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We have described dynamics of changes in nerve cells of the skin and reaction of connective tissue in the case of injection of living tulamia vaccine to guinea pigs into the skin of their nose tip and abdomen.

The process of formation of postvaccinal immunity against tularemia after immunization with the living tularemia vaccine is very radioresistant. Experiments were performed on guinea pigs. These animals were irradiated with various x-ray doses, ranging from 10–20 (30) LD to 50–60 (30) LD, at different periods before the vaccination and 24 hr after it. Such irradiation caused only a moderate reduction in intensity of the postvaccinal immunity. The process of postvaccinal immunity formation was found to be even more radioresistant in white mice, in which irradiation provoked an acute exacerbation of the vaccinal infection. The authors compare the ability of the irradiated and normal organisms to form immunity and the intensity of immunobiological reactions (antibody synthesis, inflamuratory reaction at the site of vaccine injection, the ability of the tissues to render the virulentPast.tularensis harmless, allergic reaction to tularin).

The relationship between hypersensitivity and cellular resistance to infection with facultative intracellular parasites was studied in mice by using infection-immunity in tularemia as a model system. Delayed hypersensitivity to antigenic fractions of Francisella tularensis was first detected 6 to 7 days after immunization with viable F. tularensis vaccine, at which time immunity against challenge infection developed. Both immunity and delayed-type sensitivity reached maximal levels by 9 to 10 days. Immediate hypersensitivity occurred after immunization with both viable and nonviable tularemia vaccines but could not be correlated with resistance since nonviable antigens were not protective. Attempts to relate resistance to F. tularensis with nonspecific immunity factors were unsuccessful. Immunization of mice with BCG vaccine stimulated protection against infection with F. novicida and Salmonella typhimurium but provided no protection against infection with F. tularensis. Moreover, viable tularemia vaccine, while inducing marked protection against challenge with specific organisms, afforded no protection against infection with S. typhimurium or S. enteritidis. It is concluded that cellular immunity in tularemia involves an immunologically specific component.

The reactivity of rabbits with an implanted Brown-Pearce tumor was studied following their vaccination with live tularemia vaccine. Formation of immune agglutinins and allergic reactions were investigated both in healthy rabbits and in animals with an implanted tumor. The study of these reactions demonstrated an intensified reactivity of the experimental animals during the first stage of tumor development; its marked depression up to the time marking the development of anergy was noted during metastasis.

A complement-fixing enzyme-linked immunosorbent assay (CF-ELISA) with bacterial sonicate as the antigen, human AB serum as the source of complement and alkaline-phosphatase-labelled anti-(human C3c) as the conjugate was developed for detecting antibodies to Francisella tularensis. Humoral responses after inoculation with a live attenuated tularemia vaccine were studied in 13 subjects by this method. Twelve responded with CF antibodies, which in most cases appeared 4 weeks after vaccination, the response time varying from 1 week to 2 months. The highest individual CF titres, of 140-9990, were reached 1-4 months after vaccination (average 2.4 months). The CF titres tended to decrease with time, and at 10 months two of the 12 subjects with an earlier CF positive response were negative again. CF antibodies were nevertheless still detectable in all three subjects studied 1.5 years after vaccination.

Tularemia is caused by the facultative intracellular bacterium Francisella tularensis. Attenuated live vaccines, such as F. tularensis LVS (live vaccine strain), afford good--although not complete--protection; how to judge the degree of this protection has long been a problem. Both natural infection and vaccination result in immunospecific and long-lasting humoral and cell-mediated immunity. The latter is the crucial protective mechanism, whereas the humoral response protects only against strains of reduced virulence, such as those used in the vaccines. Immune serum has been used to screen for structures of F. tularensis with the ability to induce a protective immune response. This immune serum is, however, primarily directed toward antigens different from those involved in cell-mediated immunity. Serum antibodies from primed individuals recognize carbohydrate capsule antigens of F. tularensis, whereas T lymphocytes recognize membrane polypeptides of the organism. The preparation of membrane polypeptides from F. tularensis is now facilitated by the availability of a capsule-deficient mutant of F. tularensis LVS. In vitro, several membrane polypeptides of the mutant stimulate T lymphocytes from vaccinees and from naturally infected individuals. Further studies of the mechanisms of induction of protective immunity should focus on these membrane polypeptides.

