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Vaccination with a recombinant vaccinia vaccine containing the B7-1 co-stimulatory molecule causes no significant toxicity and enhances T cell-mediated cytotoxicity
Abstract
B7-1 is a co-stimulatory molecule that provides a second signal for T-cell activation. Several studies have demonstrated that vaccination with a vector containing genes encoding B7-1 and an antigen appears to be efficacious at promoting immune responsiveness to the antigen. To evaluate the safety of such a protocol and determine the effect of the B7-1 vector on immune responsiveness, female C57BL/6 mice were administered Wyeth wild-type vaccinia virus (V-WT) or V-WT containing the gene for B7-1 (rV-B7-1) as a single s. c. injection or 3 monthly s.c. injections. Immunologic parameters were evaluated in half of the mice and general toxicity in the other half. Immunologic end points included determination of splenic lymphocyte phenotypes, mitogen-induced T- and B-cell proliferation, T-cell proliferation in response to alloantigens, cell-mediated cytotoxicity (CMC), natural killer cell activity and serum anti-nuclear antibody (ANA) titers. No significant signs of general toxicity were noted. The primary immunologic effect was an increase in the ability of spleen cells to lyse allogeneic targets and to proliferate in response to allogeneic stimulation. Numbers of splenic CD8(+) cells were also increased. These effects were more pronounced after 3 vaccinations than after a single vaccination. Minimal differences in ANA were observed between mice immunized with V-WT and rV-B7-1. In addition, no serum antibodies against B7-1 were detected in any mice. The data suggest that vaccination with rV-B7-1 augments CMC with minimal toxicity.
... Antibody vaccinia and anti-fowlpox antibody titers were reported as the reciprocal of the highest serum dilution yielding a positive signal. Anti-B7-1, anti-ICAM-1, and anti-LFA-3 antibodies were quantified by fluorescence-activated cell-sorting capture assay as described previously (45). Detection limits were 0.49, 7.8, and 15.6 ng/ml, respectively. ...
... In humans and in mice, anti-ssDNA can be present for many years and is not known to be associated with pathological lesions (50). Because normal C57BL/6 mice express increased ANA titers with age (45,48), this finding was not considered significant. ...
Many current clinical trials involve vaccination of patients with vaccines directed against tumor-associated antigens, which are, in actuality, "self-antigens" overexpressed in tumors as compared with normal tissues. As tumor vaccines become more potent through the addition of costimulatory molecules and cytokines and the use of diversified prime and boost regimes, the level of concern rises regarding the balance between antitumor immunity and pathological autoimmunity. Studies were conducted using mice bearing a transgenic self-antigen [human carcinoembryonic antigen (CEA)], which is expressed in some normal adult tissues, and tumor expressing the same self-antigen. These mice were vaccinated with recombinant poxviral vectors [recombinant vaccinia, recombinant fowlpox (rF)] encoding the CEA transgene as well as a triad of costimulatory molecules [B7-1, ICAM-1, and LFA-3 (TRICOM)]. Here we investigate the mechanism of tumor therapy and evaluate the safety of such a regimen in a self-antigen system. To our knowledge, the study reported here is the first description of a vaccine to a defined antigen where the regimen is potent enough to induce tumor therapy in the absence of autoimmunity.
CEA transgenic mice were transplanted with CEA-expressing tumors. Fourteen days later, mice were vaccinated with recombinant vaccinia-CEA/TRICOM admixed with recombinant murine granulocyte macrophage colony-stimulating factor and then given low-dose interleukin 2. Mice were boosted on days 21, 28, and 35 with rF-CEA/TRICOM admixed with rF-granulocyte macrophage colony-stimulating factor and then given low-dose interleukin 2. Mice were monitored for survival and compared with groups of mice vaccinated in a similar manner with poxviral vectors containing CEA/B7-1 or CEA transgenes. To determine the mechanism of antitumor therapy, mice were depleted of T-cell subpopulations before vaccination with the CEA/TRICOM regimen. Mice successfully cured of tumor and age-matched control mice were monitored for 1 year. At 1 year, several clinical assays were carried out involving analysis of 9 serological parameters, 11 urinalysis parameters, and 14 immunological parameters. In addition, histopathology was performed on 42 tissues/mouse.
The CEA/TRICOM vaccination regimen induced a therapeutic antitumor response as measured by increased survival, which was due largely to induced T-cell responses (both CD4(+) and CD8(+)) as determined by selective T-cell subset depletion. The CEA/TRICOM vaccination regimen induced a significant increase in proliferation of CD4(+) T cells to CEA protein and a significant increase in secretion of IFN-gamma from CD8(+) T cells in response to a defined CEA epitope. Despite CEA expression in normal adult gastrointestinal tissues, no toxicity was observed in the CEA/TRICOM-vaccinated group when an array of clinical serum and urine chemistry assays was conducted 1 year after vaccination. Moreover, a comprehensive histopathological evaluation of all tissues from these groups also showed no evidence of toxicity.
