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The implementation of PCR technology to study the incidence age distribution and etiology of bacterial meningitis in Burkina Faso a country that is representative of the “meningitis belt” of sub-Saharan Africa unearthed several important aspects of this disease. The study was conducted during an unusual epidemic of meningococcal meningitis in which serogroup W135 rather than serogroup A was the predominant pathogen. Population-based surveillance with PCR technology as well as with culture and capsular polysaccharide antigen detection permitted greater test sensitivity for the identification of pathogens. Whereas attention has been almost entirely focused on epidemic group A meningococcal meningitis endemic bacterial meningitis is a common disease that is caused by the same pathogens that cause bacterial meningitis in developing countries (i.e. pneumococci and Haemophilus influenzae type b as well as meningococci). (excerpt)

Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis media in children and of pneumonitis in adults with depressed resistance. Lipooligosaccharide (LOS) is a major surface antigen of NTHi and elicits bactericidal and opsonic antibodies. We prepared detoxified LOS (dLOS) protein conjugates from NTHi for use as experimental vaccines. LOS from NTHi 9274 was treated with anhydrous hydrazine and had its toxicity reduced to clinically acceptable levels. dLOS was bound to tetanus toxoid (TT) or high- molecular-weight proteins (HMPs) from NTHi through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratio of the dLOS to protein carriers ranged from 26:1 to 50:1. The antigenicity of the conjugates was similar to that of the LOS alone as determined by double immunodiffusion. Subcutaneous or intramuscular injection of the conjugates elicited a 28- to 486-fold rise in the level of immunoglobulin G antibodies in mice to the homologous LOS after two or three injections and a 169- to 243-fold rise in the level of immunoglobulin G antibodies in rabbits after two injections. The immunogenicity of the conjugates in mice and rabbits was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced LOS antibodies induced complement-mediated bactericidal activity against the homologous strain 9274 and prototype strain 3189. These results indicate that a detoxified LOS-protein conjugate is a candidate vaccine for otitis media and pneumonitis caused by NTHi.

The safety and immunogenicity of investigational conjugates, composed of the O-specific polysaccharides of Shigella sonnei and Shigella flexneri type 2a covalently bound to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), were evaluated in 192 Israeli soldiers. None had significant local reactions or fever. Fourteen days after injection, 90% of S. sonnei-rEPA recipients and 73 to 77% of S. flexneri-rEPA recipients had a fourfold or greater increase in serum immunoglobulin G (IgG) and IgA anti-lipopolysaccharide (anti-LPS) levels; at 2 years, these remained higher than at prevaccination (P < 0.01). There was a fourfold or greater increase in IgM anti-LPS in 20% of vaccinees at 2 weeks, but levels returned to prevaccination values at 6 to 12 months. IgG was the highest and most sustained class of LPS antibodies. Reinjection at day 42 did not boost antibody levels. Eighteen of 23 (78%) who received S. sonnei-rEPA and 13 of 19 (68%) who received S. flexneri-rEPA. had significant IgA-secreting cell responses. Significant IgG antibody-secreting cell responses were detected in 19 of 23 (83%) and 11 of 19 (58%) volunteers following vaccination with S. sonnei-rEPA and S. flexneri 2a-rEPA, respectively. On the basis of these data, further evaluation of the Shigella conjugates for protective efficacy in field trials in Israel was started.

Stereocontrolled, stepwise synthesis of decyl glycosides of α-(1 → 2)-linked di- to pentaglucosides (1–5) is described; these constitute fragments of Polysaccharide II of Mycobacterium tuberculosis. Phenyl 3,4,6- tri -O- acetyl -2-O- benzyl-1-thio-α- d -glucopyranoside (7) was used as the single key intermediate, obtained from 1,3,4,6- tetra -O- acetyl -2-O- benzyl-β- d -glucopyranose (6) and PhSSiMe3. Halogenolysis of 7 afforded the isolated β bromide (10) and β chloride (13). Solvolysis of 10 with decanol without heavy metal salts gave decyl 3,4,6- tri -O- acetyl -2-O- benzyl-α- d -glucopyranoside (14) in a highly stereoselective reaction, in high yield. Subsequent, iterative hydrogenolytic removal of the O-benzyl group and glycosylation with the β-chloride 13 under catalysis by silver salts afforded the protected di- to penta-saccharide glycosides 16, 19, 21, and 23, which were conventionally deblocked.

To the Editor: The limitations of the three licensed typhoid vaccines that precluded their use in children younger than five years old have been overcome by a Vi conjugate vaccine (Vi-rEPA, a conjugate of the capsular polysaccharide of Salmonella typhi, Vi, bound to nontoxic recombinant Pseudomonas aeruginosa exotoxin A [rEPA]).1–3 After 27 months of active surveillance in a double-blind, placebo-controlled, randomized trial involving 12,008 children two to five years old in the Mekong Delta region of Vietnam, Vi-rEPA was found to confer 91.1 percent protection against typhoid.3,4 Vi-rEPA induced an increase by a factor of 10 or more . . .

