Non-living Vaccines Research Articles

Immunochemical characterization and purification of Sm-97, a Schistosoma mansoni antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine.

Mice protected against Schistosoma mansoni infection by intradermal (i.d.) vaccination with nonliving schistosomula or soluble extracts of larval or adult schistosomes (SCHLAP and SWAP, respectively) produce antibodies that react by Western blot analysis with one antigen of Mr (X 10(-3)) 97 in SWAP prepared in the presence of protease inhibitors and two antigens of Mr (X 10(-3)) 95 and 78 in SWAP prepared in their absence. Vaccine antibodies also immunoprecipitated a single 97k molecule, with a pI of 5.5, from detergent extracts of [35S] methionine-labeled schistosomes. Three hybridomas, produced from spleen cells of i.d. immunized mice, all recognized both the 95k/78k doublet and the 97k antigen, indicating that the two lower Mr components are degradation products of the same 97k molecule. The 97k/95k/78k complex (Sm-97) was purified by affinity chromatography and found to constitute 0.5% of the total protein in SWAP. 125I-concanavalin A bound weakly to purified Sm-97, indicating that this antigen is minimally glycosylated. By indirect immunofluorescence, Sm-97 was localized to regions just below the tegumental and gut syncitia of adult worms. Mice protected by i.d. vaccination produced high titers (1:10,240) of anti-Sm-97 antibodies, whereas chronically infected mice responded at a much lower level (titer 1:640). In contrast, mice protectively immunized with irradiated cercariae and mice nonprophylactically inoculated by the i.v. route failed to produce detectable anti-Sm-97 antibodies. Competitive radioimmunoassays performed with 125I-labeled monoclonal antibodies and purified antigen defined at least two distinct epitopes on Sm-97. Antibodies from i.d. vaccinated mice recognized both monoclonal antibody-defined epitopes, whereas anti-Sm-97 antibodies in chronic infection sera recognized neither. Finally, purified Sm-97 was shown to elicit delayed-type hypersensitivity in i.d. vaccinated mice, suggesting that this molecule is also capable of evoking cell-mediated responses, a finding consistent with its proposed function as a vaccine immunogen.

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. II. Response of mouse strains with selective immune defects.

The efficacy of a new vaccination procedure against Schistosoma mansoni, involving intradermal injection of nonliving antigen combined with the bacterial adjuvant Mycobacterium bovis strain bacillus Calmette Guérin, was tested in several strains of mice. Development of protection against subsequent infection was compared with in vivo skin test reactivity and in vitro humoral reactivity to soluble and surface-associated schistosome antigens. Significant levels of resistance and immune response were displayed by many inbred mouse strains, including C57BL/6J, C3H/HeN, and CBA/J, as well as outbred Swiss-Webster mice. However, no definite correlation was observed between the level of any particular immune response and the level of resistance to challenge S. mansoni infection. Development of protective immunity was also examined in mice with various immune defects, to determine whether these responses are relevant to resistance in this model. Animals with defective specific immediate hypersensitivity response due to deficiencies in IgE (SJL/J) or mast cell (W/Wv) production displayed strong resistance as a result of immunization. Likewise, mice bearing the lpsd (C3H/HeJ) or xid (CBA/N) mutations, affecting cellular or humoral response to certain thymus-independent antigens, developed significant levels of resistance after immunization. A/J mice, with defects in cellular recognition of bacterial endotoxin as well as deficiencies in natural killer cell activity and complement function, also showed significant protective immunity. Thus, these reactivities do not appear to be essential to the resistance against S. mansoni induced by the nonliving vaccine. Two nonresponder strains were identified, P and BALB/c. P mice were defective in specific delayed hypersensitivity response as well as resistance to infection. However, BALB/c mice showed no obvious immune deficiencies at the time of challenge. These results agreed with previous findings in mice immunized by exposure to radiation-attenuated cercariae with one exception; BALB/c mice were protected by vaccination with irradiated cercariae but not by the nonliving vaccine. Thus, further examination of immune response in mice identified in this study as high and low responder strains should allow characterization of critical immune resistance mechanisms induced by the nonliving vaccine, as well as immune mechanisms operating in common between these two models of resistance to S. mansoni.

