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.

Capsulation of pneumococcus with soluble cell wall-like polysaccharide. II. Nonidentity of cell wall and soluble cell wall-like polysaccharides derived from the same and from different pneumococcal strains.

Methods are described for the separation of the C or cell wall polysaccharide from the C(s) or soluble C-like capsular polysaccharide of C(s) pneumococcal strains. Immunologic analysis has shown that both the C and C(s) polysaccharides of a variety of pneumococcal strains are heterogeneous and that the dissimilarities appear to reside in the mucopeptide portion of the molecule or in the region of its attachment to the teichoic acid moiety of the molecule rather than in the teichoic acid fraction. Differences of the type described have been observed in the C polysaccharides of wild-type capsulated strains of several types, in those of independently isolated noncapsulated variants derived from a single strain of a given capsular type, and in the C and C(s) polysaccharides of spontaneous mutant or transformed strains of pneumococci producing capsules of C(s) polysaccharide.

Capsulation of pneumococcus with soluble C-like (Cs) polysaccharide. I. Biological and genetic properties of Cs pneumococcal strains.

Capsulated mutants of pneumococcus producing a capsule of soluble polysaccharide related immunologically to the C or cell wall polysaccharide of pneumococcus have been isolated from several noncapsulated variants of this organism. The capsular material of these strains reacts with antisera both to homologous strains and to noncapsulated strains of pneumococcus and with human C-reactive protein. C-reactive protein has been shown to give a positive capsular precipitin or Quellung reaction with C(s) pneumococcal variants and to agglutinate them. The genetic locus which determines the production of C(s) polysaccharide is situated in a region of the pneumococcal chromosome distinct from that controlling normal capsular polysaccharide synthesis. Binary and ternary capsulated pneumococci, one of the capsular components of which is C(s) polysaccharide, have been isolated following DNA-mediated transformation.

Immunodeterminant specificity of human immunity to type III group B streptococcus.

The type III polysaccharides of group B Streptococcus in its native state chemically consists of glucose, galactose, glucosamine, and sialic acid. The core of this polysaccharide lacks sialic acid and precipitates with type III antiserum to give a partial identity with the precipitate between the native antigen and this serum. The core determinant is immunochemically similar to the capsular polysaccharide of type XIV Streptococcus pneumoniae, while the native type III group B streptococcal polysaccharide does not cross-react with type XIV pneumococcal antiserum. In human sera, it is antibody directed to the native antigen which correlates very highly with opsonic immunity (r = 0.94) while a poorer correlation exists between antibody to the core antigen and opsonins (r = 0.51 P less than 0.001). In natural infections, as association exists between low levels of maternal antibody to the native antigen and risk of disease in the infant. This association is not true for antibody to the core structure, where both infected infants and their mothers have much higher levels of antibody to the core than the native antigens. Infected infants are also more likely to respond to infection by developing antibody to the native antigen. Immunization of 12 adults with multivalent pneumococcal polysaccharide induced significantly better antibody response to the core antigen than to the native, and this vaccine induced opsonic activity in only one recipient. Immunization of adults with type III group B streptococcal antigens induced antibody to the native determinant which correlated with opsonic activity. Therefore, it would appear that native group B streptococcal polysaccharides will provide the best candidate antigens for immunization.

Correlation between G2m(n) immunoglobulin allotype and human antibody response and susceptibility to polysaccharide encapsulated bacteria.

To determine whether genetic factors influence the human antibody response to polysaccharides, we correlated Ig allotypes with the concentrations of antibody to 14 bacterial capsular antigens in 130 actively immunized Caucasian adults. The 88 individuals possessing G2m(n), an allotype antigen of IgG2 subclass heavy chains, had significantly higher postimmunization antibody levels to Haemophilus influenzae type b (Hib) and 8 of 11 pneumococcal types (P less than 0.05) than the 42 lacking this antigen. For Hib, pneumococcus type 14, and meningococcus group C, an increased response was observed in IgG class but not in IgM or IgA classes of antibody. The G2m(n) positive individuals also had higher preimmunization antibody levels to most polysaccharide antigens. Total IgG2 concentrations were correlated with the mean postimmunization antibody concentrations to pneumococci (P = 0.005), but this correlation was independent of G2m(n) by multiple regression analysis. To determine if the lack of G2m(n) was associated with increased susceptibility to infection, we compared the frequencies of various Ig allotypes in 98 children infected with Hib and 98 matched controls. Caucasian children with Hib infections other than epiglottitis were significantly more likely to lack the G2m(n) allotype than controls (P less than 0.05). G2m(n) negative Caucasian children less than or equal to 18 mo old have a 5.1-fold higher risk of nonepiglottitic Hib infections than G2m(n) positive children (P less than 0.01). We conclude that allotypic variants of the gamma-2 heavy chain genes, or genes in linkage equilibrium with them, exert a regulatory influence on the caucasian antibody response to a variety of immunologically distinct bacterial polysaccharide antigens. Young Caucasian children of the low responder phenotype, i.e., those lacking the G2m(n) allotype, are genetically predisposed to Hib and perhaps other bacterial infections.

