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Polysaccharide extracts of whole bacteria of the species Staphylococcus aureus, Bacillus pumilus, Lactobacillus plantarum and Streptococcus faecium were found to precipitate with Haemophilus influenzae type b (HITB) antiserum. This precipitation was due to the polyribitol-phosphate component of these bacteria reacting with the type b capsular polysaccharide antibodies of the HITB antiserum. Immunization of rabbits and burros with formaldehyde-fixed cross-reacting bacteria and blood-borne infections in humans caused by S. aureus (coagulase positive) induced precipitating and bactericidal HITB antibodies. Preliminary analysis of the HITB capsular polysaccharide revealed ribitol in addition to ribose providing an explanation for this cross-reaction. The ubiquitous distribution of Gram-positive bacteria containing polyribitol-phosphate in their cell-wall teichoic acid suggests that these bacteria may be responsible in part for the wide prevalence of antibodies to HITB capsular polysaccharide in the normal human population.

Meningococcal vaccines, composed of group A and group C polysaccharides, for group-specific prevention of meningitis are immunogenic in man but not in laboratory animals. Therefore, a control program utilizing in vitro physico-chemical, biological and serological characterization of the antigens as related to clinical potency and safety is proposed. Laboratory and clinical results on different lots of vaccines prepared by various methods have been compared. This program may serve as a model for other polysaccharide vaccines.

A capsular polysaccharide typing schema for Staphylococcus aureus , based upon the preparation of rabbit typing sera with eight prototype strains, has been reported (Karakawa and Vann, 1982). These antisera were used to classify the capsular polysaccharides of 246 S. aureus isolates from patients in a survey of hospitals in several countries and 49 consecutive blood isolates obtained over a 17-month period in a clinical study at the Boston Veterans' Administration Medical Center. Two capsular types, 5 and 8, accounted for about 70% of these isolates; most of the remaining strains could not be typed with the available antisera. The clinical study of bacteremia identified capsular types 5 and 8 among both community-acquired and nosocomial isolates and showed that strains bearing these two types caused the patterns of disease reported for staphylococcal bacteremia. There was an association between the phage type and the capsular type of these bacteremic strains. The capsular types of the “classic” encapsulated strains of S. aureus , M (type 1) and Smith (type 2), were not observed among blood isolates in this study. The observation that most clinical isolates of S. aureus belong to two recently defined capsular types provides a new focus for investigations into the virulence of this common nosocomial pathogen and suggests the potential for protective acquired immunity against staphylococcal bacteremia.

The characterization of bacterial polysaccharide antigens by molecular sizing is an important physicochemical measurement which has been correlated in some cases with clinical antigenicity. The original method developed used Sepharose® 4B; while this was adequate for the meningococcal polysaccharides then available (molecular size ∼5 × 10 5 ), it is not ideal for the larger polysaccharides developed more recently. In addition, the development of pneumococcal polysaccharide antigens has produced materials of much larger molecular size (> 10 6 ) which elute in the void volume on Sepharose 4B. Sepharose CL-2B is much better suited to characterize these antigens; however, because of the extensive work done on 4B, it is desirable to establish some correlation between the K D values observed on the two media. This is a report of experiments carried out on both media that show that the K D on CL-2B is ∼0·3 larger than that observed on 4B for a variety of bacterial polysaccharides.

The structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with that from type b Haemophilus influenzae, was determined by using a combination of chemical and spectroscopic techniques. The structure of the K100 repeating unit was found to be----3)-beta-D-Ribf-(1----2)-D-ribitol-5-(PO4----. The K100 polysaccharide is thus identical in composition to, but different in linkage from, the H. influenzae type b capsular polysaccharide, which has beta-D-Ribf-(1----1)-D-ribitol linkages.