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. III. Correlation of resistance with induction of activated larvacidal macrophages.

Mice protected against Schistosoma mansoni infection by intradermal (i.d.) immunization with nonliving larval or adult worm antigens plus bacterial adjuvant developed 24-hr skin test responsiveness to schistosome antigens with the histologic features of delayed hypersensitivity. Intraperitoneal antigen injection elicited a mononuclear cell-enriched exudative population containing macrophages activated for direct cytotoxicity against schistosomula and tumor cell targets. This was likely to be due to in vivo exposure to macrophage-activating lymphokines, since these cells were unresponsive to further lymphokine stimulation in vitro and splenocytes from immunized mice reacted to specific in vitro antigen challenge by production of lymphokines capable of conferring larvacidal activity upon control macrophages. In contrast, mice treated with schistosome antigens by i.v. injection, which were not protected against challenge infection, failed to develop delayed hypersensitivity or activated macrophages in response to specific antigen challenge in vivo, and the titers of macrophage-activating lymphokine produced by in vitro antigen-stimulated splenocytes from these mice were threefold to fourfold lower than those produced by cells from animals immunized by the i.d. route. Thus, sensitization for cell-mediated immune responses including lymphokine production and macrophage activation correlated with induction of resistance to S. mansoni in this model of vaccination.

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. IV. Fractionation and antigenic properties of a soluble adult worm immunoprophylactic activity.

An aqueous buffer-soluble, nonparticulate fraction of adult Schistosoma mansoni worms (SWAP) was separated by gel filtration on Ultragel AcA-34, and portions of the eluate were tested for their capacity to induce protective immunity against cercarial challenge when administered intradermally to mice in combination with the adjuvant BCG. All of the immunogenic activity was found in a single peak of protein excluded in the void volume of the column. This same fraction was determined by SDS-PAGE and Western immunoblotting to be unique in that it contained a component of Mr (X 10(-3) 97 (97,000) recognized monospecifically by antibodies from mice vaccinated with unseparated SWAP plus BCG. Similarly, the protective fraction was unique in possessing the capacity to elicit 24 hr delayed footpad swelling responses, as well as lymphokine production, in SWAP-BCG-immunized mice. These results suggest that the immunogenic activity of SWAP resides in a restricted population of molecules, and possibly in the 97,000 antigen detected with antibodies from vaccinated animals. Because both the protective capacity of unfractionated SWAP and the serologic reactivity of the 97,000 antigen are sensitive to digestion with protease, it is likely that the immunologic activity of these molecules is dependent on peptide-bonded structural elements.

Attempts to manipulate specific responses to induce resistance to Schistosoma mansoni in Kenyan baboons (Papio anubis).

Attempts were made to manipulate specific responses of baboons to protect them from infection with Schistosoma mansoni. In Experiment 1, eosinophilia was induced in naive baboons with Trichinella spiralis larvae given intravenously before intraperitoneal injection of globulin fractions from S. mansoni-infected baboon sera and subsequent percutaneous exposure to S. mansoni cercariae. In Experiment 2, baboons with 8- or 32-week-old primary S. mansoni infections received T. spiralis i.v. before an S. mansoni challenge. In experiments 3 to 5 respectively, naive baboons received intramuscularly before challenge: formalin-fixed S. mansoni schistosomula, with Bordetella pertussis as an adjuvant; a preparation of S. mansoni adult worm teguments; and a preparation of IgE-immune complexes obtained from S. mansoni-infected rat sera, with Freunds Complete Adjuvant. Minor, but statistically insignificant, protection was obtained in Experiments 2 (32-week infections) and 3, but was far less than that given by intact, irradiated living vaccines. There are signs on the horizon of non-living vaccines protecting rodents against S. mansoni infection and it would be prudent, as with drugs, to test these in primates before proceeding to man. The results of our experiments, though essentially negative, should help the design of any future vaccine trials in primates.

