Interleukin-12 (IL-12) is a heterodimeric cytokine originally defined by its ability to induce the maturation of cytolytic lymphocytes and by its capacity to effectively synergize with IL-2 in the induction of cytolytic activity. Recent studies in mice have demonstrated the ability of IL-12 to cause tumor regression and stimulate long-term antitumor immunity in treated animals. To examine the antitumor effect of direct gene transfer of IL-12 into tumors, we have developed retroviral vectors that coordinately express both subunits of IL-12. An MFG-based retroviral vector was used to generate a recombinant retrovirus in which a long terminal repeat (LTR)-driven polycistronic transcript encodes both subunits of human IL-12: hp35 and hp40 cDNAs are linked and coexpressed using the internal ribosome entry site (IRES) from the encephalomyocarditis virus (DFG-hIL-12). In addition, two IRES sequences were used to express both subunits of IL-12 and a neomycin resistance (neoR) selectable marker gene from the same polycistronic message (TFG-hIL-12). The amphotropic DFG-hIL-12 and TFG-hIL-12 viruses were used to infect both human and murine cell lines as well as primary tumor cultures. The production of human IL-12 by the nonselected, infected cells was measured in both a PHA blast proliferation bioassay and an ELISA and ranged from 15 to 40 ng/10(6) cells per 24 hr. Following G418 selection of TFG-hIL-12-infected cells, the level of expression of IL-12 was significantly higher (up to 120 ng/10(6) cells per 24 hr). The IL-12 protein secreted by the infected cells exhibited all of the biologic activities of recombinant hIL-12: proliferation of activated natural killer (NK) and T cells, stimulation of interferon-gamma (IFN-gamma) induction by NK and T cells, and enhancement of lymphokine-activated killer (LAK) activity. These retroviral vectors expressing human IL-12 should be useful in evaluating the biological properties of IL-12 as well as for use in clinical trials for gene therapy of patients with cancer.

The peptide corresponding to sequence 91-108 of the hemagglutinin of type A H3N2 influenza virus has been synthesized by the solid-phase peptide synthesis method and covalently attached to several macromolecular carriers. The conjugate with tetanus toxoid was used for immunization ofrabbits and mice. The immunoglobulin fraction of the rabbit antiserum showed the presence of antipeptide antibodies by both agar gel diffusion and radioimmunoassay. In the latter assay, the antibodies showed marked crossreactivity with the intact virus of the A/Texas/77 strain. The antibodies were also capable of inhibiting the hemagglutination of chicken erythrocytes by the virus; the highest hemagglutination inhibition titer (1:32) was achieved with a serumresistant strain of A/Texas/77. When the in vitro virus plaque formation assay was used with monolayers of Madin-Darby canine kidney (MDCK) cells, the number of plaques was reduced on interaction with the immunoglobulin fraction of the antiserum, which was effective up to a dilution of 1:32. Preliminary results indicate that C3H/DiSn mice immunized with the peptide-tetanus toxoid conjugate are partially protected against a further challenge with A/Texas mouse-adapted influenza virus. The results are thus indicative of the efficacy of the synthetic material in eliciting antiinfluenza immune response.The idea of using synthetic materials to replace the vaccines currently used against viruses was put forth several years ago (1, 2). According to this approach, the chemically defined vaccine should contain a unique synthetic antigenic determinant(s) capable of provoking antibodies with neutralizing efficacy against the virus. Although this issue, as discussed recently (3), raises several problems, it also offers many potential advantages and its feasibility for a model virus has been proven. Thus, early studies by Anderer (4) have shown that, by immunizing with a conjugate containing the COOH-terminal hexapeptide of tobacco mosaic virus protein, it is possible to obtain antibodies that interact to some extent with the intact virus. In a more recent study (5), an immunologically active synthetic peptide analogous to a fragment of the coat protein of another virus, MS-2 coliphage, was described. A conjugate containing this peptide provoked antibodies capable of efficiently inactivating the native bacteriophage. It seemed of interest to test this chemical approach in an animal virus system.The influenza virus provides a suitable model for an animal virus for this purpose for the following reasons. (i) Detailed information is available on the structure and function ofthis virus, as well as on its serological specificities and genetic variations.(ii) Various reliable assays of the virus are available (6-9) for evaluating the effect of the immune response on the different viral functions. (iii) Sufficient information is available on the amino acid sequence of the influenza hemagglutinin (10) and on its immunochemical properties (11) to allow the synthesis of a peptide fragment(s) t...

Four peptides have been synthesized, corresponding to different regions of the H3 influenza hemagglutinin, that are related to antigenic sites "A" and "B" of the molecule. The peptides consisted of the following sequences: 139-146, which forms the "loop" in the native hemagglutinin molecule, with either glycine or aspartic acid at position 144; 147-164, which contains part of antigenic determinant "B"; and 138-164, which comprises both the loop and the area 147-164.The peptides were conjugated to tetanus toxoid and used for immunization of rabbits and mice. All four conjugates elicited an immune response against the homologous peptides, but only the peptides 138-164 and 147-164 gave rise to antibodies that recognized and bound to the intact virus. Protection of mice against challenge infection with A/Eng/42/72 virus was achieved only by immunization with the conjugate (138-164)-TT, which led to partial protective effect. These data emphasize the role of molecular structure in determining the antigenic properties of synthetic peptides and indicate that the length of the peptide could be crucial for enforcing the right folding required to mimic the native structure.

No abstract

No abstract