Toward a new generation of vaccines: The anti-cytokine therapeutic vaccines

Pathological conditions, such as cancers, viral infections, and autoimmune diseases, are associated with abnormal cytokine production, and the morbidity associated with many medical disorders is often directly a result of cytokine production. Because of the absence of negative feedback control occurring in some pathophysiologic situations, a given cytokine may flood and accumulate in the extracellular compartment of tissues or tumors thereby impairing the cytokine network homeostasis and contributing to local pathogenesis. To evaluate whether the rise of anti-cytokine Abs by vaccination is an effective way to treat these pathological conditions without being harmful to the organism, we have analyzed each step of the cytokine process (involving cytokine production, target response, and feedback regulation) and have considered them in the local context of effector–target cell microenvironment and in the overall context of the macroenvironment of the immune system of the organism. In pathologic tissues, Abs of high affinity, as raised by anti-cytokine vaccination, should neutralize the pool of cytokines ectopically accumulated in the extracellular compartment, thus counteracting their pathogenic effects. In contrast, the same Abs should not interfere with cytokine processes occurring in normal tissues, because under physiologic conditions cytokine production by effector cells (induced by activation but controlled by negative feedback regulation) does not accumulate in the extracellular compartment. These concepts are consistent with results showing that following animal and human anti-cytokine vaccination, induction of high-affinity Abs has proven to be safe and effective and encourages this approach as a pioneering avenue of therapy.

Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.

DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.

Transloading of tumor cells with foreign major histocompatibility complex class I peptide ligand: a novel general strategy for the generation of potent cancer vaccines.

The major hurdle to be cleared in active immunotherapy of cancer is the poor immunogenicity of cancer cells. In previous attempts to overcome this problem, whole tumor cells have been used as vaccines, either admixed with adjuvant(s) or genetically engineered to express nonself proteins or immunomodulatory factors before application. We have developed a novel approach to generate an immunogeneic, highly effective vaccine: major histocompatibility complex (MHC) class I-positive cancer cells are administered together with MHC class I-matched peptide ligands of foreign, nonself origin, generated by a procedure we term transloading. Murine tumor lines of the H2-Kd or the H2-Db haplotype, melanoma M-3 and B16-F10, respectively, as well as colon carcinoma CT-26 (H2-Kd), were transloaded with MHC-matched influenza virus-derived peptides and applied as irradiated vaccines. Mice bearing a deposit of live M-3 melanoma cells were efficiently cured by this treatment. In the CT-26 colon carcinoma and the B16-F10 melanoma, high efficacies were obtained against tumor challenge, suggesting the universal applicability of this new type of vaccine. With foreign peptide ligands adapted to the requirements of a desired MHC class I haplotype, this concept may be used for the treatment of human cancers.

DNA vaccine for hepatitis B: evidence for immunogenicity in chimpanzees and comparison with other vaccines.

Vaccination of two chimpanzees against hepatitis B virus (HBV) by intramuscular injection of plasmid DNA encoding the major and middle HBV envelope proteins induced group-, subtype- and preS2-specific antibodies. These were initially of IgM isotype, and then they were of IgG (predominantly IgGl) isotype. The chimpanzee injected with 2 mg of DNA attained >100 milli-international units/ml of anti-HBs antibody after one injection and 14,000 milli-international units/ml after four injections. A smaller dose (400 microg) induced lower and transient titers, but a strong anamnestic response occurred 1 year later. Comparison with responses in 23 chimpanzees receiving various antigen-based HBV vaccines suggests that the DNA approach is promising for prophylactic immunization against HBV.

Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.

The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo.

p53 as a target for cancer vaccines: recombinant canarypox virus vectors expressing p53 protect mice against lethal tumor cell challenge.

The p53 protein is an attractive target for immunotherapy, because mutations in the p53 gene are the most common genetic alterations found in human tumors. These mutations result in high levels of p53 protein in the tumor cell, whereas the expression level of wild-type p53 in nonmalignant tissue is usually much lower. Several canarypox virus recombinants expressing human or murine p53 in wild-type or mutant form were constructed. Immunization with these viruses protected BALB/c mice from a challenge with an isogenic and highly tumorigenic mouse fibroblast tumor cell line expressing high levels of mutant p53. The tumor protection was equally effective regardless of whether wild-type or mutant p53 was used for the immunization, indicating that the immunologic response was not dependent on any particular p53 mutation and that immunization with this live virus vaccine works effectively against mutant p53 protein expressed in a tumor cell. In tumors escaping immunologic rejection, the expression of the p53 protein was commonly down-regulated.

Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine.

