsid proteins, and a conserved Adenoviridae hexon epitope. (a) PBMC from a single donor were stimulated for 7 days with 102 or 103pp/cell HAd5; 5 or 25 nM HAd5 hexon (Hx); 5 nM HAd5 penton base (PB); 5 nM HAd5 fiber (F); 5, 25, 125, or 625 nM peptide 910-924; 102 or 103pp/cell CAV-2; 5 or 25 nM CAV-2 Hx; 5 or 25 nM CAV-2 PB; or 5 or 25 nM CAV-2 F. (b and c) Combined results from eight donors using the stimuli in panel a. The horizontal bar denotes the SImean. Preliminary experiments from three donors using 0 to 50µM HAd5 hexon showed that 5 µM was in the linear range of the curve while 25 µM was saturating. Compared to 108 pp/well [(1 x 105 cells/well) (1 x 103 pp/cell)], 25 nM hexon (720 molecules/capsid) and Hx910-924 is a 2.5-fold molar excess, 25 nM penton base (60 molecules/capsid) is a 30-fold molar excess, and 25 nM F (36 molecules/capsid) is a 50-fold molar excess. All assays were performed in triplicate, and results are shown as SI.


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  		TABLE 2. SI induced by capsid proteins and hexon epitopea


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DISCUSSION
 
This study addresses a caveat for vector-mediated gene transfer. Several laboratories have questioned the clinical potential of serotype switching (39) of HAd vectors because a HAd5-induced cytotoxic TM can lyse autologous cells infected with other HAd species. Although 49 of 50 donors from a random cohort did not harbor significant titers of neutralizing CAV-2 Abs (31), it is clear that humoral immunity is only one of the obstacles to successful gene transfer. {Delta}E1 CAV-2 vectors were also less immunogenic than an HAd5 vector in the immunologically naïve rat CNS; we detected significantly fewer infiltrating CD4+ and CD8+ cells at an equivalent number of injected particles (74). We hypothesized that this was due to a combination of factors: (i) the lack of transduction of immune mediator cells (micro- and macroglia) (2), (ii) the dispersion of the vector from the site of injection via the axoplasmic transport, and (iii) possibly a lower innate immune response due in part to the lack of identifiable integrin-interacting motifs in the external CAV-2 capsid (16, 71). Using helper-dependent (HD) vectors, only the therapeutic transgene will be expressed and therefore the risk is due to the input capsid load, which should be processed and presented primarily via MHC class II molecules. Nonetheless, under some conditions, exogenous proteins can be presented via the MHC class I molecules, but the role of CD8+ TM in HAd immunosurveillance is currently poorly understood. We therefore concentrated on CD4+ TM because in the context of the possible use of CAV-2 vectors for long-term gene transfer, only HDCAV vectors (3, 74) are probably applicable.

Here, we compared the frequencies, proliferation, activation, and phenotypic characteristics of human T cells stimulated by CAV-2 virions and capsid proteins versus HAd. We tested total, CD4-depleted and CD8-depleted PBMC; and purified memory CD4+ or CD8+ cells individually or with professional antigen-presenting cells. The most notable results were that the majority of the cohort did not harbor cellular immunity to CAV-2 and, in the responders, the SImean was less than a 10-fold lower dose/cell of HAd5. We did not find a correlation between donors that had a high (>2x SImean) HAd5-induced (n = 10) versus the CAV-2-induced (e.g., 4 of the 10 were CAV-2 nonresponders) SI. In contrast, the eight donors that had a high CAV-2-induced SI were above the HAd5-induced SImean (not shown). The overall significance of this is not clear. We found a general correlation between the frequencies of T-cell proliferative responses and cytokine secretion capacities, as evaluated by standard proliferation, IFN-{gamma} ELISPOT assay, and CFSE staining. In all donors, responding T cells appeared to be CD4+, which showed a considerably homogenous expression of markers characteristic of antigen-experienced memory/effector cells of the Th1 phenotype that secreted IFN-{gamma}. Our results are consistent with those of Sester et al., who found, using whole blood, IFN-{gamma} staining, and an "Ad antigen with broad specificity," that 0.05 to 0.64% of the CD4+ cells (~1,000 to 13,000 SFC/106 PBMC) from 50% of the donors were Ad specific (65). Olive et al. reported a >10-fold lower range (34 to 294 SFC/106 PBMC) with a mean of 122 SFC/106 PBMC when using 10 µg/ml of Ad-infected cell extracts as the stimuli and assayed 48 h poststimulation (46). Both Sester et al. and Olive et al. found a reverse correlation with age and the quantity of Ad-specific CD4+ TM. While we found a positive correlation (r = 0.99) between IFN-{gamma} SFC and Ad-induced CD4+ T-cell proliferation (n = 8) (data not shown), Olive et al. did not in the four donors reported (46). Consistent with the above data, CAV-2 induced a lower level of T-cell activation as determined by CD25 and CD69 upregulation, as well as HLA-DR expression. When we compared the CAV-2-induced proliferation to five of the six HAd species, we found that CAV-2 was similar to a rare serotype. We modestly improved the sensitivity of the proliferation assay using MoDC as antigen-presenting cells, and we were unable to detect CD8+ TM proliferation or activation in these in vitro assays.

