Insect cell-derived VP2 of infectious bursal disease virus confers protection against the disease in chickens.

Infectious bursal disease virus (IBDV) has become a major problem in recent years. Conventional vaccines make use of attenuated or inactivated viral strains, but these are gradually losing their effectiveness. We investigated the possibility of using purified VP2, a subunit of IBDV structural protein expressed in insect cells, as a vaccine. The VP2 gene was cloned into pAcYM1. The cloned gene was expressed in a baculovirus system, giving rise to a high quantity of recombinant VP2 (rVP2) protein. The length of the VP2 is 453 amino acids, and it contains two additional amino acids of the baculovirus at the carboxyl terminus. The molecular mass of the protein is about 48 kD. The rVP2 protein reacted with antibodies raised against viral VP2 and had a similar molecular weight. This protein was tested in a controlled vaccination experiment and compared with an inactivated commercial vaccine. High levels of antibodies were raised by the vaccinated birds. The vaccinated birds were challenged with a pathogenic viral strain. rVP2-vaccinated chickens exhibited high resistance to the virus. No mortality or weight changes in the bursa of Fabricius were observed in the vaccinated birds, whereas in the negative control birds, vaccinated with phosphate buffer, up to 50% mortality was found. Higher levels of antibodies were found by enzyme-linked immunosorbent assay in birds vaccinated with rVP2 compared with those vaccinated with the commercial vaccine. This study suggests the potential use of the isolated rVP2 as a subunit vaccine.

Effect of a single injection of cyclophosphamide on immunization therapy of metastases remaining after excision of dermal primary tumors in guinea pigs.

Guniea pigs with established (7 or 14 days old) syngeneic dermal tumors and metastases in the draining lymph nodes were unsuccessfully treated by excision of the dermal tumors and specific immunization. The vaccines consisted of killed BCG in oil in an emulsified form admixed with mitomycin C treated or irradiated tumor cells. The therapeutic failure to eradicate the metastases was overcome by an additional treatment with a single injection of cyclophosphamide prior to excision of the primary tumor and immunization. It is assumed that cyclophosphamide destroys suppressor elements in the tumor-bearing guinea pigs and, in this way, augments the therapeutic effects of specific immunization.

Eradication by active specific immunotherapy of established tumor transplants and microscopic lymph node metastases.

Guinea pigs, each with an established syngeneic dermal line 10 tumor and microscopic lymph node metastases, were immunized by injection of a mixture of irradiated line 10 tumor cells and an oil-in-water emulsion containing heat-killed cells of Mycobacterium bovis strain Bacillus Calmette-Guérin. Squalane or squalene-in-water emulsions, prepared by ultrasonication and containing mg doses of mycobacterial cells, were effective adjuvants. Immunization eradicated established dermal tumors (about 10 mm in diameter) and prevented growth of microscopic lymph node metastases. Untreated animals, animals treated by intradermal administration of Bacillus Calmette-Guérin cells attached to oil droplets alone or with irradiated tumor cells alone, all died with progressive tumor growth.

Immunotherapy of guinea pigs with a transplanted hepatoma: comparison of intralesionally injected emulsions containing heat-killed Nocardia rubra, Mycobacterium bovis (BCG) and Mycobacterium phlei.

Heat-killed cells of Mycobacterium bovis (BCG), Mycobacterium phlei and Nocardia rubra were each tested in emulsified form for their ability to cause regression of established dermal transplants and lymph node metastases of a syngeneic hepatocarcinoma in guinea pigs. On a weight basis, BCG was superior to N. rubra in causing tumor regression. Under the conditions tested N. rubra was inferior to M. phlei in its antitumor activity. M. phlei and BCG were approximately the same in their therapeutic potency. In BCG-sensitized guinea pigs, N. rubra provoked a weaker delayed cutaneous hypersensitivity (DCH) reaction than did BCG. In N. rubra-sensitized guinea pigs, BCG provoked a weaker DCH reaction than did N. rubra. Purified protein derivative of M. tuberculosis was more active in eliciting DCH in BCG-sensitized guinea pigs than in animals sensitized with N. rubra.

A specific vaccine effective against stage I and stage II malignant disease in guinea pigs. Effect of variations in preparations and storage.