Activation of T cells directed against a tumor-associated self-antigen, sufficient to mediate therapeutic antitumor immunity, was observed in vivo without the development of autoimmunity as analyzed by a comprehensive evaluation of biochemical, immunological, and histopathological criteria. These studies demonstrate that the use of vectors containing as many as three costimulatory molecules does not induce autoimmunity or other pathology. These studies thus demonstrate that a balance can indeed be achieved between the induction of an immune response to a self-antigen, which is capable of antitumor therapy, and the absence of autoimmunity.
... On the basis of previous experimental studies in animal models showing that the addition of a triad of costimulatory molecules (B7-1, ICAM-1, and LFA-3) to recombinant vaccinia and avipox vectors expressing CEA enhances both CEA-specific T-cell immunity and antitumor immunity [76][77][78][79]89,189,[191][192][193][194], Marshall et al. conducted the first trial in humans using rV-CEA(6D)-TRICOM and rF-CEA(6D)TRICOM vaccines alone or in combination. The study was conducted with 58 patients with advanced CEA-expressing tumors (35 colorectal, 9 lung, 3 breast, 1 thyroid, 2 unknown primary tumors, 1 ovary, 7 other gastrointestinal), divided into eight cohorts. ...
Carcinoembryonic antigen (CEA), a glycosylated protein of MW 180 kDa, is overexpressed in a wide range of human carcinomas, including colorectal, gastric, pancreatic, non-small cell lung and breast carcinomas. Accordingly, CEA is one of several oncofetal antigens that may serve as a target for active anti-cancer specific immunotherapy. Experimental results obtained by employing animal models have supported the design of clinical trials using a CEA-based vaccine for the treatment of different types of human cancers. This review reports findings from experimental models and clinical evidence on the use of a CEA-based vaccine for the treatment of cancer patients. Among the diverse CEA-based cancer vaccines, DCs- and recombinant viruses-based vaccines seem the most valid. However, although vaccination was shown to induce a strong immune response to CEA, resulting in a delay in tumor progression and prolonged survival in some cancer patients, it failed to eradicate the tumor in most cases, owing partly to the negative effect exerted by the tumor microenvironment on immune response. Thus, in order to develop more efficient and effective cancer vaccines, it is necessary to design new clinical trials combining cancer vaccines with chemotherapy, radiotherapy and drugs which target those factors responsible for immunosuppression of immune cells. This review also discusses relevant patents relating to the use of CEA as a cancer vaccine.
... Results from animal studies are encouraging. [167][168][169] Conclusion Encouraging safety profiles and local-regional activity have been demonstrated with a variety of oncolytic viral therapeutics. Unfortunately, the inability to demonstrate systemic response with currently available viral constructs limits future clinical development opportunities. ...
Oncolytic virotherapy is an emerging experimental treatment platform for cancer therapy. Oncolytic viruses are replicative-competent viruses that are engineered to replicate selectively in cancer cells with specified oncogenic phenotypes. Multiple DNA and RNA viruses have been clinically tested in a variety of tumors. This review will provide a brief description of these novel anticancer biologics and will summarize the results of clinical investigation. To date oncolytic virotherapy has shown to be safe, and has generated clinical responses in tumors that are resistant to chemotherapy or radiotherapy. The major challenge for researchers is to maximize the efficacy of these viral therapeutics, and to establish stable systemic delivery mechanisms.
... One of the issues that also needs to be resolved in future studies is that of pathological autoimmunity as a result of the use of vectors containing a self-antigen and costimulatory molecules. In a previous study, it was shown that repeated vaccinations with rV-B7-1 did not induce any evidence of autoimmunity (69). In recent toxicology studies, no evidence of autoimmunity, as analyzed by histochemistry, altered blood chemistry, or antinuclear antibody, was observed when CEA-transgenic mice received four vaccinations of rV-CEA/TRI-COM or rF-CEA/TRICOM vectors. ...
