The detection of a spleen focus-forming virus neoantigen by lymphocyte-mediated cytolysis.

The existence of a nonvirion tumor-associated cell surface antigen (TASA) on cells transformed with Friend (FLV) on Rauscher (RLV) leukemia virus has been difficult to demonstrate. Antisera raised against classically defined Friend- Moloney-Rauscher antigenic determinants have been shown to react with virus structural proteins coded for by genetic information contained in the lymphatic leukemia or helper (LLV) virus genome. The recent development of nontrans-formed fibroblast cell lines which contain the replication-defective spleen focus-forming virus (SFFV) genome, free of replicating LLV, has allowed investigation of an SFFV-specific antigen. We have applied the techniques of mixed tumor-lymphocyte culture stimulation followed by lymphocyte-mediated cytolysis assays to search for the cell surface expression of an antigen coded expressly by SFFV genetic information. SFFV nonproducer-immune, in vitro activated spleen cells were capable of effecting the lysis of SFFV-containing BALB/c 3T3 and Fischer rat epithelial, cloned cell lines. Normal BALB/c 3T3 and BALB/c 3T3 cells infected with three types of ecotropic LLV were unaffected. Syngeneic FLV and RLV-induced murine leukemia cells were also killed by SFFV nonproducer-immune lymphocytes. In addition, Kirsten sarcoma virus-transformed, replication-defective and replication-rescued BALB/c 3T3 fibroblasts were not susceptible to SFFV antigen-directed cytolysis. Antibody-dependent complement-mediated cytolysis assays using monospecific goat antisera confirmed that SFFV nonproducers lacked cell surface expression of virion structural proteins. These observations suggest that the antigen detected in LMC experiments was not coded for by genetic information contained in the helper component of FLV, and that it represents a true SFFV-specific cell surface antigen. Based upon the recent molecular evaluation of the SFFV genome as consisting of both xenotropic and ecotropic virus sequences, it appears reasonable that xenotropic genetic information may be responsible for expression of the SFFV- specific antigen. Since the replication-defective SFFV genome is also responsible for the malignant transformation associated with FLV-induced erythroleukemia, one might postulate that gene sequences capable of programming transformation may also code for the TASA detected in these studies.

Intranasal vaccination with a helper-dependent adenoviral vector enhances transgene-specific immune responses in BALB/c mice.

Helper-dependent adenoviral (HDAd) vectors were developed primarily for genetic disease therapy by deleting all coding regions for attenuating the host cellular immune response to adenovirus (Ad) and long-lasting gene expression. Recently Harui et al. reported that HDAd vaccine could stimulate superior transgene-specific cytotoxic T lymphocyte (CTL) and antibody responses via the intraperitoneal route, compared to first-generation adenoviral (FGAd) vaccine. This prompted us to explore the potential of HDAd as a vaccine vector administrated intranasally. In this study, we prepared HDAd and FGAd vectors expressing enhanced green fluorescent protein (EGFP), respectively, and compared their efficacy in mice. Mice were immunized intranasally with 5x10(9) vp HDAd or FGAd vector particles. Despite stimulating similar anti-Ad antibody responses with FGAd vaccine in the prime/boost strategy, HDAd vector expressing EGFP displayed superior transgene-specific serum IgG, mucosal IgA and cellular immune response, with the characterization of balanced or mixed Th1/Th2 CD4+ T-cell responses. Meanwhile, a single dose of intranasal (i.n.) vaccine of HDAd-EGFP induced a serum IgG response with more efficacy than FGAd-EGFP. In addition, i.n. boost immunization enhanced transgene-specific humoral and cellular responses, compared to single i.n. HDAd-EGFP immunization. Our results suggest that HDAd has potential for a mucosal vaccine vector via i.n. route, which will be useful for the development of vaccines against respiratory viruses, such as respiratory syncytial virus and influenza virus.

[Advances in helper-dependent adenoviral vector--a review review].

