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A parenteral Shigella ribosomal vaccine (SRV) was investigated in animals for safety, antibody-inducing capacity, and protective activity. Ribosomal preparations from a Shigella sonnei phase I avirulent strain were obtained and shown to possess chemical, sedimentation, and other properties typical of bacterial ribosomes. No endotoxin contamination was revealed by a ketodeoxyoctonate assay, although the presence of some kind of O antigen was evidenced by serological findings and the high activity of SRV in inducing the O-antibody response and immunological memory in animals. SRV was nontoxic in mice, guinea pigs, and monkeys and induced no local reactions when injected subcutaneously in reasonable doses. Significant protection against a local Shigella infection (Sereny test) was seen in guinea pigs injected with SRV (efficiency index, about 60%) and the specificity of the protection was evident from cross-challenge experiments. The protective efficiency of SRV was especially high in rhesus monkeys challenged orally with virulent Shigella cells (89%, as calculated from the summarized data of several experiments in 71 animals). Protection in monkeys was long lasting and could be demonstrated several months after injection of SRV. An inexpensive technique can be used for the production of SRV on a large scale. The high immunogenicity of SRV is discussed in terms of the amplifying effect of the ribosome, which serves as a delivery system for polysaccharide O antigen. Further study of SRV as a candidate vaccine for humans seems justified by the data obtained.

It has been established that rabbits possess an increased sensitivity to antibiotics of the tetracycline group. On administering chlortetracycline, tetracycline, or oxytetracycline to rabbits per os in doses of from 5 to 50 mg/kg per day the majority of the animals died within 2 weeks after initiation of the treatment, with phenomena of increasing apathy, anorexia, weight reduction and profuse diarrhea.

Immune bacteriolysis test with meningococcus, group A, was used for the purpose of serum antibody study. Meningococcus cultures with a bright orange fluorescence of the colonies in oblique illumination (the I type) proved to possess the greatest lysability. Guinea pig serum sorbed with meningococcus suspension was found to be the best source of the complement. Sera obtained after 1 to 3 days of rabbit immunization, containing mostly IgM antibodies, had the greatest bactericidal capacity. Only those fractions which contained IgM possessed bactericidal activity in the hyperimmune rabbit sera with a high IgG antibody concentration. No lytic activity was displayed against meningococcus by unfractionated hyperimmune sera.

Flagellar preparations were obtained from 6 salmonellae strains by differential ultracentrifugation; they were characterized by morphological, immunochemical, and serological tests. The results of investigations showed that the preparations possessed high serological activity; somatic antigen admixture was insignificant. Flagellins extracted from the flagellae were used to prepare erythrocytic H-diagnostic agents. The results of their use in the examination of sera of healthy persons, and of those suffering from typhoid fever and salmonellosis indicated the expediency of using passive H-hemagglutination for diagnostic purposes.

The authors studied the lytic and agglutinating activity of H-antibodies against S. typhimurium. Sera containing H-antibodies proved to possess a distinct lytic activity expressed in the presence of a complement and caused by specific antibodies. In H-antibodies specific lytic activity (as per 1 agglutinating unit) was much weaker than in O-antibodies. A relatively low lytic action of H-antibodies was apparently associated with topography of corresponding antigenic determinants of the microbial cell which determined the low possibility of fatal injuries. In examination of H-bacteriolysins there was revealed the same regularity as noted in studying the O-bacteriolysins: reduction of the lytic activity of antibodies in hyperimmunization.

An electron-microscopic study of the negatively-stained S. typhimurium cells from the culture subjected to the action of a specific hyperimmune antiserum in the presence of a complement (the "complement-antibody" system) pointed to increase in the population of morphological signs of cell destruction--an increase in the number of small bacterial fragments. Many cells which retained the configuration of bacterial cells also had signs of specific affection. The determining sign of specificity of the process was the presence on the surface of the great majority of the mentioned morphological types of accumulations of bacterial fragments of the stain in the form of dark stains surrounded by a lighter ring. The stains were as a rule round with the diameter of the dark zone of about 80 A. The light framing of about 30 A in width consisted of 12 subunits. A conclusion was drawn that further experimental analysis was necessary for detalization of this process and reproduction of a complete picture of bacteriolysis.

In earlier studies Shigella sonnei ribosomal vaccine was shown to be highly protective for guinea pigs and monkeys. The object of the present study, carried out in 20 healthy volunteers, was the safety and the antigenic activity of this vaccine. The subcutaneous injection of the ribosomal vaccine in doses of 100 micrograms and 200 micrograms produced no febrile reactions nor biochemical and histological changes. The minimal local reaction was observed after injection into the subscapular region: in this case 200 micrograms of the vaccine produced neither severe, nor moderate reactions. A single injection of this dose led to a more than 4-fold rise in the levels of total and cysteine-resistant O-antibodies, as well as to the prolonged elevation of the complement level in the serum.