Several different vaccine strategies have been evaluated and combined in an attempt to amplify T-cell responses toward induction of antitumor immunity. The model tumor antigen used was carcinoembryonic antigen (CEA). While initial T-cell activation studies were conducted in conventional mice, combined vaccine strategy studies and antitumor studies were conducted in transgenic mice in which CEA is expressed in normal gastrointestinal tissue and CEA protein is found in sera. The studies reported here demonstrate: (a) A recombinant avipox (fowlpox, rF) vector expressing the signal 1 (CEA) and the B7-1 costimulatory molecule transgenes (designated rF-CEA/B7-1) is more potent in inducing CEA-specific T-cell responses than rF-CEA; one administration of recombinant fowlpox vector expressing CEA and three different costimulatory molecule transgenes (B7-1, ICAM-1, LFA-3, designated rF-CEA/TRICOM) was more potent in inducing CEA-specific T-cell responses than four vaccinations with rF-CEA or two vaccinations with rF-CEA/B7-1. Moreover, up to four vaccinations with rF-CEA/TRICOM induced greater CEA-specific T-cell responses with each vaccination. (b) A diversified prime and boost strategy using a prime with a recombinant vaccinia vector expressing CEA and the triad of costimulatory molecules (designated rV-CEA/TRICOM) and a boost with rF-CEA/TRICOM was more potent in inducing CEA-specific T-cell responses than the repeated use of rF-CEA/TRICOM alone. (c) The addition of granulocyte macrophage colony-stimulating factor (GM-CSF) to the rF-CEA or rF-CEA/TRICOM vaccinations via the simultaneous administration of a rF-GM-CSF vector enhanced CEA-specific T-cell responses. These strategies (TRICOM/diversified prime and boost/GM-CSF) were combined to treat CEA-expressing carcinoma liver metastases in CEA-transgenic mice; vaccination was initiated 14 days posttumor transplant. Antitumor effects in terms of survival and CD8(+) and CD4(+) responses specific for CEA were also observed in this CEA-transgenic mouse model. These studies demonstrate that the use of cytokines and diversified prime and boost regimens can be combined with the use of recombinant vectors expressing signal 1 and multiple costimulatory molecules to further amplify T-cell responses toward more effective vaccine strategies.
... Animal results are encouraging. [302][303][304] In summary, whether using mutant vaccinia viruses having tumor selective replication features or using vaccinia virus tumor lysates as antitumor vaccines, vaccinia virus appears to offer multiple potentially effective ways to treat cancer. ...
Molecular research has vastly advanced our understanding of the mechanism of cancer growth and spread. Targeted approaches utilizing molecular science have yielded provocative results in the treatment of cancer. Oncolytic viruses genetically programmed to replicate within cancer cells and directly induce toxic effect via cell lysis or apoptosis are currently being explored in the clinic. Safety has been confirmed and despite variable efficacy results several dramatic responses have been observed with some oncolytic viruses. This review summarizes results of clinical trials with oncolytic viruses in cancer.
Increased expression of the molecule CD200 in mice receiving renal allografts is associated with immunosuppression leading to increased graft survival, and altered cytokine production in lymphocytes harvested from the transplanted animals. Preferential production of IL-4, IL-10 and TGFbeta occurs on donor-specific restimulation in vitro, with decreased production of IL-2, IFNgamma and TNFalpha. These effects are enhanced by simultaneous infusion of CD200 immunoadhesin (CD200Fc) and donor CD200 receptor (CD200r) bearing macrophages to transplanted mice. C57BL/6 mice do not normally resist growth of EL4 or C1498 leukaemia tumour cells. Following transplantation of cyclophosphamide-treated C57BL/6 with T-depleted C3H bone marrow cells, or for the EL4 tumour, immunization of C57BL/6 mice with tumour cells transfected with a vector encoding the co-stimulatory molecule CD80 (EL4-CD80), mice resist growth of tumour challenge. Immunization of C57BL/6 mice with EL4 cells overexpressing CD86 (EL4-CD86) is ineffective. Protection from tumour growth in either model is suppressed by infusion of CD200Fc, an effect enhanced by co-infusion of CD200r+ macrophages. CD200Fc acts on both CD4+ and CD8+ cells to produce this suppression. These data are consistent with the hypothesis that immunosuppression following CD200-CD200r interaction can regulate a functionally important tumour growth inhibition response in mice.
Although the field of immunology developed in part from the early vaccine studies of Edward Jenner, Louis Pasteur and others, vaccine development had largely become the province of virologists and other microbiologists, because the model for classic vaccines was to isolate the pathogen and prepare a killed or attenuated pathogen vaccine. Only recently has vaccinology returned to the realm of immunology, because a new understanding of immune mechanisms has allowed translation of basic discoveries into vaccine strategies.
Prostate-specific antigen (PSA) is. a serine protease secreted by prostatic epithelial cells and is widely used as a marker for prostate cancer. The tissue specificity of PSA makes it a potential target for active specific immunotherapy, especially in prostate cancer patients who have undergone prostatectomy and in whom the only PSA-expressing tissue in the body resides in metastatic deposits. We report here the cloning, construction and immunological consequences of immunization of rhesus monkeys with a recombinant vaccinia virus expressing human PSA (designated rV-PSA). The prostate gland of the rhesus is structurally and functionally similar to the human prostate. While rodent and other mammalian species do not share homology with human PSA, there is 94% homology between the amino acid sequences of rhesus and human PSA. Immunization of rhesus monkeys with wild-type vaccinia virus or rV-PSA elicited the usual low-grade constitutional symptoms of vaccinia virus infection. There was no evidence of any adverse effects in any immunized monkeys. A short-lived PSA-specific IgM antibody response was noted in all rV-PSA immunized monkeys regardless of dose level. All monkeys receiving the 108pfu dose of rV-PSA demonstrated PSA-specific T-cell responses that were maintained up to 270 days. No differences in anti-PSA immune responses or toxicity were observed in animals that received prostatectomy prior to immunization. Our results thus demonstrate the safety and immunogenicity of rV-PSA in a non-human primate and have implications for potential specific immunotherapy protocols using PSA as a target.