Helper-dependent adenoviral vector (HDAd) lacking all viral coding sequences with the advantages of minimal immunogenicity, negligible chronic-toxicity, and durable transgene expression over first-generation adenoviral vector (FGAd). HDAd vehicles have demonstrated tremendous potential for gene therapy in animal models for inherited diseases, neurodegenerative diseases and cancer etc. Additionally, the large cloning capacity of HdAd, up to 37 kb, permits the delivery of whole genemic loci, multiple transgenes. In this review we characterize the basic features of HdAd and summarize some of their experimental and potential clinical applications both at present and in future.

Immune response to helper dependent adenoviral mediated liver gene therapy: challenges and prospects.

Adenovirus-mediated gene therapy holds significant potential especially for applications requiring high levels of target tissue transduction. While significant advances in clinical adenoviral gene therapy applications have been made in cancer, the clinical translation of adenoviral gene replacement therapy for genetic disease has lagged. Encouragingly, advances in vector production have led to the development of Helper-Dependent ("gutted" or "high capacity") adenoviral vectors (HDV) deleted of all viral coding genes. HDV significantly reduces the chronic toxicity associated with early generation adenoviral vectors that has been most significant after systemic administration in both small and large animal models. However, the field remains confounded by innate immune responses inherent to adenovirus, and more generally, to the adaptive immune response to transgene. Together they decrease the effective therapeutic index for any particular treatment. This review summarizes the current advances toward understanding the decisive cell and molecular mechanisms underlying the acute toxicity to systemic HDV administration. We focus on the complex immune response and consequences of systemic vector delivery in the context of liver-directed monogenic disease therapy. Future development of interventions to avoid the innate immune response, including vector and pharmacologic manipulations, should further contribute to minimizing vector toxicity while maximizing the efficacy of systemic HDV gene transfer.

Spontaneous regression of Friend virus-induced erythroleukemia. II. regression of Friend murine leukemia virus-induced lymphocytic leukemia.

We characterized several aspects of spontaneous regression of lymphocytic leukemia in mice. The disease, induced by the helper murine leukemia virus (MuLV) component obtained from the regressing Friend virus complex (RFV), was characterized by spleen and lymph node enlargement, thymus involvement, and anemia. Leukemia regression occurred in about 25% of infected mice and resulted in the return of lymphoid organs to near-normal weight and normal histology and the recovery from anemia. A tenfold to 1,000-fold decrease in virus titer was seen in those mice in which leukemia regressed when compared to leukemic animals, although infectious virus was still recoverable from apparently normal spleens. The sera of mice in which leukemia regressed contained potent virus-neutralizing activity that was associated mainly with immunoglobulins. These studies firmly supported the evidence that the regressing phenotype of RFV was due to its helper MuLV component (MuLV-RF).

Enhancement of erythroid target cells for Friend murine leukemia virus by intravenous pyran treatment.

Male BALB/c mice that received prophylactic iv treatment with pyran had significantly enhanced splenomegaly, an increased number of splenic foci induced by the spleen focus forming virus (SFFV) in the Friend murine leukemia virus (F-MuLV) complex, and a slightly decreased mean survival time as compared with untreated controls infected with F-MuLV. A corresponding increase in the lymphatic leukemia virus component of the F-MuLV complex was not observed, which suggests that the enhancement of the disease was due primarily to a selective increase in the SFFV component of the F-MuLV complex. That the enhancement was related to an increased number of target cells for SFFV was substantiated by data concerning erythropoiesis in iv pyran-treated animals. Increases in splenic hematocrits and in uptake of 59Fe in the spleens of animals treated iv with pyran provided quantitative evidence for the histologic finding of increased erythroid precursors in the spleens.

A phage display system for detection of T cell receptor-antigen interactions.

The process of T cell recognition involves a complex set of interactions between the various components of the TCR/MHC/peptide trimolecular complex. We have developed a system for exploring the specific binding interactions contributed by the constituent subunits of TCR complexes for components of their ligands. We utilized an M13 phage display system, designed for multivalent receptor display, to explore specific binding interactions between various TCR alpha chains and specific antigen in the absence of MHC. The multivalent TCR-phage display system was sensitive enough to reveal some TCR alpha chains capable of binding directly to antigen with the same fine specificity shown by the MHC-restricted T cells from which the alpha chains were derived. Cross-specificity analysis using two antigen-binding TCR alpha chains derived from T cells with different polypeptide antigen specificities confirmed the fidelity of this binding. In mixtures of antigen-binding and non-binding TCR alpha-displaying phage, specific selection was achieved at a starting frequency of 1/1000, suggesting that this system can be employed for selection and analysis of TCR-displaying phage libraries. While the binding specificities exhibited by these TCRs are unusual, they provide a novel perspective from which to study the specific binding interactions that constitute TCR antigen binding.