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine is dependent on the method of antigen presentation.

A preparation of nonliving parasite antigens containing both soluble and particulate components of frozen-and-thawed invasive larvae was used to immunize C57BL/6J mice against challenge Schistosoma mansoni infection. The method of antigen presentation was observed to be critical to the ability of this preparation to induce protective immunity, because intradermal administration in conjunction with a bacterial adjuvant (BCG) resulted in strong protection against challenge parasites (51% reduction in worm burden in six experiments), whereas i.v. injection of the same antigenic preparation was completely ineffective. Induction of resistance was accompanied by specific immune responsiveness toward schistosome antigens. Protection correlated more closely with sensitization for specific delayed hypersensitivity than with elicitation of circulating antibodies to larval surface antigens or immediate hypersensitivity in these models. These results suggest that it will be possible to design a defined vaccine against S. mansoni infection, but that identification of the route of antigen presentation that most effectively elicits relevant immune effector mechanisms will be crucial to the success of any vaccination protocol involving nonliving antigens.

Antibody Production Following Immunization with Diphtheria and Tetanus Toxoids in Children Receiving Chemotherapy During Remission of Malignant Disease

Twenty-seven children with various childhood malignancies who were in clinical remission and receiving maintenance chemotherapy were given diphtheria-pertussis-tetanus (DPT) immunizations. Antidiphtheria and antitetanus titers were drawn before and 1 month after immunization. Only one child had no antibody response to either antigen. Two other children failed to develop any detectable antitetanus antibody titer but did mount a normal antibody response to inactivated diphtheria antigen. In fact, most children made good antibody responses to both immunizing antigens, irrespective of the nature of their disease or of the treatment given. These results show that children receiving long-term chemotherapy should not be denied the protection afforded by immunization with nonliving vaccines.

Does trypsin extract soluble antigen meet the criteria of immunogenicity without allergenicity?

We were unable to confirm the finding of Crowle that TESA possesses the attribute of immunogenicity and absence of allergenicity which are desired in a non-living antituberculosis vaccine. This disagreement could be a function of the particular lot of TESA examined and/or differences in the animal test systems used for the assay of protective potency. The relative contribution of these two possibilities could be determined by the simultaneous assay of a potent TESA product in different laboratories using different animal models.

New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development

PIP: This review attempts to synthesize the new knowledge of pathogenesis of bacterial enteric infections and relate this information to vaccine development. Discussion focuses on human infections and to those in which significant strides have been made. As a general theme in the pathogenesis of bacterial enteric infections, pathogens can be characterized into 5 groups on the basis of their degree of ultimate invasiveness after ingestion by a susceptible hose: mucosal adherence and enterotoxin production; mucosal adherence and brush border dissolution -- enteropathogenic E. coli (EPEC) of classical serotypes; mucosal invasion and intraepithelial cell proliferation; mucosal translocation followed by bacterial proliferation in the lamina propria and mesenteric lymph nodes; and mucosal translocation followed by generalized infection. The review covers cholera (motility and chemotaxis, mucosal adhesion, flagellar sheath protein, hemagglutinins, outer membrane proteins, enterotoxin production, quality and duration of infection derived immunity, immune response in humans, LPS, flagellar sheath protein, cholera lectin, other cholera hemagglutinins, outer membrane protein, previous cholera vaccines, killer whole cell vaccines, toxoids, combination vaccines, attenuated versus cholerae vaccines): enterotoxigenic Escherichia coli (ETEC) (entertoxins, O:H serotypes and enterotoxin phenotypes, colonization factors, immune response in humans, vaccines against ETEC, and toxiods); EPEC (vaccines against EPEC); Shigella (smooth LPS O antigen, epithelial cell invasiveness, Shigella toxin, and Shigella vaccines); and typhoid fever (caccines against typhoid fever). The major attraction of a nonliving oral cholera vaccine is its safety. A review of available information leads to the conclusion that an oral vaccine consisting of a combination of antigens, intending to stimulate both antibacterial and antitoxic immunity, would be most likely to succeed. Current approaches to immunoprophylaxis of ETEC infection involve vaccines that stimulate antitoxic or antiadhesion immunity or both by means of killed antigens or attenuated strains. It is likely that the most effective vaccines will contain appropriate antigens intended to simultaneously stimulate both antibacterial and antitoxic immunity, thereby leading to a synergistic protective effect. Now that the speical enteroadhesive properties of EPEC have been characterized and shown to be associated with a plasmid, it should be possible to identify the phenotypic gene products responsible for this phenomenon. It is likely that fimbriae or outer membrane proteins will prove to be the organelle of adhesion. When such information becomes available, it should be possible to prepare oral vaccines consisting of the purified antigen. Efficacy has been shown for attenuated Shigella strains utilized as oral vaccines. The major thrust in the development of new immunization agensts against typhoid fever is to identify immunizing agents at least equal in efficacy to the parenteral acetone killed vaccine but which cause no adverse reactions.

