Enhancing efficacy of recombinant anticancer vaccines with prime/boost regimens that use two different vectors.

BACKGROUND: The identification of tumor-associated antigens and the cloning of DNA sequences encoding them have enabled the development of anticancer vaccines. Such vaccines target tumors by stimulating an immune response against the antigens. One method of vaccination involves the delivery of antigen-encoding DNA sequences, and a number of recombinant vectors have been used for this purpose. To optimize the efficacy of recombinant vaccines, we compared primary and booster treatment regimens that used a single vector (i.e., homologous boosting) with regimens that used two different vectors (i.e., heterologous boosting). METHODS: Pulmonary tumors (experimental metastases) were induced in BALB/c mice inoculated with CT26.CL25 murine colon carcinoma cells, which express recombinant bacterial beta-galactosidase (the model antigen). Protocols for subsequent vaccination used three vectors that encoded beta-galactosidase--vaccinia (cowpox) virus, fowlpox virus, naked bacterial plasmid DNA. Mouse survival was evaluated in conjunction with antibody and cytotoxic T-lymphocyte responses to beta-galactosidase. RESULTS: Heterologous boosting resulted in significantly longer mouse survival than homologous boosting (all P<.0001, two-sided). Potent antigen-specific cytotoxic T lymphocytes were generated following heterologous boosting with poxvirus vectors. This response was not observed with any of the homologous boosting regimens. Mice primed with recombinant poxvirus vectors generated highly specific antibodies against viral proteins. CONCLUSIONS: The poor efficacy of homologous boosting regimens with viral vectors was probably a consequence of the induction of a strong antiviral antibody response. Heterologous boosting augmented antitumor immunity by generating a strong antigen-specific cytotoxic T-lymphocyte response. These data suggest that heterologous boosting strategies may be useful in increasing the efficacy of recombinant DNA anticancer vaccines that have now entered clinical trials.

Minimal determinant expressed by a recombinant vaccinia virus elicits therapeutic antitumor cytolytic T lymphocyte responses.

Anticancer vaccine strategies can now target intracellular antigens that are involved in the process of malignant transformation, such as oncogene products or mutated tumor suppressor genes. Fragments of these antigens, generally 8-10 amino acids in length and complexed with MHC class I molecules, can be recognized by CD8+ T lymphocytes (TCD8+). To explore the possibility of using a genetically encoded, minimally sized fragment of an intracellular antigen as an immunogen, we constructed a recombinant vaccinia virus encoding an 8-residue peptide derived from chicken ovalbumin that is known to associate with the mouse H-2Kb molecule. Compared to standard methods of immunization, recombinant molecule. Compared to standard methods of immunization, recombinant vaccinia virus expressing the minimal determinant as well as full length ovalbumin were the only approaches that elicited specific primary lytic responses in C57BL/6 mice against E.G7OVA, a transfectant of the murine thymoma EL4 containing the ovalbumin gene. Stimulating these effectors in vitro with OVA257-264 peptide induced H-2Kb-restricted TCD8+ that not only lysed but also specifically secreted IFN-gamma in response to an antigen. Furthermore, when transferred adoptively, these anti-OVA257-264 TCD8+ cells significantly reduced the growth of established ovalbumin-transfected tumors in a pulmonary metastasis model system. Synthetic transfected tumors in a pulmonary metastasis model system. Synthetic oligonucleotides encoding minimal antigenic determinants within expression constructs may be a useful approach for treatment of neoplastic disease, thus avoiding the potential hazards of immunizing with full-length cDNAs that are potentially oncogenic.

Recombinant virus vaccination against "self" antigens using anchor-fixed immunogens.

To study the induction of anti-"self" CD8+ T-cell reactivity against the tumor antigen gp100, we used a mouse transgenic for a chimeric HLA-A*0201/H-2 Kb molecule (A2/Kb). We immunized the mice with a recombinant vaccinia virus encoding a form of gp100 that had been modified at position 210 (from a threonine to a methionine) to increase epitope binding to the restricting class I molecule. Immunogens containing the "anchor-fixed" modification elicited anti-self CD8+ T cells specific for the wild-type gp100(209-217) peptide pulsed onto target cells. More important, these cells specifically recognized the naturally presented epitope on the surface of an A2/Kb-expressing murine melanoma, B16. These data indicate that anchor-fixing epitopes could enhance the function of recombinant virus-based immunogens.

Generation of polyclonal rabbit antisera to mouse melanoma associated antigens using gene gun immunization.

Lymphocytes from patients with melanoma have been used to clone melanoma associated antigens which are, for the most part, nonmutated melanocyte tissue differentiation antigens. To establish a mouse model for the use of these 'self' antigens as targets for anti-tumor immune responses, we have employed the mouse homologues of the human melanoma antigens Tyrosinase, Tyrosinase Related Protein-1 (TRP-1), gp100, and MART-1. We sought to generate antisera against these proteins for use in the construction of experimental recombinant and synthetic anti-cancer vaccines, and for use in biologic studies. Using genes cloned from the B16 mouse melanoma or from murine melanocytes, we immunized rabbits with plasmid DNAs coated onto microscopic gold beads that were then delivered using a hand-held, helium-driven 'gene gun'. This strategy enabled us to generate polyclonal rabbit sera containing antibodies that specifically recognized each antigen, as measured by immunostaining of vaccinia virus infected cells. The sera that we generated specifically for TRP-1, gp100, and MART-1 recognized extracts of the spontaneous murine melanoma, B16. The identities of the recognized proteins was confirmed by Western blot analysis. The titers and specificities of these antisera were determined using ELISA. Interestingly, serum samples generated against murine MART-1 and gp100 developed antibodies that were cross-reactive with the corresponding human homologues. Recognition of human gp100 and murine Tyrosinase appeared to be dependent upon conformational epitopes since specificity was lost upon denaturation of the antigens. These antisera may be useful in the detection, purification and characterization of the mouse homologues of recently cloned human tumor associated antigens and may enable the establishment of an animal model of the immune consequences of vaccination against 'self antigens.

Which end is up? Two representations of orientation in visual search.

What is the orientation of an object? A simple line has an axis of orientation. That line, turned upside-down, is indistinguishable from the original line. Thus, the possible orientations of a line range from 0 to 180 degrees. Most objects, however, have an axis and a polarity. A polar object, turned upside-down, looks upside-down. Accordingly, the orientations of a polar object range from 0 to 360 degrees. A series of visual search experiments were run to determine if preattentive processes represent orientation in a 180 or a 360 degrees framework. Results suggest that preattentive orientation is represented in 180 degrees. Experiments 1 and 4 show that search for a target rotated 90 degrees from the distractors is more efficient than search for a target rotated 180 degrees from the distractors. Experiments 2, 3, and 5 use a variety of different stimuli to demonstrate that search for targets rotated 180 degrees from distractors is inefficient.