Effect of trypsin and ribonuclease on the immunogenic activity of ribosomes and ribonucleic acid isolated from Mycobacterium tuberculosis

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Effect of trypsin and ribonuclease on the immunogenic activity of ribosomes and ribonucleic acid isolated from Myobacterium tuberculosis. J. Bacteriol. 91:2146-2154. 1966.-The ribosomal fraction of the attenuated strain, H37Ra, of Mycobacterium tuberculosis was treated with trypsin alone, ethylenediaminetetraacetic acid (EDTA) alone, EDTA and pancreatic ribonuclease, or with trypsin and ribonuclease. After each of these treatments, the ribosomal fractions were injected intraperitoneally into male CF-1 mice to test their capacity to produce an immune response to infection with virulent tubercle bacilli, strain H37Rv. Removal of protein with trypsin left the immunogenicity unchanged; EDTA alone reduced immunogenicity in the smaller vaccinating doses; EDTA plus ribonuclease reduced the immunogenicity by approximately 50% in the highest (1.0 mg) vaccinating dose; ribonuclease alone, after treatment with trypsin, reduced immunogenicity also approximately 50%. A crude mycobacterial ribonucleic acid (RNA) was prepared by extraction of the ribosomal fraction with alcohol. This RNA preparation was as effective in producing an immune response as the ribosomal fraction from which it was prepared, unless the RNA was partially or completely degraded during the preparation. The effect of ribonuclease on the immunogenicity of the RNA was similar to that obtained with the ribosomal fractions, except that ribonuclease completely destroyed the immunogenicity of a partially degraded RNA. RNA appears to be an essential part of an immunizing substance in attenuated tubercle bacilli, which produces a high degree of immunity in mice; 50 mug (dry weight) will protect approximately 80% of the mice, and as little as 0.5 mug will protect approximately 30% of the mice. Mycobacterial RNA not incorporated in Freund's incomplete adjuvant was nonimmunogenic. Yeast RNA incorporated in Freund's incomplete adjuvant was not immunogenic.     

Factors Affecting Immunogenic Activity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations

By following careful procedures, mycobacterial ribosomal fractions and ribonucleic acid (RNA) prepared by ethyl alcohol precipitation were obtained which have immunogenic activities similar to the viable attenuated H37Ra cells of Mycobacterium tuberculosis from which they were obtained. This comparison was based on the amount of ribonucleic acid (RNA) present. These preparations consisted of approximately 63% RNA and 37% protein; no deoxyribonucleic acid or polysaccharide was detected by chemical tests. A high correlation was found between the immunogenic activity of a preparation and the per cent increase in hyperchromicity at 260 nm of a ribonuclease-hydrolyzed portion. Final concentrations of sodium dodecyl sulfate higher than 0.25% when used for the preparation of the ribosomal fractions and RNA resulted in significantly lower immune responses and greater variation between experiments. This was not related to the amount of protein present. The stability of the ribosomal and RNA preparations was tested under a variety of conditions. The need for a good protective adjuvant again was shown since mouse serum readily hydrolyzed the RNA. Equal immunity was obtained after immunization by the intraperitoneal and subcutaneous routes; however, no immune response was obtained when the intravenous route was used. Preliminary results with RNA prepared with phenol showed that it was more easily degraded during preparation. This resulted in a lower immune response than was obtained with the RNA prepared with ethyl alcohol.     

