Analytic Sensitivity of 3 Nucleic Acid Detection Assays in Diagnosis of SARS-CoV-2 Infection.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcription PCR is the primary method to diagnose coronavirus disease 2019 (COVID-19). However, the analytic sensitivity required is not well defined and it is unclear how available assays compare. METHODS: For the Abbott RealTime SARS-CoV-2 assay (m2000; Abbott Molecular), we determined that it could detect viral concentrations as low as 26 copies/mL, we defined the relationship between cycle number and viral concentrations, and we tested naso- and oropharyngeal swab specimens from 8538 consecutive individuals. Using the m2000 as a reference assay method, we described the distribution of viral concentrations in these patients. We then used selected clinical specimens to determine the positive percent agreement of 2 other assays with more rapid turnaround times [Cepheid Xpert Xpress (GeneXpert; Cepheid); n = 27] and a laboratory developed test on the Luminex ARIES system [ARIES LDT (Luminex); n = 50] as a function of virus concentrations, from which we projected their false-negative rates in our patient population. RESULTS: SARS-CoV-2 was detected in 27% (95% CI: 26%-28%) of all specimens. Estimated viral concentrations were widely distributed, and 17% (95% CI: 16%-19%) of positive individuals had viral concentrations <845 copies/mL. Positive percent agreement was strongly related to viral concentration, and reliable detection (i.e., ≥95%) was observed at concentrations >100 copies/mL for the GeneXpert but not the ARIES LDT, corresponding to projected false-negative rates of 4% (95% CI: 0%-21%) and 27% (95% CI: 11%-46%), respectively. CONCLUSIONS: Substantial proportions of clinical specimens have low to moderate viral concentrations and may be missed by methods with less analytic sensitivity.

Pre-existing tumor-sensitized T cells are essential for eradication of established tumors by IL-12 and cyclophosphamide plus IL-12.

The antitumor immune response activated by IL-12, especially by a combination of cyclophosphamide and IL-12 (Cy+IL-12), is clinically significant in certain experimental tumor models, in that a number of well-established (10-20 mm in diameter) s.c. tumors are completely eradicated. Furthermore, Cy+IL-12 treatment is also able to eradicate well-established grossly detectable experimental lung metastases and advanced ascites tumors. Despite the dramatic antitumor effects seen in some tumor models, Cy+IL-12 fails to induce regression of other established tumors. Characterization of tumor immunogenicity shows that all tumors responding to IL-12 and Cy+IL-12 treatments are immunogenic tumors, in that an antitumor immune response is detectable in tumor-bearing hosts upon tumor establishment. In contrast, none of the nonimmunogenic tumor responds to IL-12 and Cy+IL-12 treatments. Analysis of cellular requirements for successful tumor rejection through an adoptive cell transfer approach reveals that the presence of tumor-sensitized, but not naive, T cells is essential for tumor rejection by IL-12 and Cy+IL-12. Transfer of these tumor-sensitized T cells must be conducted before, but not after, IL-12 treatment in order for tumor rejection to occur. The requirement of sensitized T cells is also tumor specific. In mice bearing immunogenic tumors, the presence of pre-existing tumor-sensitized T cells is demonstrated by adoptive cell transfer experiments using purified spleen T cells from these mice. Results from our study show that Cy+IL-12-based immunotherapy of cancer may be highly effective and that pre-existing tumor-sensitized T cells are essential for the success of the therapy.

Construction and characterization of a triple-recombinant vaccinia virus encoding B7-1, interleukin 12, and a model tumor antigen.