Conjugates were prepared by carbodiimide-mediated coupling of adipic acid hydrazide derivatives of Haemophilus influenzae type b (Hib), Escherichia coli K100, and pneumococcal 6A (Pn6A) polysaccharides with tetanus toxoid (TT), as an example of a “useful” carrier, and horseshoe crab hemocyanin (HCH), as an example of a “nonsense” carrier. These conjugates were injected into NIH mice, and their serum antibody responses to the polysaccharides and proteins were characterized. As originally reported, Hib conjugates increased the immunogenicity of the capsular polysaccharide and elicited greater than the estimated protective levels of anti-Hib antibodies in most recipients after one injection and in all after the third injection (Schneerson et al., J. Exp. Med. 152 :361-376, 1980). Both Hib conjugates induced similar anti-Hib responses. The K100-HCH conjugate was more immunogenic than the K100-TT conjugate and elicited anti-Hib responses similar to the Hib conjugates after the third injection. Simultaneous injection of the K100 and the Hib conjugates did not enhance the anti-Hib response. The Pn6A-TT conjugate induced low levels of anti-Hib antibodies; when injected simultaneously with the Hib conjugates, the anti-Hib response was enhanced, as all mice responded after the first injection and with higher levels of anti-Hib than observed with the Hib conjugates alone ( P < 0.05). The Pn6A conjugates were not as immunogenic as the Hib conjugates. Pn6A-TT was more effective than was Pn6A-HCH; it elicited anti-Pn6A (>100 ng of antibody nitrogen per ml) in 6 of 10 mice after the third injection. The addition of the Hib-HCH conjugate to the Pn6A-TT conjugate increased the anti-Pn6A response with a higher geometric mean antibody titer, and 9 of 10 mice responded after the third injection. A preparation of diphtheria toxoid, TT, and pertussis vaccine increased the anti-Hib antibody levels after the first injection only in mice receiving Hib-TT, but not in mice receiving Hib-HCH, suggesting that additional carrier protein (TT) enhanced the anti-polysaccharide response. Simultaneous injection of Hib and Pn6A conjugates with the same or different carriers resulted in an enhanced serum antibody response to each polysaccharide. The anti-tetanus toxin response reached protective levels (>0.01 U/ml) in most mice after the first injection and in all mice after the second and third injections of TT conjugates. A progressive increase in the anti-HCH response with each additional injection was noted in animals receiving HCH conjugates. Animals receiving the diphtheria toxoid-TT-pertussis vaccine preparation responded with a greater increase in anti-carrier antibody than those receiving the conjugates alone. This method of synthesis provided conjugates capable of inducing protective levels of antibodies to both the polysaccharides and carrier proteins.

We have described an IgM antibody from a patient with macroglobulinemia specifically reacting with poly-alpha(2----8)N-acetyl neuraminic acid (NeuNAc) the capsular polysaccharide of two important human pathogens, group B meningococcus and E. coli K1. This antibody has a narrowly defined specificity in its interactions with polysaccharides, being unable to bind poly-alpha(2----9)NeuNAc or alternating poly-alpha(2----8)alpha(2----9)NeuNAc. However, it shows interesting crossreactivity with seemingly unrelated polynucleotides and denatured DNA, supporting the hypothesis that charged groups with a given spacing may determine the specificity of antigen-antibody interactions on otherwise dissimilar molecular structures. Despite the crossreactivity with denatured DNA and polynucleotides, the antibody does not appear to have adverse effects in the patient. The antibody protects newborn rats against E. coli K1 infection, as well as the standard horse antiserum H46, and one would expect it to prove useful in humans as an adjunct to antibiotic therapy in infections with group B meningococcus and E. coli K1. We have attempted to clone the antibody-producing cells from peripheral blood, and have shown that the relevant cells are present and can be cultured.

Rabbit and burro antisera produced by injection of an Escherichia coli (075:Kf147:H5) strain Easter reacted only with the type b capsular antigen of whole bacterial extracts of the six types of encapsulated Haemophilus influenzae and strains of H. parainfluenzae, H. hemolyticus and H. aphrophilus . The E. coli antisera had comparable agglutinin, quellung, immunofluorescence and anti type b precipitins in agar gel to H. influenzae type b typing serum. In counter current immunoelectrophoresis used to detect the type b antigen in CSF, serum and other body fluids of patients, the rabbit E. coli antisera had the same specific activity as several preparations of commercially available H. influenzae type b typing reagents. The monospecificity of this E. coli antiserum should avoid false interpretations of serologic tests among H. influenzae types due to cross-rezctions of noncapsular antigens observed at times with H. influenzae type b typing serum. Immunization with this cross-reacting E. coli provides a simple method for preparing typing serum for H. influenzae type b without the hazard of handling potentially pathogenic organisms.

In adults Hib CPS protein conjugates are much more immunogenic than the polysaccharide alone; further studies have shown that they induce a booster response in children. The antibodies produced in response to the conjugates have the same biologic properties, isotype and IgG subclass composition as those elicited by Hib CPS alone or those present in serum after convalescence from Hib disease. More recently attempts have been made to make the conjugates compatible with DTP vaccine. In this procedure DTP is absorbed onto aluminum compounds (aluminum hydroxide or phosphate), with the effect of significantly prolonging diphtheria and tetanus antibody synthesis. Adsorption of the Hib CPS conjugate under controlled conditions does not alter the total amount of antibody elicited in infant rhesus monkeys after the third or final injection. The appearance of Hib CPS antibodies after the first injection, however, is accelerated with conjugate that has been adsorbed. This is an encouraging finding, because it means that more polysaccharide conjugates can be compatible with existing DTP vaccine.