After immunization of guinea pigs with Shigella sonnei ribosomal vaccine O-antibodies appeared not only in the blood serum of the animals, but also in their lacrimal fluid. Since no correlation between the levels of serum and secretory antibodies was detected and since the time course of changes in these antibody levels was quite different (serum antibodies reached their peak on day 7 while secretory antibodies, on day 14 after vaccination), antibodies in lacrimal fluid were supposed to reflect local immune response induced by parenteral administration of ribosomal vaccine, irrespective of systemic immune response. The peak of secretory O-antibodies coincided in time with the period of the highest protection of guinea pigs from Shigella keratoconjunctivitis. The animals with a high level of secretory antibodies were better protected from Shigella infection than those with a low level of secretory antibodies. These data suggest that locally produced O-antibodies play an important role in protective immunity induced by parenteral administration of the ribosomal vaccine.

The ribosomal preparations of S. sonnei and some other bacterial species were obtained by the method of differential centrifugation, and the specificity of their protective action was studied in the keratoconjunctivitis test on guinea pigs. The ribosomal preparations were introduced parenterally in a single injection, and their protective action was determined two weeks later by the challenge of the animals with S. sonnei virulent strain and the subsequent calculation of the efficiency index (EI) by the formula: EI = C-V/C X 100, where C and V are the percentage of resistant eyes in the control and vaccinated groups of the animals respectively. For the ribosomal preparation obtained from a homologous avirulent strain this index was equal to 58%, while for the heterologous ribosomes obtained from Escherichia coli, Salmonella minnesota and S. flexneri in was close to zero. The ribosomal preparations obtained from S. sonnei R-strain which had no surface or cytoplasmic O-antigen also proved to be ineffective in rendering protection against local Shigella infection. The results of this investigation are compared with the data obtained by other authors, and the analysis of these results leads to the conclusion that the O-specific component is the indispensable factor of the protective activity of many ribosomal vaccines and its molecular properties require further study. The possible role of other components of the ribosomal vaccine is also discussed.

The difficulties arising in the study of the immunogenicity of bacterial ribosomes and in their possible use as vaccines are due to the fact that preparative ultracentrifugation, constituting a necessary stage in most of the methods used for the isolation of ribosomes, has a low productive capacity. To develop a more effective method for obtaining Shigella ribosomal vaccines, an attempt to use the method of precipitation with 10% polyethylene glycol (PEG), proposed by Expert-Bezançon et al., has been made. The serological determination of O antigen has shown that nearly contained in the supernatant fluid S-30 can be detected in precipitated ribosomes. Taking into account the wide spectrum of the biological activity of bacterial endotoxin, it must be removed from the vaccine. The study has revealed that precipitation by means of ethanol (15-35%), low pH (4,2-4,7) and PEG (4-8%) can be used for this purpose. In accordance with the chosen method, the clarified material obtained by precipitation with 10% PEG is fractionated by means of 5% PEG which causes the complete precipitation of ribosomes, thus leaving endotoxin in the solution. Centrifugation in the density gradient of saccharose and electron microscopy have demonstrated that ribosomes isolated by this method possess typical sedimentation properties and structure. The yield of ribosomes is 3 times greater than that obtained by ultracentrifugation. Fractionation with PEG may be used as the method of the mass production of ribosomal vaccines.

Shigella ribosomal vaccine was prepared by fractionation with polyethylene glycol (PEG), recently proposed as an alternative of the more expensive and labor-consuming technique of differential centrifugation. In the present investigation the biological activity of ribosomal preparations isolated by these two methods was compared. Ribosomal vaccine obtained by PEG fractionation proved to be nontoxic for mice (LD50 greater than 2 mg) and produced no local and systemic reactions in monkeys when introduced subcutaneously in a dose of 600 micrograms. Ribosomes isolated by the two methods did not differ in the antigenic potency of their O-specific component responsible for inducing antibody formation in guinea pigs and monkeys. The protective potency of Shigella ribosomal vaccines prepared by PEG fractionation and ultracentrifugation was compared by tests in guinea pigs under the conditions of intraconjunctival challenge 2 weeks after a single injection of 40-200 micrograms of ribosomes. The mean resistance rate (percentage of protected eyes) was almost the same with both preparations, 67% and 65%, while in the control (nonimmunized) group only 13% of eyes were resistant to challenge. In monkey tests two injections of ribosomal vaccine obtained by PEG fractionation ensured a high protective effect against dysentery. No clinical signs of dysentery were observed in two groups of the test animals (totaling 10 monkeys) immunized 5 and 16 weeks before oral challenge with a dose of 75 X 10(9) virulent shigellae, which caused dysentery of moderate severity in all 5 control monkeys. The low toxicity and high protective potency of ribosomes isolated from S. sonnei by PEG fractionation makes it possible to use this method for the large-scale production of ribosomal vaccine.

A study was made of the dynamics of serum H-antibodies in typhoid patients depending on the period of the disease and the severity of its course; the blood sera of chronic carriers were examined as well. H-antibodies were determined in the indirect hemagglutination test with the use of erythrocyte H-dignoasticum. Antibodies of different physico-chemical nature (of IgG and IgM classes) were obtained by fractionation in columns with DEAE-cellulose. Results of the investigations showed that the titres of sum total serum H-antibodies failed to depend on the severity of the course of the disease. The maximum value of the mean geometrical titre of H-antibodies in the blood serum was determined on the 3rd-4th week from the onset of the disease. In chronic carriers the sum total H-antibody titres were greater than in typhoid patients. The serum H-antibody titres increased on account of IgG-globulins both in the typhoid patients and in carriers.