Pectin, a plant polysaccharide, is mostly a linear homopolymer of poly(1-->4)-alpha-D-GalpA with < 5% neutral sugars: its molecular size has a broad distribution around 400 kDa, and the degree of esterification is < 5%. The structure of the capsular polysaccharide of Salmonella typhi (Vi) differs from pectin in that it is N acetylated at C-2 and O acetylated at C-3, and has a molecular size of approximately 2 x 10(3) kDa. There is no serological cross-reaction between pectin and Vi. Pectin, when O acetylated at C-2 and C-3, is antigenically identical to Vi in double immunodiffusion. Unlike Vi, O-acetylated pectin (OAcPec) is not immunogenic in mice, probably because of its comparatively low molecular weight. After storage at 3 to 8 degrees C for 3 months, there was no change in the O-acetyl content or the M(r) of OAcPec. At 60 degrees C, the M(r) of OAcPec declined more rapidly than that of Vi. OAcPec conjugated to tetanus toxoid elicited Vi antibodies in mice, and reinjection elicited a booster response. The levels of Vi antibodies elicited by OAcPec-tetanus toxoid conjugates were lower than those elicited by Vi conjugates, but these differences were not statistically significant. OAcPec has some advantages because it can be measured by standardized colorimetric assays and because it forms more soluble conjugates with proteins than does Vi. One disadvantage is that its glycosidic bond is not as stable as that of Vi. The use of a plant polysaccharide, pectin, as an immunogen for prevention of a systemic infection caused by a capsulated pathogen (S. typhi) provides a novel approach to improve the preparation and immunogenicity of polysaccharide-based vaccines.

Clinical and serum antibody responses following intramuscular injection of two formulations of Salmonella typhi Vi capsular polysaccharide (Vi) were assessed in a double-blind evaluation. Healthy adults were randomly assigned to receive a 25 micrograms dose of liquid (Vi-Liq; n = 182) or freeze-dried Vi vaccine (Vi-Lyoph; n = 55), or placebo (n = 86). Erythema and/or indurationor = 1 cm in diameter at the injection site developed in 13/182 (7%) of Vi-Liq and 3/55 (5%) of Vi-Lyoph recipients (not significant, n.s.). Fever (oral temperatureor = 100 degrees F (37.8 degrees C)) occurred in2% of vaccinees. The frequencies of rises of fourfold or greater and of maximal Vi antibody levels were similar in the two vaccine groups. Fourfold or greater rises in serum Vi antibody levels (RIA) developed in 53% of Vi-Lyoph and 60% of Vi-Liq recipients by 1 week (n.s.), and 98 and 93%, respectively, by 1 month (n.s.). The frequencies of adverse reactions and mean Vi antibody levels following booster immunization with Vi-Liq 27 to 34 months after primary immunization (n = 55) were similar to those observed following primary immunization, although subjects given a booster dose were more likely to develop local reactionsor = 1 cm in diameter than those given a first dose (10/55 versus 13/182, p = 0.013 by the chi 2 test). Primary and booster immunizations with the Vi vaccines are well tolerated in healthy adults; mean Vi antibody levels remain significantly elevated for up to 34 months after primary immunization.

One hundred forty-five infants were vaccinated with 25 micrograms of pertussis toxoid (NICHD-Ptxd) at 3, 5 and 7 or at 3, 5 and 12 months of age. One month after the third vaccination all had high serum IgG and neutralizing antibodies (antitoxin) against pertussis toxin. Vaccination at 3, 5 and 12 months resulted in higher antibody titers than vaccination at 3, 5 and 7 months. Sera obtained from 109 children at 3 years of age showed a decline of antibodies, but all had detectable antibodies. Adverse reactions were confined to local redness and swelling, which exceeded 2 cm after 17% of all injections. When the children were 3 years old, a comparison was made of the incidence of clinical pertussis in 142 of the 145 vaccinated children and in 284 age-matched controls living in the same areas. Information on symptoms of pertussis was obtained from the parents during telephone interviews. None of the vaccinated children had clinical pertussis, defined as a 6-week course of paroxysmal cough with whooping attacks or vomiting, whereas 57 controls (20%) had experienced these symptoms. Sixteen vaccinated children were exposed to pertussis in the household. Two of them had laboratory-verified Bordetella pertussis infections with cough of 2 and 4 weeks, respectively, without whooping attacks or vomiting, whereas 14 did not develop a cough. The study shows that NICHD-Ptxd is immunogenic in infants and that it most likely confers a high degree of protection against pertussis.

The objective of a vaccine for pertussis is to prevent the paroxysmal cough with its complications. Prevention of the paroxysmal cough should prevent transmission of B. pertussis. Clinical studies indicate that a critical level of antitoxin confers protection against pertussis and that the long-lived protective immunity that follows pertussis is best explained by the presence of antitoxin. Vaccine-induced protective immunity can be mediated by a critical level of antitoxin alone. In addition to preventing pertussis with vaccines, we predict this level of antitoxin will also exert epidemiologic control (herd immunity) by inhibition of colonization with B. pertussis and by prevention of the paroxysmal cough which will reduce transmission of this pathogen. Lastly, a pertussis toxoid need not have detectable pharmacologic activity to exert its protective actions: residual activity could exert a deleterious effect on glucose metabolism and exert immunomodulating effects. Accordingly the new pertussis vaccine should contain inactivated pertussis toxoid alone and there is no need to include other components. Several candidates could serve as a satisfactory pertussis toxoid. The admonition to physicians given by Hippocrates, Primum non nocere (first of all do no harm), should be heeded by those responsible for the development and use of the new pertussis vaccine.

