Use of xenogenized (modified) tumor cells for treatment in experimental tumor and in human neoplasia.

The need to modify tumor cells in order to render them more "immunogenic" was based on the assumption that normal, nonmodified tumor cells are non- or weakly immunogenic and as such are unable to raise an efficient protective immune response. Various methods for "xenogenization" (modification of tumor cells) were suggested: induction of new foreign antigens, treatment with either chemicals or enzymes and use of mutagens. Xenogenized tumor cells by their coupling to proteins, and use of chemicals like DTIC (5-[3,3-dimethyl- 1-triazeno]-imidazole-4-carboxamide), TZC (8-carbamoyl-3-methyl-imidazo[5, 1-d]- 1,2,3,5-tetrazin-4 [3H]-one 8-carbamoyl-3-[2-chloroethyl] imidazole [5,1 -d]- 1,2,3,5-tetrazin-4[3H]-one) and antiemetic drugs, were tested in experimental models of murine leukemia. Non-tumorigenic clones, xenogenization with DNA hypomethylating agents, aryl-triazine derivatives and DTIC were evaluated for their induction of protective immune response in murine lymphoma. Murine plasmacytoma cells were used for immunization after treatment with glutaraldehyde. Viral modifications of tumor cells were evaluated for their ability to induce a protective tumor response in model systems of rat fibrosarcoma, liver metastatic rat tumor cells, lymphoid tumor cells and hamster tumor cells. In the case of human cancer, attempts were reported to use DNP-conjugated melanoma cells, mutagenic triazine compounds, an autologous colon tumor cell bacillus Calmette-Guerin (BCG) vaccine and genetically engineered vaccines for immunization. The general conclusion drawn from experimental tumor models and for human cancer is, that although modified tumor cells were found to be partially effective in experimental models, it is still necessary to provide more data in order to determine the effective use of xenogenized human tumor cells for immunotherapy.

Rational approaches to reduce adverse reactions in man to vaccines containing tetanus and diphtheria toxoids.

Adverse reactions to routine vaccines are obstacles to the mass vaccination campaigns. Though the absolute safety of any injectable vaccine cannot be guaranteed, the adverse side effects to vaccines can be minimized by practicing existing scientific knowledge. Adverse side effects to tetanus and diphtheria toxoids have been known for many years and there have been ways to minimize these reactions. These procedures did not get wide acceptance, because the current partially purified tetanus and diphtheria vaccines meet the regulatory requirements and the manufacturers are reluctant to change the established procedures of production due to the amount of work involved in the regulatory issues under the current Good Manufacturing Practices (GMP). Due to the recent epidemic of diphtheria in the independent states of the former Soviet Union, and its potential for spread to other European Countries, vaccination campaigns with tetanus and diphtheria vaccines received a new boost with several international agencies. In this report, we review the causes for adverse reactions to tetanus and diphtheria vaccines and offer practical suggestions for minimizing these reactions. The major issues in minimizing adverse reactions to these vaccines include: (1) purifying the toxins before detoxification as the reactogenic accessory antigens get covalently bound to the toxins during detoxification; (2) either using well-tolerated adjuvants which do not elicit the production of antigenic specific IgE antibodies responsible for adverse reactions or by using non-adjuvanted highly immunogenic polymerized antigens; (3) checking the status of immunity by recently developed rapid serological methods or by the Schick skin-test for diphtheria to avoid allergic or Arthus-type reactions. These approaches are applicable to industrial scales and would result in a pure, less reactogenic and better characterized toxoids antigens which would be more suitable for combined vaccines comprising highly purified acellular pertussis components, polysaccharide-protein conjugates and other antigens.

Passive haemagglutination tests using purified antigens covalently coupled to turkey erythrocytes.

Passive haemagglutination tests have been developed by covalent coupling purified antigens to turkey red blood cells. Circulating antibodies can be assessed in 20 minutes using one drop of blood. False positive reactions are avoided by using highly purified antigens; sensitized erythrocytes are stable in the absence of freeze-drying and blood samples can be preserved on paper discs. This method, applied to the determination of circulating tetanus (T) and diphtheria (D) antibodies and titres compared to other in vivo or in vitro methods, gave good correlation. The titration of circulating antibodies can be applied in emergency care units and field trials to establish whether the individuals are adequately protected. Results of surveys by several health care centres have shown that tetanus immune coverage was insufficient in France. The decrease of both T and D immune coverage with age has been established. The antibody response of pregnant women, vaccinated with two different adsorbed T toxoids exhibiting a low and a high titre as expressed in international immunizing units (I.I.U.), was studied. No significant difference in circulating antibody titres was obtained after the first injection of either vaccine, but titres after second injection were much higher for the vaccine having a low value expressed in I.I.U. The activity of commercial and reference T vaccines can be evaluated in mice after immunization and titration of the antitoxin levels. This simple method is much easier than the official evolution of immunodeficiency in certain diseases. The passive haemagglutination test has also been used to measure anti-HBs and anti-gp 160 antibodies.