Analysis of the helper virus in murine retrovirus-induced immunodeficiency syndrome: evidence for immunoselection of the dominant and subdominant CTL epitopes of the BM5 ecotropic virus.

In genetically susceptible strains, such as C57BL/6 (B6) mice, LP-BM5 causes murine AIDS (MAIDS). LP-BM5 is a complex mixture of murine leukemia viruses (MuLV) that includes replication competent ecotropic (BM5eco) and mink cell focus inducing (MCF), and replication defective (BM5d) MuLV. At present, for the BM5eco virus, sequence information on only the gag region is available. In this paper, we describe for the first time the sequencing of the entire BM5eco viral genome as well as analysis of homology with two other previously sequenced and well-characterized MuLVs, Emv-11 and Emv-2, the latter constituting the parental virus for BM5eco. We propose that the detailed sequence comparisons herein provide cogent evidence that BM5eco utilizes variations in cytotoxic T lymphocytes (CTL) epitopes as an immune escape mechanism. This CTL evasion mechanism may contribute substantially to the underlying prototypic susceptibility of B6 mice to LP-BM5-induced MAIDS.

Selection of specific phage from display libraries: monoclonal antibody against VCS M13 helper phage coat protein III (gIIIp).

Screening of specific phage is often hampered by nonspecific binding either of the VCS M13 helper phage to the solid phase absorbent or to the polyclonal antibodies used for selection. The former is improved by increasing the stringency for selection. However, the available polyclonal anti-VCS M13 antibodies often react with immobilized antigen nonspecifically, making it difficult to distinguish between positive and negative clones. To improve this selection process, a monoclonal antibody (MAb) was produced which recognizes ligand-coat protein three (gIIIp) on the helper phage VCS M13. This MAb is highly sensitive and specific, and it is useful for selecting relevant clones. This reagent should find widespread application in identifying interactive clones from a variety of phage display libraries.

Liver-directed gene therapy with helper-dependent adenoviral vectors: current state of the art and future challenges.

Successful liver-directed gene therapy has the potential to revolutionize medicine. Helper-dependent adenoviral vectors (HDAds) are devoid of all viral coding sequences and have shown tremendous potential for liver-direct gene therapy. In small and large animals, hepatic transduction with HDAd has resulted in high level, long-term transgene expression without chronic toxicity in a variety of disease models. Recent advancements in the large-scale manufacture of HDAd have permitted contemplation of clinical application. However, dose-dependent activation of the host innate inflammatory response remains an obstacle for clinical translation. Recent advancements in vector capsid modifications, immune modulation regimes, as well as novel routes of vector administration may yet permit clinical liver-directed gene therapy with HDAd.

Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery.

In spite of the extensive research in the field of gene therapy, host immune responses continue to be the major barrier in translating basic research to clinical practice. Helper-dependent adenoviral (HD-Ad) vectors show great potential for pulmonary gene therapy, but the knowledge of pulmonary immune responses toward these vectors is very limited. In this study, we show that HD-Ad vectors are potent stimulators of dendritic cell (DC) maturation, thus leading to stimulation of T cell proliferation with approximately 6% of naive CD4(+) T cells from pulmonary mediastinal lymph node responding to HD-Ad-treated DCs. In contrast to the belief that HD-Ad vectors are unable to prime adaptive immune response, we show for the first time, through in vivo pulmonary studies in mice, that HD-Ad vectors can prime CD4(+) and CD8(+) T cell responses in the lung at high and substantially low doses. This indicates cross-presentation of HD-Ad-derived epitopes by DCs to prime CD8(+) T cell responses. To assess the basis of pulmonary T cell response against HD-Ad vectors, we examined the response of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the lung. In response to HD-Ad delivery, there is induction of maturation in both cDC and pDC subsets, but it is the cDCs, not pDCs, that migrate rapidly to draining lymph nodes within the first 2 days after vector delivery to prime adaptive immune response against these vectors. These findings have implications for development of strategies to prevent adaptive immune responses against gene therapy vectors.