Each of several antigen-antibody systems studied has been found to affect the coagulation mechanism in the rabbit, causing a marked shortening of the coagulation time in vitro of samples of whole blood maintained in siliconized glassware. Addition of specific antigen to the blood of actively immunized animals or addition of antigen-antibody mixtures to the blood of normal animals produced the effect. The coagulation time of plasma was not affected, indicating that the phenomenon may be mediated by an effect on platelets. This effect of antigen-antibody interaction may be involved in the production of tissue damage in vivo.

The capsular substance of H. influenzae type b was isolated in a highly purified form. Serologic identity was demonstrated among the polysaccharides of seven different strains of H. influenzae type b. The polysaccharide had a molecular weight greater than 200,000 estimated by gel filtration and 152,000 by analytic ultracentrifugation and was shown to be relatively non-toxic and non-pyrogenic for laboratory animals.

Direct contact of external secretory tissue with living, but not with dead, Salmonella typhimurium stimulated formation of high concentrations of anti-“O” antibodies in the small intestinal fluid and colostrum. Purified IgG and IgA anti-S. typhimurium “O” antibodies were isolated from these fluids and their activities measured on a molar basis. Serum and exocrine IgG molecules had identical complement-dependent bactericidal, opsonization and PCA reactions. Exocrine IgA antibodies showed little or no activity in these biologic assays but did exert a similar suppressive effect upon antibody formation after intravenous injection or oral administration, as did exocrine IgG antibodies. Both IgA and IgG molecules had similar specific agglutination activities.

Ion-exchange chromatography and 8 m urea were used to purify rabbit antibodies to acidic and basic proteins and to a hapten-protein conjugate. Antigen-antibody precipitates, formed from whole immune sera, were dissolved in 8 m urea without detectable dissociation of the antigen from the antibody. The persistence of antigen-antibody complexes in 8 m urea was shown for bovine ribonuclease and bovine serum albumin and their respective antibodies. Antibody was separated from the antigen after ion-exchange chromatography in 8 m urea. The antibodies were recovered in a good yield, had high precipitating activity, and were of the immunoglobulin G class as determined by physicochemical and immunological assays. This procedure constitutes a simple general method for the purification of antibodies to charged antigens.

Purified type b capsular polysaccharide of Hemophilus influenzae induced a serum antibody response in 20 adults with pre-existing antibodies. Using absorption experiments in mouse protection and passive hemagglutination and complement-dependent bactericidal assays, the anti-type b antibodies induced by immunization had identical characteristics to “natural” and infection-induced antibodies. Further evidence for the protective quality of anti-type b antibodies was derived from the finding of an inverse relationship between the age incidence of influenzal meningitis and the percentage of individuals of different ages with serum anti-capsular antibodies. Most infants and children with disease did not have anti-capsular antibodies during the acute phase of their disease. Patients with antibody deficiency syndromes, highly susceptible to disease with H. influenzae type b, did not have detectable serum anti-type b antibodies.

Herzberg, Mendel (University of Florida, Gainesville), Kathryn V. Kenny, and John B. Robbins . Nonabsorbable rabbit anti- Salmonella typhimurium antibody as detected by the complement-mediated bactericidal reaction. J. Bacteriol. 91: 1548–1555. 1966.—A portion of antibody active in the complement-mediated bactericidal reaction against Salmonella typhimurium from hyperimmune rabbit serum has been shown to be nonabsorbable by repeated serial absorptions with whole heat-killed or living bacteria. The first two absorptions remove 90 to 95% of the activity, but 1 to 5% cannot be removed by subsequent absorptions. The nonabsorbable antibody appears to be a macroglobulin by density-gradient centrifugation and by comparison of activity and absorbability of purified γ-M and γ-G immune globulins. Alternative hypotheses involving low avidity antibody or antibody to minor cell antigenic components are offered in explanation of the phenomenon.

Human serum α1-antitrypsin has been isolated, free of albumin and other serum proteins, by DEAE cellulose and affinity chromatography, and preparative polyacrylamide gel electrophoresis. The fractionation procedures employed did not alter the antitryptic activity of the isolated proteins. α1-Antitrypsin in native serum or as the isolated material was electrophoretically heterogeneous. Also, a second, immunologically distinct, α1-antitrypsin protein, similar to that seen in mouse serum, was demonstrated in normal human serum and in the serum of patients with homozygous deficiency of α1-antitrypsin.