T lymphocyte-mediated immunity is important for resistance to Francisella tularensis. To characterize the specificity of this immunity, we used membrane proteins and two lipopolysaccharide (LPS) preparations. Both membrane proteins were heat-modifiable, as indicated by their migration in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). One had an apparent molecular mass (Mm) of 120 kilodaltons (kDa) when solubilized in the SDS buffer at room temperature, but 17 kDa after heating. The respective values for the other protein were 35 kDa before and 40 kDa after heating. Both proteins were purified by a preparative SDS-PAGE. The LPS-containing preparations were isolated by aqueous phenol (WP) or PCP (phenol-chloroform-petroleum ether) extraction (LPS-R), and rendered protein-free by treatment with proteinase K. Lymphocytes from nine subjects immunized with a live tularemia vaccine from one to three years earlier responded specifically to both an F. tularensis whole cell antigen and the 17 kDa protein in the lymphocyte blast transformation test. By contrast, the 40 kDa protein and the two LPS preparations did not stimulate any detectable lymphocyte proliferation.

An immune response to Francisella tularensis was demonstrated in man by the lymphocyte stimulation test. Peripheral blood lymphocytes were obtained from 26 individuals vaccinated with a viable tularemia vaccine, from 29 unvaccinated individuals, and from two patients who had recently undergone tularemia. The lymphocytes were incubated in the presence of various dilutions of heat-killed bacteria of the vaccine strain. The bacteria induced a deoxyribonucleic acid synthesis in the lymphocytes from 18 of the vaccinated individuals and from the two patients which was higher than that in the lymphocytes from any of the unvaccinated individuals. The deoxyribonucleic acid synthesis was maximal after about 6 days of incubation irrespective of concentration of bacteria. Lymphocytes from vaccinated and unvaccinated individuals were stimulated by two unrelated agents, tuberculin purified protein derivative and pokeweed mitogen. Lymphocytes from the vaccinated individuals did not show a higher response to these agents than did those of the unvaccinated. This suggests that the lymphocyte response to the Francisella bacteria was not due to a nonspecific activation. The vaccine-induced lymphocyte stimulation did not correlate with serum antibodies agglutinating F. tularensis antigen.

The frequency with which Pasteurella tularensis infects hunters of rabbits and laboratory workers studying this microorganism makes vaccination of these persons desirable. However, the protective value of available nonviable vaccines is not certain. Studies on this point have been conducted by Foshay et al. 1 and Kadull et al. 2 The ideal method of evaluating a vaccine intended for protection of humans is to challenge volunteers, both vaccinated and nonvaccinated, with a reproducible known infective dose of the disease-producing agent. A study in a small vaccinated group challenged by a known infective dose can provide more specific information in a shorter time than by assembling a much larger number in a study in which vaccinated persons are "exposed" accidentally in varying degree or not at all. Pasteurella tularensis offers certain advantages in such a critical study employing human challenge with viable microorganisms. A broad base of preliminary experience is provided

SummaryStudies were made on the clinical acceptability and immunogenicity of live tularemia vaccine administered to the monkey and guinea pig by the dermal or respiratory route. Aerogenic as well as dermal vaccination proved innocuous. Animals vaccinated aerogenically showed higher levels of antibody and comparable if not greater protection against challenge with a highly virulent strain of P. tularensis than animals vaccinated dermally.

Previously reported data on the pathogenesis and immunogenicity of live vaccine strain LVS have been sufficiently encouraging to warrant an evaluation of oral vaccination with this strain. A group of eight monkeys were administered strain LVS dermally by acupuncture. Both the oral and the dermal inocula of LVS proved innocuous but 13 of the 16 animals that received strain 425 showed a transient low-grade febrile reaction. An agglutinin response was obtained in all vaccinees. Approximately 10 weeks after vaccination, these animals and a group of seven unvaccinated controls inhaled 10,000 organisms of strain SCHU. All animals became infected but 64 to 71% of the vaccinees survived in contrast to 14% of the controls. These studies indicate a potential for oral immunization with live tularemia vaccine prepared from strain LVS.

