Host Cell-mediated Immune Response Research Articles

Efficacy of tuberculin immunotherapy in verruca vulgaris: Our experience from single center from North-West India and review of literature.

Verruca vulgaris (VV) acquired through direct contact or autoinoculation of human papilloma virus (HPV). Treatment of VV are challenging as destructive modalities have variable efficacy and recurrence rates. Various immunotherapies attempt to stimulate the cell-mediated host immune response against HPV. We have assessed efficacy of intralesional tuberculin in patients with verruca vulgaris and to see the long term follow-up at 12 months. Forty patients were enrolled in the study. All patients received intralesional tuberculin injections (5 Tuberculin U/mL) in a dose of 0.1 mL/lesion every fortnightly. Patients were followed up for 12 months duration, and response to treatment assessed. Thirty patients (75%) showed complete resolution, 5 (12.5%) patients showed partial response, 3 (7.5%) showed minimal response and 2 (5%) were non-responders. Irrespective of tuberculin sensitivity test response the majority of patients showed grade 3 response. Five (12.5%) patients had a recurrence of lesions. Two had a recurrence on the same site, and three had recurrences over distant sites. Intralesional tuberculin immunotherapy is an effective modality for VV. Immunotherapy of warts is a potential area of research, and exact method and agents are still under investigation.

Talimogene laherparepvec and novel injectable oncolytic viruses in the management of metastatic melanoma

Talimogene laherparepvec (T-VEC) is an oncolytic virus (OV) therapy derived from the JS1 strain of herpes simplex virus one that was approved by the Food and Drug Administration in 2015 to be administered as direct injection therapy for patients with melanoma. The anti-tumor effects of T-VEC are due to viral-mediated tumor cell lysis at the site of administration and a local, and in some cases systemic, anti-tumor response via T cell-mediated host immune response pathways aided by GM-CSF. T-VEC has shown promising results for metastatic melanoma, particularly in patients with skin, lymph node, and soft tissue metastases (stages IIIB, IIIC, and IVa). Studies have explored the utility of T-VEC as monotherapy, neoadjuvant therapy, and in combination with other immunotherapies and targeted therapies. T-VEC has proven to improve durable response rates and overall survival with a very tolerable safety profile. More research is needed to better understand which patients are most likely to benefit from T-VEC therapy, which combination therapies are most effective, and how to sequence multimodality therapy. Additionally, new OVs are currently in development and/or being studied in clinical trials. In this review, we will discuss T-VEC as a monotherapy, neoadjuvant therapy, and combination therapy, in addition to future directions for melanoma therapy as it pertains to new OVs.

The Antimicrobial Peptide, Bactenecin 5, Supports Cell-Mediated but Not Humoral Immunity in the Context of a Mycobacterial Antigen Vaccine Model.

Bactenecin (Bac) 5 is a bovine antimicrobial peptide (AMP) capable of killing some species of bacteria through the inhibition of protein synthesis. Bac5 and other AMPs have also been shown to have chemotactic properties and can induce inflammatory cytokine expression by innate immune cells. Recently, AMPs have begun to be investigated for their potential use as novel vaccine adjuvants. In the current work, we characterise the functionality of Bac5 in vitro using murine macrophage-like cells, ex vivo using human tonsil tissue and in vivo using a murine model of vaccination. We report the effects of the peptide in isolation and in the context of co-presentation with mycobacterial antigen and whole, inert Bacillus subtilis spore antigens. We find that Bac5 can trigger the release of nitric oxide from murine macrophages and upregulate surface marker expression including CD86, MHC-I and MHC-II, in the absence of additional agonists. When coupled with mycobacterial Ag85 and B. subtilis spores, Bac5 also enhanced IFNγ secretion. We provide evidence that B. subtilis spores, but not the Bac5 peptide, act as strong adjuvants in promoting antigen-specific immunoglobulin production in Ag85B-vaccinated mice. Our findings suggest that Bac5 is an important regulator of the early cell-mediated host immune response.

Depletion of CD8αβ+ T Cells in Chickens Demonstrates Their Involvement in Protective Immunity towards Marek's Disease with Respect to Tumor Incidence and Vaccinal Protection.

