Adenovirus Hexon Protein Research Articles

Reduction of nontarget infection and systemic toxicity by targeted delivery of conditionally replicating viruses transported in mesenchymal stem cells

The fiber-modified adenoviral vector Delta-24-RGD (D24RGD) offers vast therapeutic potential. Direct injection of D24RGD has been used to successfully target ovarian tumors in mice. However, systemic toxicity, especially in the liver, profoundly limits the efficacy of direct viral vector delivery. Mesenchymal stem cells (MSC) have the ability to function as a vector for targeted gene therapy because of their preferential engraftment into solid tumors and participation in tumor stroma formation. We show that MSC-guided delivery of D24RGD is specific and efficient and reduces the overall systemic toxicity in mice to negligible levels compared with D24RGD alone. In our model, we found efficient targeted delivery of MSC-D24RGD to both breast and ovarian cell lines. Furthermore, immunohistochemical staining for adenoviral hexon protein confirmed negligible levels of systemic toxicity in mice that were administered MSC-D24RGD compared with those that were administered D24RGD. These data suggest that delivery of D24RGD through MSC not only increases the targeted delivery efficiency, but also reduces the systemic exposure of the virus, thereby reducing overall systemic toxicity to the host and ultimately enhancing its value as an anti-tumor therapeutic candidate.

Efficient Vascular Endothelial Gene Transfer Following Intravenous Adenovirus Delivery

Efficient intravascular gene delivery to the endothelium in defined vascular beds pertinent to pathological states has been a long-pursued goal for many gene therapy applications. Clear advantages include the minimally invasive route of delivery and the ability to target distal sites in the body that are relatively inaccessible surgically. Such targets are diverse and include angiogenic endothelium in cancer, the vasculature of defined organs such as the lung, and microvascular endothelium in regions of poor blood flow.

CMV Specific T Cells for Adoptive Transfer Exhibit Multiple Effector Functions Associated with Protective Immunity Including the Concurrent Production of Cytokines and Cytolytic Activity

Background: Cytomegalovirus (CMV) reactivation post allogeneic haemopoietic stem cell transplant (HSCT) causes significant morbidity. Adoptive transfer of ex-vivo generated CMV specific T cells has the potential to restore immunity, prevent CMV reactivation and circumvent the need for pharmacotherapy. Donor derived CMV specific T cells were generated for prophylactic infusion into haemopoietic stem cell transplant recipients as part of a phase I/II clinical trial aiming to reduce the incidence of CMV reactivation. T cells were expanded by co-culturing with dendritic cells transfected with Ad5F35pp65, a recombinant adenovirus promoting the presentation of epitopes derived from the immunodominant CMV antigen pp65. The aim of this study was to determine if ex vivo expanded CMV specific T cells have the capacity to produce different cytokines and chemokines associated with protective immunity.Results:Ex vivo expanded Ad5F35pp65 stimulated cultures were primarily CD3+ T cells (median 92%) with a predominance of CD8+ (14–90%) over CD4+ (2.5–57%) cells. An assay measuring antigen specific production of interferon-γ (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor (TNF) and macrophage inflammatory protein 1β (MIP-1β) by intracellular cytokine flow cytometry was established to quantify the frequency of T cells with specificity towards CMV or adenovirus and to assess the quality of responses reflected by the simultaneous production of multiple cytokines. Ad5F35pp65 stimulated cultures were greatly enriched for CMV specific T cells producing cytokines in response to pp65 (mean 59%, 19.1–90%) compared to the starting PBMC population (0.5–1.5%). Responses directed towards the adenovirus hexon protein were also detected accounting for 0.65 to 9% of T cells. CMV specific CD8 T cells predominantly produced IFN-γ and MIP-1β followed by TNF with means of 61.3%, 59% and 44% respectively. The majority of CMV specific CD8+ T cells produced both IFN-γ and MIP-1β (80–96%) with a substantial proportion of these also producing TNF (72%, 44–88.5%). IL-2 producing CD8+ T cells were less frequent, ranging from 0.5–40%. However, IL-2 producers consistently exhibited the highest level of functionality with the production of all four cytokines. Furthermore, IFN-γ producing CD8+ T cells mobilized CD107, a marker of degranulation and cytotoxic activity. In the majority of cases, fewer CD4+ T cells exhibited specificity towards CMV pp65 (30%, 4–49.5%) however greater than 80% co-produced IFN-γ, MIP-1β and TNF. Furthermore, a high proportion of CD4+ T cells also produced IL-2 (53.4%). Adenovirus specific T cell responses were detected in all cultures but were mainly confined to the CD4+ population. Finally, we examined CMV specific responses in the starting donor PBMC population. Cytokine production profiles of CMV specific CD4 and CD8 T cells closely resembled those of ex vivo generated cultures suggesting the uniform expansion of CMV specific functional subsets. In conclusion, these results demonstrate that ex vivo expanded CMV specific T cells intended for adoptive transfer perform several functions associated with protective immunity in vivo, including the capacity to kill infected cells and the simultaneous production of multiple cytokines.