Production in rabbits of high levels of anti-HIV-1 gp160 antibodies.

The production of anti-HIV-1 gp160 antibodies was obtained in rabbits given gp160 either in saline or adsorbed onto calcium phosphate. Immunization with gp160 in saline induced the formation of antibodies directed to the p18 protein, whereas the gp160 adsorbed onto calcium phosphate elicited antibodies recognizing the gp160, p55, p25 and p18 proteins. Calcium phosphate was found to be a powerful adjuvant and it should be used for potentiating candidate anti-HIV vaccines.

Adjuvant properties of aluminum and calcium compounds.

It is likely that aluminum compounds will continue to be used with human vaccines for many years as a result of their excellent track record of safety and adjuvanticity with a variety of antigens. For infections that can be prevented by induction of serum antibodies, aluminum adjuvants formulated under optimal conditions are the adjuvants of choice. It is important to select carefully the type of aluminum adjuvant and optimize the conditions of adsorption for every antigen since the degree of adsorption of antigens onto aluminum adjuvants markedly affects immunogenicity. The mechanism of adjuvanticity of aluminum compounds includes formation of a depot at the site of injection from which antigen is released slowly; stimulation of immune-competent cells of the body through activation of complement, induction of eosinophilia, and activation of macrophages; and efficient uptake of aluminum-adsorbed antigen particles by antigen-presenting cells because of their particulate nature and optimal size (< 10 microns). Limitations of aluminum adjuvants include local reactions, production of IgE antibodies, ineffectiveness for some antigens, and inability to elicit cell-mediated immune responses especially cytotoxic T-cell responses. Calcium phosphate, which has adjuvant properties similar to aluminum adjuvants, has the potential advantages of being a natural component of the body and of not increasing IgE production. There is a need for alternative adjuvants, particularly for diseases in which cell-mediated immune responses are important for prevention or cure.

Humoral response in rabbits immunized with calcium phosphate adjuvanted HIV-1 gp160 antigen.

Rabbits were immunized with either calcium phosphate adjuvanted purified HIV-1 gp160 or a fluid preparation. Circulating antibodies were detected by ELISA, RIPA and Western Blot tests. Sera of rabbits immunized with the adjuvanted preparation contained high levels of anti-gp160 antibodies, as well as antibodies recognizing p55, p25 and p18. On the contrary, rabbits immunized with the fluid preparation contained only anti-p18 antibodies. Neutralizing antibodies were also detected. It is concluded that the calcium phosphate adjuvant could be used for preparation of candidate anti-HIV vaccines, since it permits one to induce high levels of circulating antibodies, in the absence of untoward reactions as observed when aluminium adjuvants or water in oil emulsions are used.

Adjuvants--a balance between toxicity and adjuvanticity.

Adjuvants have been used to augment the immune response in experimental immunology as well as in practical vaccination for more than 60 years. The chemical nature of adjuvants, their mode of action and the profile of their side effects are highly variable. Some of the side effects can be ascribed to an unintentional stimulation of different mechanisms of the immune system whereas others may reflect general adverse pharmacological reactions. The most common adjuvants for human use today are still aluminium hydroxide, aluminium phosphate and calcium phosphate although oil emulsions, products from bacteria and their synthetic derivatives as well as liposomes have also been tested or used in humans. In recent years monophosphoryl lipid A, ISCOMs with Quil-A and Syntex adjuvant formulation (SAF) containing the threonyl derivative of muramyl dipeptide have been under consideration for use as adjuvants in humans. At present the choice of adjuvants for human vaccination reflects a compromise between a requirement for adjuvanticity and an acceptable low level of side effects.

Simplified activity evaluation of several tetanus vaccines.

The activity of several Tetanus Toxoids, Adsorbed, (commercial vaccines and references) were tested in mice in comparison with a standard, by a simple method, easier than the official challenge test (WHO and European Pharmacopoeia): the Tetanus Antitoxin level was titrated by agglutination of sensitized turkey red blood cells after immunization by the toxoids. Immuno-stimulation by the Pertussis component in associated vaccines was studied and the results with the conventional and the acellular Pertussis preparations were prepared. The method was also found to be suitable for Tetanus Toxoids, Non-Adsorbed, when a booster effect was used, except for the adjuvant-free polymerized antigen (POLAN) which did not require a booster, since it gave almost as good results as conventional adsorbed tetanus vaccines.