Human carcinoembryonic antigen (CEA) is a 180-kd glycoprotein expressed in human colorectal, gastric, pancreatic, breast, and non-small-cell lung carcinomas. Previous studies have demonstrated enhanced immune responses to other antigens presented with vaccinia virus proteins via a recombinant vaccinia virus construct. In addition, we have developed a recombinant CEA-vaccinia virus construct, designated rV(WR)-CEA, and have demonstrated humoral anti-CEA responses in mice after immunization with that virus.
The goals of this study were (a) to construct a recombinant CEA-vaccinia vaccine in a less virulent vaccinia strain that is potentially safe and effective for treatment of patients whose tumors express CEA and (b) to evaluate the ability of the recombinant CEA-vaccinia vaccine to prevent and reverse tumor growth in mice and to elicit cell-mediated and humoral anti-CEA immune responses.
Using the New York City strain of vaccinia virus, which is used in smallpox vaccination and is more attenuated for humans than rV(WR), we derived a recombinant CEA-vaccinia construct, designated rV(NYC)-CEA. The ability of this construct to induce antitumor immunity was evaluated in mice receiving subcutaneous injections of murine colon adenocarcinoma cells expressing the human CEA gene.
Administration of rV(NYC)-CEA in mice induced strong anti-CEA antibody responses, as well as CEA-specific cell-mediated responses, including delayed-type hypersensitivity, lymphoproliferative, and cytotoxic responses. Vaccination of mice with the rV(NYC)-CEA rendered them resistant to the growth of subsequently transplanted CEA-expressing tumors. Moreover, when mice were vaccinated 7 days after tumor cell injection, tumor growth was either greatly reduced or eliminated. No toxic effects were observed in any of the mice.
These studies demonstrate that antitumor activity can be induced with the use of a recombinant CEA-vaccinia virus construct derived from an attenuated vaccinia strain, and they reveal the range of cell-mediated and humoral responses induced by this recombinant vaccine.
Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein expressed on most gastrointestinal carcinomas. A 2.4-kb cDNA clone, containing the complete coding sequence, was isolated from a human colon tumor cell library and inserted into a vaccinia virus genome. This newly developed construct was characterized by Southern blotting, DNA hybridization studies, and polymerase chain reaction analysis. The CEA gene was stably integrated into the vaccinia virus thymidine kinase gene. The recombinant was efficiently replicated upon serial passages in cell cultures and in animals. The recombinant virus expresses on the surface of infected cells a protein product recognized by a monoclonal antibody (COL-I) directed against CEA. Immunization of mice with the vaccinia construct elicited a humoral immune response against CEA. Pilot studies also showed that administration of the recombinant CEA vaccinia construct was able to greatly reduce the growth in mice of a syngeneic murine colon adenocarcinoma which had been transduced with the human CEA gene. The use of this new recombinant CEA vaccinia construct may thus provide an approach in the specific active immunotherapy of human GI cancer and other CEA expressing carcinoma types.
We have shown that spontaneously occurring, organ-specific autoimmune lesions develop in aged C57BL/6 mice of both sexes, especially in 24-month-old senescent mice. The inflammatory lesions were found in the multiple organs such as salivary gland, kidney, pancreas, lung, and liver, associated with ageing process. Organ-specific autoimmune lesions first appeared in 6-month-old C57BL/6 mice, and were aggravated with advancing age. In contrast, significant inflammatory changes did not develop in the thyroid, stomach, testis, ovary, and prostate in aged C57BL/6 mice. The incidence and severity of organ-specific autoimmune lesions in this strain of non-autoimmune mice increase with advance of age. The most severely affected lesion was sialadenitis developed in the submandibular salivary gland of aged mice, and a significant difference between male and female mice was noted only in the salivary gland. The infiltrating cells within the lesions of multiple organs consisted mainly of Thy 1.2+ and L3T4+ cells. Autoantibodies were detected in the sera of the mice with each corresponding organ-specific autoimmune lesions.