Effect of cyclophosphamide on the immune response of BALB/c mice bearing an immunoglobulin M plasmacytoma (TEPC-183).

Mice bearing TEPC-183, an immunoglobulin (M(kappa)-secreting plasmacytoma, exhibit severe suppression of their immune responses. Since chemotherapy with cyclophosphamide (CY) is used in the treatment of myeloma, the present study was undertaken to determine its effect on the immune response. CY was tested at different doses in order to establish whether a tumor-lytic dose could be established which would minimally impair the immune response. In addition, CY was injected at different time intervals in respect to antigen administration, and its short- and long-term effects on the immune response were determined. It was found that 1 mg CY suppressed the six-day primary immune response to the type 3 pneumococcal polysaccharide SSS-III, and even a secondary response 30 days later. Three mg CY were required to suppress the primary as well as the secondary immune response elicited by 2,4-dinitrophenyl-hemocyanin. One injection of 1 mg CY per mouse resulted in complete regression of all tumors. Splenectomy did not aid in the recovery of the immune response to SSS-III but accentuated the impairment. The time of CY administration played a crucial role in affecting the immune response. When CY was administered simultaneously with or two days after antigen injection, it was totally suppressive, whereas the immune response was enhanced when CY was given four days prior to antigen injection. Minimal effects were observed when CY was given at other time intervals.

Effect of TEPC-183 plasmacytoma on resistance of passively or actively immunized BALB/c mice to infection with Streptococcus pneumoniae.

This study focuses on the effect of TEPC-183 (tetramethylpentadecane)-induced plasmacytoma on resistance of mice to infection with Streptococcus pneumoniae type III. Vaccination of normal or of TEPC-183-bearing mice with either a polyvalent pneumococcal vaccine (Pnu-Imune) or purified polysaccharide type III protects both against challenge with live S. pneumoniae. However, a 38-fold increase in susceptibility of the tumor-bearing mice over that of controls was observed. Immunized splenectomized mice were ten times more susceptible to infection than were immunized normal mice. However, this increased susceptibility of splenectomized mice could be overcome by passively administered antibody. In passive protection experiments, antisera obtained from normal and TEPC-183-bearing or from cyclophosphamide-treated mice were almost equally protective. A 6-fold increase of antibody (133 ng of antibody), however, was required to protect TEPC-183-bearing mice against challenge with 500 organisms, as compared with the 22 ng of antibody required to protect normal mice, indicative of defective host defense mechanisms in addition to the lower production of antibody in the TEPC-183-bearing mice. In contrast, mice bearing an equal tumor load of Sarcoma 37 behaved similarly to normal mice, showing that the increased susceptibility to infection was due to TEPC-183, a particularly immunosuppressive tumor.

Effect of splenectomy on the immune response of BALB/c mice bearing an immunoglobulin M plasmacytoma (TEPC-183).

Mice bearing TEPC-183, an immunoglobulin M(kappa)-secreting plasmacytoma, exhibit severe suppression of their immune responses to both thymus-dependent and thymus-independent antigens, 2,4-dinitrophenyl, and the type 3 pneumococcal polysaccharide SSS-III. This immunosuppression is not lifted by splenectomy of the tumor-bearing mice or prevented by removal of the spleen prior to tumor injection. On the contrary, splenectomy either before or after tumor implantation further accentuates the immunosuppressed state of tumor bearers and even depresses the immune response of normal mice. A secondary immune response of normal mice 34 to 51 days after splenectomy is still reduced. Thus, spleen cells may play a dual role. While splenectomy may remove a source of suppressor cells in tumor-bearing mice, it also eliminates a major source of antibody-producing cells and results in reduced immune responses of normal and TEPC-183-bearing mice. These findings have clinical relevance since splenectomy is used as a therapeutic and diagnostic procedure in neoplastic lymphoproliferative disorders.