Finally, the CAV-2 hexon, penton base, and fiber, as well as a conserved hexon epitope, were used in our assays to determine their individual contribution. Our data are consistent with the fact that the primary hexon sequence is highly conserved in Adenoviridae and antigenic. Souberbielle and Russell also compared the HAd2 and capsid protein-induced proliferation in a single donor and found that the complete capsid induced a higher SI than individual proteins (70). It is not surprising that the fiber induced a lower SImean: the antigenic divergence in this protein may be the greatest among the serotypes. Although our data resemble the results of Olive et al. (45, 46), their data are inconsistent with our overall results using CAV-2, HAd17, and HAd12 virions (Fig. 1 and 5). While MHC class II loading in vitro is efficient using proteins and peptides, this poorly mimics the antigen processing and presentation of virion. It is conceivable that HAd5 virions are more efficiently degraded in the lysosome, and although we are unaware of any direct data to support this in the cells used in our assays, we cannot exclude this scenario. Three of the other serotypes (HAd4, HAd12, and HAd17) tested here, as well as CAV-2, either interact with similar receptors (coxsackievirus and adenovirus receptor) or, like HAd11, use another receptor(s) and are significantly more efficient at infecting PBMC (8, 15, 48, 54, 59, 60). One would predict that greater binding and uptake by HAd11 (species B) in PBMC would lead to greater antigen presentation and proliferation. Shayakhmetov et al. showed using fiber chimeric vectors that HAd5 escaped more efficiently from the endosomal/lysosomal compartments than a HAd5 capsid with a species B fiber knob (from HAd35) (66). In addition, a significant portion of the latter chimeric vector particles appeared to be recycled back to the cell surface in HeLa cells. It is unknown if the entire HAd35 virion—or species B HAd in general—behaves similarly or if the serotypes tested here from species A, D, and E are representative or exceptions.

While TM currently pose an unquantifiable risk in the context of viral vector-mediated gene therapy, our results must also be viewed in the context of the innate response, the memory humoral response, the interpatient haplotype variation, the route of delivery, and the target organ (the immune response in skeletal muscle will be much different than in the CNS). To add another layer of complexity, children, adolescents, and young and aged adults will have different immunological pasts. If children or adolescents are targeted, they may have the highest and most varied anti-Ad TM response. Memory T cells are functionally and phenotypically heterogeneous and can be divided into central-memory (TCM) and TEM (56, 57). Each has a different steady-state and antigen-induced proliferation rate (38, 57) as well as a variable response to cytokines (20). TEM probably divide more quickly in response to pathogens, while TCM may serve as an expandable pool in secondary lymph nodes to generate effector cells. The decrease in virus-specific immunity in adults may reflect the elimination of a pathogen and the concomitant lack of necessity to maintain high-level immunity. Alternatively, it may also indicate a decreased susceptibility toward that particular virus. Studies of other viruses indicated that the presence or absence of virus-specific TM might be triggered by their persistence. The immune response toward varicella zoster virus (VZV) showed an age-dependent waning of VZV-specific cellular immunity. This is compatible with an increased susceptibility to VZV reactivation and morbidity (4). A therapy-induced decline in human immunodeficiency virus load leads to loss of both virus-specific CD4+ and CD8+ T cells (49, 64). However, cytomegalovirus-specific T-cell response can be detected irrespective of age (63) and is indicative of a continued antigenic stimulation and persistent infection. In the case of Ad, however, there is no evidence to support an increased incidence of infection with age. An evolving hypothesis is that HAd are childhood pathogens (11), due to the lack of memory immunity and the expression and accessibility of its receptor(s) during development (26, 27). Immunosuppression probably leads to the escape of latent virus due to the disruption of the well-balanced equilibrium between Ad-specific TM responses and viral replication and thus correlates with morbidity.

It is premature to predict the global or specific risks patients may face, but monitoring of Ad-specific TM responses (as well as the memory humoral response) before gene transfer may identify patients who are at risk. A priori, our data suggest that there is no correlation between NAb titer (98% of a random cohort were negative) and TM (55% negative). However, in light of our results, it seems prudent to thoroughly determine the presence and avidity of nonneutralizing antibodies that recognize the CAV-2 capsid. Using serum from a subset of the donors in this study, we found no correlation between anti-CAV-2 or anti-HAd5 NAb, IgG, IgA, or IgM titers versus the SI (M. Perreau and E. J. Kremer, unpublished data). Furthermore, if Hx910-924 is the key to TM immunity, we do not know if the hexon will tolerate mutations here (located in the eight-stranded ß-barrel viral jelly roll sequence at the internal base [55, 77]). An increased understanding of Adenoviridae and the underlying immune responses will lead to improved therapeutic strategies.


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ACKNOWLEDGMENTS
 
This work was supported by the association Vaincre les Maladies Lysosomales (VML), the Association Française contre les Myopathies (AFM), and Vaincre la Mucoviscidose. M.P. is an AFM/VML fellow. E.J.K. is an INSERM Director of Research.

We acknowledge the exceptional aid of C. Frapaise and M. Bichouard from the Montpellier EFS. We thank F. Mennechet, O. Billet, H. Wodrich, V. Kalatzis, J. Hernandez, V. Pinet, and M. Villalba for technical help, suggestions during the course of this work, and critical readings of the manuscript. We thank H. Yssel for access to the cell harvester, G. Wadell for the wild-type adenoviruses, P. Boulanger for the HAd5 penton base and fiber, G. Nemerow for the HAd5 hexon, O. Coux for help with protein purification, and V. Juillard for help with the ELISPOT assays. Hx910-924 peptide was synthesized and purified by Synt:em.

We have no conflicting financial interests.


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FOOTNOTES
 
* Corresponding author. Mailing address: IGMM CNRS 5535, 1919 Route de Mende, 34293 Montpellier, France. Phone: (33) 4 67 61 36 72. Fax: (33) 4 67 04 02 31. E-mail: eric.kremer{at}igmm.cnrs.fr. Back


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Journal of Virology, December 2005, p. 14595-14605, Vol. 79, No. 23
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.23.14595-14605.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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