Rabbit gammaM- and gammaG-anti-S. typhimurium antibodies were isolated by combined immune specific and physical methods and some of their properties in immunological systems were measured. gammaM-Antibody was detectable at lower concentrations and revealed a higher specific activity than the gammaG-globulins. Indirect studies indicated that the gammaG-globulin was the more "avid" immunoglobulin. Treatment of the gammaM-globulin with the reducing agent 2-ME decreased but did not destroy the immune activity of the subunits. The results confirm the necessity of analysis of purified immunoglobulin antibodies to evaluate the significance of their biological properties secondary to their interaction with antigens.

The quantitative immunochemical determination of human serum transferrin was automated with a 100-µl sample. The estimated standard deviation from the true value was 7.9 mg/100 ml in 21 samples of serum, which had transferrin concentrations of 139-325 mg/100 ml.

An Escherichia coli antigen was shown to be cross-reactive with the capsular polysaccharide of Hemophilus influenzae type b. The cross-reactive antigen was identified as a thermostable, acidic, capsular polysaccharide consistent with its designation as E. coli “K” antigen (type A). Antibodies raised by intravenous injection of formaldehyde-treated E. coli possessing the cross-reactive antigen precipitated with the H. influenzae type b capsular polysaccharide and had “protective” activity against H. influenzae type b in biologic assays. The cross-reactive antigen was identified in E. coli with various O and H serotypes and did not appear to be associated with pathogenicity.

By intensive stimulation with large amounts of Salmonella flagellar antigen, newborn rabbits were induced to form high titer flagellar agglutinins usually by the 7th to 10th day of life. Characterization of the agglutinins at various times during the first 30 days of life revealed that the earliest antibody which appeared was a gamma-1 macroglobulin, and that 7S gamma-2 globulins did not appear until the 4th or 5th week of life. In contrast, the adult animals produced macroglobulin antibodies for only 3 to 5 days before the lower molecular weight variety appeared. The infant macroglobulin appears to be similar in all respects to adult macroglobulin antibodies. These data are interpreted to indicate that the newborn and adult rabbit differ in their response to this type of stimulus not in timing of macroglobulin antibody production, but chiefly in the prolonged interval, which precedes the development of the capacity for the 7S type response in the newborn animal.

Summary An immunoglobulin was isolated from rabbit colostrum which differed from γG-immunoglobulin in its β-electrophoretic mobility, content of unique antigenic sites, higher sedimentation coefficient (10.8S) and higher carbohydrate content (3.2% hexose and 3.2% hexosamine). The colostral immunoglobulin appeared to be homologous with γA-immunoglobulins of other species. A yield of 3 to 4.5 mg of γA-immunoglobulin was obtained per milliliter of colostrum. The rabbit γA-immunoglobulin was used to prepare a specific anti-α chain reagent in the goat. The serum concentration of γA-immunoglobulin was found to be 55 to 217 µg/ml for various rabbits by quantitative precipitation with anti-α chain. The Ab4 allotypic sites controlled by the b locus were present on γA-immunoglobulin but allotypic sites controlled by the a locus (Aa1 and Aa2) could not be identified on this protein.

Light polypeptide chains of immunoglobulin G, its fractions obtained upon chromatography in DEAE-Sephadex, as well as purified antibodies to proteins, synthetic polypetides and hapten conjugates of proteins and synthetic polypeptides, were subjected to acrylamide gel electrophoresis. The light chains from all the preparations studied resolved into six to eight distinct bands. Significant differences were found in the electrophoretic mobility of bands from light chains derived from antibodies against antigens with different net charge and from normal rabbit immunoglobulin G fractions which were separated on DEAE-Sphadex. Small differences were also observed in the relative intensities of the bands resolved from light chains of defferent preparations.

In two separate experiments, newborn rabbits were fed a live suspension of either of two Escherichia coli strains which possess a “K” antigen cross-reactive with the capsular polysaccharide of Haemophilus influenzae type b. Both feedings were harmless and resulted in fecal excretion of the fed E. coli in most animals as well as active immunization of fed animals toward H. influenzae type b. Feeding non-enteropathogenic, cross-reacting E coli to newborns may be a method for inducing active immunity toward H. influenzae type b diseases by accelerating the acquisition of “natural” immunity.