BACKGROUND: Construction of recombinant viruses that can serve as vaccines for the treatment of experimental murine tumors has recently been achieved. The cooperative effects of immune system modulators, including cytokines such as interleukin 12 (IL-12) and costimulatory molecules such as B7-1, may be necessary for activation of cytotoxic T lymphocytes. Thus, we have explored the feasibility and the efficacy of inclusion of these immunomodulatory molecules in recombinant virus vaccines in an experimental antitumor model in mice that uses Escherichia coli beta-galactosidase as a target antigen. METHODS: We developed a "cassette" system in which three loci of the vaccinia virus genome were used for homologous recombination. A variety of recombinant vaccinia viruses were constructed, including one virus, vB7/beta/IL-12, that contains the following five transgenes: murine B7-1, murine IL-12 subunit p35, murine IL-12 subunit p40, E. coli lacZ (encodes beta-galactosidase, the model antigen), and E. coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene). The effects of the recombinant viruses on lung metastases and survival were tested in animals that had been given an intravenous injection of beta-galactosidase-expressing murine colon carcinoma cells 3 days before they received the recombinant virus by intravenous inoculation. RESULTS: Expression of functional B7-1 and IL-12 by virally infected cells was demonstrated in vitro. Lung tumor nodules (i.e., metastases) were reduced in mice by more than 95% after treatment with the virus vB7/beta/IL-12; a further reduction in lung tumor nodules was observed when exogenous IL-12 was also given. Greatest survival of tumor-bearing mice was observed in those treated with viruses encoding beta-galactosidase and B7-1 plus exogenous IL-12. CONCLUSION: This study shows the feasibility of constructing vaccinia viruses that express tumor antigens and multiple immune cofactors to create unique immunologic microenvironments that can modulate immune responses to cancer.

gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand.

Many tumor-associated antigens are nonmutated, poorly immunogenic tissue differentiation antigens. Their weak immunogenicity may be due to "self"-tolerance. To induce autoreactive T cells, we studied immune responses to gp100/pmel 17, an antigen naturally expressed by both normal melanocytes and melanoma cells. Although a recombinant vaccinia virus (rVV) encoding the mouse homologue of gp100 was nonimmunogenic, immunization of normal C57BL/6 mice with the rVV encoding the human gp100 elicited a specific CD8(+) T cell response. These lymphocytes were cross-reactive with mgp100 in vitro and treated established B16 melanoma upon adoptive transfer. To understand the mechanism of the greater immunogenicity of the human version of gp100, we characterized a 9-amino acid (AA) epitope, restricted by H-2Db, that was recognized by the T cells. The ability to induce specific T cells with human but not mouse gp100 resulted from differences within the major histocompatibility complex (MHC) class I-restricted epitope and not from differences elsewhere in the molecule, as was evidenced by experiments in which mice were immunized with rVV containing minigenes encoding these epitopes. Although the human (hgp10025-33) and mouse (mgp10025-33) epitopes were homologous, differences in the three NH2-terminal AAs resulted in a 2-log increase in the ability of the human peptide to stabilize "empty" Db on RMA-S cells and a 3-log increase in its ability to trigger interferon gamma release by T cells. Thus, the fortuitous existence of a peptide homologue with significantly greater avidity for MHC class I resulted in the generation of self-reactive T cells. High-affinity, altered peptide ligands might be useful in the rational design of recombinant and synthetic vaccines that target tissue differentiation antigens expressed by tumors.

Vaccinia virus for human gene therapy.

Recombinant vaccinia virus may be an ideal viral vector for cancer gene therapy. It has a large genome that is able to be engineered for the insertion and simultaneous expression of multiple genes. It reliably infects a large variety of tumors and expresses high levels of the gene(s) of interest. Although naturally cytopathic, it can be engineered to be noncytopathic but still infectious. Recently, it has been used in preclinical trials of animals and patients with cancer.

Immune response against large tumors eradicated by treatment with cyclophosphamide and IL-12.

Previous studies have demonstrated eradication of small (4-8 mm) established murine MCA207 sarcomas by treatment with systemic IL-12. Analysis of the mechanism has revealed a cellular and molecular immune response at the tumor typical of a Th1 cell-mediated, macrophage-effected, delayed-type hypersensitivity (DTH) response. In the current study we investigate the immune response against long term established, large MCA207 tumors induced by combined treatment with IL-12 and cyclophosphamide (Cy), an agent known to potentiate the DTH response. Our results demonstrate that s.c. large MCA207 tumors (15-20 mm) that are refractory to treatment by either IL-12 or Cy alone can be completely eradicated by the combination of Cy and IL-12. IL-12 is apparently the only cytokine capable of mediating tumor eradication, and the effect is dependent on IFN-gamma. The contribution of Cy is probably due to immunopotentiation of DTH rather than to direct cytotoxicity to the tumor. The regression of these large tumors takes >4 wk and, in many cases, is self-sustained, in that little or no additional IL-12 is needed beyond the initial week of administration. Analysis of the cellular and molecular events at the tumor site suggests that the mechanism is a Th1-mediated antitumor immune response.