Marek’s disease (MD) is a lymphoproliferative disease in chickens caused by Marek’s disease virus (MDV), a highly oncogenic alphaherpesvirus. Since 1970, MD has been controlled through widespread vaccination of commercial flocks. However, repeated and unpredictable MD outbreaks continue to occur in vaccinated flocks, indicating the need for a better understanding of MDV pathogenesis to guide improved or alternative control measures. As MDV is an intracellular pathogen that infects and transforms CD4+ T cells, the host cell-mediated immune response is considered to be vital for controlling MDV replication and tumor formation. In this study, we addressed the role of CD8+ T cells in vaccinal protection by widely-used monovalent (SB-1 and HVT) and bivalent (SB-1+HVT) MD vaccines. We established a method to deplete CD8+ T cells in chickens and found that their depletion through injection of anti-CD8 monoclonal antibodies (mAb) increased tumor induction and MD pathology, and reduced vaccinal protection to MD, which supports the important role of CD8+ T cells for both MD and vaccinal protection.

Co-expulsion of Ascaridia galli and Heterakis gallinarum by chickens

Worm expulsion is known to occur in mammalian hosts exposed to mono-species helminth infections, whilst this phenomenon is poorly described in avian hosts. Mono-species infections, however, are rather rare under natural circumstances. Therefore, we quantified the extent and duration of worm expulsion by chickens experimentally infected with both Ascaridia galli and Heterakis gallinarum, and investigated the accompanying humoral and cell-mediated host immune responses in association with population dynamics of the worms. Results demonstrated the strong co-expulsion of the two ascarid species in three phases. The expulsion patterns were characterized by non-linear alterations separated by species-specific time thresholds. Ascaridia galli burden decreased at a daily expulsion rate (e) of 4.3 worms up to a threshold of 30.5 days p.i., followed by a much lower second expulsion rate (e = 0.46), which resulted in almost, but not entirely, complete expulsion. Heterakis gallinarum was able to induce reinfection within the experimental period (9 weeks). First generation H. gallinarum worms were expelled at a daily rate of e = 0.8 worms until 36.4 days p.i., and thereafter almost no expulsion occurred. Data on both humoral and tissue-specific cellular immune responses collectively indicated that antibody production in chickens with multispecies ascarid infections is triggered by Th2 polarisation. Local Th2 immune responses and mucin-regulating genes are associated with the regulation of worm expulsion. In conclusion, the chicken host is able to eliminate the vast majority of both A. galli and H. gallinarum in three distinct phases. Worm expulsion was strongly associated with the developmental stages of the worms, where the elimination of juvenile stages was specifically targeted. A very small percentage of worms was nevertheless able to survive, reach maturity and induce reinfection if given sufficient time to complete their life cycle. Both humoral and local immune responses were associated with worm expulsion.

Combining forces: the promise and peril of synergistic immune checkpoint blockade and targeted therapy in metastatic melanoma.

Both immune checkpoint inhibitors and molecularly targeted agents have dramatically improved clinical outcomes for patients with metastatic melanoma. These two therapeutic approaches harness distinct mechanistic pathways-on the one hand, monoclonal antibodies against the immune checkpoints CTLA-4 and PD-1/PD-L1 stimulate the T cell mediated host immune response, while targeted inhibitors of the proto-oncogenes BRAF and MEK disrupt constitutive kinase activity responsible for tumor growth. The prospect of combining these two treatment modalities has been proposed as a potential way to increase overall response rate, extend durability of the anti-tumor response, and circumvent the immune-mediated resistance to targeted therapy. This review explores the preclinical rationale-building upon a wealth of in vitro and in vivo studies-for improved anti-tumor efficacy from combined immune checkpoint inhibition and targeted therapy. In the process, we detail the early clinical trials that have assessed the compatibility of combining these two therapies and the unexpected challenges faced from studies showing increased toxicity from these regimens. Ultimately, with more clinical data expected to mature and accrue in the near future, we elucidate a potentially novel and promising strategy for patients with advanced melanoma.

MTOR Regulation of Lymphoid Cells in Immunity to Pathogens.

Immunity to pathogens exists as a fine balance between promoting activation and expansion of effector cells, while simultaneously limiting normal and aberrant responses. These seemingly opposing functions are kept in check by immune regulators. The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that senses nutrient availability and, in turn, regulates cell metabolism, growth, and survival accordingly. mTOR plays a pivotal role in facilitating immune defense against invading pathogens by regulating the differentiation, activation, and effector functions of lymphoid cells. Here, we focus on the emerging and sometimes contradictory roles of mTOR in orchestrating lymphoid cell-mediated host immune responses to pathogens. A thorough understanding of how mTOR impacts lymphoid cells in pathogen defense will provide the necessary base for developing therapeutic interventions for infectious diseases.