Critical factors for liposome-incorporated tumour-associated antigens to induce protective tumour immunity to SL2 lymphoma cells in mice

Physical and immunogenic properties of reconstituted membranes designed for the presentation of tumour-associated antigens (TAA) to the immune system are described. Proteins and lipids of crude membranes of SL2 murine lymphosarcoma cells were partially solubilized with octylglucoside. Reconstituted membranes, consisting mainly of unilamellar vesicles with a diameter of 0.03–0.15 μm, were formed by detergent removal and were purified by floatation in a discontinuous sucrose gradient to remove non-lipid-bound protein. Subcutaneous immunization of syngeneic mice with reconstituted membranes or with purified reconstituted membranes induced protection against an intraperitoneal challenge with 10³ viable SL2 cells. Reconstituted membranes were more immunogenic than crude membranes in immunoprotection experiments when compared on the basis of protein dose. Detergent removal was required to obtain an immunogenic presentation form of SL2 membrane antigens and to avoid toxicity associated with the detergent. Reconstitution of SL2 membranes in the presence of exogenous phospholipid slightly increased the fraction of protein that associated with the reconstituted membranes. However, the immunogenicity of the solubilized membrane TAA was not significantly affected by the presence of exogenous phospholipid. The reconstitution procedure described may be useful in identifying membrane factors required for the induction of immune responses against TAA. The versatility of the system may be employed to develop safe alternatives for whole-cell vaccines.     

Characteristics of the immunologic response of animals to Clostridium perfringens anatoxin]

Experiments were conducted on guinea pigs, rabbits and mice (mongrel and inbred); immunogenic properties of Cl. perfringens toxoids of different purity were studied. Toxin neutralization and passive hemagglutination tests were used to determine the antitoxic immunity level. It appeared that in the immunization of guinea pigs and rabbits the degree of immunogenicity of the preparations increased with the elevation of their specific activity. Under the same conditions both the mongrel and the inbred mice displayed the maximum immune response in the immunization with the least purified preparations, and the minimum after the injection of a highly purified antigen.     

Immunogenic dialyzable factor derived from a ribosomal fraction of Salmonella typhimurium. I. Preparation of the protective dialyzable factor from the ribosomal fraction by the freeze-thaw procedure

The preparation, properties, and immunogenicity of the dialyzable factor from a ribosomal fraction of Salmonella typhimurium are described. The ribosomal fraction was purified to eliminate O-antigenic components, by affinity chromatography (Sepharose-anti-O antibody conjugates used as immunoadsorbent). The dialyzable factor was obtained in the concentrated dialysate of the purified ribosomal fraction which was alternately frozen in dry-ice acetone and thawed in an 80 C water bath, for a total of five or six cycles. When this preparation was tested for its ability to protect mice against challenge with 1,000 LD50 of the homologous bacteria, it afforded 100% protection at a dose equivalent to 5.0 micrograms of RNA. The protection conferred by this factor was mainly cell mediated but immune serum enhanced this immunity despite the fact that no antibodies were detected in it. The protective activity of this factor was sensitive to RNase digestion but resistant to proteolytic enzymes. Ion exchange chromatography of this factor with DEAE-Sephadex A-25 (in 7 M Urea-0.02 M Tris-HCl buffer, pH 7.5) resulted in a single A260 peak which was found to be immunogenic. Chemical analysis of this peak after it was concentrated and desalted revealed that this immunogenic fraction was composed mainly of mixed nucleotides. The data indicate that protective immunity conferred by a ribosomal vaccine is associated with RNA but may not require the intact RNA molecule.     

Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulfate

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Preparation of highly immunogenic ribosomal fractions of Mycobacterium tuberculosis by use of sodium dodecyl sulfate. J. Bacteriol. 91:2139-2145. 1966.-Ribosomal fractions of Mycobacterium tuberculosis, strain H37Ra, were prepared by treatment of the intracellular particulate fraction with 0.25 or 0.5% sodium dodecylsulfate (SDS) followed by centrifugation at 144,700 x g for 3 hr. This procedure has greatly simplified the preparation of ribosomal fractions and has given fractions composed of approximately 50% ribonucleic acid (RNA) and 15 to 20% protein. When incorporated into Freund's incomplete adjuvant and injected intraperitoneally into CF-1 mice, the SDS ribosomal fractions were more immunogenic than the particulate fractions from which they were prepared. They were as much as 100 times more immunogenic than ribosomal fractions prepared by differential centrifugation, 1 mug (dry weight) per mouse being sufficient for the induction of some immunity. However, none of these ribosomal preparations, in comparable doses, was as immunogenic as the living cells from which they were prepared. It was also shown that the addition of 10(-4)m MgCl(2) to the final diluent increased immunogenic activity, whereas larger concentrations (10(-3)m) reduced immunogenic activity. Preparation of the ribosomal fraction from ruptured cells in one continuous process during the course of 1 day increased the activity. Two-week-old H37Ra cells contained more RNA and were more immunogenic than the older cultures which have been used in the past.     