O -(2,4-Di- O -chloroacetyl-α- l -rhamnopyranosyl)-(1 → 2)- O -(3,4,6-tri- O -benzoyl-α- d -galactopyranosyl)-(1 → 3)- O -(2-acetamido-4,6-di- O -acetyl-2-deoxy-α- d -glycopyranosyl)-(1 → 3)-2,4-di- O -benzoyl-α- l -rhamnopyranosyl trichloroacetimidate ( 1 ) was synthesized in a stepwise manner, using the following monosaccharide units: 2-(trimethylsilyl)ethyl 2,4-di- O -benzoyl-α- l -rhamnopyranoside, 2-azido-4,6- O -benzylidene-3- O -chloroacetyl-2-deoxy-β- d -glycopyranosyl chloride, methyl 3,4,6-tri- O -benzoyl-2- O -(4-methoxybenzyl)-1-thio-β- d -galactopyranoside, and 2,4-di- O -benzoyl-3- O -chloroacetyl-α- l -rhamnopyranosyl chloride. Compound 1 corresponds to a complete tetrasaccharide repeating unit of the O-specific polysaccharide of the lipopolysaccharide of Shigella dysenteriae type 1.

Two clinical trials of the Vi vaccine provided evidence that serum antibodies to the capsular polysaccharide of Salmonella typhi confer protective immunity to typhoid fever. The immunogenicity of the Vi has been improved by binding this polysaccharide to a protein. in laboratory animals, this conjugate had increased immunogenicity compared to the Vi alone and elicited booster responses. Because there are as yet no vaccines for non-typhoidal Salmonellae, we synthesized a non-toxic conjugate of the O-SP of Salmonella typhimurium O:4,12 which conferred protective immunity in the murine model at doses and routes of immunization which are clinically acceptable. The rationale for developing conjugate vaccines for prevention of other enteric bacterial infections is discussed.

Immunoprophylaxis for bacterial enteric diseases is hindered because the protective immune mechanism(s) against nontyphoidal salmonellae or shigellae in humans are not established. On the basis of the similarities between the clinical signs, epidemiology, pathogenesis, and pathology of as well as protective immunity to salmonellae and shigellae, we propose that serum IgG antibodies to the O-specific polysaccharide (O-SP) of their lipopolysaccharides (LPSs) will confer protective immunity to these two pathogens. Critical to this notion is that (1) the virulence of these two pathogens requires full expression of their LPS; (2) active or passive immunization with serum IgG O-SP antibodies confers protection of mice against Salmonella typhimurium (there are no comparable data for humans); and (3) in humans, convalescence from shigellosis confers type (O-SP) -specific protective immunity, and indirect evidence shows a correlation between the level of serum LPS antibodies and resistance to shigellosis. We designed conjugate vaccines to elicit high levels of long-lived serum IgG O-SP antibodies to nontyphoidal salmonellae and shigellae to test this hypothesis.

Meningitis and other systemic infections caused by group B Neisseria meningitidis and Escherichia coli K1 remain important problems. The capsular polysaccharides (CPs) of these pathogens (poly[(2----8)-alpha-N-acetylneuraminic acid] or poly(alpha 2-8NeuNAc] are identical and are virulence factors and protective antigens for both. CP vaccines for these pathogens are not available because poly(alpha 2-8NeuNAc) alone, as a complex or a conjugate, is poorly immunogenic. Because oligomers of poly(alpha 2-8NeuNAc) in fetal brain and other tissues bind antibodies in vitro, it has been suggested that antibodies to this CP might be pathologic. We synthesized conjugates of this CP with tetanus toxoid under conditions that avoid lactone formation. Using this scheme, we also synthesized conjugates of group C meningococcal CP (poly[(2----9)-alpha-N-acetylneuraminic acid] or poly(alpha 2-9NeuNAc] and of E. coli K92 CP [poly(alpha 2-8, alpha 2-9NeuNAc)]. When injected s.c. in saline into mice, conjugates of poly(alpha 2-8NeuNAc) or poly(alpha 2-9NeuNAc) elicited homologous antibodies. E. coli K92 conjugates elicited both poly(alpha 2-8NeuNAc) and poly(alpha 2-9NeuNAc) antibodies. Both components of the conjugates expressed T-dependent immunologic properties under conditions and dosages acceptable for clinical evaluation. Poly(alpha 2-8NeuNAc) antibodies elicited by the homologous or the K92 conjugates had lower binding activities at 37 degrees C than at 22 degrees C. "Natural" poly(alpha 2-8NeuNAc) antibodies were present in almost all matched pairs of human maternal and cord sera; most cord levels were higher than in corresponding maternal sera. These findings suggest that increased levels of poly(alpha 2-8NeuNAc) IgG antibodies elicited by our conjugates will confer protective immunity to group B meningococci and E. coli K1 and will not be pathologic.

