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For prevention of Streptococcus pneumoniae (pneumococcus) infections in infancy, protein-conjugated capsular polysaccharide vaccines provide serotype-specific, antibody-mediated immunity but do not cover all of the 90+ capsule serotypes. Therefore, microbiologists have sought protective noncapsular antigens common to all strains. Alternatively, we investigated killed cells of a noncapsulated strain, which expose many such common antigens. Given to mice intranasally, this vaccine elicits antibody-independent, CD4+ T lymphocyte-dependent accelerated clearance of pneumococci of various serotypes from the nasopharynx mediated by the cytokine IL-17A. Such immunity may reproduce the natural resistance that develops in infants before capsular antibodies arise. Given by injection, the killed cell vaccine induces bifunctional immunity: plasma antibodies protective against fatal pneumonia challenge, as well as IL-17A–mediated nasopharyngeal clearance. Human testing of this inexpensive candidate vaccine by intramuscular injection is planned. Bacterial cellular vaccines are complex—a challenge for reproducibility. However, when several known protective antigens were deleted, the killed pneumococcal vaccine was still protective. This antigenic redundancy may prevent vaccine escape variants by recombinational loss, which is frequent in pneumococcus. Biochemically defined immunogens with bifunctional activity have also been devised. These immunogens are three-component conjugates in which cell wall teichoic acid (a common antigen capable of T cell activation) is coupled to a genetic fusion of two common pneumococcal proteins: a protective surface antigen and a derivative of pneumolysin, which provides TLR4 agonist activity and induces antitoxic immunity. Such constructs induce accelerated clearance when given intranasally and induce both immune mechanisms when injected. The defined composition permits analysis of structure-function activity.

Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxin-related adjuvants, has been shown to confer multi-serotype protection against colonization of the nasopharynx and middle ear in mice. However, because novel mucosal immunization strategies may be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 was engineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutant pneumolysoid. Liter-scale and 60-l Good Manufacturing Practice (GMP) cultures were grown in bovine-free soy-based medium, killed with chloroform or beta-propiolactone, and injected into C57Bl/6 mice without or with aluminum adjuvant. The adjuvant Al(OH)(3) strongly increased responses, particularly if pre-treated with phosphate. Protection was found in several tested model infections: nasal colonization with a serotype 6B strain and fatal aspiration-sepsis with strains of serotype 3 and 5. Protection against colonization was mechanistically dependent on the presence of CD4+ T cells at the time of challenge; in contrast, in the type 3 aspiration-sepsis model, CD4+ T cells were not required for protection at the time of challenge, suggesting that antibody alone was sufficient to protect against death in this model. Rabbits receiving sequential intramuscular injections in a pilot toxicity study displayed local reactogenicity at injection sites but no clinical signs. The rabbit antiserum thus produced was active in an in vitro phagocytic killing assay and passively protected mice in the type 3 aspiration-sepsis model. Approval is being sought for human trials of this vaccine.

Cell wall polysaccharide (CWPS), pneumolysin, and surface adhesin A (PsaA) are antigens common to virtually all serotypes of Streptococcus pneumoniae (pneumococcus), and all have been studied separately for use in protection. Previously we showed that protection against nasopharyngeal (NP) colonization by intranasal vaccination of mice with killed pneumococci is mediated by T H 17 cells and correlates with interleukin-17A (IL-17A) expression by T cells in vitro; we have also shown that CWPS and other species-common antigens protect against colonization by a similar mechanism. Here we made a fusion protein of PsaA with the pneumolysin nontoxic derivative PdT and then coupled CWPS to the fusion protein, aiming to enhance immune responses to all three antigens. When given intranasally with cholera toxin adjuvant, the fusion conjugate induced higher serum antibody titers and greater priming for IL-17A responses than an equimolar mixture of the three antigens. The conjugate administered intranasally protected mice against experimental NP colonization by a strain of serotype 6B, while mice immunized with the mixture or with bivalent conjugates were not protected. Subcutaneous immunization with the conjugate and alum adjuvant likewise induced higher antibody titers than the mixture, primed for IL-17A responses, and reduced colonization. The conjugate, but not the antigen mixture, fully protected mice from fatal pneumonia caused by a highly virulent serotype 3 strain. Thus, a covalent construct of three antigens common to all serotypes exhibits protection with both mucosal and systemic administration.

Serotype-specific immunity toStreptococcus pneumoniaeis conferred by antibodies to the capsular polysaccharides, which define the 90 known serotypes. Whether antibody to the species-common cell wall polysaccharide (C-Ps) is protective has been a matter of controversy. Here we show that C-Ps given intranasally with mucosal adjuvant increased the resistance of mice to experimental nasopharyngeal colonization by capsulatedS. pneumoniaeof serotype 6B. This immunity could be induced in mice congenitally lacking immunoglobulin but was dependent upon CD4+T cells. Elimination of the charged amino group on the polymer backbone by N acetylation of C-Ps reduced the immunity, as did treatment of the mice with antibody to the cytokine interleukin-17A at the time of challenge, both consistent with the hypothesis of T-cell activation due to the zwitterionic motif of the polymer. C-Ps also protected in a model of fatal aspiration pneumonia by heavily capsulated serotype 3. These findings suggest a novel immunization strategy againstS. pneumoniae.