Chikungunya Virus Vaccine Prepared by Tween-Ether Extraction

Chikungunya virus vaccines prepared by Tween 80 and ether inactivation of virus grown in green monkey kidney cell cultures were shown to be as immunogenic as comparable Formalin-inactivated vaccines. Both types of vaccine stimulated hemagglutination-inhibiting, complement-fixing, and neutralizing antibody and afforded protection to mice against a live virus challenge. It was shown after Tween-ether treatment of chikungunya virus that the infectivity of the virus was lost and the hemagglutinin titer was increased. By characterization of the resultant hemagglutinin by sucrose and cesium chloride density gradient centrifugation, it was found that the extracted particle was smaller in size and greater in density than the parent virus particle. Removal of lipid may account for the alterations in the physical characteristics of the infectious virus particle. Conditions for treatment of chikungunya virus with Tween and ether were found that preserved high titers of hemagglutinin as well as the immunogenicity of the virus preparations.     

Immunologic assessment of preparations of the RS virus at the stages of its purification and concentration

The work demonstrates the immunogenic potency of some preparations of RS virus, differing in the degree of their purification and introduced by multiple intranasal administration. The immunogenic potency of these preparations was manifested by the synthesis of antibodies determined in the coagglutination test (the specificity of these antibodies was confirmed in the reaction of the neutralization of RS virus), as well as by sensitization determined in the leukocyte migration inhibition test. Immunization with concentrated virus was more effective than immunization with the preparation of virion fraction. The intranasal administration of live RS virus to mice induced the appearance of a morphologically determined disease which was not prevented by the development of immune response accompanying the disease. The conclusion was made that further improvement in the system of testing the preparations of RS virus was necessary.     

Liposome-Based Cancer Vaccines

Abstract

Specific active immunotherapy employing crude or purified tumour cell extracts containing tumour-associated antigens (TAA) is of interest as a therapeutic modality for cancer treatment. Weak immunogenicity of TAA preparations and difficulties associated with the extraction of TAA from tumour cells have been major problems, but may be overcome by various strategies. In this context, we studied the potential of well-characterized reconstituted tumour cell membranes to induce protective tumour immunity. The weakly immunogenic SL2 lymphosarcoma syngeneic to DBA/2 mice was used as a tumour model. It was found that immunization with reconstituted membranes induces specific and systemic tumour immunity. Presentation of TAA on a membrane structure was essential. The tumour rejection potency of the reconstituted membranes was dramatically enhanced by the addition of muramyl tripeptide phosphatidyl-ethanolamine (MTP-PE) and interleukin-2. In addition, the effect of liposome encapsulation of IL-2 on its immunomodulating effect was monitored. Finally, insight was obtained into the immunological basis of the protective tumour immunity. On the basis of these findings, suggestions are made to improve liposome-based cancer vaccines.     