Extract: Differences in sialic acid content and not genetic polymorphism determine the observed electrophoretic heterogeneity of human lactoferrin, the iron-binding protein of external secretions. Lactoferrin molecules have at least one to four sialic acid residues. The asialo derivative is electrophoretically homogeneous.

TO account for the change in health of previously "normal" persons, characterized by susceptibility to infection and low serum immunoglobulins, the term "acquired" hypogammaglobulinemia has been proposed.1 2 3 The validity of this classification was substantiated by morphologic evidence recently offered by Charache et al.4 These workers documented the change of a normal lymphoid architecture, observed in an appendix removed in surgery, eight years before the appearance of the histologic lesion characteristic of the hypogammaglobulinemic state. To provide further data consistent with this concept of "acquired" hypogammaglobulinemia, we report a case in which, during the past four years, nearly complete loss of . . .

Employing a sensitive and immunoglobulin-specific assay method based upon antiglobulin augmentation, quantitative and qualitative aspects of the primary and secondary response of the rabbit to Salmonella typhimurium O antigens have been evaluated. These studies examine the validity of the method of assay for detecting and measuring γG- and γM-antibodies produced in response to whole organisms or its lipopolysaccharide. The results show that during the primary response γG-antibodies, not detectable by usual techniques, are produced in a pattern similar to that reported in animals stimulated by other classes of antigens. Moreover, the γG response following reinjection is characteristic of a secondary-type response. In contrast, γM-antibody levels after both primary and secondary stimulation rose equally to levels between 1 and 4 mg/ml. Despite increased sensitivity of detection and quantitative estimates of the actual molar concentration of each immunoglobulin, the minimal interval between γM and γG appearance in serum was not less than 1.5 days. The variable degree of augmentation of agglutination by antiglobulin reagent found during the immune response severely limits the quantitative usefulness of the methods developed. However, the data suggest that qualitative changes in anti-O antibodies interpretable as changes in avidity occur regularly during the immune response.

Two active components of mouse serum alpha 1-antitrypsin have been isolated by DEAE-cellulose chromatography, removal of albumin by affinity chromatography, and selective stacking in preparative polyacrylamide gel electrophoresis. Physicochemical and antigenic properties of mouse alpha 1-antitrypsin are described and compared to those of human serum alpha 1-antitrypsin. Mouse alpha 1-antitrypsin is similar to human alpha 1-antitrypsin and should be usable as a model system for future physiological studies.

A purified capsular antigen, polyribophosphate, has been prepared in a nontoxic form and found to induce serum antibodies in adults and children that have bactericidal and opsonic activity. Antibody response is age dependent, and maximum in 3 wk with a single injection. Further studies will provide a basis for an efficacy trial.

After a single injection of purified Vi polysaccharide vaccine against typhoid fever, serum titres were followed in student volunteers by passive haemagglutination assay and by radioimmunoassay. Elevated Vi antibody titres were still present after 36 months. This preliminary study should be followed by further investigations on the extent and duration of protection provided by Vi vaccine, and on volunteers in endemic areas.

A polysaccharide, antigenically related to group C meningococcus, has been isolated from Escherichia coli strain Bos-12 (016; K92; NM). Like groups B and C meningococcal polysaccharide, the Bos-12 antigen is a pure polymer of sialic acid. 13C NMR studies on the meningococcal group B and C polysaccharides have indicated that the former consists of sialic acid units linked 2 leads to 8- alpha, whereas the latter contains the sialic acid residues linked 2 leads to 9-alpha (Bhattacharjee, A.K., Jennings, H.J., Kenny, C.P., Martin, A., and Smith, I.C.P. (1975), J. Biol. Chem. 250, 1926). Comparison of natural abundance 13C NMR spectra of the Bos-12 polysaccharide with group B and C meningococcal polysaccharides established that Bos-12 was either (a) an equimolar mixture of 2 leads to 8-alpha linked sialic acid homopolymers or (b) a 2 leads to 8-alpha/2 leads to 9-alpha heteropolymer. These possibilities were distinguished in the following manner. The fact that Bos-12 polysaccharide precipitated with anti-group C serum but not with anti-group B serum would seem to exclude a. Further, chemical studies (periodate oxidation followed by tritiated NaBH4 reduction) gave saccharides with a radioactive-labeling pattern expected for alternating 2 leads to 8-alpha/2 leads to 9-alpha sialic acid linkages. Bos-12 is thus an 2 leads to 8/2 lead to 9-alpha heteropolymer.