Listeria monocytogenes and the Inflammasome: From Cytosolic Bacteriolysis to Tumor Immunotherapy.

Inflammasomes are cytosolic innate immune surveillance systems that recognize a variety of danger signals, including those from pathogens. Listeria monocytogenes is a Gram-positive intracellular bacterium evolved to live within the harsh environment of the host cytosol. Further, L. monocytogenes can activate a robust cell-mediated immune response that is being harnessed as an immunotherapeutic platform. Access to the cytosol is critical for both causing disease and inducing a protective immune response, and it is hypothesized that the cytosolic innate immune system, including the inflammasome, is critical for both host protection and induction of long-term immunity. L. monocytogenes can activate a variety of inflammasomes via its pore-forming toxin listeriolysin-O, flagellin, or DNA released through bacteriolysis; however, inflammasome activation attenuates L. monocytogenes, and as such, L. monocytogenes has evolved a variety of ways to limit inflammasome activation. Surprisingly, inflammasome activation also impairs the host cell-mediated immune response. Thus, understanding how L. monocytogenes activates or avoids detection by the inflammasome is critical to understand the pathogenesis of L. monocytogenes and improve the cell-mediated immune response generated to L. monocytogenes for more effective immunotherapies.

Balanced TH1 and TH2 immunopotentiating effects of silicates partly containing nanoparticles present in calcined serpentine

Calcined Serpentine (CS) is used in various formulations of alternative systems of medicine as a tonic to vital organs and as an anti-inflammatory agent. The process of calcination or incineration is believed to render non-toxic, gently absorbable, adaptable and digestible properties to the mineral compounds. The present study characterized CS and also evaluated its immunostimulatory potential. CS was characterized by using transmission electron microscopy (TEM), X-ray powder diffraction, atomic absorption spectroscopy and CHNS analysis. The characterized CS was further evaluated for its immunomodulatory potential in Swiss mice. X-Ray diffraction analysis revealed that the CS contained silicates of magnesium, calcium and iron as major minerals. Elemental composition and heavy metal analyses showed a presence of various inorganic elements/heavy metals, albeit at levels well below daily permissive intake values. TEM analysis of the test CS revealed a presence of nano particles with an average size of 10–20 nm (≈ 26% of total material). Oral administration of CS to mice at 50, 75, 100 or 200 μg/kg body weight for 10 days led to enhanced levels of total IgG, IgG1, IgG2a and IgG2b in ovalbumin-immunized mice as well as ex vivo lymphocyte proliferation and levels of TH1 (IL-2, IFNγ) and TH2 (IL-4, IL-10) cytokines produced by their cultured splenocytes. Similarly, CS treatment resulted in enhanced delayed-type hypersensitivity responses in GRBC-primed hosts. CS also activated host peritoneal macrophages, as indicated by increases in phagocytic activity and in TLR-2, CD80 and CD86 expression. The CS did not affect liver, kidney and spleen histology. Taken together, the results indicated that absorbed CS was stimulatory of host cell-mediated immune responses. It is hypothesized for now that the immunomodulatory effect of CS may have been due, in part, to a presence of nanoparticles on the CS; further study is required to validate this viewpoint.

105. A Single Administration of Low Dosage (5 x1010 vp/kg) hFX/HDAd Complex Achieves Saturated hFVIII Levels in the Blood of Mice