Artificial antigen-presenting cells engineered by recombinant vaccinia viruses expressing antigen, MHC class II, and costimulatory molecules elicit proliferation of CD4+ lymphocytes in vitro.

The current study was designed to test the ability of recombinant Vaccinia virus (rVV) encoding essential components of an artificial antigen-presenting cell to activate antigen-specific T cells in vitro. We have constructed a set of rVV encoding separately or in combination a CD4+ T cell-specific epitope (the 133-145 peptide of chicken conalbumin), the MHC class II molecule I-Ak, and costimulatory molecules (mB7-1 and mB7-2). Cultured cells infected with rVV encoding both the antigen and the presenting MHC, but not either one alone, could activate cloned CD4+ T cells specific for the virus-encoded epitope. Additional co-expression of mB7-1 and mB7-2 resulted in further enhancement of T cell response. Thus, our rVV vector expressing four different foreign gene products elicited the highest proliferation rates of antigen-specific cloned T cells.

Identification of a Kb-restricted CTL epitope of beta-galactosidase: potential use in development of immunization protocols for "self" antigens.

The use of recombinant and synthetic vaccines in the treatment of cancer has recently been explored using model tumor associated antigens (TAA), many of which do not model the immunological state of affairs in which the TAA is expressed by normal tissues. One potentially useful model Ag is beta-galactosidase (beta-gal). Because the activity of this enzyme is so easily detectable, this gene has been inserted into a large number of recombinant viruses and tumors useful to the cancer vaccinologist. In addition, numerous transgenic mouse colonies that have tissue-specific expression of beta-gal have been developed, enabling the modeling of tolerance to "self" Ags. Since most of these mice have an H-2b background, we generated cytotoxic T lymphocytes (CTL) capable of recognizing beta-gal-expressing tumor cells of C57BL\6 origin and have determined that their restriction element is the K(b) molecule. Using an allele-specific epitope forecast to generate a panel of candidate peptides, we have determined that the K(b)-restricted sequence is DAPIYTNV and corresponds to amino acids 96-103 of the intact beta-gal molecule. A recombinant vaccinia virus (rVV-ES beta-gal96-103) was constructed that encoded the peptide epitope preceded by an endoplasmic reticulum insertion signal sequence. Tumor cells infected with this rVV were recognized by the original CTL that had been used to identify the epitope. Furthermore, splenocytes of mice immunized with a rVV encoding the full-length beta-gal molecule and restimulated with the DAPIYTNV peptide specifically recognized tumor cells expressing beta-gal. The identification of this immunogenic beta-gal sequence enables the modeling of immunization strategies in animal models of malignant disease in which the target antigen is a "self" protein.

IL-12 induces T helper 1-directed antitumor response.

Although IL-12 possesses the most potent single-cytokine antitumor efficacy, the mechanism by which IL-12 exerts its antitumor activities remains unclear. Using a complete tumor regression model induced by IL-12 treatment, we demonstrate that the antitumor response induced by IL-12 is mediated by a Th1 cell-directed process, with the macrophage as the effector cell and nitric oxide produced by the activated macrophage as the effector molecule. The induction of the Th1 response by IL-12 depends on the existence of a host T cell response to the tumor before IL-12 administration. IL-12 treatment causes the complete regression of 10-day established s.c. tumors (4-8 mm). Associated with the induction of tumor necrosis, activated macrophages expressing high levels of inducible nitric oxide synthase were found surrounding the tumor. The importance of nitric oxide as the effector molecule was further confirmed by the delay and loss of tumor regression in the presence of a nitric oxide synthase inhibitor in vivo. Examination of tumor-associated T cells indicates that IL-12 induces production of the Th1 cytokine IFN-gamma and suppresses production of IL-2, IL-4, and IL-10 at the tumor site, where these are found to be the predominant cytokines produced by tumor-associated T cells before IL-12 treatment. These findings demonstrate that IL-12 plays an essential role in the induction of an effective Th1 type of cell-mediated immune response against established tumors.