Immune response to porin in cattle immunized with whole cell, outer membrane, and outer membrane protein antigens of Brucella abortus combined with trehalose dimycolate and muramyl dipeptide adjuvants.

The immune response of cattle to nonliving vaccines derived from Brucella abortus rough strain 45/20 was studied. Vaccines contained trehalose dimycolate and a derivative of muramyl dipeptide. N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine. A factorial experiment was designed to test the effects of type of antigen, quantity of antigen, and quantity of mineral oil on the immune response to porin. Muramyl dipeptide was kept constant at 5 mg per dose, and 1 part of trehalose dimycolate was incorporated for two parts of dry matter. Over a 10-week period, blastogenesis responses to porin were largest in cattle immunized with outer membranes; the highest antibody titers to the porin-lipopolysaccharide complex were achieved by immunization with detergent-extracted outer membrane proteins. There was no advantage in the use of 25, rather than 5, mg of any of the antigens, but antibody responses were improved by increasing the quantity of oil from 0.6 to 1.8 ml per dose. In other animals, blastogenesis and antibody responses were sustained at high levels longer than 3 months after two vaccinations with outer membrane proteins. Intradermal injection of porin evoked inflammatory reactions histologically consistent with delayed-type hypersensitivity. Cross-reactions in cases of delayed-type hypersensitivity occurred with porin derived from a smooth strain of B. abortus but were less extensive than in the blastogenesis test. The magnitude of the delayed-type hypersensitivity and blastogenesis responses induced by vaccination exceeded those observed after natural or experimental infections. No ill effects were observed after vaccination. These findings provide a basis for the use of trehalose dimycolate and muramyl dipeptide adjuvants in evaluating nonviable vaccines for bovine brucellosis.

New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development.