The development of helper-dependent adenoviral vectors (HDAds) devoid of all functional viral genes has largely overcome cellmediated host immune responses associated with Ad viral proteins. However, a dose-related acute toxicity associated with systemic delivery has been a major obstacle to the clinical translation of Adbased vectors. Since HDAd has a large transgene cargo (up to 34 kb), we recently succeeded in producing an HDAd encoding two codon-optimized hFVIII variant N6 expression cassettes (HDAd2xcoN6). We evaluated whether HDAd2xcoN6 improves hFVIII expression and activity in vivo. Hemophilia A (HA) mice were injected with a sub-lethal (human) dose (5 x1011 vp/kg) of HDAd2xcoN6 or HDAd1xcoN6 encoding a single expression cassette. HA mice injected with HDAd2xcoN6 exhibited over 10-fold higher hFVIII expression and activity compared to those injected with an equal titer of HDAd1xcoN6. Thus, we evaluated hFVIII expression after a systemic administration of 5 x1010 vp/kg (one log-lower than a human sub-lethal dose) of HDAd2xcoN6 in non-human primates. One primate showed 2% hFVIII levels in plasma compared to pretreatment. Though that expression was transient, it represents the first time that an Ad-based vector has shown a detectable level of therapeutic gene expression in the blood after systemic administration with such a low dosage in a large animal model. However, this result also suggests that the improvement of transduction of HDAd and transgene expression in the liver is required for long-lasting, high hFVIII expression in a large animal model.Previous reports have revealed that the adenoviral capsid interacts with coagulation factor X (FX) in the blood after systemic administration, and the interaction leads to transduction of this complex into the liver in conjunction with highly expressing HSPGs (receptor of FX/Ad5 complex). The complex of Ad5 with human FX (hFX/HDAd) has a greater affinity to HSPGs than mouse FX does. We therefore evaluated forming a complex of HDAd with hFX in order to improve HDAd liver transduction. Mice injected with 5 x1010 vp/kg of hFX/ HDAd2xcoN6 complex sustained 100% hFVIII levels in the blood until 4 weeks after injection. In contrast, mice injected with same dosage of HDAd2xcoN6 alone had undetectable levels of hFVIII in the blood 4 weeks post-injection, similar to our observations in non-human primate. Although mice injected with the hFX/HDAd complex showed 10-fold higher transgene products in vivo compared to mice treated with HDAd alone, HDAd vector copies in the livers of mice injected with the hFX/HDAd complex were only 2-fold higher. Interestingly, mice injected with the hFX/HDAd complex exhibited 10-fold higher transgene expression at mRNA level in the liver compared to mice injected with HDAd alone. These results suggest that the hFX/HDAd complex can lead not only to high liver transduction, but also to improving transgene expression at the transcriptional level after systemic administration. We are now investigating how the hFX/HDAd complex improves transgene expression at the transcriptional level.

Chondroitin nanocapsules enhanced doxorubicin induced apoptosis against leishmaniasis via Th1 immune response

Current leishmaniasis treatment is strangled due to concealed residence of parasite and reduced host cell mediated immune response. To circumvent above challenges, novel macrophage targeted oily core polymeric shell based doxorubicin (DOX) loaded nanocapsules (NCAPs) were fabricated employing chondroitin sulphate (CHD) for complimentary immunotherapy coupled chemotherapy against leishmaniasis. Excellent encapsulation efficiency along with pH dependent drug release was demonstrated by NCAPs. Improved cell cycle arrest at G1-S phase (1.56 folds) and apoptosis against promastigotes (6.26 folds), support the remarkable in-vitro antileishmanial activity of NCAPs (IC50: 0.254±0.038μg/ml) compared to free DOX (IC50: 0.543±0.012μg/ml). In-vivo antileishmanial activity in hamsters represented a significantly enhanced parasitic inhibition by NCAPs (1.42 folds). Improved activity was mediated via immunotherapeutic activity of NCAPs which up-regulated Th1 immune response (IL-12, INF-γ, and TNF-α) and down-regulated Th2 immune response (IL-4, IL-10, and TGF-β). In conclusion, current novel nano-formulation could be a viable option against leishmaniasis.

Biomarkers for the diagnosis of bacterial infections: in pursuit of the 'Holy Grail'.

Biomarkers for the diagnosis of bacterial infections: in pursuit of the 'Holy Grail'.

Inflammatory infiltration into placentas of Neospora caninum challenged cattle correlates with clinical outcome of pregnancy

Infection with Neospora caninum stimulates host cell-mediated immune responses, which may be responsible for placental damage leading to bovine abortion. The aim of this study was to compare immune responses in the bovine placenta, following experimental infection in different stages of pregnancy. Placentomes were examined by immunohistochemistry and inflammation in early gestation was generally moderate to severe, particularly in the placentas carrying non-viable foetuses, whereas it was milder in later stages, mainly characterised by the presence of CD3+, CD4+ and γδ T-cells. This distinctive cellular immune response may explain the milder clinical outcome observed when animals are infected in later gestation.

