Virulent Pneumococci Research Articles

Purification and immunogenicity of genetically obtained pneumolysin toxoids and their conjugation to Streptococcus pneumoniae type 19F polysaccharide

As part of an ongoing study concerned with improving human vaccines against Streptococcus pneumoniae, the genes for two defined pneumolysin (PL) toxoids (pneumolysoids), Pd-A (PL with a Cys----Gly substitution at amino acid 428) and Pd-B (PL with a Trp----Phe substitution at position 433), were inserted into the high-expression vector pKK233-2 in Escherichia coli and the pneumolysoids were purified. Groups of mice which had been immunized with either Pd-A, Pd-B, or native PL purified from S. pneumoniae were then challenged either intranasally or intraperitoneally with virulent pneumococci. Mice in all immunized groups survived significantly longer than sham-immunized controls. Both pneumolysoids were more effective than PL as protective immunogens. Pneumolysoid Pd-B was conjugated covalently with pneumococcal type 19F capsular polysaccharide (19F PS), and the immunogenicities of both the protein and the PS moieties of the conjugate in mice were determined. Significant anti-PL titers were obtained, and the immunogenicity of the 19F PS moiety was markedly enhanced compared with that of unconjugated PS. Conjugation also appears to have converted the 19F PS into an antigen capable of inducing a booster effect. These results support the notion that the efficacy of human, PS-based antipneumococcal vaccines might be improved by supplementation with pneumolysoid in the form of a covalent pneumolysoid-PS conjugate.

A comparison of antibody concentration measured by mouse protection assay and radioimmunoassay in sera from patients at high risk of developing pneumococcal disease

The radioimmunoassay (RIA) of pneumococcal capsular polysaccharide antibodies is dependent on the association of radiolabeled antigen and pneumococcal antibody. However, it is not known whether the ability of the antibody to complex with antigen correlates with in vivo protection against infection. A method for evaluating protective ability of antibody vis-à-vis binding ability is to passively transfer a measured quantity of antibody into recipient mice followed by a lethal challenge with virulent pneumococci. Protection against a fatal outcome is then correlated with the amount of antibody (as measured by RIA) passively transferred. This comparison of quantitation by RIA and biological protection in mice was performed on 30 sera from humans. The sera were obtained from vaccinated healthy persons and vaccinated persons at high risk of developing pneumococcal infection, including people with nephrotic syndrome, chronic obstructive pulmonary disease and various forms of cancer. The results of these studies indicate that antibody as measured by RIA correlates with protective antibody against pneumococcal infection. These studies were conducted on pneumococcal serotype 3.

Changes in the Serum of Mice Infected with Streptococcus Pneumoniae that Stimulate In-vitro Multiplication of Virulent but not Avirulent Strains

During pneumococcal infection of mice, nucleic-acid products, including deoxynucleotides, may be released into the serum from cellular disintegration in at least three organs, the lungs, spleen and liver. The serum, after sterilisation to remove contaminating pneumococci, stimulated multiplication of virulent but not avirulent pneumococci in vitro. It also stimulated growth of virulent pneumococci in serum from uninfected animals and could be replaced, at least in part, by certain nucleic-acid degradation products at concentrations found in infected serum. The effects of the serum were lost after dialysis or dilution.

Some biological properties of Pneumococcus type 37 and the chemistry of its capsular polysaccharide.

Pneumococcus Type 37, like pneumococcus Type 3, is characterized by the production of large mucoid colonies on the surface of solid media and by its very large capsule. It differs from the highly virulent pneumococcus Type 3 in that it is only slightly virulent for mice and rats and is isolated infrequently from man. A study of the behavior of pneumococcus Type 37 in systems comparable to those used in the study of pneumococcus Type 3 and an examination of the chemical structure of the capsule of pneumococcus Type 37 are described. The capsular polysaccharide of pneumococcus Type 37 is a viscous, optically inactive polymer composed of 95% hexose. Glucose is obtained in 88% yield upon acid hydrolysis. Periodate oxidation studies and the behavior of the polysaccharide on acid hydrolysis suggest that the molecule consists of a core of repeating units of 1,3 glucosyl-glucose to which short chains of glucose are attached at frequent intervals. Isolation of a disaccharide, the properties of which are identical with those of sophorose (beta1 --> 2 glucosyl-glucose), and of a trisaccharide are described. A tentative structure for the capsular polysaccharide of pneumococcus Type 37 is proposed.