Induction of antigen-presenting capacity in tumor cells upon infection with non-replicating recombinant vaccinia virus encoding murine MHC class II and costimulatory molecules.

The possibility of inducing antigen-presenting capacity in cells normally lacking such capacity, currently represents a major goal in vaccine research. To address this issue we attempted to generate 'artificial' APC able to stimulate CD4+ T cell responses when tumor cells were infected with a single, recombinant, vaccinia virus (rVV) containing the two genes encoding murine MHC class II I-Ak and a third gene encoding the murine B7-1 (mB7-1) costimulatory molecule. To minimize the cytopathic effect and to improve safety, in view of possible in vivo applications, we made this rVV replication incompetent by Psoralen and long wave UV treatment. Tumor cells infected with rVV encoding I-Ak alone, pulsed with hen egg white lysozyme peptide (HEL46-61), induced IL-2 secretion by an antigen-specific T hybridoma. Tumor cells infected with the rVV encoding mB7-1 provided costimulation for activating resting CD4+ T cells in the presence of ConA. Tumor cells infected with the rVV encoding I-Ak and mB7-1, and pulsed with chicken ovotransferrin peptide (conalbumin133-145), induced a significantly higher response in a specific Th2 cell clone (D10.G4.1) as compared to cells infected with rVV encoding I-Ak molecules only. Thus, this replication incompetent rVV represents a safe, multiple gene, vector system able to confer in one single infection step effective APC capacity to non-professional APCs.

Non-replicating recombinant vaccinia virus encoding murine B-7 molecules elicits effective costimulation of naive CD4+ splenocytes in vitro.

Using a series of new insertion/expression vectors, we constructed a set of recombinant vaccinia viruses (recVV) encoding the murine T cell costimulatory molecules mB7-1 or mB7-2, or both together in the same construct. On infection with replication incompetent and non-cytopathic recVV, several tumour cell lines expressed the respective molecules and bound to CTLA-4. The highest binding capacity was found when both mB7 molecules were co-expressed. Mouse B16.F10 melanoma cells expressing mB7-1 or mB7-2 provided effective costimulation for proliferation of resting CD4+ T cells in the presence of concanavalin A and plate-bound anti-T cell receptor antibodies, respectively. If mB7-1 and mB7-2 were delivered together on the same cell, the proliferative response of CD4+ T cells increased further. The costimulatory effect could be blocked with CTLA-4, the soluble ligand for B7 molecules. The possibility of engineering tumour cells using recVV holds implications for the future design of vaccination strategies.

Expression of secreted platelet-derived growth factor-B by recombinant nonreplicating and noncytopathic vaccina virus.

OBJECTIVE: The purpose of this study was to develop a noncytopathic vector for transient delivery of biologically active platelet-derived growth factor (PDGF) to wounds. BACKGROUND: Topical application of the protein PDGF-B has improved wound healing in experimental studies of healing-impaired wounds. However, use of PDGF-B has been limited by availability of recombinant protein, short half-life, and inability to reliably apply to the wounded area. One approach to supply local PDGF-B is through transient gene transfer and expression. METHODS: Treatment of vaccinia virus with psoralen and long-wave ultraviolet irradiation makes it noncytopathic and nonreplicative. The authors inserted various transgenes encoding different forms of PDGF into recombinant vaccinia virus at the hemaglutinin locus by homologous recombination. Because the PDGF-B expressed from full length cDNA is not secreted because of the membrane retention sequence at the C-terminal end of the polypeptide, the authors inserted a 3'-truncated form of human PDGF-B cDNA in recombinant vaccinia virus to achieve secretion. To avoid interference in bioassays by a virally encoded epidermal growth factor homologue called vaccinia growth factor (VGF) in wild type vaccinia virus (CR-19), we used a VGF-negative strain to express PDGF-B (vSC20PDGF-B). Biologic activity of PDGF was tested by measuring proliferation of a 3T3 fibroblast cell line. RESULTS: Supernatant from CR-19-infected cells (VGF+) and from truncated vSC20PDGF infected cells caused mild and marked proliferation of 3T3 cells, respectively, whereas supernatant from full-length vSC20PDGF virally infected cells did not. Furthermore, in vitro infection of a confluent 3T3 monolayer with noncytopathic and nonreplicative vaccinia encoding either VGF or truncated PDGF also caused similar proliferation. CONCLUSIONS: These results provide important preliminary evidence for the ability to treat nonhealing wounds with nonreplicating and noncytopathic recombinant vaccinia viruses encoding cytokine growth factors.