Pneumococcal immunization in adult diabetics.

The antibody responses of 102 adult insulin-treated diabetics who received 14-valent pneumococcal polysaccharide vaccine were measured. Grand mean preimmunization antibody levels were similar for diabetics, 255 ng protein N/ml, and controls, 234 ng protein N/ml. Postimmunization, the values in the diabetics, 1009 ng protein N/ml, and 834 ng protein N/ml in 48 healthy controls, were not significantly different. The height of antibody response in the diabetic group did not correlate with age, sex, duration of diabetes, insulin dose, concentration of glycosylated hemoglobin, fasting or 2-h postprandial glucose concentrations, or the presence of retinopathy. Side effects were minimal and occurred in 26%. Antibody response to pneumococcal polysaccharide vaccine is not impaired in adult diabetics. Pneumococcal immunization is safe and may reduce the frequency of pneumonia and its complications in the diabetic population.

Response to pneumococcal vaccine in renal transplant and hemodialysis patients.

Because of the risk of serious pneumococcal infections in patients receiving a renal transplant, a study was undertaken to determine if pneumococcal vaccine could be administered before or after transplantation. Vaccine was given to recipients of transplants and to patients who were undergoing dialysis. Both groups responded to the vaccine, and although the mean antibody levels were lower than those reported for normal populations, the levels were in the range thought to be protective for most pneumococcal types. Antibody levels, both before and after vaccination, were substantially lower in patients with recent transplants than in patients who were undergoing hemodialysis. Patients who are awaiting renal transplantation can be immunized while they are undergoing hemodialysis. Further study is needed to determine how long antibody levels will persist after vaccination in both patients undergoing hemodialysis and those receiving a transplant.

Revaccination of renal transplant and hemodialysis recipients with pneumococcal vaccine.

Two years after pneumococcal vaccine was given to patients on a university renal transplant and hemodialysis service, vaccine failures began to occur. Serologic studies showed a threefold decrease in antibody levels during this period, from 913 ng of antibody nitrogen per milliliter to 315 ng/mL. The decrease was greater in patients undergoing hemodialysis than in renal transplant recipients (879 to 215 ng/mL vs 932 to 385 ng/mL). The lowest antibody levels were to types 4, 6A, and 19F. Patients were revaccinated, without serious reactions, and pneumococcal infections decreased as they had after the original vaccination program. After revaccination, there was a twofold increase in antibody levels (315 to 602 ng/mL), but the levels did not reach those seen after primary vaccination. The increase was greater in hemodialysis than in renal transplant recipients (215 to 757 ng/mL vs 385 to 536 ng/mL). This experience indicates that pneumococcal vaccines may be effective in patients undergoing hemodialysis and in asplenic renal transplant recipients, but these groups will require revaccination sooner than normal subjects to maintain immunity.

Immunogenicity of pneumococcal revaccination in patients with chronic disease.

BACKGROUND: To prevent serious pneumococcal infections, 23-valent pneumococcal polysaccharide vaccine is recommended for individuals over 24 months of age with chronic predisposing diseases and for healthy older adults. This nonrandomized controlled study in rural Alaska assessed the immunogenicity of revaccination in adults. METHODS: Twenty-six adults, 33 to 88 years of age, vaccinated a mean of 7.4 years before this study, were matched to 26 previously unvaccinated subjects by age, number of chronic diseases, sex, and ethnicity. One or more chronic diseases were validated in 62% of subjects (32 of 52). All received a first or second intramuscular dose of pneumococcal vaccine. Antibody levels were determined by radioimmunoassay for 12 pneumococcal capsular serotypes immediately before and 20 to 84 days after vaccination. RESULTS: Six to 9 years after primary vaccination, over one third of serotype-specific antibody levels were below 500 ng of antibody nitrogen per milliliter, equal to the percentage in unvaccinated subjects of similar age. Antibody levels against all pneumococcal serotypes rose to similar levels after primary vaccination and revaccination, and 54% and 55%, respectively, of subjects who received primary vaccination and revaccination had at least a 1.4-fold increase in antibody levels. Only the antibody level for serotype 4 remained low. Neither gender nor age affected peak response. For those with chronic diseases, there was a trend toward fewer low antibody levels against three or more serotypes after revaccination (two subjects [13%]) than after primary vaccination (five subjects [31%]). CONCLUSIONS: Following the initial immunization of high-risk and elderly patients with pneumococcal polysaccharide, pneumococcal antibody levels appear to wane with time. Primary vaccination and revaccination 6 or more years after a first dose of pneumococcal vaccine stimulate comparable mean antibody levels.