Utilization of deoxyribonucleosides by virulent and avirulent pneumococci

The utilization of deoxyribonucleosides was studied in virulent and avirulent pneumococci. It was found that virulent pneumococci incorporated five [ 14C]deoxyribonucleosides into DNA at a greater rate and extent than avirulent pneumococci. However, when the polysaccharide capsule surrounding virulent cells was removed by a hydrolytic enzyme, incorporation was reduced considerably. 1. 1. The rate of uptake of [ 14C]deoxyribonucleosides was greater in virulent than avirulent pneumococci, but the differences were greater for pyrimidine deoxyribonucleosides than for purine deoxyribonucleosides. 2. 2. Almost all of the radioactivity found in DNA of virulent and avirulent cells after incorporation of deoxycytidine, deoxyuridine and thymidine was localized in the thymine moiety. No significant radioactivity was detected in the cytosine moiety. The pathway of deoxycytidine, deoxyuridine and thymidine utilization, for which all of the intermediates were detected in studies with cell-free extracts, was as follows: ▪ No deoxycytidine kinase or dCMP deaminase were detected. More radioactivity was localized in the thymine moiety of DNA when deoxycytidine was used as the substrate than when thymidine was employed, despite the same initial specific activity. 3. 3. Radioactivity from deoxyadenosine, deoxyguanosine and deoxyinosine that was incorporated into DNA of virulent cells was found both in the adenine and guanine moieties. In avirulent cells, only the radioactivity from deoxyguanosine was found in these two purine moieties, while label from deoxyadenosine or deoxyinosine was found exclusively in the adenine moiety of avirulent DNA. Not all of the intermediates were found in cell-free extracts for any single pathway involving purine deoxyribonucleosides but by combining the results obtained with the three of them, an almost complete picture of metabolism was revealed. 4. 4. Whereas virulent cells were capable of converting deoxyinosine (or dIMP) to deoxyxanthosine or dXMP, and the latter to deoxyguanosine (or dGMP), avirulent cells could not carry out these reactions. 5. 5. Almost 70–80% of the radioactivity found in the nucleic acid (DNA and RNA) fraction after incorporation of purine deoxyribonucleosides was removed by alkaline hydrolysis and subsequent acid precipitation of the DNA. This was not the case with pyrimidine deoxyribonucleosides.

Effects of deoxyribonucleic acid products on respiration of virulent and avirulent pneumococci.

Firshein, W. (Wesleyan University, Middletown, Conn.). Effects of deoxyribonucleic acid products on respiration of virulent and avirulent pneumococci. J. Bacteriol. 82:181-186. 1961.-In the presence of deoxyribonucleic acid (DNA) + deoxyribonuclease + mixtures of deoxynucleosides and deoxynucleotides (supplement-1) which had previously been shown to enhance DNA synthesis in virulent (S) pneumococci without affecting such synthesis in avirulent (R) pneumococci, a significant enhancement of glucose oxidation was observed over control levels in S cells of types I, II, and III. In contrast, this supplement either depressed oxygen uptake or had no effect when R or weakly virulent (I) pneumococci were present. In the absence of glucose, S cells were unable to oxidize supplement-1 extensively, whereas R cells exhibited definite activity in this respect. Supplement-1 enhanced glucose oxidation specifically and was not oxidized in the process. The selective advantage produced by the DNA products in S cells could not be explained on the basis of nitrogen content, since a substance containing twice as much of this element as that found in the entire supplement did not enhance oxygen uptake to the extent that was observed with supplement-1.

