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.

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.

Preparation and use of calcium phosphate adsorbed vaccines.

Simple or combined D,T,P and inactivated polio vaccines adsorbed onto calcium phosphate are prepared according to two procedures. Antigens can be dialysed in a sodium phosphate solution and quickly mixed with an equal volume of an equimolar solution of calcium chloride, the pH is adjusted to 6.8-7. Toxoids added to the phosphate solution are in this way entrapped within the 3-dimensional network during the formation of the precipitate. Antigens can also be added to a calcium phosphate gel suspension prepared in advance. Results of animal experiments and of field trials using either combined vaccines or simultaneous immunization with diphtheria, tetanus, pertussis, meningococcal and several viral vaccines: polio, rabies, hepatitis-B, etc., will be presented. Several programs have been studied in developing countries, mainly with the aim at simplifying vaccination campaigns. The efficiency of a two-dose regimen with DT vaccine has been ascertained, this has also been applied to pregnant women. Adsorbed tetanus toxoid was successfully used as a diluent for freeze-dried measles, meningococcal polysaccharide and rabies vaccines. Levels of circulating antibodies and potency of vaccines have been measured by new in vivo and in vitro methods. The choice of detoxified purified toxins is desirable for the preparation of vaccines in order to prevent the occurrence of adverse reactions. Local and generalized reactions have been studied. Adverse Arthus-type reactions have been encountered and related with the presence of high levels of circulating tetanus antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

[Studies on the bactericidal activity of glutaraldehyde (author's transl)]. / Etude du pouvoir bactéricide du glutaraldéhyde.

Bactericidal activity of aqueous glutaraldehyde has been determined in comparison with aqueous sodium hypochlorite. Several bacterial strains have been tested and it was found that the activity of the glutaraldehyde solution was superior or equal to that of the hypochlorite solution with the exception of the action on mycobacteria.

Employment of toxins bound to porous silica beads for isolation of pure antibodies by immunoadsorption.

A simple technique for the isolation of pure antibodies by use of bacterial toxins or toxoids bound to porous silica beads "Spherosil" is described. Employment of toxins instead of toxoids has the advantage of resulting higher yields of purified antibodies.

[Results of simplified immunization schedules in the developing countries]. / Resultats de calendriers de vaccinations simplifiees dans des pays en voie de developpement.

The results of studies on the immunization of people in developing countries with vaccines adsorbed on calcium phosphate are presented in this paper. Several programs which were studied with various organizations cooperating and which were intended to simplify vaccination campaigns brought us to the following conclusions: --that immunization against diphtheria and tetanus by two injections with an interval of one year between them is effective, --that two injections of inactivated trivalent poliomyelitis vaccine followed up by booster one year later ensure that 90% of the infants are protected, --that it is possible to vaccinate pregnant women by one or two injections of tetanus vaccine and protect both mothers and newborn babies against tetanus, and --that combined vaccines which enable simultaneous immunization of subjects against a maximum of infections are possible.

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.

[Allergens adsorbed on aluminum hydroxide preparation, standardization controls and therapeutic results]. / Allergènes adsorbés sur hydroxyde d'aluminum: preéparation, standardisation, côntroles et résultats thérapeutiques

Specific desensitizing treatments with house dust extract purified and adsorbed on aluminium hydroxyde gave 79% good and very good results in 355 patients. Grass pollen extracts adsorbed on the same adjuvant gave 76% good and very good results in 166 patients. Desensitization by the adsorbed preparations requires fewer injections than the usual methods, while offering security by the absence of anaphylactic reactions, particularly with pollen extracts. The activity of the preparations is tested after appropriate dilution, taking account of the total nitrogen content of the dust extracts and the weight of raw materials used in the manufacture of the pollen extracts. The results of the intradermal tests undertaken with fluid preparations and compared with those obtained with a standard preparation allow to standardize the allergenic activity of the extracts. It is thus possible to guarantee the pharmaceutical quality of the allergens both for diagnostic tests and for treatments. Chemical analyses and control of acute, delayed or chronic toxicity ensure the innocuousness of the preparations.

Studies on culture conditions of Bordetella pertussis and relationship to immunogenicity of vaccines.