Phenotypic characterisation of the cellular immune infiltrate in placentas of cattle following experimental inoculation with Neospora caninum in late gestation

Despite Neospora caninum being a major cause of bovine abortion worldwide, its pathogenesis is not completely understood. Neospora infection stimulates host cell-mediated immune responses, which may be responsible for the placental damage leading to abortion. The aim of the current study was to characterize the placental immune response following an experimental inoculation of pregnant cattle with N. caninum tachyzoites at day 210 of gestation. Cows were culled at 14, 28, 42 and 56 days post inoculation (dpi). Placentomes were examined by immunohistochemistry using antibodies against macrophages, T-cell subsets (CD4, CD8 and γδ), NK cells and B cells. Macrophages were detected mainly at 14 days post inoculation. Inflammation was generally mild and mainly characterized by CD3+, CD4+ and γδ T-cells; whereas CD8+ and NK cells were less numerous. The immune cell repertoire observed in this study was similar to those seen in pregnant cattle challenged with N. caninum at early gestation. However, cellular infiltrates were less severe than those seen during first trimester Neospora infections. This may explain the milder clinical outcome observed when animals are infected late in gestation.

Levels of peripheral CD4+FoxP3+ regulatory T cells are negatively associated with clinical response to adoptive immunotherapy of human cancer

AbstractCD4+FoxP3+ regulatory T cells (Tregs) have been shown to suppress T cell–mediated host immune responses against self- and nonself-antigens; however, the impact of CD4+ Tregs on human antitumor immune responses and their influence on cancer treatment are unknown. In the present study, we explored the factors that influence CD4+ Treg reconstitution in patients receiving adoptive immunotherapy following conditioning regimens designed to enhance T-cell function and evaluated potential associations between CD4+ Treg levels and clinical responses to therapy. The analysis of 4 trials employing nonmyeloablative chemotherapy with or without total body irradiation (TBI) before adoptive T-cell transfer revealed that the percentage and number of reconstituting CD4+FoxP3+ Tregs observed in the peripheral blood was higher in nonresponders than in responders. The addition of TBI resulted in a further depletion of CD4+ Tregs, and the degree of depletion was dependent on the TBI dose. The number of administered doses of IL-2 was found to be positively associated with peripheral Treg reconstitution. These observations provide strong evidence that endogenous CD4+ Tregs have a negative impact on cancer therapy, and suggest that strategies reducing Treg levels may provide clinical benefit to cancer patients. All 5 clinical trials are registered at www.clinicaltrials.gov as NCT00001832, NCT00096382, NCT00335127, NCT00509496, and NCT00513604.

Long-Term Survival of Photoreceptors Transplanted into the Adult Murine Neural Retina Requires Immune Modulation

Stem cell therapy presents an opportunity to replace photoreceptors that are lost as a result of inherited and age-related degenerative disease. We have previously shown that murine postmitotic rod photoreceptor precursor cells, identified by expression of the rod-specific transcription factor Nrl, are able to migrate into and integrate within the adult murine neural retina. However, their long-term survival has yet to be determined. Here, we found that integrated Nrl.gfp(+ve) photoreceptors were present up to 12 months post-transplantation, albeit in significantly reduced numbers. Surviving cells had rod-like morphology, including inner/outer segments and spherule synapses. In a minority of eyes, we observed an early, marked reduction in integrated photoreceptors within 1 month post-transplantation, which correlated with increased numbers of amoeboid macrophages, indicating acute loss of transplanted cells due to an inflammatory response. In the majority of transplants, similar numbers of integrated cells were observed between 1 and 2 months post-transplantation. By 4 months, however, we observed a significant decrease in integrated cell survival. Macrophages and T cells were present around the transplantation site, indicating a chronic immune response. Immune suppression of recipients significantly increased transplanted photoreceptor survival, indicating that the loss observed in unsuppressed recipients resulted from T cell-mediated host immune responses. Thus, if immune responses are modulated, correctly integrated transplanted photoreceptors can survive for extended periods of time in hosts with partially mismatched H-2 haplotypes. These findings suggest that autologous donor cells are optimal for therapeutic approaches to repair the neural retina, though with immune suppression nonautologous donors may be effective.