Effects of deoxyribonucleic acid products on deoxyribonucleic acid synthesis of virulent and avirulent pneumococci.

Firshein, W. (Wesleyan University, Middletown, Conn.). Effects of deoxyribonucleic acid products on deoxyribonucleic acid synthesis of virulent and avirulent pneumococci. J. Bacteriol. 82:169-180. 1961.-Cell suspensions of virulent pneumococci synthesize a greater amount of deoxyribonucleic acid (DNA) per cell in the presence of DNA + deoxyribonuclease + mixtures of deoxynucleosides and deoxynucleotides (supplement-1) than unsupplemented suspensions. Under identical conditions, avirulent and weakly virulent pneumococci do not respond to these DNA breakdown products. Glucose and casitone must be present for maximal effects to occur.A DNA turnover occurs in virulent cells. This has been demonstrated by a decrease in specific activity of H(3)-DNA extracted from virulent (S) cells exposed to nonradioactive supplement-1.

The survival of staphylococci within human leukocytes.

In a study of the phagocytosis of staphylococci by human leukocytes it has been observed that strains of staphylococci producing human infection were phagocytized by human polymorphonuclear leukocytes in vitro under conditions in which virulent pneumococci, streptococci, or Klebsiella were rarely engulfed. In the presence of human leukocytes in plasma there was a rapid fall in the numbers of viable staphylococci of both pathogenic and non-pathogenic strains, the beginning of which was detectable in 10 to 15 minutes. The fall in culturable pathogenic microorganisms was considerably less marked, however, and a rapid resurgence of growth occurred in 4 to 8 hours, whereas the number of culturable non-pathogenic microorganisms remained low for 18 to 24 hours. These differences appear to be explained by the observation that a significant number of microorganisms of pathogenic strains were able to survive within human leukocytes. Such intracellular survival was found to be associated with evidence of destruction of the leukocytes. In contrast, non-pathogenic strains of staphylococci failed to survive within human polymorphonuclear leukocytes following ingestion.

The reaction of the normal and the allergic sinus to virulent pneumococci: Frank, I., Blahd, M., and Howell, K. M.: Ann. Otol., Rhin. & Laryng. 52: 81, 1943

The reaction of the normal and the allergic sinus to virulent pneumococci: Frank, I., Blahd, M., and Howell, K. M.: Ann. Otol., Rhin. & Laryng. 52: 81, 1943

Studies on the Mechanism of Species-Specific Immunity against Pneumococcal Infection

Summary There is demonstrable in rabbits an active, non-type-specific immunity against intrapleural infection with a highly virulent strain of Pneumococcus Type 1. This immunity may be produced by prolonged immunization with rough Pneumococcus Type 2. This non-type-specific immunity could not be passively transferred to normal rabbits. However, the immunity could be passively transferred by serum to other rabbits if the site of infection (pleural cavity) were already the site of a 72-hour aleuronat-starch inflammation. This active immunity seems to depend on the ability of the tissues of the immunized animal to dispose of the injected organisms promptly. While neither blood nor serum from actively immunized animals would kill virulent pneumococci, the pleural exudate from rough Pneumococcus 2 immune rabbits was markedly bactericidal for virulent Type 1 pneumococcus. Exudate from animals immunized with virulent Type 1 organisms was not bactericidal to a significantly larger degree. Normal exudate was not bactericidal. Minced splenic tissue from animals immunized with rough Pneumococcus Type 2 was bactericidal for large numbers of virulent Type 1 pneumococci. Normal splenic tissue was only feebly bactericidal. Cells obtained from immunized animals, when transferred to normal animals, would not protect against infection with Pneumococcus 1, whereas normal rabbit-cells, resuspended in supernate from the pleural exudate of Pneumococcus 2 (rough) immune animals produced a high degree of protection against Pneumococcus 1. Immune supernate, when passively transferred, did not protect. Pleural exudates from immunized animals showed a higher percentage of macrophages than did normal exudates. These macrophages were more highly phagocytic than the macrophages of normal exudate; however, this increased phagocytic power was non-specific. The antiserum produced by immunization with rough Type 2 pneumococcus had no opsonic effect on the highly virulent Pneumococcus Type 1 used for infection. The protective value of species-specific antiserum could not be related either to the “C” polysaccharide antibody or to heterophile antibody present in rough pneumococcal antiserum. The possible manner in which species-specific antibody exerts its protective power is discussed.