Midkine promoter-based conditionally replicative adenovirus therapy for midkine-expressing human pancreatic cancer

BackgroundTo develop a novel therapeutic strategy for human pancreatic cancer using a midkine promoter-based conditionally replicating adenovirus.MethodsWe examined midkine mRNA expression and midkine protein expression by seven human pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, MIAPaCa-2, PANC-1, and Suit-2), as well as by non-cancerous pancreatic tissue and pancreatic cancers. Midkine promoter activity was measured in cancer cell lines by the dual luciferase reporter assay. Adenoviral transduction efficiency was assessed by fluorescent staining of cancer cell lines using adenovirus type 5 containing the green fluorescent protein gene (Ad5GFP). Replication of adenovirus type 5 containing the 0.6 kb midkne promoter (Ad5MK) was assessed by the detection of E1 protein in cancer cell lines. The cytotoxicity of Ad5MK for cancer cells was evaluated from the extent of growth inhibition after viral infection. Infection and replication were also assessed in nude mice with subcutaneous Suit-2 tumors by intratumoral injection of Ad5MK, Ad5GFP, or vehicle. E1a mRNA expression in the treated tumors and expression of the replication-specific adenoviral hexon protein were evaluated. Finally, the anti-tumor activity of Ad5MK against intraperitoneal xenografts of Suit-2 pancreatic cancer cells was examined after intraperitoneal injection of the virus.ResultsBoth midkine mRNA expression and midkine protein expression were strong in AsPC-1 and CFPAC-1 cell liens, moderate in BxPC-3, HPAC, and Suit-2 cell lines, and weak in PANC-1 and MIAPaCa-2 cell lines. Expression of midkine mRNA was significantly stronger in pancreatic cancers than in non-cancerous pancreatic tissues. The relative luciferase activity mediated by the 0.6 kb midkne fragment in AsPC-1, PANC-1, and Suit-2 cell lines was approximately 6 to 20 times greater than that in midkne-negative MIAPaCa-2 cell lines. Pancreatic cancer cell lines exhibited a heterogeneous adenoviral transduction profile. E1A expression was higher in cell lines with strong midkine expression than in cell lines with weak midkine expression. Ad5MK showed much greater cytotoxicity for midkine-expressing Suit-2 and PANC-1 cell lines than for midkine-negative MIAPaCa-2 cell lines. In the Suit-2 subcutaneous xenograft model, expression of E1A was detected in Ad5MK-treated tumors, but not in untreated and Ad5GFP-treated tumors. In the Suit-2 intraperitoneal xenograft model, the Ad5MK group survived for significantly longer than the Ad5GFP, PBS, and untreated groups.ConclusionAd5MK has an anti-tumor effect against human pancreatic cancer cell lines that express midkine mRNA. Midkine promoter-based conditionally replicative adenovirus might be a promising new gene therapy for pancreatic cancer.

T-cell Lines Specific for Peptides of Adenovirus Hexon Protein and Devoid of Alloreactivity Against Recipient Cells can be Obtained From HLA-haploidentical Donors