This review attempts to synthesize the new knowledge of pathogenesis of bacterial enteric infections and relate this information to vaccine development. Discussion focuses on human infections and to those in which significant strides have been made. As a general theme in the pathogenesis of bacterial enteric infections pathogens can be characterized into 5 groups on the basis of their degree of ultimate invasiveness after ingestion by a susceptible hose: mucosal adherence and enterotoxin production; mucosal adherence and brush border dissolution -- enteropathogenic E. coli (EPEC) of "classical" serotypes; mucosal invasion and intraepithelial cell proliferation; mucosal translocation followed by bacterial proliferation in the lamina propria and mesenteric lymph nodes; and mucosal translocation followed by generalized infection. The review covers cholera (motility and chemotaxis mucosal adhesion flagellar sheath protein hemagglutinins outer membrane proteins enterotoxin production quality and duration of infection derived immunity immune response in humans LPS flagellar sheath protein cholera lectin other cholera hemagglutinins outer membrane protein previous cholera vaccines killer whole cell vaccines toxoids combination vaccines attenuated versus cholerae vaccines): enterotoxigenic Escherichia coli (ETEC) (entertoxins O:H serotypes and enterotoxin phenotypes colonization factors immune response in humans vaccines against ETEC and toxiods); EPEC (vaccines against EPEC); Shigella (smooth LPS O antigen epithelial cell invasiveness Shigella toxin and Shigella vaccines); and typhoid fever (caccines against typhoid fever). The major attraction of a nonliving oral cholera vaccine is its safety. A review of available information leads to the conclusion that an oral vaccine consisting of a combination of antigens intending to stimulate both antibacterial and antitoxic immunity would be most likely to succeed. Current approaches to immunoprophylaxis of ETEC infection involve vaccines that stimulate antitoxic or antiadhesion immunity or both by means of killed antigens or attenuated strains. It is likely that the most effective vaccines will contain appropriate antigens intended to simultaneously stimulate both antibacterial and antitoxic immunity thereby leading to a synergistic protective effect. Now that the speical enteroadhesive properties of EPEC have been characterized and shown to be associated with a plasmid it should be possible to identify the phenotypic gene products responsible for this phenomenon. It is likely that fimbriae or outer membrane proteins will prove to be the organelle of adhesion. When such information becomes available it should be possible to prepare oral vaccines consisting of the purified antigen. Efficacy has been shown for attenuated Shigella strains utilized as oral vaccines. The major thrust in the development of new immunization agensts against typhoid fever is to identify immunizing agents at least equal in efficacy to the parenteral acetone killed vaccine but which cause no adverse reactions.

Oral immunization of dogs with purified cholera toxin, crude cholera toxin, or B subunit: evidence for synergistic protection by antitoxic and antibacterial mechanisms.

The immunogenicity and safety of purified cholera toxin (CT), its B subunit, and a crude culture filtrate of toxigenic Vibrio cholerae (CrT) were compared in dogs immunized orally and challenged with virulent V. cholerae. CT and CrT caused marked protection in two- or three-dose regimens. Protection due to CT occurred only with doses that caused transient, sometimes severe, diarrhea in most dogs; this protection was proportional to the peak antitoxin response in jejunal mucosa and lasted at least 15 weeks. In contrast, minimum protective doses of CrT contained much less cholera toxin, caused very mild diarrhea in only 21% of the dogs, and evoked protection that was greater than predicted from the modest jejunal antitoxin response. B subunit caused smaller jejunal antitoxin responses than did similar doses of CT and was poorly protective, the 50% protective dose being >40-fold greater than that of CT. Two observations indicated that protection due to CrT involved synergy between antibacterial and antitoxic immune responses. First, the 50% protective dose of CrT was 24-fold and >36-fold smaller than the 50% protective doses of its CT and non-CT antigenic components, respectively, when tested separately. Second, protection was greater in CrT-immunized dogs than in CT-immunized dogs for a given mucosal antitoxin response. Low doses of CrT evoked serotype-specific protection, indicating that the serotype-specific O somatic antigen contributed significatly to antibacterial protection. These results suggest that a simple, effective, nonliving oral vaccine for cholera based on combined antibacterial and antitoxic immunity can probably be achieved. However, further studies are needed to determine how a protective antitoxic response can be evoked without causing diarrhea during immunization.

Delayed hypersensitivity responses of experimental animals to histoplasmin from the yeast and mycelial phases of Histoplasma capsulatum.