Guinea pigs, each with an established, syngeneic dermal line 10 tumor and lymph node metastases, were immunized by intradermal injection of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG. Immunization eradicated 7- or 10-day-old dermal tumors (about 10 or 12 mm in diameter, respectively) and prevented growth of microscopic lymph node metastases. Fourteen-day-old dermal tumors (about 15 mm in diameter) were not rejected by immunization. Guinea pigs with stage II disease (21-day-old dermal tumors and palpable metastases in the first draining lymph node) were treated by excision of the dermal tumor and the first draining lymph node, and by specific immunization. This treatment eliminated tumor cells remaining in the second draining lymph nodes. The surgical treatment alone was not curative, palpable metastases in the second draining lymph nodes progressed and the animals died (some with visible lung metastases). Emulsions containing killed BCG were good adjuvants even after prolonged storage at 4 degrees C, but lost most of their adjuvant activity after autoclaving or freezing.

Treatment by limited surgery and specific immunization of guinea pigs with stage II experimental cancer.

The malignant disease produced in guinea pigs by intradermal inoculation of line-10 was allowed to progress to stage II, at which time the dermal tumor and the first draining lymph node were grossly evident. At that stage, the external appearance of the next draining lymph node was normal, but it contained tumor cells. Limited surgery consisting of excision of the dermal tumor and first draining lymph node was not curative; palpable metastases developed in the second and other draining lymph nodes, and at autopsy, some animals were found to have gross, visible lung metastases. Immunization of guinea pigs with a mixture of irradiated syngeneic tumor cells plus mycobacterial cell walls in an oil-in-water emulsion eradicated tumor cells remaining in lymph nodes after limited surgery for stage II experimental cancer and prevented progression of the disease to stage III. Tumor intravenously implanted in the lungs of animals after limited surgery for stage II disease was also eliminated by immunization.

Immunotherapy of experimental cancer with a mixture of synthetic muramyl dipeptide and trehalose dimycolate.

The antitumor activity of a mixture of synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and trehalose-6,6'-dimycolate (TDM) (MDP+TDM) in emulsified form was studied in guinea pigs, each with a syngeneic dermal tumor and microscopically detectable metastases in regional lymph nodes. A single intralesional administration of an ultrasonically prepared emulsion containing MDP+TDM in squalane or in mineral oil caused tumor regression and elimination of lymph node metastases. Similar emulsions of MDP+TDM made with squalene or hexadecane were immunotherapeutically inactive.

Regression by active specific immunotherapy of established dermal tumor transplants and lymph node metastases in guinea pigs.

Guinea pigs, each with an established, syngeneic dermal tumor (line-10) and microscopic lymph node metastasis, were treated by intradermal inoculation of living line-10 tumor cells admixed with emulsified heat-killed Mycobacterium bovis BCG cells. This treatment caused complete regression of established dermal tumors (about 10 mm in diameter) and prevented the growth of microscopic lymph node metastases in 25 of 39 treated animals (64%). All control animals treated by intradermal inoculation with heat-killed M. bovis BCG cells attached to oil droplets died with progressive dermal and lymphatic tumor growth.

Influence of type of oil and surfactant concentration on the efficacy of emulsified Mycobacterium bovis BCG cell walls to induce tumor regression in guinea pigs.

The influence of mineral ol, squalane, squalene, hexadecane, or peanut oil and of the concentration of Tween 80 on the immunotherapeutic capability of emulsified Mycobacterium bovis Bacillus Calmette-Guérin (BCG) cell walls was studied in guinea pigs, each with an established dermal transplant of a syngeneic hepatocarcinoma and tumor cells in the draining lymph node. Immunotherapy consisted of an intratumoral injection of emulsified cell walls. Conditions were established under which therapeutically effective emulsions could be made with mineral oil, squalane, squalene, or hexadecane. Emulsions made with peanut oil failed to cause tumor regression. Emulsions of squalene or hexadecane were effective substitutes for mineral oil as carriers of cell walls in the absence of added Tween or at a Tween concentration one-hundredth of that used to stabilize the mineral oil-containing emulsions. Cell wall emulsions made with squalane were therapeutically effective over the same range of Tween concentrations used to prepare emulsions containing mineral oil. Cell wall emulsions made without added Tween demonstrated effective antitumor activity even after autoclaving. Emulsions made with Tween separated after autoclaving. Emulsions of whole killed BCG were immunotherapeutically as active as those made with cell walls.

Immunotherapy of experimental cancer by intralesional injection of emulsified nonliving mycobacteria: comparison of Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis.

Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis were each tested in emulsified form for their potency to cause regression of transplants of a syngeneic murine fibrosarcoma and of a syngeneic guinea pig hepatoma. On a weight basis, M. phlei and M. smegmatis were as effective as BCG in causing tumor regression. M. phlei and M. smegmatis were comparable to BCG in provoking delayed cutaneous hypersensitivity reactions in guinea pigs sensitized to M. phlei or M. smegmatis. In BCG-sensitized guinea pigs, M. phlei and M. smegmatis provoked weaker delayed cutaneous hypersensitivity reactions than did BCG. Purified protein derivative of M. tuberculosis was more active in eliciting delayed cutaneous hypersensitivity in BCG-sensitized guinea pigs than in animals sensitized with M. phlei or M. smegmatis.