Conditions for growing Bordetella pertussis bacteria in fermentors were studied for the purpose of producing highly protective vaccines. Bacteria with high protective potency were obtained in fermentors when the inoculum consisted of a dense suspension of young B. pertussis cells grown on Bordet-Gengou agar plates. When the time of cultivation in fermentors exceeded 30 h, bacteria harvested showed lower protective potency activity and lower levels of histamine sensitizing factor (HSF) and leukocytosis promoting factor (LPF), indicating that the optimal time for collection is at the end of the logarithmic growth phase. In fermentors, the ATP concentration in the bacterial cells increased from the time of inoculation until the end of the logarithmic growth phase. At the late logarithmic growth phase, the amounts of ATP, HSF, LPF and protective antigen declined rapidly. ATP kinetics thus provides a way of predicting the immunogenicity of vaccines. These results may be applicable to the production of vaccines exhibiting high protective activity at low bacterial concentrations, and to the preparation of soluble vaccines resulting from high-yield extraction of protective antigens. A correlation between the potency and LPF activity of vaccines was established for use in the rapid screening of vaccines for protective activity.

Studies on untoward reactions to diphtheria and tetanus toxoids.

Adverse reactions to diphtheria and tetanus toxoids have frequently been reported. The cause of these reactions has been attributed to different factors, including sensitization by prior vaccination. However, this explanation does not hold for primary vaccination. In this case, undesirable reactions to diphtheria toxoid are most likely due to bacterial cellular fractions present in the vaccines. In fact, we were able to demonstrate that fortuitous contact with atoxigenic diphtheria strains can induce sensitization. Consequently, vaccination should be carried out with highly purified toxoids, so as to prevent untoward reactions due to the presence of impurities. It is also preferable to detoxify purified toxins, rather than to purify crude toxoids.

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.

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.

Prevention and treatment of L1210 mouse leukemia by immunization with xenogenized tumor cells combined with immunostimulation by the P40 fraction of C. granulosum and chemotherapy.

The effect on L1210 leukemia in mice of immunostimulation, in combination or not with chemotherapy with either daunorubicin or mitomycin, was studied. Immunostimulation with the immunomodulator P40 isolated from C. granulosum, together with xenogenized syngeneic tumor cells (GA-L1210-Tet: L1210 tumor cells inactivated with glutaraldehyde and coupled with tetanus toxoid), on days -14 and -7 before and days 2 and 9 after tumor inoculation, resulted in significant increase of the mean survival time as compared to control group with or without chemotherapy. Administration of P40 or P40 + GA-L1210-Tet cells, before or before and after inoculation of L1210 cells partly inactivated in vitro with antineoplastic agents, leads to a marked prolongation of mean survival time and to inhibition of ascitic tumor growth in a high percentage of mice. About 50% of the mice treated with P40 + GA-L1210-Tet cells and surviving the first challenge were resistant to rechallenge, providing that P40 + cells were reinjected before the second challenge, whereas all mice treated with P40 alone and surviving the first inoculation, were susceptible to rechallenge. The major conclusion is that treatments of combining chemotherapy, active immunization and nonspecific immunostimulation in the applied sequence are more effective than single treatments for control of L1210 mouse leukemia.

[Prevention and therapy of L1210 leukemia in mice, using inactivated and grafted tumor cells, in association with the immunomodulator P40, and Antineoplastic Agents (author's transl)]. / Prévention et traitement de la leucémie L1210 de la souris á l'aide de cellules tumorales inactivées et greffées, en association d'un immunomodulateur, la fraction P40, et d'agents antitumoraux.

The effect on L1210 in Mice of an immunostimulation with P40 fraction isolated from C. granulosum and inactivated L1210 cells coupled with tetanus toxoid, in combination or not with chemotherapy with either daunorubicin or mitomycin, has been looked for using various modalities. Cells partly inactivated by action of the drugs in vitro have also been used for grafting the Mice. The strongest inhibition of tumour growth was observed when the following treatment sequence was applied: Immunostimulation, tumor grafting, chemotherapy, immunostimulation. The significance of the reported results for the treatment of Human neoplasia is discussed.