Eighty-five children received three injections of a vaccine consisting of Haemophilus influenzae type b (Hib) capsular polysaccharide (CPS) conjugated to tetanus toxoid (TT) (Hib-TT) at 3, 5 and 12 months of age according to the vaccination schedule for Swedish children. Diphtheria-tetanus toxoid vaccine was concurrently injected at another site. Two dosages, 7.5 and 15 micrograms, of Hib CPS were studied. No serious reactions occurred. Hib-TT elicited fewer local reactions than diphtheria-tetanus toxoid vaccine. Significant increases in Hib CPS serum antibodies occurred after all injections in both dosage groups with virtually no differences between the two groups. After the first and second injections geometric mean serum antibody concentrations of both dosage groups combined increased to 0.49 and 3.71 micrograms/ml and 81 and 99% of the vaccinees, respectively, had concentrations greater than 0.15 micrograms/ml. After the third dose geometric mean concentrations increased to 13.7 micrograms/ml and all had concentrations greater than 0.15 micrograms/ml. The geometric mean Hib CPS antibody concentrations decreased to 1.24 micrograms/ml 18 months after the third injection, but 97% still had concentrations greater than 0.15 micrograms/ml. The rise of Hib CPS antibodies was mostly in the IgG class. The most pronounced increase was seen in the IgG1 subclass but there were also increase in IgG2 and IgG3. Protective concentrations of TT antibodies were found in all postimmunization sera. In conclusion Hib-TT is safe and immunogenic in infants and should be protective from 6 to 30 months and probably longer thereafter.

Only indirect evidence has been cited to document that lipopolysaccharide-mediated virulence at the bacterial level and serum antibodies to the O-specific side chain of the lipopolysaccharide molecule may prevent shigellosis. Our proposed use of the B subunit of Shiga toxin as a carrier protein is based upon evidence (even more indirect) that serum antitoxin may reduce the severity of dysentery and diarrhea. Because animal models of disease may provide information inapplicable to the prediction of vaccine-induced protective immunity, we suggest that clinical trials in the population at risk should be started after successful completion of the safety and immunogenicity phases of vaccine development in laboratory animals and in the target population. Clinical studies of shigella vaccines are difficult because of the many causes of dysentery in a population with a high rate of intestinal disease. The component(s) of shigellae (and related bacteria) and of the host immune system that confer protection against enteric diseases caused by these microorganisms remain unclear. That prevention is a worthwhile objective in both developing and industrialized nations is clear from the high frequency, morbidity, and mortality associated with shigellosis in individuals of all ages [1]. Most investigators have concluded that shigellosis is confined to the intestinal tract, without systemic involvement. Efforts at inducing mucosal immunity with attenuated strains of Shigella are under way [2-4]. We are developing vaccines for the prevention of shigellosis that are composed of the O-specific side chains of Shigella lipopolysaccharide (LPS) covalently bound to Shigella toxins; these vaccines are to be administered by injection. This experimental approach has been derived from our experience with the Vi antigen of Salmonella typhi as a vaccine for typhoid fever [5-8]; from the studies by Svenson and Lindberg [9] and by Carlin et al. [10-12] with 0-specific side chains of S. typhimurium and S. flexneri; from the studies by workers at the Walter Reed Army Institute of Research on the genetics of shigellae [13, 14]; and from the experiments by Branham and her colleagues [15, 16] and by Morgan and Schiitze [17] on S. dysenteriae type 1 infection of mice.

The safety and immunogenicity of Haemophilus influenzae type b-tetanus toxoid conjugate vaccine (Hib-TT) were evaluated in 77 healthy infants receiving injections at 3, 5, 7, and 18 months of age. No serious local or systemic reactions were noted. After the first injection the geometric mean Hib antibody level rose to 0.55 micrograms/ml, and each subsequent injection elicited a statistically significant rise in the geometric mean. The percentage of vaccinees with Hib antibody levels greater than 0.15 micrograms/ml serum was 75.5% after the first, 97.4% after the second, and 100% after the third Hib-TT injection. This percentage fell to 90.9% at 18 months of age but rose again to 100% after the fourth injection. Control infants (n = 10) injected with diphtheria-tetanus toxoid-pertussis vaccine only had nondetectable levels after the second injection. Hib-TT elicited increases of Hib antibody in all isotypes: IgG greater than IgM greater than IgA. Among IgG subclasses the highest increases were of IgG1. All vaccinated subjects had greater than 0.01 U/ml of TT antibody (estimated protective level) throughout the study. We conclude that Hib-TT, injected at 3, 5, 7, and 18 months, is safe and induces protective levels of antibodies during the age of highest incidence of meningitis caused by Hib.