Plasmodium falciparum malaria parasites were transformed with plasmids containing P. falciparum or Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (dhfr-ts) coding sequences that confer resistance to pyrimethamine. Under pyrimethamine pressure, transformed parasites were obtained that maintained the transfected plasmids as unrearranged episomes for several weeks. These parasite populations were replaced after 2 to 3 months by parasites that had incorporated the transfected DNA into nuclear chromosomes. Depending upon the particular construct used for transformation, homologous integration was detected in the P. falciparum dhfr-ts locus (chromosome 4) or in hrp3 and hrp2 sequences that were used in the plasmid constructs as gene control regions (chromosomes 13 and 8, respectively). Transformation by homologous integration sets the stage for targeted gene alterations and knock-outs that will advance understanding of P. falciparum.

Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides.

We studied inhibition of growth of the malaria parasite Plasmodium falciparum in in vitro culture using antisense (AS) oligodeoxynucleotides (ODNs) against different target genes. W2 and W2mef strains of drug-resistant parasites were exposed to AS ODNs over 48 hr, and growth was determined by microscopic examination and [3H]hypoxanthine incorporation. At ODN concentrations of 1 microM, phosphorothioate (PS) ODNs inhibited growth in a target-independent manner. However, between 0.5 and 0.005 microM, ODNs against dihydrofolate reductase, dihydropteroate synthetase, ribonucleotide reductase, the schizont multigene family, and erythrocyte binding antigen EBA175 significantly inhibited growth compared with a PS AS ODN against human immunodeficiency virus, two AS ODNs containing eight mismatches, or the sense strand controls (P < 0.0001). The IC50 was approximately 0.05 microM, whereas that for non-sequence-specific controls was 15-fold higher. PS AS ODNs against DNA polymerase alpha showed less activity than that for other targets, whereas a single AS ODN against triose-phosphate isomerase did not differ significantly from controls. We conclude that at concentrations below 0.5 microM, PS AS ODNs targeted against several malarial genes significantly inhibit growth of drug-resistant parasites in a nucleotide sequence-dependent manner. This technology represents an alternative method for identifying malarial genes as potential drug targets.

Sphingolipid synthesis as a target for chemotherapy against malaria parasites.

The human malaria parasite Plasmodium falciparum contains sphingomyelin synthase in its Golgi apparatus and in a network of tubovesicular membranes in the cytoplasm of the infected erythrocyte. Palmitoyl and decanoyl analogues of 1-phenyl-2-acylamino-3-morpholino-1-propanol inhibit the enzyme activity in infected erythrocytes. An average of 35% of the activity is extremely sensitive to these drugs and undergoes a rapid, linear decrease at drug concentrations of 0.05-1 microM. The remaining 65% suffers a slower linear inhibition at drug concentrations ranging from 25 to 500 microM. Evidence is presented that inhibition of the sensitive fraction alone selectively disrupts the appearance of the interconnected tubular network in the host cell cytoplasm, without blocking secretory development at the parasite plasma membrane or in organelles within the parasite, such as the Golgi and the digestive food vacuole. This inhibition also blocks parasite proliferation in culture, indicating that the sensitive sphingomyelin synthase activity as well as the tubovesicular network may provide rational targets for drugs against malaria.

Cancer vaccines: the interleukin 2 dosage effect.

Cancer vaccines genetically engineered to produce interleukin 2 have been investigated intensively in a series of animal models and are at the point of entering into clinical trials. In this study we demonstrate a strong correlation between the rate of interleukin 2 production and the protection efficiency of murine S91 melanoma cell (clone M-3) vaccines. Best immunization is achieved with vaccines producing medium interleukin 2 levels of 1000-3000 units per 10(5) cells per day. Reduced interleukin 2 production evokes a corresponding decline in the number of successfully treated animals. Unexpectedly, when interleukin 2 expression is raised to high levels of 5000-7500 units per 10(5) cells per day, protection is completely absent because of impaired generation of tumor-specific cytotoxic T lymphocytes. In comparison, granulocyte-macrophage colony-stimulating factor as immunomodulator induces substantial immunization even at a moderate level of secretion and protects all animals at the maximal obtainable level of secretion. Our findings demonstrate the importance of the interleukin 2 level produced by genetically modified tumor cells and may have substantial impact for the clinical application of cancer vaccines.

Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites.

Immunization of rodents and humans with irradiation-attenuated malaria sporozoites confers preerythrocytic stage-specific protective immunity to challenge infection. This immunity is directed against intrahepatic parasites and involves T cells and interferon gamma, which prevent development of exoerythrocytic stages and subsequent blood infection. The present study was undertaken to determine how protective immunity is achieved after immunization of rodent hosts with irradiated Plasmodium berghei sporozoites. We present evidence that irradiated parasites persist in hepatocytes of rats and mice for up to 6 months after immunization. A relationship between the persistence of parasites and the maintenance of protective immunity was observed. Protective immunity was abrogated in irradiated-sporozoite-immunized rats following the application of chemotherapy to remove preexisting liver parasites. Additionally, protective immunity against sporozoite challenge was established in rats vaccinated with early and late hepatic stages of irradiated parasites. These results show that irradiation-attenuated sporozoites produce persistent intrahepatic stages in vivo necessary for the induction and maintenance of protective immunity.