Human adenovirus (HAdV) infection may cause life-threatening complications in recipients of hematopoietic stem cell transplantation (HSCT), the highest risk being observed in children given T-cell depleted haploidentical allografts. The effectiveness of pharmacologic therapy for HAdV infection is suboptimal. Recently, cell therapy was demonstrated to offer a unique opportunity to restore antiviral immune surveillance, leading to clearance of infection and prevention/treatment of disease. However, infusion of insufficiently selected HAdV-specific T cells in haplo-HSCT may increase the risk of graft-versus-host disease. We conducted scale-up experiments to validate a method of in vitro culture to expand T cells specific for HAdV from donor peripheral blood mononuclear cells (PBMC), based on stimulation with a pool of five 30-mer peptides derived from HAdV5 hexon protein, for use in recipients of haplo-HSCT. A total of 21 T-cell lines that included a majority of CD4 T lymphocytes, were generated. Nineteen of the 21 T-cell lines proliferated specifically against HAdV. The 2 nonspecific, and 3 T-cell lines with lower specific activity, included a median of 48% CD8 T cells. The 19 HAdV-specific T-cell lines showed a median 357-fold decrease in alloreactivity, compared with proliferation of noncultured donor PBMC in response to recipient PBMC, only 4/19 T-cell lines showing residual alloreactivity. Our data indicate that HAdV-specific CD4 T-cell lines with efficient in vitro antiviral response and low/undetectable alloreactivity against recipient targets may be expanded from PBMC of most human leukocyte antigen-haploidentical HSCT donors after stimulation with HAdV hexon protein-derived peptides. These T cells may be safely employed for adoptive treatment of HAdV complications.

Clinical Grade Generation of Hexon-specific T Cells for Adoptive T-cell Transfer as a Treatment of Adenovirus Infection After Allogeneic Stem Cell Transplantation

Adenovirus infection after allogeneic hematopoietic stem cell transplantation is still causing significant morbidity and mortality, especially in children. It has been demonstrated that a sufficient host T-cell response is essential to clear the virus. Adoptive transfer of specific T-cell immunity from the donor to the recipient has become a new treatment option for patients with systemic adenoviral infection who lack specific T-cell responses. The adenoviral hexon protein was shown to be an immunodominant T-cell target. We describe here a Good Manufacturing Practice-compatible generation of hexon-specific T cells developed by isolating interferon-gamma-secreting T cells after stimulation of mononuclear cells ex vivo with hexon protein. Phenotypical and functional characterization of the generated, specific T-cell product resulted in a mixed population of CD4 and CD8-positive T cells with an intermediate effector memory phenotype. Isolated hexon-specific T cells showed high expansion potential in vitro and specific cytotoxicity. T-cell lines, directed against type 5 hexon protein showed good crossreactivity against viral strains from other adenovirus species. The availability for isolation of hexon-specific T cells among 76 hematopoietic stem cell transplantation donors showed in > 72% a sufficient T-cell response (0.05% of T cells). In conclusion, Good Manufacturing Practice-grade selection of adenovirus-specific T cells for adoptive immunotherapy by hexon-induced secretion of interferon-gamma has been established. Adoptive T-cell transfer could potentially restore T-cell immunity against adenovirus after allogeneic stem cell transplantation.

Baculovirus-based Vaccination Vectors Allow for Efficient Induction of Immune Responses Against Plasmodium falciparum Circumsporozoite Protein

Baculovirus vectors are able to transduce a large variety of mammalian cell types and express transgenes placed under the control of heterologous promoters. In this study, we evaluated the potential of baculovirus vectors for malaria vaccination. To induce efficient CD4(+) and CD8(+) T-cell responses, we produced a series of vectors that display the Plasmodium falciparum circumsporozoite (CS) protein in the virion envelope and/or allow for CS expression upon transduction of mammalian cells. We found that baculovirus vectors can transduce professional antigen-presenting cells and trigger their maturation, which is a prerequisite for efficient antigen presentation. Upon intramuscular injection into mice, the vector that both displayed and expressed CS induced higher anti-CS antibody titers (of the immunoglobulin (IgG)1 and IgG2a type) and a higher frequency of interferon-gamma-producing T cells specific to CS, than the vectors which either only displayed or only expressed CS. The baculovirus CS display/expression vector was also superior in inducing CS-specific CD4(+) and CD8(+) T-cell responses in vitro using human peripheral blood mononuclear cells from naive donors. This, together with the absence of pre-existing immunity to baculoviruses in humans, the absence of viral gene expression in mammalian cells, and the relative low immunogenicity of baculovirus virions, makes these vectors promising tools for vaccination. Furthermore, the ability to produce large amounts in serum-free medium at a low cost adds a further advantage to this vector system.

Hexon Specific T-Cells for Adoptive T-Cell Transfer as a Treatment of Adenovirus Infection after Allogeneic Stem Cell Transplantation.