Development of a Testing Battery to Assess Chemical-Induced Immunotoxicity: National Toxicology Program's Guidelines for Immunotoxicity
Evaluation in Mice. Luster, M. I., Munson, A. E., Thomas, P. T., Holsapple, M. P., Fenters, J. D., White, K. L., Jr., Lauer,
L. D., Germolec, D. R., Rosenthal, G. J., and Dean, J. H. (1988). Fundam. Appl. Toxicol..
Twenty-nine patients with a positive antimitochondrial antibody titer greater than or equal to 1/40, who were detected during screening for other autoimmune disease, are described who had a normal serum bilirubin, alkaline phosphatase and transaminase and who had no symptoms of liver disease at presentation. Liver biopsies in 12 of the 29 fulfilled diagnostic criteria for primary biliary cirrhosis; a further 12 were consistent with primary biliary cirrhosis, but only 2 were normal. There was a high incidence of other autoantibodies and autoimmune diseases, especially thyroid antibodies and disorders. Sixteen of these patients have been followed for over 4 years since diagnosis (mean = 6 years, range = 4 to 9 years) and for a mean of 8.7 years since initial detection of the antimitochondrial antibody (range = 4 to 13). Five of 16 developed symptoms suggestive of primary biliary cirrhosis, and 11 of 16 developed elevation of alkaline phosphatase. The antimitochondrial antibody activity in these patients was in the same IgG subclasses (predominantly IgG1 and IgG3) as that seen in a group of 23 patients with clinically, biochemically and histologically advanced primary biliary cirrhosis. All showed the same abnormalities on quantitative estimation of the total IgG subclasses in serum; relative excess of IgG3 and, to a lesser extent, IgG2 was exhibited. It is concluded that, in this study, the finding of an antimitochondrial antibody titer greater than or equal to 1/40 is strongly suggestive of primary biliary cirrhosis even in the absence of symptoms and the presence of a normal alkaline phosphatase.
The in vitro cytotoxic effect of spleen cells of mice immunized by tumour allografts was studied by measuring target cell inactivation as a function of release of radioactive label (51Cr) or loss of cloning efficiency. When sensitized lymphoid cells were incubated with target cells at a ratio of 100:1, up to 90 per cent of the incorporated label was released within 6–9 hours, while the number of clone-forming cells was reduced by up to 99 per cent in the same time period. Isoantiserum from the graft recipients, as well as its 19S and 7S fractions, protected target cells against the toxic effect of the spleen cells, but a lipoprotein antigen isolated from the tumour cells failed to inhibit the cytotoxic reaction. Target cell lysis as measured by specific release of 51Cr was partially inhibited by actinomycin-D and by cycloheximide at concentrations which effectively blocked DNA-dependent RNA and protein synthesis.
Activation of T cells requires at least two signals: an antigen-specific signal delivered through the T-cell receptor and a costimulatory signal mediated through molecules designated B7-1 and B7-2. Previous studies have shown that introduction of B7-1 and B7-2 into tumors using retroviral vectors has led to enhanced antitumor effects. A limiting factor for potential clinical applications using this approach is the low efficiency of infection of retroviral vectors and consequent manipulations of infected cells. Vaccinia virus thus represents an alternative vector for B7 gene expression in tumor cells. In this report we describe the construction and characterization of recombinant vaccinia viruses containing the murine B7-1 and B7-2 genes (designated rV-B7-1 and rV-B7-2). Infection of BSC-1 cells with these constructs results in rapid and efficient cell surface expression of both B7-1 and B7-2 (> 97% of cells at 4 h). Infection of murine carcinoma cells with low multiplicity of infection of wild-type vaccinia virus leads to the death of the host following tumor transplantation. In contrast, inoculation of rV-B7-1- or rV-B7-2-infected tumor cells into immunocompetent animals resulted in no tumor growth. These studies demonstrate the utility of recombinant vaccinia viruses to deliver B7 molecules to tumor cells for potential gene therapy and recombinant approaches to cancer immunotherapy.
At least two signals are required for the activation of naive T cells by antigen-bearing target cells: an antigen-specific signal, delivered through the T-cell receptor, and a costimulatory signal delivered through the T-cell surface molecule CD28 by its natural ligand B7-1. The immunological benefit of coexpression of B7 with target antigen has been demonstrated with the use of several retroviral systems to transfect antigen-bearing cells. Although engineering recombinant constructs with genes for two or more antigens can mediate the dual expression of those antigens, disadvantages of this approach include the time for construction of each desirable combination and the inability to control differential expression levels of each gene product. An alternative approach would utilize separate constructs that could be admixed appropriately before administration. In this report we describe the functional consequences of the admixture of recombinant vaccinia murine B7-1 (rV-B7) to recombinant vaccinia expressing the human carcinoembryonic antigen gene (rV-CEA). Coinfection of cells resulted in high levels of cell surface expression of both the CEA and B7 molecules. Immunization of mice with various ratios (1:3, 1:1, 3:1) of rV-CEA and rV-B7 demonstrated that an admixture of rV-CEA and rV-B7 at a 3:1 ratio resulted in the generation of optimal CEA-specific T-cell responses. Next, we examined the efficacy of this admixture on antitumor activity. Typically, injection of murine carcinoma cells expressing CEA leads to the death of the host. One immunization of C57BL/6 mice with rV-CEA:rV-B7 (3:1) resulted in no tumor establishment. In contrast, administration of rV-CEA or rV-B7 alone had little or no antitumor effects. These studies demonstrate the advantages of the use of recombinant vaccinia viruses to deliver B7 molecules in combination with a tumor-associated antigen. The availability of the rV-B7 single construct and the ability to alter the B7 ratio could also have potential utility when coinfecting rV-B7 with recombinant vaccinia viruses containing genes for infectious agents or other tumor-associated antigen genes.