Pneumococcal vaccination of recipients of bone marrow transplants.

A 14-valent pneumococcal vaccine was administered to 39 recipients of allogeneic bone marrow transplants, and their type-specific antibody responses were compared with normal control subjects. Preimmunization antibody levels in patients were twofold to 12-fold lower than those levels in control subjects for all serotypes. Mean postimmunization antibody levels for each serotype were also considerably lower in patients (range, 56 to 859 ng of antibody nitrogen per milliliter) than in control subjects (range, 727 to 5,626 ng/mL). Poor antibody responses were primarily associated with early vaccination after transplantation, corticosteroid therapy for graft-v-host disease and other illnesses, and the male sex. Antibody responses of patients not given corticosteroids and vaccinated more than seven months after transplantation improved with time after transplantation. Postvaccination infection occurred in five patients who were vaccinated early after transplantation. Pneumococcal vaccination has limited potential for providing protection in marrow transplant recipients except in the cases of those patients who are not receiving corticosteroids and are vaccinated more than seven months after transplantation.

Comparison of an enzyme-linked immunosorbent assay with radioimmunoassay for the measurement of pneumococcal capsular polysaccharide antibodies.

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure antibodies against pneumococcal polysaccharides of the IgA, IgG and IgM isotypes. Antibodies against pneumococcal polysaccharide types 1, 3, 6A, 8 and 9N were measured by ELISA and radioimmunoassay. Similar antibody responses were observed when comparing both assays. The study included 39 persons at high risk of developing pneumococcal infection and 13 healthy adults. Within 1 month after immunization IgM was the principle isotype. After 1 month, IgG was the principle isotype. Very low levels of IgA were detected in the post-immunization serum. The ELISA procedure described can be used to study the immune response to pneumococcal vaccines.

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.

Antibody response to pneumococcal polysaccharides in insulin-dependent diabetes mellitus.

Twenty-one insulin-dependent diabetics and 11 healthy control children were immunized with polyvalent pneumococcal polysaccharide vaccine. Serum antibody to pneumococcal polysaccharides was measured by radioimmunoassay before and after immunization. Although there were some differences in type-specific antibody concentrations between diabetic and control subjects, the overall antibody concentrations preimmunization, 3-4 wk postimmunization, and 6-7 wk postimmunization were similar in both populations. In both groups antibody response to immunization correlated strongly with preimmunization antibody concentration. Among the diabetic subjects there was no correlation between antibody responses and duration of disease, insulin dose, or concentration of glycosylated hemoglobin. Insulin-dependent diabetic subjects have a serum antibody response to pneumococcal polysaccharides equivalent to that of controls, and in both populations the magnitude of the antibody response correlates with preimmunization antibody levels.

A radioimmunoassay for immunologic phenomena in pneumococcal disease and for the antibody response to pneumococcal vaccines. I. Method for the radioimmunoassay of anticapsular antibodies and comparison with other techniques.

A radioimmunossay is described which uses a 14C biosynthetically internally labeled antigen. This modified Farr technique has been standardized by quantitative precipitation and compared with hemagglutination and mouse protection. Specificity was established by use of heterologous hyperimmune sera and by use of unlabeled pneumococcal polysaccharides for inhibition. Reproducibility has been evaluated for different preparations of antigens and varying storage conditions of sera. The system has been applied to a wide variety of studies requiring analysis of human and animal sera.

Immunogenicity of the 23-valent pneumococcal polysaccharide vaccine in Alaska Native chronic alcoholics compared with nonalcoholic Native and non-Native controls.