Adenovirus infection (ADV) after allogeneic hematopoietic stem cell transplantation (HSCT) is an emerging pathogen causing relevant morbidity and mortality, with preponderance in children. Since a sufficient host T-cell response has been shown essential to clear the virus, diagnostic procedures for detection of virus-specific T-cells have recently developed to asses the specific cellular immune response. Furthermore adoptive immunotherapy is a new treatment option for patients with absent specific T-cell response and present systemic adenoviral infection. The possibility of an adoptive T-cell transfer depends on the availability of GMP compatible protocols and reagents. The adenoviral hexon protein was shown a immunodominant T-cell target within the viral capsid. In the present study we investigate the presence of Hexon-specific T-cell responses in HSCT donors, i.e. the availability of donors for an adoptive T-cell transfer. Secondly, the emergence of Hexon-specific T-cells in recipients post HSCT was analyzed. Thirdly, a protocol was established under GMP-conditions for adoptive T-cell immunotherapy through isolation of IFN-γ secreting T-cells after ex-vivo stimulation with the adenoviral Hexon protein. This procedure resulted in a mixed population of CD4 and CD8 positive T-cells with an effector memory phenotype and Th1 cytokine pattern (n=8). Isolated Hexon-specific T-cells show a strong expansion potential in vitro as well as specific cytotoxic activity. The availability of a donor was evaluated in 76 HSCT donors. Only 17.1% of donors had no ADV-specific T-cell response and 72.4% of donors were eligible for an adoptive T-cell transfer using the presented approach. The Hexon protein was responsible for almost the complete response to ADV, since no significant difference was seen against ADV lysate and the Hexon protein. In 76% HSCT recipients Hexon directed T-cell responses were evaluated and were shown to be responsible for clearance of the viral infection. In conclusion feasibility of an adoptive T-cell transfer for the treatment of ADV infection post HSCT is shown in accordance to current GMP regulations.

Treatment of Adenovirus Infection by Adoptive Immunotherapy: Adenovirus Capsid Hexon Is the Main Target Protein of Adenovirus-Specific Cellular Immunity.

Introduction: Adenovirus (AdV) infection is a severe complication after hematopoietic stem cell transplantation, particularly in paediatric patients. Control of AdV infection particularly seems to be dependent on CD4+ T-cells but their main AdV antigenic targets have remained unknown so far. In order to design protocols for targeted adoptive immunotherapy the identification of the main AdV antigenic targets is a fundamental prerequisite.Methods: We here adapted a novel technique to directly assess the entire repertoire of CD4+ T-cells specific AdV antigens according to antigen-induced CD154 expression after short-term ex vivo stimulation. AdV-lysate-specific and AdV-hexon-specific CD4+ T-cells were isolated, expanded in vitro and further assessed for their fine specificities using recombinant AdV-proteins and AdV-lysates from various AdV-serogroups. The cytokine profile of AdV-lysate- and AdV-hexon-specific CD4+ T-cells were assessed by intracellular analysis of AdV-induced CD154+ CD4+ T-cells.Results: AdV-lysate-specific CD4+ T-cells reacted predominantly with AdV-hexon capsid protein. Furthermore, AdV-hexon (serogroup B) specific CD4+ T-cells crossreacted with recombinant hexon protein derived from various other AdV-serotypes and were characterized by a Th1-like cytokine profile.Conclusion: Our results prove the effectiveness of antigen-induced CD154-expression for assessment of the entire repertoire of CD4+ T-cells specific for pathogens, for the identification of immunodominant target antigen from pathogens. We demonstrate that adenovirus hexon protein is a suitable candidate antigen for the ex vivo generation of adenovirus-specific, serogroup cross-reactive CD4+ T-cells with a Th1-like cytokine profile for adoptive T-cell therapies.

Generation of Multi-Specific CTL Lines Using Overlapping Pentadecapeptide Pools of Aspergillus f16 and Adenovirus Hexon Antigens Presented on Epstein Barr Virus-Transformed B Cells as Antigen Presenting Cells.