B7-CD28 costimulation is essential for the activation of CD4+ T helper cells, mainly by regulating IL-2 and other cytokine production. The requirement for this costimulatory pathway in the activation of CD8+ T cells, however, is still poorly understood. Here we analyzed the role of B7-CD28 costimulation in the differentiation of Ag-specific CTL precursor. We found that the activation of not only IL-2 production but also cytotoxic function in CD8 T cells requires B7 costimulation. The costimulatory signal, which cannot be replaced by exogenous IL-2, is directly implicated in the activation of the lytic machinery in CD8 T cells. Moreover, B7-CD28 costimulation appears to play a critical role in the accumulation of mRNA encoding at least one of the granzymes required for cytolytic function, granzyme B or CTLA-1. The production of IFN-gamma by CD8 T cells, however, does not appear to require costimulation.
The human carcinoembryonic antigen (CEA), which is expressed in several cancer types, is a potential target for specific immunotherapy using recombinant vaccines. Previous studies have shown that when the CEA gene is placed into vaccinia virus, the recombinant vaccine (rV-CEA) can elicit T-cell responses in both rodents and non-human primates.
Our objective was to determine if rVCEA could elicit CEA-specific T-cell responses in humans with appropriate human leukocyte antigen (HLA) motifs.
Peripheral blood lymphocytes (PBLs) obtained from patients with metastatic carcinoma, both before and after vaccination with rV-CEA, were analyzed for T-cell response to specific 9- to 11-mer CEA peptides selected to conform to human HLA class I-A2 motifs.
While little or no T-cell growth was seen from preimmunization PBLs of patients pulsed with CEA peptides and interleukin 2 (IL-2), T-cell lines were obtained from PBLs of patients after vaccination with one to three cycles of stimulation. Cytolytic T-cell lines from three HLA-A2 patients were established with a 9-amino acid peptide (CAP-1), and the CD8+/CD4+ double-positive T-cell line (V24T) was chosen for detailed analysis. When autologous Epstein-Barr virus (EBV)-transformed B cells were either incubated with CAP-1 peptide or transduced with the CEA gene using a retroviral vector, they were lysed by the V24T cell line, but allogeneic non-A2 EBV-transformed B cells were not. The SW403 human colon carcinoma cell line, which is CEA positive and HLA-A2 positive, was also lysed by the V24T cell line, while two non-HLA-A2 CEA-positive colon carcinoma cell lines were not. To further confirm the class I HLA-A2 restricted nature of the V24T cytotoxicity, the non-HLA-A2 SW837 CEA-expressing colon carcinoma cell line was infected with a recombinant vaccinia virus expressing the HLA class I-A2 gene, and it became susceptible to V24T lysis. Cells infected with vector alone were not lysed.
This study demonstrates for the first time (a) the ability to generate a human cytolytic T-cell response to specific epitopes of CEA, (b) the class I HLA-A2 restricted nature of the T-cell mediated lysis, and (c) the ability of human tumor cells to endogenously process CEA to present a specific CEA peptide in the context of major histocompatibility complex for T-cell-mediated lysis.
These findings have implications in the development of specific second-generation cancer immunotherapy protocols.
Small, resting human peripheral blood T cells are able to mediate anti-CD3 redirected lysis against murine P815 cells transfected with human B7, a ligand of CD28. We demonstrate that cytotoxicity is mediated by preexisting cytotoxic effectors within the small, resting "memory" T cell population and by the de novo generation of additional CTL within both the "memory" and "virgin" T subsets. This conclusion is based on analysis of the kinetics of the response and the effects of metabolic inhibitors on the generation of CTL function. Memory CD45RO+ T cells demonstrated cytotoxicity within 4 h of coculture with anti-CD3 mAb and B7+ P815 cells and cytolysis was only partially prevented by inhibitors of protein synthesis. By contrast, virgin CD45RO- T cells demonstrated anti-CD3-induced lysis against B7+ P815 targets only after 6 or 8 h of coculture and cytotoxicity was completely prevented by inhibiting protein synthesis. Induction of cytotoxicity was B7 dependent in that parental P815 cells and P815 cells transfected with CD72 and vascular cell adhesion molecule-1, ligands for T cell-associated membrane receptors CD5 and very late activation antigen-4, respectively, did not initiate cytotoxicity. Our studies also revealed cooperation between the CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 pathways in the generation of CTL from small, resting T cells. However, after CTL generation, the CD28-B7 interaction was not required for cytotoxic effector cell function. These observations may have important physiologic implications because this would permit activated CTL to lyse targets in vivo that do not express B7, after the CTL were generated by APC that do express B7 or possibly other costimulatory molecules.