Allogeneic administration of fetal membrane-derived mesenchymal stem cells attenuates acute myocarditis in rats

We reported previously that the autologous administration of bone marrow-derived mesenchymal stem cells (BM-MSC) significantly attenuated myocardial dysfunction and injury in a rat model of acute myocarditis by stimulating angiogenesis and reducing inflammation. Because BM aspiration procedures are invasive and can yield low numbers of MSC after processing, we focused on fetal membranes (FMs) as an alternative source of MSC to provide a large number of cells. We investigated whether the allogeneic administration of FM-derived MSC (FM-MSC) attenuates myocardial injury and dysfunction in a rat myocarditis model. Experimental autoimmune myocarditis (EAM) was induced in male Lewis rats by injecting porcine cardiac myosin. Allogeneic FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (5×105 cells/animal) were injected intravenously into Lewis rats one week after myosin administration. At day 21, severe cardiac inflammation and deterioration of cardiac function were observed. The allogeneic administration of FM-MSC significantly attenuated inflammatory cell infiltration and monocyte chemoattractant protein 1 expression in the myocardium and improved cardiac function. In a T-lymphocyte proliferation assay, the proliferative response of splenic T lymphocytes was significantly lower in cells obtained from FM-MSC-treated EAM rats that reacted to myosin than in cells obtained from vehicle-treated rats with EAM. T-lymphocyte activation was significantly reduced by coculture with FM-MSC. The allogeneic administration of FM-MSC attenuated myocardial dysfunction and inflammation, and the host cell-mediated immune response was attenuated in a rat model of acute myocarditis. These results suggest that allogeneic administration of FM-MSC might provide a new therapeutic strategy for the treatment of acute myocarditis.

Effects of Peg-Interferon-Alpha-2A on Schistosoma mansoni Infection in Mice

In some regions of the world, co-existence of schistosomiasis and hepatitis C (HCV) infection is common. Because the morbidity in human schistosomiasis is primarily due to host cell-mediated immune response, it was of interest to determine the effects on Schistosoma mansoni infection of an immune stimulator used in the standard treatment of HCV infection. Schistosoma mansoni -infected mice were treated with PEG-interferon-alpha-2a (PEG-IFN-alpha) by subcutaneous injection. Groups 1, 2, and 3 received 0.2 microg, 0.6 microg, and 1 microg PEG-IFN-alpha/wk, respectively, while group 4 received saline. The total worm burden was lower in all treated groups, with a maximal reduction of 35% after 9 wk of treatment with 1 microg PEG-IFN-alpha. Interferon treatment also increased the proportion of single worms over pairs. Ova counts in intestine and liver, as well as the number of liver granulomas, were greatly decreased at all time points for all treated groups. PEG-IFN-alpha also had inhibitory effects on the size of granulomas after 4 wk of treatment. The results suggest that PEG-IFN-alpha may be worth investigating for the treatment of human schistosomiasis when standard oral agents cannot be used, or when rapid inhibition of granuloma formation may be a priority.

Smoking and Lung Cancer: The Role of Inflammation

Worldwide over 1 million people die due to lung cancer each year. It is estimated that cigarette smoking explains almost 90% of lung cancer risk in men and 70 to 80% in women. Clinically evident lung cancers have multiple genetic and epigenetic abnormalities. These abnormalities may result in activation of oncogenes and inactivation of tumor-suppressor genes. Chronic inflammation, which is known to promote cancer, may result both from smoking and from genetic abnormalities. These mediators in turn may be responsible for increased macrophage recruitment, delayed neutrophil clearance, and increase in reactive oxygen species (ROS). Thus, the pulmonary environment presents a unique milieu in which lung carcinogenesis proceeds in complicity with the host cellular network. The pulmonary diseases that are associated with the greatest risk for lung cancer are characterized by abundant and deregulated inflammation. Pulmonary disorders such as chronic obstructive pulmonary disease (COPD)/emphysema are characterized by profound abnormalities in inflammatory and fibrotic pathways. The cytokines and growth factors aberrantly produced in COPD and the developing tumor microenvironment have been found to have deleterious properties that simultaneously pave the way for both epithelial-mesenchymal transition (EMT) and destruction of specific host cell-mediated immune responses. Full definition of these pathways will afford the opportunity to intervene in specific inflammatory events mediating lung tumorigenesis and resistance to therapy.

Fusion of Epstein-Barr Virus Nuclear Antigen-1-Derived Glycine–Alanine Repeat to Trans-Dominant HIV-1 Gag Increases Inhibitory Activities and Survival of Transduced CellsIn Vivo

Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.