The innate immune system responds to pathogen infection by eliciting a nonspecific immune response following the recognition of various pathogen-associated molecular patterns. TLRs and the RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 recognize foreign nucleic acid within endosomal and cytoplasmic compartments, respectively, initiating a signaling cascade that involves the induction of type I IFN through the transcription factors IFN regulatory factor (IRF) 3 and NF-κB. However, a recent paradigm has emerged in which bacterial DNA and double-stranded B-form DNA trigger type I IFN production through an uncharacterized TLR- and RIG-I-independent pathway. We have previously described a response in primary fibroblasts wherein the entry of diverse RNA- and DNA-enveloped virus particles is sufficient to induce a subset of IFN-stimulated genes and a complete antiviral response in an IRF3-dependent, IFN-independent manner. In this study, we show that the innate immune response to virus particle entry is independent of both TLR and RIG-I pathways, confirming the existence of novel innate immune mechanisms that result in the activation of IRF3. Furthermore, we propose a model of innate antiviral immunity in which exposure to increasing numbers of virus particles elevates the complexity of the cellular response from an intracellular, IFN-independent response to one involving secretion of cytokines and activation of infiltrating immune cells.

Although antitumor activity of herpes simplex virus 1 (HSV-1) ICP0 null oncolytic vectors has been validated in murine breast cancer models, oncolytic virus treatment alone is insufficient to break immune tolerance. Thus, we investigated enhancing efficacy through combination therapy with the immunogenic cell death-inducing chemotherapeutic drug, mitoxantrone. Despite a lack of enhanced cytotoxicity in vitro, HSV-1 ICP0 null oncolytic virus KM100 with 5 mmol/L mitoxantrone provided significant survival benefit to BALB/c mice bearing Her2/neu TUBO-derived tumors. This protection was mediated by increased intratumoral infiltration of neutrophils and tumor antigen-specific CD8 þ T cells. Depletion studies verified that CD8-, CD4-, and Ly6G-expressing cells are essential for enhanced efficacy of the combination therapy. Moreover, the addition of mitoxantrone to KM100 oncolytic virus treatment broke immune tolerance in BALB-neuT mice bearing TUBO-derived tumors. This study suggests that oncolytic viruses in combination with immunogenic cell death-inducing chemotherapeutics enhance the immunogenicity of the tumorassociated antigens, breaking immunologic tolerance established toward these antigens.

An expression cassette designed for high-level production of rabies virus glycoprotein (RG) could not be rescued into a replication-defective, adenovirus-based vector using standard procedures. To overcome this difficulty, a 293-based cell line, designated 293LAP13, was constructed that contained and expressed a derivative of the lac repressor protein. The lac operator sequence, to which the repressor binds, was incorporated into an expression cassette, containing a promoter and intron, designed for high-level production of RG. Insertion of a single operator sequence immediately downstream of the transcription start site and the use of the 293LAP13 cell line allowed recombinant viruses that could not be isolated with 293 cells to be rescued efficiently. The operator-containing virus reached higher titres in 293LAP13 than in parental 293 cells and also produced plaques more efficiently in 293LAP13 cells. Moreover, in non-complementing human and canine cell lines, adenovirus vectors with a promoter-intron expression cassette expressed RG at much higher levels than vectors lacking the intron. These observations, together with the demonstration that expression of RG by operator-containing vectors was repressed markedly in 293LAP13 cells and that this inhibition was relieved at least partly by IPTG, suggest that the 293LAP13 cell line may be useful for the rescue and propagation of many vectors in which high expression of the desired protein prevents vector rescue in 293 cells.

Helper-dependent (HD) adenovirus vectors devoid of all viral coding sequences have a large cloning capacity and provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of producing HD vectors involves coinfecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by loxP sites. Cre-mediated packaging signal excision renders the helper virus genome unpackageable but still able to replicate and provide helper functions for HD vector propagation. Typically, helper virus contamination is <1% pre-and <0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. However, this objective has not been realized since the Cre/loxP system was first developed. This lack of progress is due, at least in part, to our lack of understanding of the origins of the contaminating helper virus, thus rendering its reduction or elimination difficult to achieve. This study was designed to investigate the possible sources of contaminating helper virus persisting during HD vector amplification. The results revealed that Cre is limiting in helper virus-infected Cre-expressing 293 cells, thereby permitting helper viruses to escape packaging signal excision and propagate. The results of this study should provide a foundation for developing rational strategies to further reduce or possibly eliminate the contaminating helper virus.Helper-dependent (HD) adenovirus vectors are devoid of all adenovirus coding sequences, retaining only ϳ500 bp of adenovirus sequences required in cis for HD vector propagation. These comprise the inverted terminal repeats (ITRs) necessary for DNA replication and the packaging signal () necessary for viral DNA encapsidation (20,26,28).

Alloreactive and soluble antigen-reactive, I-A-restricted T cell clones were examined for their ability to recognize hybrid I-A antigens. Several clones that recognized hybrid I-A(b)/I-A(k) molecules on (C57BL/6 x A/J)F(1) [(B6A)F(1)] spleen cells were studied. We were able to distinguish clones that recognized hybrid I-A molecules of the A(b)(a)A(k)(β) type from those that recognized A(k)(a)A(b)(β) molecules. We reached this conclusion by considering data from three independent types of experiments. (a) Monoclonal antibodies were used to inhibit T cell stimulation. Antibodies 10.2.16 and H116.32 distinguished two mutually exclusive "families" of T cell clones. One group of clones was inhibited by 10-2.16 and not H116.32, the other group exhibited reciprocal inhibition. (b) T cell proliferation was assayed using antigen-presenting cells from B6.C-H-2(bml2) (bml2) and [bml2 × B10.A(4R)]F(1) mice. Because the bml2 strain has a mutation that results in an altered A(b)(β) polypeptide chain (A(bm12)(β)), we reasoned that clones that could recognize the [bm12 × B 10.A(4R)]F(1) cells were recognizing A(b)(a)A(k)(β) molecules. Alternatively, clones not recognizing [bml2 × B10.A(4R)]F(1) cells had specificity for A(k)(a)A(b)(β) molecules. (c) I-A molecules immunoprecipitated from radiolabeled (B6A)F(1) splenocyte extracts were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These experiments confirmed an earlier report that antibody 10.2.16 recognized determinants on the A(k)(β) chain (12). Antibody H116.32 immunoprecipitated products consistent with recognition of A(k)(a) determinants. Taken together, these three types of results offer conclusive evidence that T cell clones recognizing "hybrid" I-A molecules use either A(b(k)A(k)(β) or A(k)(a)A(b)(β) molecules as recognition or restriction sites. Clones whose proliferation was supported by [bm 12 x B10.A(4R)]F(1) cells and blocked by anti-I-A(k) antibody 10-2.16 recognized A(b)(a)A(k)(β) B molecules. Clones that were blocked by antibody H116.32 and did not recognize [bml2 X B10.A(4R)]F(1) cells use a recognition site(s) on A(b)(a)A(k)(β) molecules. Thus, we can demonstrate both functionally and biochemically that hybrid F(1) I-A molecules of the structure A(k)(a)A(b)(β) and A(b)(a)A(k)(β) both exist on (B6A)F(1) splenocytes and that both configurations are used in immune recognition phenomena.