Allogeneic hematopoietic progenitor cell transplantation (HPCT) is the optimal treatment for several hematological malignancies, aplasia or other disorders of the hematopoietic or immune system. Post HPCT, patients undergo a prolonged period of immune incompetence during which they are highly susceptible to viral and fungal infections. Epstein Barr Virus (EBV), Adenovirus (Ad) and Aspergillus fumigates are among the most common and lethal infections during this period. We have described the use of overlapping pools of pentadecapeptides pulsed onto monocyte-derived dendritic cells (DC) to generate cytolytic T-cell (CTL) lines specific for an A. fumigatus-derived antigen, Asp f16 (Ramadan et. al., 2005, J Clin Immunol 139:257; Ramadan et. al., 200 J Clin Immunol 140:81). We have further demonstrated in 5 donors, the generation of CTL lines recognizing peptides contained within the conserved region of the Adenovirus hexon protein using a similar approach (Keever-Taylor, et. al, Cytotherapy 8, Suppl 1, 122). To reduce the need for large numbers of DC to prime and expand the CTL cultures, we modified our procedures to use peptide pool (pp)-pulsed EBV-transformed B lymphoblastoid cell lines (BLCL) as antigen presenting cells (APC) following 2–3 rounds of initial priming with pp-DC. This approach results in lines that are predominately reactive to Asp f16 or Ad hexon, and which are moderately reactive to EBV. We expanded our studies to determine the feasibility of generating CTL lines that recognize all three infectious agents. Peripheral blood lymphocytes (PBL) from donor RD0309 were primed weekly ×3 with autologous DC (10:1 PBL to DC ratio) pulsed with a pool of both Asp f16 (104 individual peptides) and Ad hexon (105 individual peptides) pentadecapeptides, then switched to Asp/Ad-pulsed BLCL as APC. The cultures were screened one week following restimulation for lysis of non-pulsed (np-BLCL) and Asp f16-pp-pulsed (Asp-BLCL) and Ad hex-pp-pulsed (Ad-BLCL) BLCL targets. Weak reactivity to Ad-BLCL was detected after 3 primings with DC alone that increased following one round of Asp/Ad-BLCL priming. Asp f16-specific CTL activity was not clearly detected until one week after 3DC plus 3BLCL primings, at which time there was also clear reactivity to np-BLCL targets (26.4% np-BLCL, 37% Asp-BLCL, and 53.9% Ad-BLCL at 50:1), indicating CTL activity to all three infectious agents. With an additional priming, Aspergillus specific reactivity exceeded that towards Adenovirus and EBV-specific reactivity declined. These data show that a pool of overlapping peptides from two different antigens presented on DC followed by BLCL as APC can result in multi-specific cell lines that can be potentially used in adoptive immunotherapy protocols for the treatment or prevention of common opportunistic infections following HPCT.

322. Syngeneic Cotton Rat Cancer Model for Replicating Adenoviral Vectors

Background: Oncolytic adenoviruses are under study as a potential anticancer treatment. Their preclinical antitumor activity; however, has not been effectively translated to the clinic. A contributing factor may be the clinical relevance of immunodeficient mouse tumor xenograft models used to study these vectors. While these demonstrate viral replication in the xenograft, they are limited by the lack of natural host immune responses to the virus, associated toxicity, dissemination and systemic viral replication. Cotton rats (Sigmodon hispidus) are semi-permissive for the replication of human adenovirus and have been used to study these vectors. Until recently this model lacked a transplantable tumor to assess oncolytic activity. We characterized three syngeneic transplantable cotton rat tumor cells lines that support adenovirus replication and oncolysis in this immunocompetent rodent model. Methods: Three tumor cell lines: CCRT, LCRT and VCRT were derived from a spontaneously arising osteosarcoma, and fibrosarcomas of the breast and jaw in animals from an inbred cotton rat colony. The ability of the cells to be infected with adenovirus was examined using an adenovirus expressing green fluorescent protein (Ad.GFP). Viral burst assays were used to determine titers of infective adenovirus produced by each cell line at 4 and 72 hours following infection with wild type adenovirus type-5 (Ad5). Transmission electron microscopy (TEM) was used to visualize the relative number of virions at each time point. We also evaluated replication and oncolysis of the cell lines in vivo. Results: Using Ad.GFP, the cell lines were readily infected and demonstrated GFP expression comparable to human A549 cells. All cell lines showed significant higher (P<0.05) intracellular titers of adenovirus and in supernatants 72 hours after infection compared to 4 hours after infection with Ad5 indicating viral replication and increased number of functional virions. In addition, all the cell lines exhibited greater expression of adenoviral hexon protein at 72 hours than at 4 hours after infection, indicating late transcriptional unit gene activation. The cotton rat cell lines formed tumors in cotton rats when injected subcutaneously. On days 5 and 8, PCR demonstrated 2 to 3-log greater number of Ad5 genome copies in tumors injected with Ad5 compared to those injected with a non- replicating adenovirus (Ad.null). This was consistent for CCRT, LCRT and VCRT. Ad5 slowed tumor growth in cotton rats subcutaneously injected with CCRT and VCRT cells. Despite in vitro oncolysis, no anti-tumor effect was seen in the LCRT tumors with the virus rapidly cleared from the tumor. Conclusions: CCRT, LCRT and VCRT, three novel transplantable cotton rat tumor-derived cell lines demonstrate replication of Ad5 both in vitro and in vivo. In addition, CCRT and VCRT demonstrated in vivo oncolysis. The varied antitumor effect in this model mirrors the results of human clinical trials highlighting the potential relevance of these cotton rat tumor models for assessing replication competent adenovirus vectors.