A direct comparison of human papillomavirus type 16 L1 particles reveals a lower immunogenicity of capsomeres than viruslike particles with respect to the induced antibody response

Capsomeres are considered to be an alternative to viruslike particle (VLP)-based vaccines as they can be produced in prokaryotic expression systems. So far, no detailed side-by-side comparison of VLPs and capsomeres has been performed. In the present study, we immunized mice with insect cell-derived human papillomavirus type 16 VLPs and capsomeres. VLPs induced consistently higher antibody titers than capsomeres but the two forms induced similar CD8 T-cell responses after subcutaneous, intranasal, and oral immunization, and at least 20 to 40 times more L1 in the form of capsomeres than in the form of VLPs was needed to achieve comparable antibody responses. These results were confirmed by DNA immunization. The lower immunogenicity of capsomeres was independent of the isotype switch, as it was also observed for the early immunoglobulin M responses. Although there were differences in the display of surface epitopes between the L1 particles, these did not contribute significantly to the differences in the immune responses. capsomeres were less immunogenic than VLPs in Toll-like receptor 4 (TLR4)-deficient mice, suggesting that the lower immunogenicity is not due to a failure of capsomeres to trigger TLR4. We observed better correlation between antibody results from enzyme-linked immunosorbent assays and neutralization assays for sera from VLP-immunized mice than for sera from capsomere-immunized mice, suggesting qualitative differences between VLPs and capsomeres. We also showed that the lower immunogenicity of capsomeres could be compensated by the use of an adjuvant system containing MPL. Taken together, these results suggest that, presumably because of the lower degree of complexity of the antigen organization, capsomeres are significantly less immunogenic than VLPs with respect to the humoral immune response and that this characteristic should be considered in the design of putative capsomere-based prophylactic vaccines.     

Isolation of different variants of Pseudomonas aeruginosa anatoxin and their characteristics

The conditions for the detoxification of the crude preparations of P. aeruginosa exotoxin A, obtained by the cultivation of strain PA-7 in Martin's broth, have been studied, and the schemes for obtaining nontoxic, stable, specifically antigenic preparations of toxoid from exotoxins A with different degrees of purification have been developed. Toxoid obtained by formalin treatment on the level of a crude preparation with its subsequent purification and additional detoxification with formalin in the presence of lysin has been shown to possess high immunogenic potency. The preparation has been found to induce immune response and to ensure the protection of experimental animals challenged not only with the lethal dose of exotoxin A, but also with P. aeruginosa toxigenic and protease-producing strains.     

On the Immunogenicity of Ribosomes and Ribosomal Proteins Isolated from Klebsiella pneumoniae and Streptococcus pneumoniae

The two pathogenic species Streptococcus pneumoniae and Klebsiella pneumoniae were used to analyze the immunogenic role of proteins in ribosomal preparations. The protective activity of ribosomes prepared from either strain and further purified by washing with high-salt concentrations, followed or not by sucrose gradient separation of the particles, was identical to that of crude unwashed ribosomes. Similarly, no substantial alteration of the level of protection was observed after treatment with the antibiotic puromycin. Therefore, the immunizing efficacy of ribosomes does not appear to be due either to the nonribosomal proteins adsorbed at the surface of organelles or to the growing polypeptide chain. It seems rather to be attributable to the structural ribosomal proteins themselves, which were indeed shown to induce alone a significant level of protection.     

Reconstituted membranes of tumour cells (proteoliposomes) induce specific protection to murine lymphoma cells

Summary Antigens presented on cell membranes or on liposomes are usually more immunogenic than antigens in soluble form, this being one of the reasons for the weak immunogenicity of extracted tumour-associated transplantation antigens (TATA). The main objective of this study is to solubilize TATA from tumour cells and to present them on a membrane-like structure to the immune system. Crude tumour cell membranes of SL2 lymphosarcoma cells (a spontaneously arising, weakly immunogenic tumour) were solubilized with octylglucoside or sodium deoxycholate, and reconstituted membranes (proteoliposomes) were prepared by detergent removal. Mice immunized s.c. with reconstituted membranes were protected against an i. p. challenge with tumour cells. Although octylglucoside solubilized only 41% of the membrane proteins, the reconstituted membranes were as immunoprotective as crude membranes. (Glyco)proteins were probably the major membrane components in the reconstituted membranes that induce immunoprotection, as mice immunized with preparations constituted of (glyco)lipids from SL2 cells could not reject SL2 cells. If Freund's complete adjuvant was used with the first immunization injection, no potentiation of the elicited immune responses was observed. Besides the membrane TATA, SL2 cells contained an apparently non-membrane-bound TATA, which was found in the cytoplasm. It is concluded that detergent solubilization of membranes and subsequent preparation of reconstituted membranes can be used to obtain membrane tumour-associated antigens that retain activity for induction of protective tumour immunity. The major advantage of this method is that membrane proteins are solubilized and are subsequently presented on a membrane-like structure that resembles the tumour cell membrane. On theoretical and practical grounds it provides a promising alternative for whole-cell vaccines.     