Modulation of TNFalpha, a determinant of acute toxicity associated with systemic delivery of first-generation and helper-dependent adenoviral vectors.

Understanding the determinants of the host innate immune response to systemic administration of adenoviral (Ad) vectors is critical for clinical gene therapy. Acute toxicity occurs within minutes to hours after vector administration and is characterized by activation of innate immune responses. Our data indicate that in mice, indicators of vector toxicity include elevations of cytokine levels, liver transaminase levels and thrombocytopenia. To discern potential targets for blunting this host response, we evaluated genetic factors in the host response to systemically administered first-generation Ad vectors (FGV) and helper-dependent Ad vectors (HDV) containing beta-galactosidase expression cassettes. A preliminary screen for modulation of vector-induced thrombocytopenia revealed no role for interferon-gamma, mast cells or perforin. However, vector-induced thrombocytopenia and interleukin 6 (IL-6) expression are less evident in tumor necrosis factor alpha (TNFalpha)-deficient mice. Moreover, we also demonstrated that TNFalpha blockade via antibody or huTNFR:Fc pretreatment attenuates both thrombocytopenia (>40% increase in platelet count) and IL-6 expression (>80% reduction) without affecting interleukin 12 , liver enzymes, hematological indices or vector transduction in a murine model. Our data indicate that the use of HDV, in combination with clinically approved TNFalpha immunomodulation, may represent an approach for improving the therapeutic index of Ad gene therapy for human clinical trials.

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus.

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance-i.e., 100% of treated animals showing sustained expression after 4 months-was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.

Spleen focus-forming virus: specific neutralization by antisera to certain gag gene-encoded proteins.

Immunization of rats with syngeneic cells infected with spleen focus-forming virus (SFFV) but not with its helper, Friend murine leukemia virus (FMuLV), produces antisera which specifically neutralize SFFV, and not FMuLV, in the Friend virus complex. To determine which SFFV-encoded protein molecule bears the antigen recognized by these neutralizing antibodies, we studied different lots of rat anti-SFFV antiserum by immunoprecipitation and virus neutralization assays. The ability of these sera to neutralize SFFV correlated with the titer of antibodies to p45gag and not with the titer of those to gp52, suggesting that the neutralizing antibodies recognize the p45gag molecule. To verify this specificity for p45gag, we tested antisera to various MuLV gag gene-encoded proteins for neutralization of SFFV. Goat anti-Rauscher murine leukemia virus (RMuLV) p30 and goat anti-RMuLV p10 sera neither precipitated p45gag from SFFV-infected nonproducer cells nor neutralized SFFV. In contrast, goat anti-RMuLV Pr65gag and goat anti-RMuLV p12 sera precipitated p45gag from SFFV-infected cells and also specifically neutralized SFFV in the Friend virus complex. These findings suggest that, unlike the gag proteins coded for by FMuLV, the proteins coded for by defective SFFV are incorporated into the envelope of virions carrying the SFFV genome, but not into the envelope of those carrying the helper FMuLV genome.

Natural human antibodies reactive with primate type-C viral antigens.

A survey of human sera from healthy individuals revealed the presence of naturally occurring antibodies that react in radioimmunoprecipitation assays with proteins of mammalian type-C viruses. Of 39 sera tested, 100% showed reactivity against baboon endogenous virus, whereas only 49% showed reactivity against simian sarcoma-associated virus. Polyacrylamide gel electrophoresis of immune precipitates revealed one to three bands that comigrate with the virus structural proteins. There were low, but detectable, levels of antibody to the major internal protein of murine leukemia virus, but no activity against the structural proteins of avian sarcoma virus.

Reticuloendotheliosis virus and disturbance in immune regulation.

Reticuloendotheliosis virus (REV-T) is a replication-defective retrovirus which induces a rapidly-fatal lymphoma. The helper virus, reticuloendotheliosis-associated virus (REV-A), induces a splenic suppressor cell population which cytostatically inhibits the proliferation of cytotoxic cells capable of lysing REV-T tumour cells.