Conjugates of an uronic acid-containing capsular polysaccharide (CP), pneumococcous type 12F (Pn12F) bound to diphtheria toxoid (DT), were studied for safety and immunogenicity in adult volunteers. In mice, these conjugates, prepared with the same lot of DT and Pn12F-40234-006, a homogenous CP of high molecular weight, or Pn12-812408, a polydisperse CP with lower-molecular-weight material, were more immunogenic than the Pn12F alone and had T-cell dependent properties (A. Fattom, W. F. Vann, S.C. Szu, A. Sutton, X. Li, B. Bryla, G. Schiffman, J. B. Robbins, and R. Schneerson, Infect. Immun. 56:2292-2298, 1988). Adult volunteers, randomized into three groups, were injected either with one of these two conjugates or with Pnu-Imune, the 23 valent pneumococcus vaccine containing 25 micrograms of Pn12F as one of its components. Volunteers were injected two times, 4 weeks apart, with the Pn12F-DT conjugates and once with the Pnu-Imune. Side reactions following injection of the conjugates of Pnu-Imune were mild and short-lived. At 4 weeks and at 7 months after the first injection, higher levels of Pn12F antibodies were found in the volunteers injected with the conjugates than in the Pnu-Imune group (P less than 0.001). The conjugate prepared with the higher-molecular-weight Pn12F elicited higher levels of antibodies than the conjugate prepared with a lower-molecular-weight Pn12F preparation (P = 0.05). Both conjugates elicited about a 13-fold rise in DT antibodies.

Landsteiner1 and then Avery and Goebel2 discovered how to increase the ability of saccharides (haptens) to elicit antibodies by chemically binding them to proteins to form conjugates. About three q...

Safety and Immunogenicity of Experimental Shigella O-Specific Polysaccharide Conjugate Vaccines in Children • 754

The safety and immunogenicity of Haemophilus influenzae type b (Hib) capsular polysaccharide (CPS) alone, or covalently bound to tetanus toxoid in saline solution (Hib-TT) or adsorbed onto AI(OH)3 (Hib-TT ads), were evaluated after one injection into 18- to 23-month-old healthy children in Sweden. No side reactions were elicited by Hib CPS; side reactions elicited by the two conjugates were similar and comparable to those reported for diphtheria and tetanus toxoids adsorbed. Hib-TT was the most immunogenic of the three vaccines, eliciting about 10-fold higher antibody levels than Hib CPS; of 28 vaccinees, all had greater than 1.0 microgram Ab/mL serum after immunization with Hib-TT. Increases of Hib CPS antibodies within immunoglobulin classes induced by the three vaccines were, in decreasing order, IgG greater than IgM greater than IgA. Within IgG subclasses, rises in IgG1 Hib CPS antibodies were the most frequent, followed by IgG2; some vaccinees with high postimmunization levels also had rises in IgG3 and one in IgG4. Immunization-induced Hib CPS antibodies were bactericidal. Hib-TT also elicited higher levels of tetanus toxoid antibodies than Hib-TT ads; these tetanus toxoid antibodies neutralized tetanus toxin in vivo.

Levels of antibodies against Haemophilus influenzae type b capsular polysaccharide were determined in acute-phase and convalescent-phase serum samples obtained from 21 children with invasive H. influenzae type b infections and from 44 children vaccinated with two H. influenzae type b vaccines. Amounts of immunoglobulin G (IgG), IgM, and IgA antibodies were measured by direct and indirect enzyme-linked immunosorbent assay (ELISA), and the total amount of antibodies was measured by radioimmunoassay (RIA). Results obtained by ELISA were calculated by multiple-point parallel-line comparison and by endpoint analysis. A very good correlation was obtained between direct and indirect ELISA values. In the lower range of antibody concentrations, the correlation between ELISA values obtained by endpoint analysis and those obtained by multiple-point parallel-line comparison was poor, since the latter method of calculation yielded values of up to 1 microgram/ml in sera that were negative according to endpoint analysis. These sera with negative endpoint titers also had undetectable or very low antibody concentrations as measured by RIA. Consistent with this finding, in acute-phase and prevaccination sera with undetectable or low antibody concentrations as measured by RIA, ELISA values calculated by multiple-point parallel-line comparison were much higher. In sera with higher antibody concentrations, however, parallel-line comparisons showed good correlation between RIA and ELISA values. Although no reference method for measuring true antibody concentrations is available, ELISA values as calculated by multiple-point parallel-line comparison appear to overestimate antibody concentrations in sera containing low antibody concentrations, whereas ELISA values obtained by endpoint analysis are less well correlated with RIA values at higher concentrations.