Cytokine production and antitumor effect of a nonreplicating, noncytopathic recombinant vaccinia virus expressing interleukin-12.

BACKGROUND: Cancer gene therapy with interleukin-12 (IL-12) has generated much interest because of the potent antitumor effects of this cytokine. The purpose of this study was to construct a nonreplicating, noncytopathic recombinant vaccinia virus (irecVV) encoding murine IL-12 (ivKT0327mIL-12) and to assess its biologic activity and antitumor effects. METHODS: ivKT0327mIL-12 was constructed by inserting the genes encoding mIL-12 into the VV genome by homologous recombination. Psoralen and long-wave ultraviolet light were used to render the virus incapable of replication or cell lysis. Cytokine production was assessed by infecting tumor cell lines and measuring mIL-12 in the culture supernatants by using a bioassay. C57BL/6 mice were injected subcutaneously in a randomized, blinded fashion with 5 x 10(5) MCA 101 fibrosarcoma cells or 5 x 10(5) PAN 02 pancreatic tumor cells infected in vitro with either ivKT0327mIL-12 or ivKT033, a control irecVV containing no cytokine genes. Emergence of tumor and tumor size were measured. RESULTS: Tumor cells infected with ivKT0327mIL-12 produced large amounts of biologically active mIL-12 (up to 793 ng/10(6) cells/24 hr) but continued to proliferate in vitro with no cytopathic effect. Mice injected with MCA 101 infected with ivKT0327mIL-12 had significantly delayed tumor emergence (p < 0.03) and decreased tumor size (p < 0.003) compared with mice injected with ivKT033-infected MCA 101. Tumor growth was also significantly smaller in a similar experiment with PAN 02 (p < 0.01). CONCLUSIONS: Tumor cells infected with irecVV encoding mIL-12 express high levels of biologically active mIL-12 in vitro and exhibit delayed tumor establishment, significant tumor growth inhibition, or both, in vivo.

Endogenous interferon-gamma acts directly on tumor cells in vivo to suppress growth.

BACKGROUND: Recent evidence implicates endogenous interferon-gamma (IFN-gamma) in the host response to an immunogenic tumor (MCA105); antibody blockade of host IFN-gamma increased tumor growth rate (Doherty et al., Ann. Surg. Oncol. 3: 198-203, 1996). Those experiments did not attempt to determine the site of IFN-gamma activity (the host, the tumor, or both). MATERIALS AND METHODS: MCA101 murine tumor cells were transfected with a plasmid expression vector containing an antisense construct to the IFN-gamma receptor (IFN-gamma R) or a control construct. Clones were isolated and tested for IFN-gamma stimulation of MHC I expression, sensitivity to IFN-gamma growth effects in vitro, and specific [125I]IFN-gamma binding. RESULTS: The antisense strategy was successful in decreasing the number of cell-surface IFN-gamma binding sites and in vitro response to IFN-gamma. Finally, in vivo experiments demonstrated significantly increased untransfected tumor growth rate in animals after blockade of endogenous IFN-gamma by a single dose of anti-IFN-gamma antibody and more rapid growth of the IFN-gamma R-deficient cells compared to controls. CONCLUSION: endogenous IFN-gamma has a direct effect on this less immunogenic tumor in vivo, which serves to slow growth and which is, at least partially, mediated through interferon-gamma receptors on the tumor cell.