A toxoid vaccine, composed of purified pertussis toxin inactivated with H2O2 (NICHD-Ptxd), was developed on the basis of evidence that serum neutralizing antibodies (antitoxin) would confer immunity to pertussis. In vivo and in vitro assays of NICHD-Ptxd showed only trace or nondetectable levels of pyrogenic, adenosine diphosphate-ribosyltransferase, binding and pharmacologic activities. Nevertheless, about 40% of the antigenicity of pertussis toxin was retained. Adult volunteers were injected, two times 6 weeks apart, with either 10 (n = 21), 50 (n = 25), or 75 (n = 30) micrograms/dose of one lot, Ptx-06, adsorbed onto AI(OH)3. Neither fever nor changes in the levels of leukocytes, lymphocytes, fasting blood glucose, or insulin were observed in the volunteers. The optimal immunizing dose, 50 micrograms, induced levels of antitoxin (geometric mean (GM) 302 U) comparable to those found in eight adults convalescent from pertussis (GM 269 U) and greater than those found in 18-month-old children after their fourth dose of diphtheria and tetanus toxoids and pertussis vaccine (GM 20.0 U, p less than 0.001). These data indicate that NICHD-Ptxd is safe and immunogenic in adults, and they justify its evaluation in infants and children.

Three structurally and antigenically similar capsular polysaccharides, two derived from group C Neisseria meningitidis (O-acetyl-positive and O-acetyl-negative variants) and one from Escherichia coli K92, which cross-reacts with polysaccharide from group C N. meningitidis, were compared for their ability to induce anticapsular and bactericidal antibodies to group C N. meningitidis in adult volunteers. All three vaccines elicited group C-specific serum antibodies. The vaccine derived from the O-acetyl-negative variant was the most immunogenic of the three vaccines. With use of radiolabeled O-acetyl-positive group C N. meningitidis polysaccharide antigen, the geometric mean titers of antibody in serum were 41.7 microgram/ml to the O-acetyl-negative variant, 22.8 microgram/ml to the O-acetyl-positive variant, and 7.1 microgram/ml to E. coli K92. Antibodies induced by all three vaccines were bactericidal for both of the group C N. meningitidis polysaccharide variants. An inverse relation between the comparative immunogenicity of the O-acetyl-negative polysaccharide and the virulence of group C N. meningitidis was found.

Glucose and galactose contents of pneumococcal capsular polysaccharides were measured by enzymatic oxidation following acid hydrolysis. The variables of acid hydrolysis and enzyme activities under various conditions were studied in detail for pneumococcal polysaccharide type 8 because its structure is comparatively simple and well characterized. The maximum glucose release from type 8 occurred with 4–6 n -HCl for 1 h at 100 °C (93·1% of theoretic value). Maximum galactose release occurred following hydrolysis in 1–4 n -HCl, 100 °C for 1 h (92·3–94·3% of theoretic value). The maximum glucose and galactose oxidase activities were observed at pH 7·5 and 37 °C. Glucose oxidase activity increased rapidly and proportionally up to 30 min reaction time: in contrast, galactose oxidase activity increased gradually up to 60 min. Under present experimental conditions, measured and theoretic glucose values were in close agreement for pneumococcal polysaccharide types 2, 6, 8, 9 and 19. For galactose, measured and theoretic values were in close agreement for types 6, 8, 14, 23 and 51.

Examination of 77 strains of Escherichia coli from the cerebrospinal fluid of neonates with meningitis revealed 65 (84 per cent) with the capsular (K1) polysaccharide. The Esch. coli K1 ca...

The currently United States-licensed group C Neisseria meningitidis vaccine, composed of the O-acetyl-positive capsular polysaccharide, is poorly immunogenic and does not afford protection from disease to infants and young children. Group C N. meningitidis O-acetyl-negative polysaccharide vaccine induces higher titers in adults than does the O-acetyl-positive vaccine. We compared the immunogenicity of these vaccines in 2-year-old children. Reactions were minimal and did not differ between the two vaccines. The postvaccination geometric mean titer was twofold greater in the O-acetyl-negative group (1.58 versus 0.73 micrograms of antibody per ml). The rates of decline in titer were similar in both groups. Further study regarding immunogenicity of and the anamnestic response to the O-acetyl-negative vaccine is warranted in the age group (less than 18 months) at highest risk for invasive meningococcal disease.