The conclusions from our studies to date may be summarized as follows. (1) Invasive E. coli strains causing neonatal meningitis are encapsulated. At least 80% of those strains inducing mengitis are K1 and approximately 40% of those strains isolated from infants with septicemia but without meningitis are also K1. Invasiveness is best related to the K1 antigen and not to E. coli O and H antigens. (2) The capsular content of CSF strains is not related to their invasiveness. In contrast to observations reporting higher K capsular polysaccharide content and molecular weight of E. coli invading the renal parenychma as compared with those E. coli confined to the bladder or in the stool, there were no differences among DSF K1 strains. Sepculation as to the mechanism of the invasive properties conferred by acidic capsular polysaccharides may be derived from the literature. Unencapsulated or "rough bacteria" are susceptible to the bactericidal action of agammaglobulinemice sera (15, 53). When injected into precolostral (agammaglobulinemic but complement containing), cesarian-delivered, and antigen-deprived piglets, unencapsulated bacteria are rapidly cleared from the circulation. In contrast, smooth bacteria injected into these same animals circulate without detectable splenic or hepatic clearance, multiply, and result in the death of these animals. The mechanism of the resistance of encapsulated bacteria has been postulated to be due to the inaccessibility of the deep somatic antigen structures capable of activating the alternate complement pathway system. Thus, opsoninization and other host complement-dependent activities may of necessity be antibody mediated for encapsulated bacteria. This complement resistance of encapsulated organisms may be quanititative and studies should be done to determine differences among various K1 E. coli strains. (3) K1 strains are widely prevalent among infants, children, and adults and are quickly transmitted to infants. In most cases the source of the infecting strain in diseased infants is the mother. However, transmission from attendants, demonstrable in our studies, is also a possible mechanism. (4) A protective role of serum anticapsular antibodies in animal models has been demonstrated. Our initial observations indicating low serum K1 antibodies in the general population and the finding that K1 antibodies are predominantly IgM in two animal species studied so far suggest that colostral K1 antibodies may be important in conferring immunity to this disease.

Serum Haemophilus influenzae Type b (HITB) anticapsular antibodies were induced in adult volunteers by feeding of either of two strains of a cross-reacting Escherichia coli 075:K100:H5. In all the fed volunteers colonization for a finite period lasting up to eight weeks occurred, without adventitious reactions. Nine of 14 normal volunteers and one of two volunteers previously immunized with the Type 6 H. influenzae polysaccharide responded with a greater than twofold increase in serum Type 6 H. influenzae antibodies. These antibodies induced by Esch. coli were specific for the capsular polysaccharide and had bactericidal activity. The safety of this procedure, the comparable results in laboratory animals and the identification of other cross-reacting strains of Esch. coli with meningococcal and pneumococcal capsular polysaccharides suggest that colonization with these nonpathogenic organisms at birth may provide a general method of preventive immunization to diseases caused by encapsulated bacter...

We studied 174 strains of the genus Bacillus for cross-reacting antigens to the capsular polysaccharides of groups A and C meningococcus, types I and III pneumococcus, and Haemophilus influenzae type b. Cross-reactions were detected by immunodiffusion in agarose gel by using type-specific antisera and confirmed by absorption and inhibition experiments. Of 20 Bacillus pumilis strains, six had an antigen cross-reacting with group A meningococcal polysaccharide. Other cross-reactions included one strain of B. pumilis with H. influenzae type b, one of B. cereus var. mycoides with pneumococcus type III, and one of B. alvei with both type b and SIII polysaccharides. These cross-reacting antigens are polysaccharides of vegetative cells and may be extracellular in location. Because these bacilli have antigens cross-reacting with the virulence factors of pyogenic bacteria, they may, as normal flora, be an antigenic stimulus for “natural” serum anti-capsular antibodies to the type b Haemophilus and group A meningococcus polysaccharides.

The effect of molecular weight or size of the components on the immunogenicity of polysaccharide-protein conjugates prepared with the native Vi capsular polysaccharide (Vi) (approximately 3 x 10(3) kilodaltons) or lower-molecular-weight Vi (Vis; approximately 46 kilodaltons) abound to cholera toxin (CT) or to its B subunit (CTB) was studied. In mice, Vi-CT, Vi-CTB, and Vis-CTB elicited higher Vi antibody levels than the Vi alone (P less than 0.0001). Vi-CT and Vi-CTB were more immunogenic than Vis-CTB (P less than 0.01). CT or Vi-CT elicited higher levels of CT antibodies, as measured by enzyme-linked immunosorbent assay, than did CTB or Vi-CTB. In rhesus monkeys, the Vi conjugates elicited higher Vi antibody levels than the Vi alone (P less than 0.01). Vi-CTB elicited higher levels of Vi antibody after each injection than did Vis-CTB. Similar levels of CT antibodies, as measured by enzyme-linked immunosorbent assay, were elicited by all three conjugates. In contrast, Vi-CT elicited higher levels of neutralizing antibodies than Vi-CTB or Vis-CTB when either CT or the related heat-labile toxin of Escherichia coli was used as the antigen. These results indicate that the holotoxin and the native Vi provide the most immunogenic components for conjugates designed to induce both Vi and CT antibodies.