BALB/c ByJ mice develop a lupus-like syndrome characterized by anti-nRNP/Sm and Su autoantibodies and immune complex glomerulonephritis after a single i.p. pristane injection. In contrast, mercuric chloride induces anti-fibrillarin Abs only in SJL and other H-2s mice, and not in BALB/c (H-2d) mice. In the present study, the specificities of autoantibodies induced by pristane and HgCl2 were compared in SJL and BALB/c mice to examine whether these strains are "programmed" to make different sets of autoantibodies in response to nonspecific immune stimulation. Unexpectedly, the predominant autoantibodies induced by pristane in SJL mice were neither those characteristic of HgCl2-treated SJL mice nor those associated with pristane-induced disease in BALB/c mice but, rather, anti-ribosomal P, another lupus-related specificity. The autoantibodies were strongly reactive with the C-terminal 22 amino acids of the ribosomal P2 protein, indicating that they exhibited similar fine specificities to anti-P Abs in human SLE and MRL/Ipr mice. Like BALB/c mice, pristane-treated SJL mice developed severe glomerulonephritis characterized by proteinuria, mesangial proliferation, and glomerular immune complex deposits. This is the first evidence that the induction of a lupus-like syndrome by pristane is not restricted to BALB/c mice. The predominance of anti-P Abs in SJL mice contrasts sharply with the predominance of anti-nRNP/Sm and Su, in pristane-treated BALB/c mice, even though the renal lesions were similar in both strains. The data suggest that H-2s does not program mice to produce anti-fibrillarin Abs in response to nonspecific immune stimulation, arguing that autoantibody induction by pristane involves Ag-specific mechanisms.
Tumor-associated antigens have considerable promise not only as diagnostic or prognostic markers but also as targets for active or passive immunotherapy. DF3/MUC1 is a tumor-associated antigen that is overexpressed with an abnormal glycosylation pattern in breast, ovarian, lung, and pancreatic cancers. The major extracellular portion of MUC1 is composed of tandem repeat units of 20 amino acids. Recombinant vaccinia viruses encoding mucin molecules have been constructed by several groups. However, these recombinants have met with limited success in protecting animals from MUC1-expressing tumors because of the vaccinia genome being subject to high-frequency homologous recombination, therefore being unstable in expression of the tandem repeats. In light of these studies, two concurrent strategies were used to improve immune responses to MUC1: a recombinant vaccinia virus was constructed containing a modified "mini" MUC1 gene containing only 10 tandem repeat sequences to minimize vaccinia-mediated rearrangement (designated rV-MUC1); and an admixture was used containing rV-MUC1 and a recombinant vaccinia virus containing the gene for the murine T-cell costimulatory molecule B7-1 (rV-B7). The rV-MUC1 gene product maintained a consistent molecular weight throughout several passages, indicating stability of the inserted gene. Mice inoculated with rV-MUC1 demonstrated MUC1-specific cytolytic responses that were further enhanced by admixture with rV-B7. In a MUC1-expressing pulmonary metastases prevention model, mice inoculated two times with rV-MUC1 were protected from the establishment of metastases. No additive effect on antitumor immunity (> 90% with rV-MUC1 alone) was observed in mice primed with an admixture of rV-MUC1 and rV-B7 and boosted with rV-MUC1. When rV-MUC1 was used to treat established MUC1 positive metastases, however, three administrations of rV-MUC1 were not sufficient to confer antitumor effects. In contrast, when tumor-bearing mice were primed with an admixture of rV-MUC1 and rV-B7, followed by two boosts with rV-MUC1, there was a significant reduction in pulmonary metastases (p = < 0.0001), which correlated to 100% survival. Coexpression of the B7 molecule, although not necessary for the induction of an immune response of sufficient magnitude to prevent MUC1 tumors, was thus essential in a treatment setting.