A Comparative Study of Phagocytosis and Digestion of Pneumococci by Macrophages and Polymorphonuclear Leucocytes in Normal and Immune Dogs

Summary A comparative study was made of the phagocytosis and digestion of pneumococci by macrophages and polymorphonuclear leucocytes in normal and immune dogs. Cellular exudates of the two types of cell were produced in dogs by the intrapleural injection of gum arabic on the one hand, and aleuronat on the other. Observations were carried on both in vivo and in vitro employing a highly virulent Pneumococcus type I. It was found that while the macrophages showed a somewhat greater phagocytic activity than the polymorphonuclears their ability to digest pneumococci was much more pronounced than that of the latter cells. This difference was most marked in the presence of opsonic fluids of relatively low concentration. The use of suspensions of macrophages which had been concentrated by differential sedimentation brought out the superior antipneumococcal activity of these cells most clearly. Macrophages secured from the pleural exudates of dogs highly immune to pneumococcus as well as from the lungs of dogs recovering from experimental pneumonia were found to be just as dependent on opsonins for the phagocytic activity as were cells of normal animals.

The failure of allergic inflammation to protect rabbits against infection with virulent pneumococci: Cannon, P. R., and Hartley, G., Jr.: Am. J. Path. 14: 87, 1938

The failure of allergic inflammation to protect rabbits against infection with virulent pneumococci: Cannon, P. R., and Hartley, G., Jr.: Am. J. Path. 14: 87, 1938

Grossmann, N. On the pathogenesis of croupous pneumonia. Experimental study of pneumococcal infection in rabbits (Ztschr. F. Jmrnunitätsf., Bd. 90, 1937)

For many years, pathologists and bacteriologists have been trying to experimentally solve the question of the pathogenesis of croupous pneumonia. The authors' attempt to induce "real" pneumonia in laboratory animals by introducing virulent pneumococci by various methods: intratracheally, by inhalation, or directly into the nose after ether anesthesia (Neufeld and Kuhn) is particularly successful.

THE PROTECTIVE ACTION OF TYPE I ANTIPNEUMOCOCCUS SERUM IN MICE

Observations are reported which concern the nature of the infectious process resulting from the intraperitoneal injection of mice with virulent pneumococci. The course of the infection has been figuratively reconstructed on the basis of the following data: The rate of bacterial multiplication, the numbers of cells present in the peritoneal cavity, the character of these cells at various stages, and the rate of phagocytosis. The significant alterations in this infectious process brought about by the administration of type specific immune serum are described, and the general significance of the findings discussed with reference to the functions of the immune serum and the rôle of phagocytes in protection.

THE EXTENT OF LOCAL DISPERSION OF INFECTIOUS AGENTS AS A FACTOR IN RESISTANCE TO INFECTION

Progressively decreasing quantities of bacteria of some 20 strains were utilized in experiments upon the effect of dispersing the organisms in the rabbit skin through the agency of an extract of testicle or an invasive staphylococcus. The same was done with 6 strains of filterable viruses. The bacterial lesions were enhanced by spreading when the organisms introduced were above a certain number or quantity (minimal effective concentration) and on the other hand were partially or totally suppressed when their number was less than this. Virulence and minimal effective concentration were observed to be in inverse relationship. The lesions due to the filterable viruses studied were, on the other hand, enhanced by the spreading factor even when the quantity of virus approached the minimal infective dose. This happened irrespective of whether the virus caused severe lesions or slight ones. The highly virulent Pneumococcus Type I, injected into normal rabbits together with the spreading factor, yielded enhanced lesions even at practically its minimal infective dose; but when the resistance of the animal was raised with specific antiserum the lesions were totally suppressed by the experimental dispersion of the bacteria. When such an experiment was repeated on a filterable virus, vaccinia, no suppression took place as a result of the dispersion of the infective agent. The significance of the differences in the bacterial and virus phenomena is discussed.