Group 9 organisms (types 9N, 9A, 9L, and 9V) account for about 3 to 4% of pneumococcal disease isolates throughout the world. Types 9N and 9V comprise about 90% of the group 9 disease isolates. Type 9N is more common than type 9V in adults, and type 9V predominates in infants and children. In the United States there have been eight reported cases due to group 9 pneumococci in individuals previously vaccinated; six were type 9V and two were type 9N. To ascertain the cross-immunogenicity of group 9 polysaccharides, volunteers were injected with vaccines of monovalent types 9N, 9A, 9V, or 9L, or bivalent (9N and 9A) or trivalent (9N, 9A, and 9V) polysaccharide vaccines. Monovalent types 9N, 9V, and 9L each stimulated a 5.8- to 7.5-fold geometric mean rise, and at least 80% of the volunteers responded with a twofold or greater homologous antibody rise. Type 9V induced a 5.8-fold geometric mean rise, but only 66% of the volunteers responded with a twofold or greater homologous antibody rise. Type 9N induced only a 2.1-fold geometric increase, and only 54% of the volunteers responded with a twofold or greater rise in anti 9V antibodies. Types 9L and 9A were the most cross-immunogenic. The trivalent preparation (9N, 9A, and 9V) gave the highest geometric mean titer and seroconversion rate to each of the group 9 polysaccharides. These results suggest that the polyvalent pneumococcal vaccine with its type 9N does not induce a satisfactory anti-type 9V response and should contain additional components in order to achieve greater protection against group 9 organisms.

We conducted a pilot study followed by a large clinical trial in Nepal of the use of the capsular polysaccharide of Salmonella typhi (Vi) as a vaccine to prevent typhoid fever. In the pilot study, involving 274 Nepalese, there were no significant side effects of the Vi vaccine; about 75 percent responded with a rise in serum antibodies of fourfold or more. In the clinical trial, residents of five villages were given intramuscular injections of either Vi or, as a control, pneumococcus vaccine dispensed in coded, randomly arranged, single-dose syringes. There were 6907 participants, of whom 6438 were members of the target population (5 to 44 years of age); each was visited every two days. Those with temperatures of 37.8 degrees C or higher for three consecutive days were examined and asked to give blood for culture. Typhoid was diagnosed as either blood culture-positive or clinically suspected on the basis of bradycardia, splenomegaly, and fever, with a negative blood culture. Seventeen months after vaccination, the codes were broken for the 71 patients meeting the criteria for either culture-positive or clinically suspected typhoid. The attack rate of typhoid was 16.2 per 1000 among the controls and 4.1 per 1000 among those immunized with Vi (P less than 0.00001). The efficacy of Vi was 72 percent in the culture-positive cases, 80 percent in the clinically suspected cases, and 75 percent in the two groups combined. These data provide evidence that Vi antibodies confer protection against typhoid. Surveillance continues to determine the duration of Vi-induced immunity.

Ultrasonic irradiation (u.i.) has been used to depolymerize biopolymers including DNA, dextran, and the Vi capsular polysaccharide from Citrobacter freundii . Representative bacterial polysaccharides were subjected to u.i. and the effect of this energy upon their molecular weight and chemical structure was characterized. U.i. depolymerized a neutral polysaccharide (dextran) and acidic polysaccharides containing either a phosphoric diester linkage [ Haemophilus influenzae type b (Hib) and pneumococcus types 6A and 6B] or a uronic acid moiety (pneumococcus type 9N). Prolonged u.i. depolymerized all the polysaccharides to a finite and similar molecular mass (∼50 000 daltons). The rate of depolymerization induced by u.i. depended on the viscosity of the solvent and the concentration of the polysaccharide. 13 C-N.m.r. data of the native Hib polysaccharide and its depolymerized products indicated that u.i. did not alter the chemical structure of the repeating units. Determination of the monophosphate terminal residues by 31 P-n.m.r. spectroscopy and of the reducing end groups by the Park-Johnson reaction indicated that both the phosphoric diester and the glycosidic linkages were cleaved. The Vi polysaccharide, prepared as an investigational vaccine, could not be analyzed for its chemical structure by 13 C-n.m.r. spectroscopy owing to its high viscosity but depolymerization by u.i. permitted this analysis. The finite molecular weight of the products observed after prolonged u.i. is best explained by the postulation that the mechanical torque necessary to rupture the linkages is dependent upon the length of the polysaccharide. The method of u.i. for depolymerization is useful for the preparation of homogeneous, low-molecular-weight polysaccharides without alteration of the chemical structure of the repeating units.

A method is presented for covalently bonding Haemophilus influenzae type b capsular polysaccharide (HIB Ps) to several proteins. The method is efficient and relies upon the use of adipic dihydrazide as a spacer between the capsular polysaccharide and the carrier protein. In contrast to the poor immunogenicity of the purified HIB Ps in mice and rabbits, the HIB Ps-protein conjugates induced serum anti-type b antibodies having bactericidal activity at levels shown to be protective in humans when low doses were injected subcutaneously in a saline solution. The antibody response in mice was related to the dose of the conjugates, increased with the number of injections, and could be primed by the previous injection of the carrier protein. The HIB Ps-protein conjugates were immunogenic in three different mouse strains. The importance of the carrier molecule for the enhanced immunogenicity of the HIB Ps-protein conjugates was shown by the failure of HIB Ps hybrids prepared with either the homologous polysaccharide or pneumococcus type 3 polysaccharide to induce antibodie in mice. Rabbits injected with the HIB Ps-protein conjugates emulsified in Freund's adjuvant produced high levels of serum anti-type b antibodies which induced a bactericidal effect upon H. influenzae type b organisms. It is proposed that the HIB Ps component of the polysaccharide protein conjugates has been converted to a thymic-dependent immunogen. This method may be used to prepare protein-polysaccharide conjugates with HIB Ps and other polysaccharides to be considered for human use.