PURPOSE: This study was designed to evaluate the immunogenicity of the 23-valent pneumococcal polysaccharide vaccine in Alaska Native chronic alcoholics and compare these responses with those in age- and sex-matched nonalcoholic, Native and non-Native control subjects. PATIENTS AND METHODS: Native alcoholic patients were recruited from the inpatient medical service and outpatient clinics. Healthy age- and sex-matched Alaska Native and non-Native nonalcoholics were recruited from hospital employees. At the initial visit, a standardized questionnaire, the Alcohol Dependency Scale, was administered to all participants. Participants were examined for liver diseases; blood was drawn for liver function tests and prevaccination pneumococcal antibody levels. Charts of all Native participants were reviewed for alcohol-related diseases. Participants received one dose of the 23-valent pneumococcal vaccine at the time of the initial visit and returned 20 to 55 days after immunization for liver function tests and pneumococcal antibody level measurement. Serotype-specific pneumococcal antibody levels were measured by radioimmunoassay. Logistic regression analysis was used to examine the proportion of persons whose serotype-specific antibody level doubled following vaccination. A model including adjustments for age, sex, and initial antibody level was used to examine the effect of alcohol status and ethnicity on response to the vaccine. Eighty-five persons completed the study. Of these, 41 were chronic alcoholics and 44 were nonalcoholic. Of these, 21 were Alaska Natives and 23 were non-Natives. RESULTS: Before vaccination, the geometric mean titers (GMTs) were similar in all 3 groups but were slightly higher in Native alcoholic participants for 11 of 12 serotypes tested. For 11 or more serotypes tested, 46% of alcoholics and 27% of nonalcoholics had total antibody levels at or above 500 nanograms of antibody nitrogen per milliliter (p = 0.11). After vaccination, the GMTs were higher in nonalcoholic than in alcoholic participants for serotypes 3, 7F, and 19F (p < 0.05). When Natives and non-Natives were compared, non-Natives had higher antibody levels than Native participants for 10 of 12 serotypes. After vaccination, 83% of alcoholics and 91% of nonalcoholics had pneumococcal antibody levels of more than 500 nanograms of antibody nitrogen per milliliter for at least 11 serotypes. When responses consisting of a twofold or greater increase in antibody level were compared, a greater proportion of nonalcoholics than alcoholics responded to serotypes 3, 4, 7F, 8, and 19F. This difference was significant for types 3 and 19F only (p < 0.05). In alcoholics there was a direct correlation between pneumococcal antibody level and age both before and after vaccination. This was significant before vaccination for serotypes 4, 6B, 18C, and 23F, and after vaccination for these types and for types 1 and 19F. In nonalcoholics there was a correlation between age and antibody response, following vaccination, for serotype 9N and 18C. Alcoholic males had antibody levels higher than that in females for most serotypes, but significantly so only for serotype 12F before vaccination, and for type 14 after vaccination. There were no sex differences seen among nonalcoholics, and no differences in response to vaccine could be detected in patients with or without liver dysfunction. CONCLUSION: In this study of Alaska Natives with chronic alcoholism, Native and non-Native participants responded adequately to immunization with the 23-valent pneumococcal vaccine, although significant differences in some serotypes were evident.

Increased risk of pneumococcal infections in cardiac transplant recipients.

We observed 5 episodes of pneumococcal infection among 129 cardiac transplant patients between March 1985 and December 1987, giving an estimated incidence of 36 cases per 1000 patient-years. Infections occurred a mean of 58 days after transplantation and included bacteremia with empyema, bacteremia alone, and pneumonia. All patients recovered from their infections. There was no correlation between infection and age, sex, immunosuppression, or rejection episodes. We also measured antibody levels to 12 pneumococcal antigens in 6 unvaccinated, uninfected patients before and after cardiac transplantation, to see if baseline antibody levels decreased. Protective levels of antibody were defined as greater than or equal to 300 ng of anticapsular antibody nitrogen per milliliter serum. Before transplantation patients had protective antibody levels to a mean of 8.7 +/- 1.2 pneumococcal serotypes; after transplantation, the number of presumably protective antibody levels decreased to 6.5 +/- 1.4 (P = 0.021). One of these patients subsequently developed pneumococcal pneumonia. Cardiac transplant patients are at increased risk of pneumococcal infections. Vaccinating transplant candidates prior to transplantation may provide protection after transplantation.

Bone marrow transplantation in the Wiskott-Aldrich syndrome. Complete hematological and immunological reconstitution.

A 21-month-old boy with the Wiskott-Aldrich syndrome conditioned with cyclophosphamide and dimethyl myleran received bone marrow from an HLA-matched sibling. Complete hematological chimerism was achieved. During the first 3 months after transplantation, in vitro B cell function, measured by a direct plaque assay, was abnormal, T cell helper activity impaired, and suppressor T cell function was excessive. These abnormalities resolved gradually over 16 months. Antibody responses to the T-dependent antigen, bacteriophage phi X174, were initially low, then became normal; antibody responses to keyhole limpet hemocyanin (KLH) and to 4 of 12 type-specific pneumococcal polysaccharide antigens were adequate when studied 9 months after transplantation. The clinical response was excellent: the patient has been free of infection, no longer has a bleeding tendency, and has shown normal growth and development.