Controlled yeast lysate (CYL) and controlled mycelial lysate (CML) histoplasmins were produced from Histoplasma capsulatum grown in a nutritionally lean, chemically defined medium. The lysates were assayed for skin-test activity in guinea pigs sensitized by infection with the homologous organism. In some studies, nonliving vaccine preparations were employed also. Inter-lot biological variation was minimal, and 20 lots of the CYL reagent elicited strong dermal reactions with high specificity. Further, CYL reagents were nonreactive in guinea pigs infected with Coccidioides immitis, whereas the commercial Food and Drug Administration preparations cross-reacted to some degree. The CML histoplasmins were generally less reactive than the CYL preparations and exhibited somewhat more inter-lot variation in sensitivity and specificity. No correlation between potency and protein:polysaccharide ratios were observed with either reagent. An intradermal test with the CYL reagent did not induce significant changes in the complement-fixing titer of sensitized guinea pigs. Such changes in sensitized animals were elicited by a skin test with commercial histoplasmin.

Epidemiology and ecology of leishmaniasis in Latin-America

Of the diseases caused by protozoal parasites, leishmaniasis is probably second in importance only to malaria. Chemotherapeutic drugs are toxic, expensive and not 100% effective. This, and the absence of any non-living vaccine against the disease, means that control depends on eliminating either reservoirs or insect vectors, or both. Recently, a greatly increased knowledge of the Leishmania species involved, and of their natural hosts, has helped to define the nature and extent of the problem.

Biologically active components from mycobacterial cell walls: V. Granuloma formation in mouse lungs and guinea pig skin

The ability of certain mycobacterial cell wall components, when attached to minute droplets of oil, to induce granulomatous inflammation in mouse lung and guinea pig skin has been studied for clues to the mechanisms by which these components protect mice against aerosol challenge with virulent tubercle bacilli and are useful in immunotherapy of dermal tumors of guinea pigs. Fractions examined were the following: the cell wall skeleton (CWS-I), which consisted of a polymeric peptidoglycolipid; wax D, which contained mono- or oligomeric peptidoglycolipid; and trehalose mycolates (P3). A combination of CWS-I and P3 induced more granuloma in mouse lungs than either of these two entities alone. Similarly, when wax D was separated into a chloroform-methanol-insoluble portion (CMI), which contained the mono- or oligomeric peptidoglycolipid, and the soluble portion (CMS), which contained P3, it was found that both moieties were required to produce maximal granulomatous inflammation. The degree of granuloma induced by these nonliving vaccines, as measured by an increase in lung weight following intravenous inoculation, could be correlated with the degree of protection produced in mice against airborne infection with M. tuberculosis H37Rv and with the degree of regression of skin tumors in guinea pigs upon intralesional inoculation. Even though quantitation of the granulomatous response in the skin of guinea pigs is difficult, it would appear that intradermal inoculation of cell walls, CWS-I, or CMI produced comparable granuloma, whereas CMS or P3 alone was essentially inactive.

The relationship of delayed hypersensitivity to acquired antituberculous immunity: II. Effect of adjuvant on the allergenicity and immunogenicity of heat-killed tubercle bacilli

Mice were injected subcutaneously on two occasions with a heat-killed, lyophilized suspension of M. tuberculosis H 37R v dispersed in a water-in-oil emulsion or as a saline suspension. The levels of tuberculin sensitivity were followed for 30 days, at which time the mice were infected intravenously with approximately 10 6 living BCG (Montreal). At intervals thereafter, groups of mice were challenged with 10 LD 50's Listeria monocytogenes so that the development of nonspecific resistance could be followed in relation to the fate of BCG and the state of hypersensitivity in the different treatment groups. Nonvaccinated controls, and mice receiving adjuvant alone, required 10–12 days to respond to the eliciting dose of BCG with the development of detectable tuberculin sensitivity and cellular resistance to the Listeria challenge. They were also unable to limit the growth of BCG in the lung. Mice receiving H 37R v in saline behaved like the normal controls; but animals receiving H 37R v in adjuvant quickly developed high levels of tuberculin sensitivity, and their resistance to Listeria and BCG both appeared much sooner. Seven months later, mice of this group showed a marked rise in tuberculin sensitivity and an accelerated onset of cellular immunity in response to the BCG infection. The significance of these findings is discussed in relation to the mechanism of antituberculous immunity and the evaluation of nonliving vaccines.