Studies were undertaken to gain insight into the virulence of type b in contrast to the other Haemophilus influenzae capsular types. A relationship was found between the comparative virulence of H. influenzae types in humans and their resistance to the bactericidal effect of antibody-free complement. Type b was most resistant to the bactericidal effect of complement. The other types could be divided into three groups based upon their susceptibility to complement; this grouping was also related to their structural similarities. No association between virulence and either the biotype, source of isolate, in vitro association with peripheral polymorphonuclear leukocytes, or the total amount of capsular polysaccharide was found. However, among the type b strains, higher levels of cell-associated polysaccharide were associated with increased resistance to complement. The relative virulence of the six H. influenzae types in the infant rat model was generally similar to that in humans. After intraperitoneal challenge, type b and type a strains had the lowest 50% effective doses for bacteremia, removed by several logs from the values of the other types. By intranasal challenge, type b strains produced higher rates and levels of bacteremia than did type a strains. High levels of natural bactericidal antibodies to types c and e were found in adult female rats; this finding alone could not account for the differences in virulence among the H. influenzae types in the infant rat model. We propose that the virulence of type b strains is due to their greater resistance to the bactericidal activity of serum complement alone. Resistance to type b disease requires serum antibody to induce the complement-mediated reaction.

The comparative immunogenicity of the two cross-reacting group 6 pneumococcal capsular polysaccharides, type 6A(6) and type 6B(26), was studied with hyperimmune rabbit typing antisera and with sera from adult volunteers injected with polyvalent pneumococcal vaccines containing either 50 μg of type 6A (U.S. designation, type 6) or 50 μg each of type 6A and type 6B (U.S. designation, type 26) polysaccharides. Both group 6 polysaccharides were linear copolymers composed of 1 mol each of d -galactose, d -glucose, l -rhamnose, and d -ribitol phosphate. They differed only in that type 6A had a rhammopyranosyl-(1 → 3)- d -ribitol bond and the type 6B had a rhamnopyranosyl-(1 → 4)- d -ribitol bond. Quantitative precipitation and absorption analyses with rabbit hyperimmune antisera induced by simultaneous injection with type 6A and type 6B organisms revealed extensive cross-reactions between the two group 6 polysaccharides. There was less, although still quite extensive, cross-reactivity between the two group 6 polysaccharides examined with antisera from rabbits injected with only one of the group 6 pneumococci. In a radioimmunoassay, using 14 C internally labeled type 6A or type 6B polysaccharide antigens, there was no difference in the serum antibody level to either type of volunteer injected with polyvalent pneumococcal vaccines containing type 6A or both type 6A and type 6B polysaccharides. These studies indicate that the structural similarity of the pneumococcal group 6 polysaccharides confers extensive cross-reactivity with hyperimmune typing antisera prepared with whole organisms or after injection of purified polysaccharides in adult volunteers. With our current polysaccharides, it appears that a polyvalent pneumococcal vaccine formulation that contains only type 6A will serve to induce the maximum amount of serum antibodies to both group 6 organisms.

A polysaccharide antigenically related to that of group C Neisseria meningitidis was isolated from Escherichia coli strain Bos-12 (O48: K91:NM). Like the polysaccharides of groups B and CN. meningitidis, the Bos-12 antigen was shown to be a pure polymer of sialic acid. The 13C-nuclear magnetic resonance studies of the groups B and C polysaccharides indicated that the former consists of units of sialic acid joined in alpha-2 leads to 8 linkages, whereas the latter contains sialic acid residues linked by alpha-2 leads to 9 glycosidic bonds. Chemical and nuclear magnetic resonance studies of the polysaccharide of E. coli strain Bos-12 established that it is a heteropolymer containing both alpha-2 leads to 8 and alpha-2 leads to 9 linkages. Physical parameters including partial specific volume, reduced viscosity, diffusion and sedimentation coefficients, shape, weight-average molecular weight, and the Stokes radius of the polysaccharides of groups A, B, and C N. meningitidis have been determined. The results indicate that the polysaccharides are highly asymmetric and exist in solution as rigid rods; aggregates are formed by the association of these rods. The polysaccharides appear to have "restricted" length.

IgG subclasses differ not only in their biochemical and biologic properties, but also in their occurrence as selective deficiencies, alone and in combination. The normal range of IgG subclass concentrations in children and adults is compared with deficiencies found in individuals with repeated respiratory tract infections. Concomitant IgA deficiency is often noted. It is suggested that decreased IgG2 and IgG3 levels in IgA-deficient individuals may increase the risk of recurrent respiratory infection and lung damage. Finally, the efficacy of immunoglobulin prophylaxis in reducing incidence of infection is anecdotally related.