Specificity of Acquired Resistance Produced by Immunization with Listeria monocytogenes and Listeria Fractions

Mice were immunized with 1.0 mg of an attenuated strain of Listeria monocytogenes to determine the period of protection afforded by this strain when the mice were challenged intravenously with 5 MLD of listeria. Protection appeared 2 days after immunization and was still apparent 4 weeks after immunization. If the challenge dose was decreased to 1 MLD, protection was apparent at 10 weeks. Mice immunized with a comparable dose of mycobacterial cells and challenged intravenously with 1 MLD of listeria showed no protection at 10 weeks. The magnitude of the immune response to listeria challenge was not increased in mice immunized with the same virulent strain as that used for challenge. It was also found that resistance to listeria challenge appeared early after listeria immunization if the immunizing dose was large. As the immunizing dose was decreased and the challenge dose increased, resistance appeared later. Listeria killed by heat or ultraviolet irradiation, living but nonmultiplying streptomycin-dependent listeria, or listeria ribosomal fraction gave no protection against listeria challenge. The magnitude of the immune responses after listeria immunization to listeria challenge and to mycobacteria challenge were compared. It was found that protection after listeria challenge was of longer duration. In addition, a 100-fold larger vaccinating dose was required to give comparable protection against tuberculous infection.     

Streptococcus mutans ribosomal preparations: purification and properties

Ribosomal preparations were obtained from Streptococcus mutans. Sucrose density gradient analyses showed the ribosomes to be 70S and dissociated subunits to be 56S and 34S. The ribosomal preparation contained 57.4% RNA and 42.6% protein and gave an absorption maximum at 260 nm and a minimum at 235 nm and ribosomal particles were approximately 150-180 X 190-220 A as determined by electron microscopy. Immunodiffusion analysis of pooled antiserum raised by injecting the ribosomal preparation into rabbits disclosed precipitin lines with glucosyltransferase and lipoteichoic acid preparations from S. mutans. Gas chromatography showed rhamnose and glucose to be present in the ribosomal preparation indicating the presence of nonribosomal carbohydrate materials. The ribosomes were able to synthesize precipitable polypeptides when exogenous mRNA and tRNA were added and anti-ribosomal antibodies reduced this activity. Protease treatment rendered the ribosomal preparation less immunogenic in rats and less antigenic when the ribosomal preparation was used to coat erythrocytes for passive haemagglutination assays, while RNase treatment of the ribosomal preparation had no effect, suggesting that a protein(s) is the principal immunogenic moiety of the ribosomal antigen. Polyacrylamide gel electrophoresis of the ribosomal preparation revealed 27 protein bands of which five were found to react with hyperimmune rabbit antisera to the S. mutans ribosomal preparation by Western blot analysis. Washing the ribosomal preparation with 1 M NH4Cl did not remove any of the five immunogenic ribosomal protein antigens indicating that these were innate ribosomal proteins.     