The pathogenesis of bacterial meningitis was studied in infant rats. Intranasal intoculation of greater than 10(3) Haemophilus influenzae type b resulted in an incidence of bacteremia that was directly related to the size of hte challenge inoculum. The temporal and quantitative relationship of bacteremia to meningitis indicated that bacteria spread to the meninges by the hematogenous route and that the magnitude of bacteremia was a primary determinant in the development of meningitis. In a sparate series of experiments, infant rats that were fed Escherichia coli strain C94 (O7:K1:H-) became colonized and developed bacteremia and meningitis, but invasive disease was rare when rats were fed E. Coli strain Easter (O75:K100:H5). A comparison of intranasal vs. oral challenge indicated that the nasopharynx was the most effective route for inducing H. influenzae bacteremia, whereas the gastrointestinal route was the more effective challenge route for the E. coli K1 serotype.

A quantitative immunoelectrophoretic method, using one-dimensional electrophoresis into antibody-containing agarose gels was standardized for measurement of the individual pneumococcal capsular polysaccharides in the final polyvalent container. The method is simple, rapid, inexpensive and provides both a qualitative and quantitative method for control of the polyvalent vaccine. The addition of Triton X-100 for type 7F(51) and treatment with galactose oxidase for type 14 were necessary modifications for extending this assay system to these two neutral polysaccharides.

Concentrated culture filtrate of Escherichia coli O78:H11, strain H10407, was applied to an affinity column prepared with IgG antibodies to the toxin of Vibrio cholerae. Elution of the retained material with 3 M KCNS yielded a nonenterotoxic protein that precipitated with antiserum to V. cholerae toxin and had three major protein components on sodium dodecyl sulfate gels. After treatment with 2-mercaptoethanol, two protein components were observed. Elution of the affinity column with 5 M guanidine yielded an enterotoxic protein that precipitated with antiserum to V. cholerae toxin. After treatment with 2-mercaptoethanol, only one protein component, with mobility identical to that of the slower component of the eluate (treated with 3 M KCNS and 2-mercaptoethanol), was observed.

Five cases of HITB maningitis occurred within six months in an enclosed population of 28 to 32 chronically ill children. Studies of nasopharyngeal carriage and serum HITB anticapsular antibodies were started after the third case occurred. Two patients had low (less than 0.04 and 0.05mug/ml) antibodies and were not carriers when studied prior to onset of their disease. The carriage rate was approximately 20% among the children. Carriage was usually prolonged, and acquisition was not prevented by high antibody levels. Attempts to arrest this outbreak with type b polysaccharide immunization and ampicillin therapy are discussed in the context of HITB meningitis as a contagious disease.

Antigenic structures may be shared among naturally occurring polymers, including proteins and polysaccharides. Proteins are polymers of amino acids. Cross-reactions between proteins are due to similarities in their overall shape rather than their individual amino acid components. Cross-reactions have been demonstrated among proteins with similar evolutionary development and structure, such as serum albumins or immunoglobulins. Polysaccharides are polymers of monosaccharides. In contrast to proteins, antigenic specificities may be conferred by mono-, di-, and trisaccharides. Since there are about 150 known naturally occurring monosaccharides, it is not unexpected that cross-reactions are demonstrable between polysaccharides from widely divergent sources.

Approximately 16,000 children, from two months to five years of age, were vaccinated with the capsular polysaccharide of either Haemophilus influenzae type b or group C Neisseria meningitidis. Immunizations were carried out in a double-masked, randomized manner; the doses of immunogens used were 10 microgram of H. influenzae type b polysaccharide and 25 micron g of the group CN. meningitidis polysaccharide. Immunogenicity of the two vaccines was measured in single, random specimens of blood taken from vaccinees of all ages at various intervals after immunization. A positive effect on formation of serum antibody was observed in children of all ages vaccinated with N. meningitidis polysaccharide, but increased levels of serum antibody to H. influenzae type b were observed only in recipients of that vaccine who were three years of age or older. No untoward reactions to either vaccine were noted, and both vaccines retained their original molecular size after storage for three years. Too few cases of disease have been studied for a definitive assessment of vaccine efficacy; however, a slightly protective effect against meningitis was observed for the H. influenzae type b vaccine in infants up to one year of age.

Using the technique of direct immunofluorescence, ligandin, and abundant soluble organic anion binding protein was localized to the cytoplasm of hepatocytes, proximal renal tubular epithelium, and epithelial cells of the proximal, mid and distal small intestine of rat, hamster and man. The localization and abundance of ligandin, variety of organic anions that are bound, and its activity as a gluthathione transferase suggest that this cytoplasmic protein is important in determining the net intracellular content of organic anions and in nonoxidative detoxification.

An antiserum agar medium was found to provide a rapid, sensitive, and highly specific method for pharyngeal culture and quantitation of HIB.