Prevention of murine breast cancer by vaccination with tumor cells modified by cytokine-producing recombinant vaccinia viruses.

BACKGROUND: Cancer gene therapy expressing specific levels of multiple genes has not been adequately tested. This study investigates the antitumor effects of recombinant vaccinia viruses (recVVs) that express predictable levels of single and multiple cytokines in a passive immunization murine breast cancer (C3HBA) model. METHODS: Seventeen recVVs encoding different cytokine combinations under weak and strong VV promoters were constructed. Cytokine production was measured in vitro by enzyme-linked immunosorbent assay. Mice were immunized with infected C3HBA cells and rechallenged 10 days later with 10(6) parental tumor cells. Controls were treated with saline or recVVs not expressing cytokines. Tumors were measured for 24 days. Data were analyzed using Fisher's exact test and the Breslow-Gehan-Wilcoxon test. RESULTS: recVVs encoding multiple cytokines induced secretion of each cytokine at predictable levels corresponding to VV promoter strength. Ten of 10 controls developed tumors by day 11 after rechallenge. recVVs producing large but not small amounts of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) alone resulted in complete protection in all immunized mice (p = 0.0003) for 24 days and disease-free survival (DFS) was significantly prolonged versus controls (p < 0.001). GM-CSF under a weak promoter was also effective in combination with human interleukin-1 beta (hIL-1 beta; p = 0.0220; DFS, p = 0.031) or interferon-gamma (IFN-gamma; p = 0.0037; DFS, p = 0.003). Effectiveness of hIL-1 beta and IFN-gamma vaccines depended on cytokine combinations and not the amount of cytokine produced. IL-2 or tumor necrosis factor-alpha vaccines had no significant antitumor effect. CONCLUSIONS: (a) recVVs can simultaneously express controllable levels of two cytokines; (b) tumor cells modified by recVVs generate systemic antitumor immunity; and (c) strength of immunity appears to be related to the amounts and specific cytokine(s) produced.

Gene expression and cytopathic effect of vaccinia virus inactivated by psoralen and long-wave UV light.

Induction of the cytopathic effect (CPE) in cells infected with poxvirus seems ubiquitous in that it has been associated with all different strains and preparations of poxviruses, regardless of the replicating status of these viruses. The study of the mechanisms by which CPE is induced by nonreplicating poxviruses is hampered by the lack of any noncytopathic mutant strains and preparations. In this paper, we report on the patterns of gene expression and induction of CPE by vaccinia viruses treated by limited cross-linking with psoralen and long-wave UV light (PLWUV). We show that treatment of cell-free virus with PLWUV could inactivate viral replication without abolishing the ability of the virus to infect cells. Viral transcription as indicated by reporter genes was generally enhanced and prolonged under early viral promoters and abolished under late promoters. Furthermore, increasing the levels of cross-linking with PLWUV resulted in a decrease and abolishment of viral expression of a large reporter gene and a concomitant loss of the induction of CPE. Cells infected with such a virus were able to express the reporter genes and proliferate. The generation of nonreplicating and noncytopathic recombinant vaccinia viruses may help in studies of the mechanisms of CPE induction by poxvirus and may facilitate the use of poxviral vectors in broader areas of research and clinical applications.

High cytokine production and effective antitumor activity of a recombinant vaccinia virus encoding murine interleukin 12.

We have constructed a recombinant vaccinia virus (recVV), vKT0334 mIL-12, containing the genes encoding the p35 and p40 subunits of murine interleukin-12 (mIL-12). In vitro experiments demonstrated that vKT0334 mIL-12 efficiently infected a variety of murine and human tumor cell lines and produced very high amounts (1.5 micrograms/10(6) cells/24 h) of biologically active mIL-12. Mice injected s.c. with 10(6) MCA 105 sarcoma cells, followed by injection at the same site with saline or a control recVV, vKT033, containing no mIL-12 genes, all developed progressively growing tumor, whereas 60% of animals injected with vKT0334 mIL-12 remained tumor free (P < 0.0005). Furthermore, tumor growth was significantly reduced in the remaining mice treated with vKT0334 mIL-12 that did develop tumor compared with mice treated with vKT033 (P < 0.03) or saline (P < 0.0001). We conclude that recVV expressing high levels of mIL-12 offers an effective in vivo method of cytokine gene delivery and expression in tumors with subsequent antitumor effect.