Determination of antibodies to pertussis toxin in working reference preparations of anti-pertussis sera from various national control laboratories.

Working reference preparations of anti-pertussis sera from various National Control Laboratories were assayed for anti-PT antibodies by standardized ELISA and toxin neutralization (Nt) test. Both the ELISA and Nt tests gave highly reproducible results for various preparations when these preparations were assayed repeatedly on different days. Various working reference preparations were assigned units against the proposed International standard for anti-pertussis serum (JNIH-10) assuming its unitage of 250. Assigning unitage to various preparations would help in comparing results of ELISA and Nt tests for anti-PT antibodies reported in various studies.

Simultaneous injection of hepatitis B vaccine with BCG and killed poliovirus vaccine.

In most developing countries, hepatitis B virus is endemic and prevention has to be carried out early in life and on a mass scale. In these regions, simultaneous administration of multiple antigens is normal practice. We have therefore investigated the interaction of hepatitis B vaccine with BCG and inactivated polio vaccine. The serological antibody response to poliovirus and HBsAg as well as the cellular immune response to tuberculin post BCG immunization were assessed. The immune responses to HBsAg, BCG and polio vaccines injected simultaneously were comparable to those observed after separate administration of each vaccine. Moreover, no increase of adverse reactions was noted. Results confirmed that HB vaccine could be introduced into the WHO expanded programmes on immunization without impairing the expected protective efficacy against the targeted vaccine-preventable diseases.

Determination of circulating antibodies directed to pertussis toxin and of agglutinogens in children vaccinated with either the whole cell or component pertussis vaccine in France, Japan and Senegal.

The antibody response to pertussis toxin (PT) and agglutinogens of children vaccinated in Japan, France and Senegal with either whole cell or component pertussis vaccine was determined at various times after immunization. Agglutinin titres were almost similar in sera of Japanese children vaccinated with either whole cell or component pertussis vaccine whereas anti-PT antibody levels were found to be higher after vaccination with whole cell vaccine than with component vaccine. The geometric mean (GM) agglutinin titres in sera of Japanese children amounted to 45.0 and 45.7, respectively, and neutralization GM titres to 71.6 and 22.6, respectively, following vaccination with the whole cell and component pertussis vaccines. Sera of French children receiving three doses of whole cell vaccine exhibited a GM agglutinin titre of 17.8, whereas only 16% of sera contained neutralizing antibodies against PT. Following the booster dose the GM agglutinin titre rose to 213.5 and 68% of the sera contained neutralizing antibodies to PT (GM titre 48.0). Sera of Senegalese children receiving three doses of whole cell vaccine exhibited a GM agglutinin titre of 18.7, whereas anti-PT neutralizing antibodies were hardly detected. Agglutinins and anti-PT antibody in sera of French and Senegalese children turned out to be lower than were found 25 years ago in sera of children immunized with the French whole cell pertussis vaccine.

Adverse reactions after injection of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine are not due only to pertussis organisms or pertussis components in the vaccine.

Reactions to adsorbed diphtheria-pertussis-tetanus (DPT) vaccine have mostly been attributed to the pertussis organisms or pertussis components in the vaccine. Nevertheless reactions may also be due to other factors such as sensitization induced by aluminium adjuvants and impurities present in crude toxoids that cannot be removed by purification of toxoids after formalinization. Aluminium compounds such as aluminium phosphate and aluminium hydroxide are the most commonly used adjuvants with vaccines for human use. Due to the increasing concern about the toxicity of aluminium, other adjuvants like calcium phosphate may be evaluated as an alternative to aluminium adjuvants. To minimize reactions after immunization with DPT vaccine due to impurities in the toxoids, the use of toxoided purified toxins is suggested.

Antibody response of pregnant women to two different absorbed tetanus toxoids.