Adenovirus Capsid Hexon Is the Main Target Protein of Adenovirus-Specific CD4+ T-Cells: Fundamentals for Targeting Adenovirus by Adoptive Immunotherapy.

Introduction: Increasing evidence has emerged that adenoviruses (AdV) can cause severe complications in immunocompromised hosts especially following hematopoietic stem cell transplantation (HSCT). So far protective immune responses in adenovirus infection have not been assessed in detail, but control of infection seems to be dependent on CD4+ T-cells. However, the main antigenic adenovirus targets for specific CD4+ T-cells are unknown.Methods: We here use a novel technique to directly assess the entire repertoire of CD4+ T-cells specific for a defined antigen according to antigen-induced CD154 expression after short-term ex vivo stimulation. AdV-lysate-specific and AdV-hexon-specific CD4+ T-cells were isolated, expanded in vitro and further assessed for their fine specificities using recombinant AdV-proteins and AdV-lysates from various AdV-serogroups. Additionally, we evaluated the cytokine profile of AdV-lysate- and AdV-hexon-specific CD4+ T-cells.Results: AdV-lysate-specific CD4+ T-cells reacted efficiently with AdV-hexon and penton proteins. Furthermore, CD4+ T-cells specific for hexon protein derived from AdV-serogroup B crossreact with recombinant hexon protein derived from various other AdV-serotypes and are characterized by a Th1-like cytokine profile.Conclusion: Our results imply that adenovirus hexon protein is a suitable candidate antigen for the ex vivo generation of adenovirus-specific, serogroup cross-reactive proinflammatory CD4+ T-cells for adoptive T-cell therapies.

Neutralizing Antibodies to Adenovirus Serotype 5 Vaccine Vectors Are Directed Primarily against the Adenovirus Hexon Protein

The utility of recombinant adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1 and other pathogens will likely be limited by the high prevalence of pre-existing Ad5-specific neutralizing Abs (NAbs) in human populations. However, the immunodominant targets of Ad5-specific NAbs in humans remain poorly characterized. In this study, we assess the titers and primary determinants of Ad5-specific NAbs in individuals from both the United States and the developing world. Importantly, median Ad5-specific NAb titers were >10-fold higher in sub-Saharan Africa compared with the United States. Moreover, hexon-specific NAb titers were 4- to 10-fold higher than fiber-specific NAb titers in these cohorts by virus neutralization assays using capsid chimeric viruses. We next performed adoptive transfer studies in mice to evaluate the functional capacity of hexon- and fiber-specific NAbs to suppress the immunogenicity of a prototype rAd5-Env vaccine. Hexon-specific NAbs were remarkably efficient at suppressing Env-specific immune responses elicited by the rAd5 vaccine. In contrast, fiber-specific NAbs exerted only minimal suppressive effects on rAd5 vaccine immunogenicity. These data demonstrate that functionally significant Ad5-specific NAbs are directed primarily against the Ad5 hexon protein in both humans and mice. These studies suggest a potential strategy for engineering novel Ad5 vectors to evade dominant Ad5-specific NAbs.

979. Antigen-Transduced T Cell Blasts Specifically Delete Antigen-Specific Cytotoxic T Cells and Induce a CD4+-CD25+- Regulatory T Cell Response

Introduction: Antigen presentation by T cells has been shown to induce anergy and apoptosis in responding antigen-specific T cells. This phenomenon might be useful in targeting antigen-specific T cells for the treatment of autoimmune disease and allogeneic organ transplantation. Therefore, we examined the immune response to antigen-presenting T cell blasts (T-APC) transduced with known antigens (CMV pp65, EBV-LMP2 or adenovirus hexon protein).