THE RESPIRATION MECHANISM OF PNEUMOCOCCUS. III

From an examination of Tables I to III, and Figs. 1 to 4 and the consideration of various observations, the following facts are evident. A virulent pneumococcus on being transformed into its-avirulent form consumes many times more oxygen than the parent organism; but this gain of activity is a temporary property. After a time it degenerates into a form which consumes very much less oxygen than either the virulent or the recently derived avirulent form. In a comparative study of the metabolic functions, and oxidation products of various virulent and avirulent pneumococci, these phenomena should receive consideration. The change that takes place in the structure of the enzyme responsible for carbohydrate biosynthesis during the shift from the virulent to the avirulent form may be associated with the changes in the enzyme structure already demonstrated in connection with these metabolic studies.

Oral Administration of Pneumococcus Antigen as a Therapeutic Agent in Experimentally Infected Rats

The object of the experiments reported here was to determine whether or not rats inoculated with lethal doses of pneumococci could be successfully immunized by means of oral vaccination begun after the introduction of the infecting dose. Various investigators1 have demonstrated that young rats can be immunized against many lethal doses of virulent pneumococci by means of feeding certain pneumococcus antigens. Such experiments have all been along prophylactic lines; we have seen no reports of experiments testing the therapeutic value of pneumococcus antigens. Since it is possible to render young white rats resistant, by oral means, to comparatively large intraperitoneal or subcutaneous inocula of pneumococci, and since this resistance manifests itself within 24-48 hours, these animals were considered to be suitable for investigating the therapeutic value of oral immunization.The method consisted of intraperitoneal injection of 100 lethal doses of Type I pneumococci into young white rats (60-100 gm.), follo...

AN ANALYSIS OF THE OPSONIC AND TROPIC ACTION OF NORMAL AND IMMUNE SERA BASED ON EXPERIMENTS WITH THE PNEUMOCOCCUS

1. In normal unheated human serum, virulent pneumococci may be prepared for phagocytosis by two separate antibodies, acting in conjunction with complement. One of these is the type-specific anticarbohydrate antibody reacting with the carbohydrate fraction of the pneumococcus. The other is probably also a type-specific antibody, but quite distinct from the former, and therefore must react with a different antigenic constituent of the bacterium. 2. In the normal human serum heated to 56 degrees C., these two antibodies may, after prolonged contact with the organism, promote phagocytosis of the pneumococcus without the adjuvant action of complement. 3. Although these two antibodies are equally effective in the phagocytosis of 24 hour culture organisms by normal blood, the anticarbohydrate antibody tends to become the predominant factor as the pneumococci approach the state in which they exist in the animal body. 4. In so far as we have been able to show, the anticarbohydrate antibody is the only antibody in immune serum which can induce phagocytosis. This substance by itself is active in a phagocytic system, but just as in the normal serum, complement enhances its effect. The failure to demonstrate the presence in the immune serum of an antibody, distinct from the anticarbohydrate antibody, analogous to that found in the normal serum, may be due to the experimental difficulty of removing all the anticarbohydrate antibody from a concentrated immune serum. 5. Thus it is seen that a single well defined antibody (the anticarbohydrate antibody) may be responsible for the phagocytic action of normal unheated serum, normal heated serum, inactivated immune serum, and immune serum activated by complement. These facts appear to us to invalidate Neufeld's division of the phagocytic antibodies into (a) bacteriotropins (antibodies, the phagocytic titre of which is not raised by the addition of complement); (b) opsonic antibodies (antibodies, comparable to the lysins, which are only active in the presence of complement). 6. Complement alone is incapable of inducing phagocytosis of the pneumococcus. In the phagocytic process, it appears simply to increase the speed at which the reaction takes place. Its role may be compared to that of a catalyst in a chemical reaction. 7. On the basis of these findings, it is proposed that the term "tropin" be discarded as misleading and unnecessary, and that the term "opsonin" be retained to denote any heat-stable antibody which prepares bacteria for phagocytosis. Contrary to current usage, it would not suggest a combination of antibody with complement.