Influence of surfactant concentration on the antitumor activity of emulsified components of mycobacteria.

The influence of Tween 80 content on the antitumor activity of emulsified mycobacterial components administered intralesionally was studied in mice. The number of treated animals in which there was complete regression of tumor depended on the concentration of Tween in each of the emulsions. An additional variable was the size distribution of the mineral oil droplets, which depended on whether the emulsions were prepared by ultrasonication or by mechanical grinding. Emulsions of mycobacterial components (cell walls of Bacillus Calmette-Guérin and trehalose-6,6'-dimycolate) prepared by ultrasonication contained smaller oil droplets than did those prepared by grinding. Ground emulsions retained antitumor activity over a wider range of Tween concentrations than did ultrasonically prepared emulsions. The latter required Tween at concentrations in an optimal range above which the tumor-regressive potency was diminished. Emulsions of trehalose-6,6'-dimycolate containing the lowest concentration of Tween needed to produce a relatively stable preparation contained larger oil droplets and were immunotherapeutically less active than were those prepared with an optimal concentration of Tween. Emulsions of B. Calmette-Guérin cell walls retained antitumor activity even in the absence of added Tween.

Regression of a murine fibrosarcoma after intralesional injection of a synthetic C39 glycolipid related to cord factor.

Intratumoral injection of ultrasonically prepared emulsions of the synthetic glycolipid methly 6-O-(2-tetradecyl-3-hydroxyoctadecanoyl)-alpha-D-glucopyranoside (designated C39) induced complete regression of transplants of a syngeneic murine fibrosarcoma in most of the treated animals as did 6,6'-di-O(2-tetradecyl-3-hydroxyoctadecanoyl)-alpha,alpha,-trehalose (designated C76) in a previous study. The C76 compound, about twice the molecular weight of C39, was more effective therapeutically than the smaller molecule. Ultrasonically prepared emulsions of C39 and C76 were not toxic when given intravenously. Intravenously administered emulsions of C39 prepared by mechanical grinding were more toxic, but less granulomagenic, than those containing C76. Squalane and squalene, but not peanut oil, were effective substitutes for mineral oil as carriers of C39 in the treatment of the tumor.

Immunotherapy of guinea pigs with a transplanted hepatoma: comparison of intralesionally administered killed BCG cells and BCG cell walls.

Heat-killed whole BCG cells (KC) and BCG cell walls (CW) were each tested in emulsified form for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basis, KC were at least as effective as CW in causing tumor regression and elimination of microscopic lymph node metastasis, and they, as well as purified protein derivative of mycobacteria, provoked delayed cutaneous hypersensitivity reactions in animals immunized with CW or with KC. On a weight basis, KC were as active as CW in eliciting delayed cutaneous hypersensitivity in sensitized guinea pigs whether the animals were immunized with CW or with KC. In unimmunized animals the inflammatory response to intradermally administered KC was similar to that induced by CW. Because KC are easier to prepare than CW, it is suggested that whole killed BCG might be used instead of CW in clinical trials of cancer treatment requiring administration of nonliving mycobacteria.

Local tumor regression after intralesional injection of croton oil.

Intralesional administration of emulsified croton oil into established syngeneic transplants of murine firosarcoma no. 1023 caused complete regression of the injected tumors in C3H mice without recurrence during the period of observation. In Sewall Wright strain 2 guinea pigs, in contrast to BCG cell wall vaccine which eradicated regional lymph node metastasis as well as dermal transplants, croton oil treatment only delayed the development of metastatic disease despite the fact that the injected skin tumors did not recur. 12-O-Tetradecanoylphorbol 13-acetate (TPA), the active principle of croton oil, incorporated in mineral oil droplets in aqueous suspension, caused regression of murine tumors when injected intralesionally. Aqueous suspensions of TPA failed to eliminate the tumors. Our results suggest that tumor regression induced by croton oil of TPA emulsions was due to indiscriminate destruction of the injected tissue.

Effect of sulfolipid I on trehalose-6,6'-dimycolate (cord factor) toxicity and antitumor activity.

Emulsified mixtures of sulfolipid I and trehalose-6,6'-dimycolate (cord factor) had tumor-regressive activity comparable to, but less toxic than, emulsified trehalose-6,6'-dimycolate alone.