Conserved CTL epitopes on the adenovirus hexon protein expand subgroup cross-reactive and subgroup-specific CD8+ T cells

AbstractAdenoviruses often cause lethal infections in immunocompromised individuals. Adoptive transfer of immune T cells offers a therapeutic option, but this strategy has been hindered by the paucity of information on molecular targets of cellular immunity and by the immunologic heterogeneity of the 51 human adenoviruses, which are grouped from A to F on the basis of genome size, composition, homology, and organization. Clonal analysis of the adenovirus-specific cytotoxic T lymphocyte (CTL) responses of seropositive individuals identified 5 novel CD8+ T-cell epitopes, all located in conserved regions of the capsid protein hexon. Reactive T cells were cross-reactive between 2 to 4 groups, while no T cells specific for a single subgroup were detected. Thus, by exploiting these peptide targets, it is possible to prepare a T-cell population capable of reacting with most adenoviruses that cause disease in immunocompromised patients.

Adenovirus nephritis in hematopoietic stem-cell transplantation.

Although adenovirus (ADV) infections may involve many different organs, kidney infection is seldom reported in association with hematopoietic stem-cell transplantation (HSCT). In the present study, the diagnosis of ADV nephritis was established by the culture isolation of adenovirus or the immunocytochemical (ICC) demonstration of the adenoviral hexon protein. The clinical description of ADV nephritis was derived from retrospective review of clinical records to identify signs, symptoms, outcomes, and associated complications. ADV nephritis was characterized as a pathologic entity by the histologic and ICC analysis of tissue from the kidney and all other major organs to establish the distribution of the virus and the associated gross and microscopic alterations. ADV nephritis was diagnosed in 21 HSCT patients, in 2 by biopsy and in 19 at autopsy. Focal signs of BK nephropathy were present in only one patient. Twenty had received allogeneic marrow and one had undergone autologous transplantation. Graft-versus-host disease was a risk factor. ADV nephritis was associated with acute renal failure in 90% of the infected patients. Prodromal symptoms included fever, hematuria, and flank pain. Adenoviruria was present in 78% of the patients. Kidney infection as determined by viral antigen ICC predominantly involved the tubular epithelial cells. ADV organ tropism was striking, with sero-types from subgenus B, cluster 2, primarily responsible for cases involving predominantly the urinary system. ADV infection was a major cause of death in 17 patients. ADV nephritis is a specific renal complication in HSCT patients that can be diagnosed by renal biopsy in patients with hematuria and adenoviruria.

T-cell response to adenovirus hexon and DNA-binding protein in mice.

The successful development of adenovirus vectors for vaccines and gene therapy will require a better understanding of the host immune response. Using the ELISPOT assay to measure IFN-gamma-secreting CD8(+) cells, we identify immunodominant epitopes of the adenovirus hexon and DNA-binding protein in BALB/c and C57BL/6 mice. The T-cell response to the intramuscular administration of adenovirus serotype 5 peaks within a few weeks and gradually declines but is still detectable after 12 weeks. A second administration did not substantially increase the number of reactive T cells. The CD8(+) T-cell response was also similar between wild type and E1-deleted adenovirus. When B-cell-deficient mice were injected with adenovirus encoding the gene for secreted alkaline phosphatase, sera phosphatase activity was reduced more quickly in mice pre-exposed to adenovirus. These results add to the evidence that cell-mediated immunity is a substantial barrier to therapeutic adenoviral vectors and provide more quantitative tools to measure cellular immune responses to adenovirus.