The antibody response in pregnant women vaccinated with either of two different adsorbed tetanus toxoids has been studied. One vaccine (A), prepared by toxoiding purified tetanus toxin followed by its adsorption onto calcium phosphate, exhibited a low titre expressed as international immunizing units, 69 IIU/0.5 ml. The other vaccine (B), prepared by purifying formalinized crude tetanus toxin and adsorbing it onto aluminium phosphate showed a high titre, 212 IIU/0.5 ml. No significant differences between titres of circulating antibodies were obtained after the first injection of either vaccine, but titres after the second injection were much higher for vaccine A as compared with those obtained using vaccine B. The results showed that the immune response in human beings is not correlated to titres expressed in IIU. These results confirm that other methods should be adopted for evaluating the potency of vaccines. A simplified technique based on the comparison of circulating antitoxin levels after vaccination of mice has recently been proposed.

[A simple method for assaying the activity of adsorbed tetanus toxoids]. / Méthode simple de contrôle de l'activité des anatoxines tétaniques adsorbées.

The authors have developed a simplified activity test for Tetanus Toxoids Adsorbed, based on the comparison of antitoxin levels in mice 4 weeks after injection of a reference toxoid and of the vaccine to be tested. Titration of tetanus antitoxin is achieved by passive agglutination of turkey RBC sensitized by means of glutaraldehyde. After preliminary experiments establishing feasibility of this method, the authors have obtained reproducible and quantitative results. They observed an increase of the immune response by a booster and an immunostimulation when pertussis component is present. They have found close correlation in immunized mice between the titre of circulating antibodies and the survival/death response after challenge by tetanus toxin as done in the official control. This simplified method using a reduced number of animals, yields, nevertheless, quantified results with confidence limits. Thus it is suitable for laying down a norm and can in many cases take the place of the official potency test which is tedious, expensive and often criticized.

Induction of protective antitumor immune response against a murine plasmacytoma not curable by alkylating-drug treatment.

Immunization with viable tumor cells followed by subsequent administration of glutaraldehyde-treated tumor cells induced a protective antitumor immune response in the host toward the alkylating-drug resistant RPC-5 plasmacytoma. This was proven by resistance to challenge with RPC-5 tumor cells, neutralization in Winn tests, by effectiveness of combined chemotherapy with melphalan plus immunotherapy with spleen cells from RPC-5 immunized mice and in vitro by cytotoxicity tests. The specificity of the immune response was ascertained in vivo by comparison with the response toward MOPC-315 plasmacytoma. However, in vitro cytotoxicity tests revealed the occurrence of shared antigens between the RPC-5 and MOPC-315 tumor cells. It is concluded that the ineffectiveness of alkylating-drug treatment toward the RPC-5 tumor is not due to the inability of this tumor to induce a specific antitumor immune response, and that cross-antigenic relationship as revealed by in vitro cytotoxicity tests does not necessarily reflect cross-protection between various plasmacytomas.

THF-gamma 2, a synthetic thymic hormone, increases effectiveness of combined chemotherapy and immunotherapy against RPC-5 murine plasmacytoma.

The effect of a synthetic thymic hormone, THF-gamma 2, on the anti-tumor activity of spleen cells was studied in mice immunized against the RPC-5 tumor. Following two courses of the THF-gamma 2 treatment, the mean RPC-5 specific cytotoxic response of immune spleen cells was significantly increased when compared to normal cells (P less than 0.001) and to untreated immune spleen cells (P less than 0.04). In addition, THF-gamma 2 treatment improved the competence of immune spleen cells in adoptive immunotherapy (AIT) when performed in combination with chemotherapy by melphalan. Recipients of spleen cells from THF-gamma 2 treated mice showed a 35% increase in survival when compared to AIT with immune cells alone. The results suggest that THF-gamma 2 treatment of donors for AIT might be applicable to cancer therapy in humans.

Synergy between low-dose chemotherapy and immunotherapy in mouse L1210 leukemia.

A high dose of 4 mg/kg of daunorubicin (DAU) given in combination with immunostimulation by the P40 immunomodulatory fraction of Corynebacterium granulosum and glutaraldehyde (GA)-treated tumor cells coupled with tetanus toxoid (P40 + GA-L1210-Tet) was more effective than DAU alone for treatment of L1210 mouse leukemia. A combination of low doses of DAU (0.0625-0.25 mg/kg) and P40 + GA-L1210-Tet was more effective than either P40 + GA-L1210-Tet or DAU alone. The cured mice were resistant to challenge with a high tumorigenic dose of L1210 tumor cells. A combination of various doses of mitomycin (1-4 mg/kg) with P40 + GA-L1210-Tet was more effective than mitomycin alone and was at least as effective as P40 + GA-L1210-Tet alone. Administration of DAU (0.5-4 mg/kg) to noninoculated C57BL/6 X DBA/2 mice resulted in increase of stimulation in vitro of collected spleen cells by mitogens.