Immunogenicity of modified by dextran recombinant fragment of Bac protein of group B streptococci

Opportunity to increase of immunogenicity of recombinant polypeptide P6 constructed on the basis of surface protective Bac protein by its chemical conjugation with dextran (D) 40 was studied. 3 preparations with different quantity of protein and polysaccharide components were obtained. Their testing with standard serum showed that antigenic determinants of the polypeptide were preserved although partly enclosed and structure of antigenic determinants did not significantly changed. On the model of subcutaneous immunization of mice it has been shown that two preparations--P6D2 and P6D3--have improved immunological characteristics. Conjugation of polypeptide P6 with dextran let to increase of immune response to P6 and affinity of P6-specific antibodies. Injection of nonconjugated P6 and dextran mixture showed that free dextran is not immunogenic and it suppress synthesis of P6-specific antibodies without effect on their affinity. Intranasal administration of nonconjugated P6 did not lead to P6-specific IgG in serum. After conjugation with dextran polypeptide P6 was recognized as an antigen and stimulated production of small quantity of antibodies. Technological process of chemical binding of protein antigen with polysaccharides, which let to regulate protein and polysaccharide components ratio, can be the effective method to increase immunogenicity of recombinant polypeptides.     

Comparative study on the immunogenic properties of Clostridium perfringens type A toxoid

The paper presents the results of a study on the immunogenic properties of toxoid preparations from Cl. perfringens type A obtained using the routine method of detoxifying alpha = toxin in the culture medium (commercial preparations) and by means of detoxifying a previously purified alpha = toxin (experimental preparations). When tested in immunized guinea pigs, the immunogenicity of experimental preparations was found to be 4.5 to 6 times that of commercial preparations. In mice, there was no difference in the immunogenic properties of the two types of preparations as determined by the ED30 of the antigen and the serum levels of Cl. perfringens antitoxin. The possibility is discussed of using the guinea pig as a laboratory animal model due to its ability to reflect most clearly the differences in the immunogenicity of Cl. perfringens type A toxoid preparations.     

Immunogenicity and thymus-dependence of the polymerized alpha-toxoid of Clostridium perfringens]

Polymeric alpha-toxoid with a molecular weight of 450 000--600 000 was obtained by condensation of alpha-toxoid of Cl. perfringens, type A, with glutaric aldehyde. Experiments on guinea pigs showed that in the adsorbed preparations the immunogenic properties of both monomeric and polymeric alpha-toxoids are practically identical. The primary immune response after the immunization with nonadsorbed antigens was 3 times greater than that induced by the monomer. Polymerization of alpha-toxoid failed to change its thymus dependency.     

Construction of an encapsulated ESAT-6-based anti-TB DNA vaccine and evaluation of its immunogenic properties

Experimental preparations based on a DNA vaccine encoding the ESAT-6 antigen of Mycobacterium tuberculosis have been obtained (KpONE6) and studied for immunogenic effects in the murine model. The core of the preparation contains DNA of the recombinant plasmid pONE6 encapsulated within a spermidine-polyglucin conjugate, thereby protecting the DNA vaccine from degradation. KpONE6 induces a proliferative T-cell immune response in mice upon intramuscular immunization.     

Homogeneous Cl. perfringens alpha-anatoxin: its physicochemical and immunogenic properties]

The authors present a method of obtaining relatively homogeneous preparations of alpha-toxoid of Cl. perfringens, type A, including the primary conception of the alpha-toxin proteins, their chromatography on DEAE-cellulose, fractionation with (NH4)2SO4, detoxication, with the subsequent gel-filtration through sephadex and isoelectric focussing. Sedimentation coefficient of the preparation proved to be 3.8 S, isoelectric point-4.83 +/- 0.07. In studying the immunogenic properties of alpha-toxoid in experiments on guinea pigs and rabbits their high immunogenicity, exceeding that of the industrial toxoid 8- and 6-fold, respectively, was established. Homogeneous preparations of alpha-toxoid provided intense anti-microbial immunity. Interlinear differences in the levels of the immune response of inbred mice, highly-reactive (BALB/c) and low-reactive (C57BL/6) to alpha-toxoid, reached 20-fold; in combination with the high immunogenicity of this antigen for mice this permits to recommend it for immunogenic studies.