Eigelsbach , H. T. (Fort Detrick, Frederick, Md.), J. J. Tulis, M. H. McGavran and J. D. White . Live tularemia vaccine. I. Host-parasite relationship in monkeys vaccinated intracutaneously or aerogenically. J. Bacteriol. 84: 1020–1027. 1962.—Bacteriological, histological, immunohistochemical, and serological studies were made on monkeys administered live tularemia vaccine strain LVS by either of two routes. Comparative data are presented on nonvaccinated monkeys exposed via the respiratory route to a highly virulent strain of Pasteurella tularensis . Tissue changes resulting from either aerogenic or intracutaneous vaccination were mild, and consisted primarily of the proliferation of histiocytes without the formation of granulomas. The vaccine strain was isolated from the site of vaccination of animals inoculated dermally, from the lungs of animals vaccinated aerogenically, and from the regional lymph nodes, liver, and spleen of both groups; it was not isolated from the blood or bone marrow. Proliferation of the vaccine strain at the site of dermal inoculation and in the lungs of animals exposed aerogenically was observed within 24 hr; in both groups, the maximal viable population was reached within 3 days and maintained through the 10th day. A reduction in the number of viable vaccine organisms had begun by the 14th day; isolations were obtained only from the regional lymph nodes on the 28th day, and the vaccine strain was not isolated from any of the tissues cultured on the 90th day. Because the monkey is less resistant to tularemia than is man, the benign response of this animal to live tularemia vaccine indicates that the vaccine might also be safe for man when administered by either the dermal or respiratory route.

IT is characteristic of tularemia that most patients develop a high degree of natural resistance after infection occurs. The mortality is low. Recovery confers lifelong immunity which protects against subsequent massive exposures with an effectiveness seldom encountered in bacterial diseases. Second attacks of disease are unknown. Local reinfections occur occasionally among immunes subjected to repeated exposures to virulent strains but, as Francis first indicated, they are either asymptomatic or associated with brief and mild constitutional symptoms.' One of us has recorded apparent immunity, due to vaccination, which successfully withstood accidental massive exposures to strains of high virulence.2 Although tularemia is of minor importance as a cause of death it has considerable social and economic importance in areas of high endemicity as a cause of prolonged morbidity and disability. Its capacity to disrupt normal activities of military encampments is recognized.3 The extreme ease with which infection is acquired, and the high infection rates among butchers and market men, scientific investigators, rabbit hunters and their wives and cooks who prepare game for the table, indicate the desirability of better methods of protection than those now in use. Preliminary experiments with heat killed and formalinized Bacterium tularense * suspensions demonstrated clearly that vaccines made by these methods, from either virulent or avirulent strains, provoked severe and extensive constitutional and local reactions upon initial injection into normal individuals. Reasonably well tolerated doses were so small that several months of daily injections were required to produce agglutinin titers to 1:320, a scheme tolerable for a few laboratory workers but impracticable for large scale use. The successful efforts of Ramon and his associates to diminish primary toxicity of toxins and of various bacteria without significant impairment of their antigenicity., by various chemicals, led us to try similar methods. We established the following specifications for an acceptable vaccine

Previous studies from these laboratories demonstrated that man can readily be infected by intracutaneous inoculation with approximately 10 Pasteurella tularensis organisms (SCHU S4 strain). 1 Prior vaccination with killed Foshay vaccine did not prevent local lesions, but did reduce the incidence of systemic manifestations of infection. Review of accidental laboratory infection indicates that the respiratory route may serve as a portal of entry. 2 Experimental respiratory infections can easily be induced in both vaccinated and nonvaccinated monkeys, and response to therapy is good. 3 This present report describes the response to respiratory challenge with P. tularensis of nonvaccinated volunteers and of volunteers who received either killed vaccine or a viable attenuated vaccine. Materials and Methods Volunteers were inmates of the Ohio State Penitentiary, 21 to 35 years of age. Criteria for selection and conditions of volunteering have been described. 1 Vaccination with Foshay killed tularemia vaccine was conducted as previously

To delineate differences in the resistance of irradiated and nonirradiated animals to live tularemia vaccine, the chronological appearance and growth rate of LVS in the lung, liver, spleen, and blood of guinea pigs were studied. Nonirradiated controls and guinea pigs having reveived 140 R three days previously were exposed via the respiratory route to 100,000 cells of LVS and sacrificed at intervals from one to 21 days. No major differences were noted in the time of appearance, growth rate, maximum organism content, or time of clearance of LVS from the tissues of irradiated and nonirradiated animals; hence, there was no evidence of a change from a self-limiting to a fulminating type of infection resulting from irradiation of the animals. Also, no appreciable difference in the time of appearance of agglutinins or maximal titer was noted in the two groups of animals. The production of agglutinins in irradiated animals in response to vaccination with LVS is in contrast to the reported inhibition of antibody response in animals inoculated with killed organaganisms or purified antigens subsequent to irradiation.