Immunization of rabbits with viable BCG and nonliving cell wall vaccines.

Immunization of rabbits with viable BCG and nonliving cell wall vaccines.

Effective nonliving vaccine against experimental tuberculosis in mice.

Ribi, Edgar (Rocky Mountain Laboratory, Hamilton, Mont.), Carl Larson, William Wicht, Robert List, and Granville Goode. Effective nonliving vaccine against experimental tuberculosis in mice. J. Bacteriol. 91:975-983. 1966.-Antituberculosis vaccines were prepared in one of three manners: lyophilized BCG suspended in light mineral oil was disrupted in a Sorvall pressure cell and the "oil disruption product" was collected by centrifugation; BCG was disrupted in water, lyophilized, and worked into a paste with a small amount of oil (about 0.16 ml per 50 mg); BCG was disrupted in water, and the cell wall fraction was isolated, lyophilized, and prepared in an oil paste. These vaccines were suspended in Tween-saline to a concentration of 5 mg/ml and heated at 65 C for 30 min. In protection tests based on pulmonary infection with Mycobacterium tuberculosis H37Rv, the median number of virulent organisms in lung tissue of mice immunized with a few hundred micrograms of these three vaccines was 3 to 4 logs lower than in unvaccinated control mice. A similar dose of viable BCG standard vaccine reduced the lung count 1 to 2 logs below the controls. Protection afforded by nonviable, whole BCG, with or without oil, was of only borderline significance. Since oil-treated fractions containing cell walls produced effective immunity, while the oil-treated protoplasm or whole cells were not active, the protective antigen appeared to be an inner component of the cell wall, exposed when the cell was disrupted, and activated by oil. Extraction of oil from immunogenic disruption products resulted in loss of ability of the products to confer protection against the aerosol challenge, whereas high protection against the conventional challenge by intravenous infection with up to 1.4 x 10(8) cells of M. tuberculosis H37Rv was retained. Retreatment with oil of these nonimmunogenic products restored the immunogenicity if the oil was applied to dried products. The consistent finding that moisture interferes with the enhancement of the vaccine potency by oil suggested that such enhancement may not be the same as that ordinarily produced by water-in-oil emulsions.

STUDIES ON THE DEVELOPMENT OF GAMMA-IRRADIATED VACCINE AGAINST TUBERCULOSIS ( 1) Oxidative metabolism of Gamma-irradiated M. Tuberculosis H37Rv

In our country, B.C.G. has been used as one of the most effective vaccines against tuberculosis. Non-living vaccines against tuberculosis have, however, been extensively studied by many workers so that the living vaccine, B.C.G. may be replaced with them, for various side reactions, decrement of titer and inconveniences of handling B.C.G. are serious problems yet to be solved. Gamma irradiated tubercle vaccine, at this point, is the one we long hoped for, because it is non-living and as effective as B.C.G. in protecting experimental animals against tuberculosis. In the present experiment, the characteristics of gamma-irradiated tubercle bacillus, the oxidative metabolism, were investigated through manometric studies and the following results were obtained. 1. Lethal dosage of gamma rays for lyophilized tubercle bacillus was 1.5×10<sup>6</sup>r which was double of the one required for the bacillus of liquid suspension. Yet, the oxygen uptake rate of lyophilized cell was as much as 2.5 time the one of suspension cell. 2. Pretreatment of tubercle bacillus with lactic acid or pyruvic acid brought about greater protection of respiratory enzymes from the gamma irradiation damage than non-treated one, though no difference in lethal dosages of gamma rays was observed between them.

A field trial of enterotoxaemia vaccines

Abstract Extract In New Zealand, the sale of some brands of non-living vaccines is in the hands of laymen. The veterinary clinician lacks the scientific knowledge to confirm the claims of excellence or superiority as advertised for any brand. This paper reports an attempt to assess the variation between brands and batches bought and administered as they would be by a typical farmer.