Adenovirus Hexon Protein Enhances Nuclear Delivery and Increases Transgene Expression of Polyethylenimine/Plasmid DNA Vectors

Inefficient nuclear delivery restricts transgene expression using polyelectrolyte DNA vectors. To increase transfer from the cytoplasm to the nucleus, we have covalently linked adenovirus hexon protein to polyethylenimine (PEI, 800 kDa). Activity of the conjugate was compared with PEI and PEI linked to albumin. Hexon-containing complexes gave 10-fold greater transgene expression in HepG2 cells than PEI/DNA or complexes containing albumin, without increasing cell uptake. Following cytoplasmic injection into Xenopus laevis oocytes, hexon-containing complexes showed reporter gene expression to be elevated by 10-fold compared with PEI/DNA. The ability of hexon to promote nuclear delivery of PEI/DNA nanoparticles was compared with that of classical nuclear localization sequences (NLS) by measuring transgene expression following intracytoplasmic microinjection of hexon–PEI/DNA complexes and NLS–albumin–PEI/DNA complexes in rat-1 fibroblasts. The resulting nuclear transfer efficiency was in the following order: hexon–PEI/DNA>NLS–albumin–PEI/DNA>PEI/DNA>DNA alone>albumin–PEI/DNA. The activities of both NLS–albumin–PEI and hexon–PEI were abolished by co-injection of wheat germ agglutinin, suggesting that both act by means of the nuclear pore complex (NPC); in contrast, excess free NLS–albumin abolished transgene expression with NLS–albumin-PEI/DNA, but only partially inhibited hexon–PEI/DNA. Nuclear transfer efficiency following cytoplasmic injection was dependent on DNA concentration for all materials, although hexon conjugates showed much better activity than NLS–albumin at low DNA doses (500–1000 plasmids/cell). Our data are consistent with hexon mediating nuclear delivery of plasmid complexes by means of the NPC, using mechanisms that are only partially dependent on the classical NLS import pathway. The hexon-mediated mechanism of nuclear import enables subst antially better transgene expression, particularly when DNA concentrations in the cytoplasm are limiting.

T cell Responses to Enterovirus Antigens and to β-cell Autoantigens in Unaffected Children Positive for IDDM-Associated Autoantibodies

Enterovirus infections have been implicated in the pathogenesis of IDDM in a number of studies. The aim of the present study was to evaluate whether the cellular immune response to enterovirus antigens is abnormal in children who test positive for IDDM-associated autoantibodies. Lymphocyte proliferation responses were analysed to enterovirus antigens and to a panel of β-cell autoantigen preparations in 31 non-diabetic ICA and/or GAD65 antibody-positive children and in 19 ICA/GAD65-negative control children. The responses to highly purified enteroviruses did not differ between autoantibody (AA)-positive and -negative subjects. However, proliferation responses to coxsackievirus-infected cell lysate, which also included non-structural proteins of the virus, were higher in AA-positive than in AA-negative subjects (P<0.05). This difference was most marked in children carrying the HLA-DQB1*02 allele (P=0.01). AA-positive subjects also had higher responses to one of the three GAD65 antigen preparations compared to AA-negative subjects (P<0.05). Proliferation responses to the adenovirus hexon protein did not differ between the groups. These results show that the increased responses to virus infected cell lysates are associated with early phases of β-cell autoimmunity.

Enterovirus infections as a possible risk factor for myocardial infarction.

An increasing body of evidence suggests that, in addition to the well-known classic risk factors, some microbial infections may be associated with the development of atherosclerosis and myocardial infarction (MI). The aim of our study was to evaluate the possible role of enteroviral infections in the pathogenesis of MI. Stored sera, collected in Eastern Finland in 1977, from a set of 12 155 randomly selected men and women aged 25 to 64 years were used in prospective, nested case-control study. The study sample comprised 183 men and 81 women with MI and matched controls. The sera were tested for IgG antibodies to a newly identified enterovirus-common (EVC) antigen, to heat-denatured coxsackievirus B5 (CBV-5), and to adenovirus hexon protein. Raw data from enzyme immunoassays were converted to relative units before analysis. In univariate analysis, EVC antibodies were significantly associated with the risk of MI in men (P=0.009) but not in women. Men with MI had a significantly higher mean level of EVC antibodies than matched controls (P=0.014). High antibody levels to EVC were associated with an increased risk of MI in men aged 25 to 49 years (relative risk [RR] 4.34, P<0.001) but not in older men (>50 years of age). Women with MI also showed a trend toward higher antibody levels than control women, but the difference was not statistically significant. Antibody levels to whole CBV-5 or adenovirus hexon protein appeared to be no different among case patients versus control subjects. If we assume that a high level of EVC antibodies reflects a history of relatively frequent enterovirus infections, the present observation might suggest that enterovirus infections increase the risk of MI at least in middle-aged men. Further studies are needed to understand possible clinical significance of this observation.