Reports from this laboratory have demonstrated that exposure of guinea pigs to sublethal X-irradiation three days before the administration of innocuous respiratory doses of live tularemia vaccine resulted in maximal mortality (23 per cent) in comparison with other X-irradiation schedules investigated. However, agglutinin production and development of immunity was not markedly altered in these animals by the X-irradiation procedure. The present study was performed to determine if the subcutaneous route of vaccination of irradiated animals would result in a decrease in the mortality rate and yet provide resistance to subsequent virulent challenge. Guinea pigs received 140 roentgens (r) of whole-body X-irradiation at intervals ranging from 12 days before to 3 days after subcutaneous vaccination with 10(exp5) viable cells of live tularemia vaccine. Serological studies of animals from each group were performed at nine post-vaccination intervals. The over-all mortality rate attributable to the combined irradiation-vaccination was fourfold less than that observed among irradiated animals vaccinated by the respiratory route. Three weeks following vaccination 20 animals of each irradiated-vaccinated group and the various control groups were challenged by a respiratory exposure to virulent P. tularensis SCHU S4. The serological study de%or%strated that there was no significant inhibition of agglutinin production among the irradiated-vaccinated groups, regardless of the time of irradiation. All vaccinated animals, both irradiated and nonirradiated, developed comparable resistance to virulent challenge.

Investigation was made in vaccinated monkeys exposed via the respiratory route to 1000 cells of highly varulent Pasteurella tularensis SCHU S4 and serially sacrificed from one hour to 28 days thereafter. Earlier and more extensive dissemination as well as greater proliferation of the challenge strain occurred in nonvaccinated controls than in vaccinees. Focal accululation of monocytic cells in the pulmonary parenchyma was first observed in nonvaccinated controls, animals vaccinated dermally, and animalsnimals vaccinated aerogenically, on the first, third, and fifth day respectively, after challenge. In contrast to septicemia and fatal diesease in nonvaccinated controls within seven days after challenge, a reduction in number of SCHU S4 organisms occurred in the lungs, tracheobronchial lymph nodes, spleen, and liver of vaccinees between 14 and 28 days. Nonvaccinated controls and animals vaccinated dermally exhibited enlarged caseous tracheobronchial lymph nodes and moderate pulmonary involvement by the fifth day whereas animals vaccinated aerogenically did not show comparable pathology until seven to ten days after challenge.

The usefulness of plasma electrophoretic patterns as one of the major markers of the physical condi tion of the Macaca mulatta was demonstrated in studies with monkeys vaccinated with the live tularemia vaccine and irradiated 24 hours after respiratory challenge with Pasteurella tularensis SCHU S4. The analysis of the extent of infection as well as differentiation between the treated groups was based on clinical signs, mortality rate, and hematological, serological, bacterio logical, and pathological studies. The vaccinated challenged-irradiated group (VCX) in their re sponses more nearly resembled the challenged group (C) than the vaccinated-challenged group (VC). The VC group, with milder disease symptomatology, showed a less marked drop in albumin than in the C or VCX groups. In addition, the return toward normal values was most rapid in the VC group and least rapid in the C group. Changes in the alpha 1 peak were less marked, with only an occasionalincrease in the VCX group. The alpha 2 peak of the VCX group increased signifi cantly and remained high longer than the alpha 2 peaks of the other groups. The beta peak rose in both vaccinated groups, VC and VCX, but remained constant at a normal level in the C group. In contrast, however, the gamma globulin response was markedly increased in the C group.

Clinical and laboratory studies in trichophytin allergy, including experiments in sensitization, hyposensitization and immunization, have received attention for three reasons: (1) because of the influence of these phenomena on the course of mycotic infections, including their possible therapeutic effect on certain fungous diseases; (2) because of their general significance in the theoretical conceptions of immune biology, and (3) because trichophytin belongs to a large and universally important group of allergens of practical significance in infectious diseases (e. g., tuberculin, luetin, the Frei vaccine, leprolin, mallein, sporotrichin, oidiomycin and tularemia vaccine). For the two reasons last mentioned, studies and experiments with trichophytin merit attention far beyond that which might be accorded them if their results were applicable only to fungous diseases. There are such close analogies, particularly between trichophytin and tuberculin allergy, that it may be stated that what is done with trichophytin may usually be repeated