A STUDY OF THE THERAPEUTIC MECHANISM OF ANTIPNEUMOCOCCIC SERUM ON THE EXPERIMENTAL DERMAL PNEUMOCOCCUS INFECTION IN RABBITS

1. Antipneumococcic serum contains in addition to the antibodies against the various bacterial constituents, a non-antibacterial therapeutic factor. 2. The non-antibacterial factor has been separated from the antibacterial antibodies by absorption with homologous, heat-killed, virulent pneumococci. 3. The non-antibacterial factor is not type-specific; it is probably species-specific; it is not neutralized by concentrated pneumococcus broth culture filtrate. 4. The therapeutic effect of the non-antibacterial factor was demonstrated on the experimental, dermal pneumococcus infection of rabbits; this effect is demonstrable when it is administered per seor in conjunction with a certain subeffective dose of serum. 5. Evidence is presented for the assumption that the ultimate outcome of pneumococcus infection depends to a considerable extent upon this non-antibacterial factor.

THE ANTIPNEUMOCOCCUS PROPERTIES OF NORMAL SWINE SERUM

Certain interesting facts have been obtained regarding the action of normal swine serum, but in the light of present knowledge it is not easy to interpret them. It has been found that the normal serum possesses the property of protecting mice from infection with virulent pneumococci, and of agglutinating virulent S pneumococi and also avirulent R pneumococci. The protective action of the serum is specific, that is, absorption of the serum with pneumococci of one type removes or destroys only the property of the serum which is responsible for its protective action against pneumococci of that particular type, leaving the serum still active against pneumococci of other types. In this particular, therefore, the swine serum resembles a polyvalent antipneumococcus serum produced by artificial immunization. In other particulars, however, the swine serum differs from that produced byartificialimmunization. Inthefirstplace, theprotectiveaction exhibited by normal swine serum against type-specific pneumococci is very slight compared with that of animals which have been artificially immunized, even though the latter have received only a very few injections of the specific antigen. Moreover, the protective properties of the swine serum are destroyed by heating to 65 degrees C., and they disappear after a few weeks, even if the serum is kept in the cold. Also the protective action of the serum is diminished after the serum has been mixed with non-type-specific avirulent R pneumococci and the latter have been removed by centrifugalization. A further difference between the action of swine serum and that of the antipneumococcus serum produced by artificial immunization is that although the swine serum is type-specific in its action in protecting mice, nevertheless when this serum is mixed with a purified type-specific polysaccharide, neither precipitation nor fixation of complement results; and, finally, the action of the serum in protecting mice against pneumococci of a particular type is not inhibited by the addition of the homologous polysaccharide to the serum. These latter facts relating to the specific polysaccharide, and especially the fact that the specific protective action disappears after applying the so called absorption technique with non-type-specific organisms, are most difficult to harmonize with the present conceptions of the nature of pneumococcus type specificity. It is not proposed at the present time to offer any theoretical explanation of these phenomena. The studies have also confirmed the observations of Robertson and Sia, that, if a proper technique be employed, normal swine serum exhibits the property of agglutinating type-specific pneumococci, and that the agglutinins for pneumococci of one type may be specifically absorbed, leaving those for pneumococci of other types unchanged. Finally, it has been shown that swine serum causes the agglutination of avirulent R pneumococci. This property of the serum is not destroyed by heating at 65 degrees C., and therefore probably depends on factors other than those responsible for the agglutination of S pneumococci, and for the protective action of the serum in mice.