Human neonates are uniquely susceptible to serious infections due to Escherichia coli. Investigation of the serotypes of E. coli isolated from neonates with meningitis revealed that greater than 80% of the isolates possessed the capsular polysaccharide antigen designated K1. Cultures of stool from healthy infants, children, and adults have shown that K1 organisms are commonly found in individuals of all ages. Studies of mother-infant pairs have demonstrated transmission of K1 strains from mother to infant shortly after birth. In the rare infant who develops meningitis due to E. coli K1, serotype analysis frequently reveals that the same organism was isolated from the infant's cerebrospinal fluid and from the stools of both mother and infant. Preliminary investigations of humoral immunity demonstrated an age-related acquisition of antibodies to the capsular polysaccharide of E. coli K1 in healthy infants and children. An animal model was developed in which feeding of E. coli K1 to infant rats resulted in colonization, bacteremia, and meningitis in the animals.

Bo A. Claesson, MD, Rachel Schneerson, MD, John B. Robbins, MD, Jan Johansson, MD, Teresa Lagerga rd , PhD, John Taranger , MD, Dolores Bryla, MPH, Lily Levi, MSc, Tad Cramton , MS, and Birger Trollfors, MD From the Departments of Infectious Diseases, Pediatrics, and Medical Microbiology, University of Gothenburg, the Department of Pediatrics, Boras Hospital, Pediatric Outpatient Clinic, Vastra Frolunda, Sweden, and the Laboratory of Developmental and Molecular Immunity, and Biometry Branch, National Institute of Child Health and Human DevelopmenL National Institutes of Health, Bethesda, Maryland

A method is described for typing Staphylococcus aureus capsular polysaccharides that is based on direct bacterial cell agglutination and immunoprecipitation of cell extracts with monospecific antisera. Encapsulated strains were identified by their inagglutinability with teichoic acid antisera. The typing sera reacted specifically with extracts of eight prototype strains.

The role of the Vi antigen, the capsular polysaccharide of Salmonella typhi, in the pathogenesis of and immunity to typhoid fever remains the subject of controversy. Vi-positive S. typhi resist phagocytosis and the action of serum complement, both of which actions are initiated by antibodies to Vi antigen. Both the laboratory potency in mice and the clinical effectiveness of whole-cell vaccines were related to their content of immunogenic Vi antigen. A Vi polysaccharide used for immunizing humans against experimental challenge with S. typhi failed to prevent typhoid fever; experimental conditions used to prepare this ineffective Vi antigen were shown to denature it and to reduce its immunogenicity. Assay of serum antibodies to Vi antigen with purified Vi antigen is a reliable method for diagnosis of typhoid fever and asymptomatic carriage of S. typhi. Vi polysaccharides prepared by modern techniques passed the requirements for meningococcal polysaccharide vaccines and had approximately 13 times the protective activity in the mouse potency assay as did the US Standard 6A whole-cell typhoid vaccine.

Studies were conducted on the characterization of Haemophilus influenzae type b polysaccharide (HITB-PS) and its mitogenic activity upon peripheral lymphocytes. This capsular polysaccharide was found to contain hexosamines and hexoses in addition to the main components of ribose and ribitol phosphate. The molecular weight of HITB-PS was determined as 585,000. The affinity constant of HITB-PS to unfractionated lymphocytes was 3.13 X 10(3) M-1 with 1.11 X 10(4) binding sites per cell. HITB-PS was found to be mitogenic for both human T and B lymphocytes. At optimum doses, a three to five fold increase in 3H-thymidine incorporation into T and B cells was observed. Higher than optimum doses resulted in suppression of this mitogenicity. The effect of concanavalin A (Con A) mitogenicity was detected in T and B cells treated with effective as well as suppressive doses of HITB-PS; the mitogenic activities of Con A and HITB-PS were found to be independent of each other.

Hyperimmune horse serum from a single animal (horse 46) immunized with group B (strain B-11) meningococcal vaccine provides a standardized, readily available diagnostic reagent used in primary isolation medium and for serogrouping of meningococci. Identification of the heavy-chain isotypes of specific anticapsular polysaccharide and anti-lipopolysaccharide isolated from horse 46 serum revealed a differential distribution in the occurrence of immunoglobulin classes. Meningococcal anticapsular antibodies of horse 46 serum were restricted predominately to the immunoglobulin M (IgM) class, with only trace amounts of IgGa present, whereas anti-lipopolysaccharide concomitantly produced showed a heterogeneity in its heavy-chain isotypes, consisting of IgM, IgGa, IgGb, moderate amounts of IgB, and a small amount of IgA.