In recent retrospective studies, it was shown that subtypes of antimitochondrial antibodies (AMA) can help to discriminate between a benign [only anti-M9 and/or anti-M2 positive by enzyme-linked immunosorbent assay (ELISA)] and a rather progressive course (anti-M2, -M4 and/or -M8 positive). According to different constellations of these AMA subspecificities in ELISA and complement fixation test (CFT), four AMA profiles (A-D) were defined. In 1984 we started a prospective study based on 200 PBC patients with known AMA profiles in order to correlate the antibody pattern with the clinical outcome. Progression was defined primarily as the necessity of liver transplantation and death due to hepatic failure or variceal bleeding. At entry, 18 (9%) of the 200 patients had AMA profile A (only anti-M9), 57 (29%) profile B (only anti-M2 with or without anti-M9), 74 (37%) profile C (anti-M2 in association with anti-M4/-M8 by ELISA), and 51 (26%) profile D (anti-M2/-M4/-M8 by ELISA and CFT). At the beginning of the study, 177 patients had PBC stage I/II. During the observation period of ten years, ten patients died and in 18 orthotopic liver transplantation (OLT) was performed; all these patients belonged to profile C/D. Furthermore, 44% of the patients with profile C and 31% of the patients with profile D progressed to late stages, as defined by histology and clinical manifestations such as portal hypertension and increase of bilirubin, while only one of the patients with profile B and none of the profile A-patients developed late stage PBC. A significant increase of bilirubin was observed only in C/D-patients. AMA profiles did not change during the follow-up. In conclusion, AMA profiles discriminate between a benign and a progressive course of PBC already at early stages.
The interaction of the TCR with MHC class I-bound Ag is insufficient for the priming of CTL unless secondary costimulatory signals are provided. To ascertain the minimum elements required to activate an Ag-specific CTL response in vivo, we injected mice intradermally or i.m. with plasmid DNA encoding a MHC class I-restricted peptide Ag (minigene) and different membrane-bound costimulatory ligands. The minigene-encoded epitope only primed a specific CTL response if injected in the vicinity of an ectopically expressed costimulatory ligand. Vector encoding B7-1 was repeatedly more potent at stimulating a cytolytic response than vector encoding B7-2. In contrast the B7-2-encoding plasmid preferentially enhanced Ag-specific Ab responses when injected with either protein or a cDNA expression vector. Gene vaccination with plasmids encoding OVA and B7-1, but not B7-2, prolonged survival in mice challenged with an OVA-transfected tumor. These results show that functional B7-1 transfection can be achieved in vivo and induces the selective induction of CTL. The data suggest that B7-1 plasmids should be coadministered with naked DNA vaccines that aim to induce tumor-specific cellular immunity.
Several recombinant vaccinia viruses are currently being evaluated to induce antigen-specific immunity to a variety of infectious disease agents and tumor associated antigens. T-cell costimulation is extremely important in enhancing T-cell responses, and recombinant vaccines have now been shown to be effective vectors to express a range of these molecules. Both combination vaccines (an admixture of a recombinant vaccinia virus expressing a specific target antigen and a recombinant vaccinia virus expressing a costimulatory molecule) and dual-gene vaccines expressing both transgenes on the same vector have been shown capable of effectively enhancing antigen-specific responses via T-cell costimulation. In this report, we compare for the first time the use of both types of approaches to enhance antigen-specific T-cell responses, and we demonstrate the importance of route of vaccine administration and vaccine dose in attaining optimal T-cell responses. These studies should have direct bearing on the design of vaccine clinical trials for infectious agents and/or tumor associated antigens, in which T-cell costimulatory molecules will be employed to enhance antigen-specific T-cell responses via the use of either combination or dual-gene vaccinia vaccines.
To identify the frequency and characteristics of clinical activity with serological quiescence (CASQ) in a large cohort of patients with systemic lupus erythematosus (SLE) followed prospectively at a single center.
Patients followed at the Lupus Clinic between 1991 and 1995 who on at least 3 consecutive visits had clinical activity in the absence of a low complement and elevated DNA binding were identified. Demographics, disease characteristics, and therapy for the CASQ periods, as well as prior and subsequent disease course until April 2002 were analyzed.
Of 514 patients followed at the Lupus Clinic according to a standard protocol, 62 patients had at least one episode of CASQ lasting a 9.8 +/- 6.4 months. During these periods, patients showed evidence of clinical disease activity with a SLEDAI-2K of 8.9 +/- 5.3. Major organ involvement (central nervous system, renal, vasculitis) occurred in 43 patients. Forty-four patients were treated with prednisone 16.7 +/- 11.4 mg/day, 21 were on immunosuppressive medication and 30 on antimalarials. Of the 58 patients who had followup after their last CASQ defining visit, 9 remained CASQ for 39 +/- 23 months. Of the remaining 49 patients, 23 became inactive, 21 became clinically and serologically active, and 5 were serologically active but clinically quiescent (SACQ).
Clinical laboratory correlation in SLE is a heterogeneous relationship. The majority of patients have clinical-serological concordance. The minority have discordance between clinical and serological status, and are either SACQ or CASQ. Therefore, monitoring both clinical and serological features in patients with SLE is important.