In vivo gene therapy of a murine pancreas tumor with recombinant vaccinia virus encoding human interleukin-1 beta.

BACKGROUND: Recombinant vaccinia virus (VV) encoding human interleukin-1 beta (vMJ601hIL-1 beta) given intravenously persists in tumor tissue and expresses hIL-1 beta for at least 9 days after treatment and is associated with significant retardation of tumor growth. To document the significance of this approach and to further elucidate the mechanism, this study compares the antitumor effect of vMJ601hIL-1 beta administered either intravenously or intratumorally and intravenous recombinant hIL-1 beta protein. METHODS: C57BL/6 mice with established subcutaneous pancreatic tumors were randomized to treatment with intravenous or intratumoral vMJ601hIL-1 beta, wild-type VV, saline solution, or intravenous recombinant hIL-1 beta protein in a blinded fashion. Toxicity and tumor size were measured. Data were analyzed with the Kruskal-Wallis and Wilcoxon tests. RESULTS: Treatment with intratumoral vMJ601hIL-1 beta repeatedly resulted in significant reduction in tumor size as compared with saline treated controls (p < 0.001). Tumor growth inhibition was consistently similar after intravenous or intratumoral vMJ601hIL-1 beta administration (p > 0.52). Wild-type VV given intratumorally or intravenously had no antitumor effect versus saline controls (p > 0.30). No significant toxicity or deaths resulted from vMJ601hIL-1 beta treatment. Recombinant hIL-1 beta protein administered intravenously caused severe toxicity (median lethal dose approximately 100 micrograms/kg), and no significant antitumor effect was observed at sublethal doses versus saline controls (p = 0.19). CONCLUSIONS: Direct, in vivo hIL-1 beta gene delivery and expression by recombinant VV given intravenously or intratumorally results in significant tumor growth inhibition, which appears to be a consequence of local, intratumoral vaccinia infection and production of hIL-1 beta.

Prolongation of skin allografts by recombinant tumor necrosis factor and interleukin-1.

OBJECTIVE: The hypothesis is that systemic administration of recombinant tumor necrosis factor-alpha (TNF-alpha) and/or recombinant interleukin-1 alpha (IL-1 alpha) can decrease the rejection of a skin allograft. SUMMARY BACKGROUND DATA: Tumor necrosis factor and IL-1 are pluripotent cytokine hormones that are central to the host immunologic response to foreign substances. Cytokine effects and toxicity may be reduced by systemic administration of recombinant cytokines. The authors previously have demonstrated that pretreatment with cytokines such as IL-1 or TNF can reduce the lethality of endotoxin (lipopolysaccharide), gram-negative sepsis, cancer cachexia, and oxygen toxicity. METHODS: Skin grafts from the tails of Balb/c mice were placed on the backs of C57Bl/6 mice. Mice were treated with daily intraperitoneal saline, recombinant m-TNF (Genentech, South San Francisco, CA) or h-IL-1 (Hoffman LaRoche, Nutley, NJ) from postgraft day 1 to postgraft day 28. Tumor necrosis factor and IL-1 high doses were chosen because they protected mice from the lethality of lipopolysaccharide. Animals were examined daily for toxicity and graft rejection. Graft survival was plotted in a Kaplan-Meier plot and analyzed by the log-rank test. Comparison of proportions was done using the Fisher's exact test. RESULTS: Either TNF or IL-1 alone significantly prolonged skin graft survival compared with saline control. Furthermore, the combination of TNF and IL-1 prolonged skin graft survival longer than either cytokine alone. Mice on the highest dose TNF and IL-1 combination did not reject skin grafts during the 28-day treatment period. Significant toxicity was associated with cytokine treatment. Similar significant proportions of death occurred with IL-1 alone and the highest combination of TNF and IL-1. CONCLUSION: Both TNF and IL-1 can be effective as suppressors of skin allograft rejection in mice.