An interlaboratory study was conducted to determine whether an enzyme-linked immunosorbent assay (ELISA) with an antigen preparation composed of various-sized fragments of Haemophilus influenzae type b polysaccharide conjugated to human serum albumin could be standardized across laboratories and whether the ELISA-derived results from different laboratories are equivalent to those obtained by the standard radioactive antigen binding assay (RABA) for quantitation of anti-H, influenzae type b polysaccharide antibodies. Twenty coded human serum samples were quantitated by ELISA in 11 laboratories and by RABA in 5 laboratories. The mean RABA-derived values served as the basis for all comparisons. While the overall correspondence of antibody values between the two methods was good, significant differences were found among some of the 11 ELISA data sets and among the mean RABA values. Seven laboratories generated higher ELISA antibody values for low-titered sera. Four laboratories generated antibody concentrations that were not statistically different between the two assay methods. The results therefore indicate that the ELISA can tolerate substantial variations in protocol, such as the use of different plates and different antibody reagents, without affecting the quantitation of serum antibodies. However, attention should be focused on low-titered sera, as some assay conditions may yield spurious results. This ELISA is a serologic assay which can serve as an alternative to the RABA for quantitation of antibodies to H. influenzae type h polysaccharide.

Injecting mice with killed cells of non-capsulated strain RM200 adsorbed on Al(OH)3 (pneumococcal whole-cell vaccine; WCV) reduces nasopharyngeal colonization by capsular serotype 6B and prevents fatal aspiration pneumonia by serotype 3 or serotype 5 strains. To further examine the potential for omni-strain immunity, we here examined a panel of clinical isolates and a library of capsule-switch variants in the TIGR4 background. IgG binding to these bacteria in sera of rabbits injected with WCV or Al(OH)3 alone was assayed by ELISA without and with adsorption with cell-wall polysaccharide, a species-common antigen. The examined strains were 23 primary isolates including at least 10 different MLS types and 13 serotypes; 15 of these strains were invasive isolates, subsequently mouse-passed. Additionally, to investigate the effect of capsulation, TIGR4 strain constructs with the capsulation genes of 20 different serotypes were evaluated. In ELISA all strains showed a large difference in IgG binding due to the immunization, of which most of the antibody typically was not CWPS-adsorbed and presumably directed to exposed protein antigens. Increased binding of IgG in the WCV-immunized serum to the 20 isogenic capsule-switch strains was shown also by flow cytometry. Further, all these 20 strains elicited IL-17A in T cells of WCV-vaccinated mice, a cytokine known to accelerate pneumococcal clearance. Thus WCV induced both humoral and T H 17 cell-mediated immunity against all tested strains.

We previously reported that cell wall polysaccharide (CWPS) given to mice intranasally with adjuvant induces serotype-independent immunity to pneumococci. Some strains make CWPS with one phosphocholine group (CWPS/1), but most express two per tetrasaccharide repeat unit (CWPS/2). Here, CWPS/1 and CWPS/2 were equally protective against colonization by CWPS/2-type pneumococci, but the related Streptococcus mitis polymer lacking phosphocholine was non-protective. Previously the protection was shown to be CD4+ T cell-dependent, abrogated by antiserum to interleukin (IL)-17A, and demonstrable in antibody-defective mice. Here, CWPS failed to protect IL-17A receptor-knockout mice, further indicating IL-17A-dependence. When commercial CWPS/1 was size-fractionated preparatively, the larger exceeded the smaller molecules in their capacity to prime for IL-17A responses, and only the larger protected against pneumococcal colonization. However, a CWPS-tetanus toxoid conjugate -- despite raising high titers of phosphocholine antibody -- was non-protective, confirming the irrelevance of humoral immunity in this model. The results strengthen the concept that IL-17A-mediated T cell immunity is inducible by zwitterionic polysaccharides with sufficient chain-length to provide coiled secondary structure. Coupling CWPS to protein, which paradoxically prevents protection, may occlude this regular linear conformation. We suggest that mucosal immunization with CWPS primes TH17 cells, which - upon contact with the phosphocholine of colonizing pneumococci - elaborate IL-17A, enhancing phagocytosis.

Although anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell–dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to naïve RAG1−/− mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-γ or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. In vitro expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection in vivo. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation in vitro could help in the development of candidate pneumococcal vaccines., Author Summary The bacterium Streptococcus pneumoniae (pneumococcus) causes serious disease in children and the elderly, including pneumonia and meningitis (inflammation of the brain). Carriage of pneumococcus in the nose is a necessary first step for most infections. As children age, they carry pneumococcus for shorter periods of time and their risk of disease decreases also. The mechanisms underlying this age-related decrease of carriage are not well understood. A deeper understanding of resistance to colonization would enable us to develop better pneumococcal vaccines. Using experimental mouse models, we show that repeated exposure to pneumococci leads to a subsequent reduction in duration of pneumococcal carriage, similar to what is observed in humans. We identify the immune cells that are responsible for this process, so-called TH17 cells, which release a factor that enables human blood cells to kill pneumococcus more efficiently. We show that these TH17 cells exist in adults and children, but not in newborn babies, which suggests that they may arise as a consequence of humans being exposed to pneumococcus. We describe an assay for the measurement of these cells in humans. Such an assay could facilitate the development of novel vaccines directed against pneumococcal carriage.

Incubation of Haemophilus influenzae type b at less than or equal to 10(7) CFU/ml with serum ultrafiltrate induces a phenotypic conversion in which complement-mediated bactericidal activity by somatic antibodies decreases while killing by capsular antibody is unchanged. Conversion had been shown to occur in a capsule-deficient (b-) mutant of strain Eag (thus appearing independent of capsulation), to include an increase in lipopolysaccharide content, and to be inhibited by chloramphenicol or puromycin. In the present study, in several strains not previously examined, conversion was not inhibited by the drugs and the corresponding b- mutants did not convert. Incubation in ultrafiltrate was also found to increase capsulation, as detected by radioassay, only 1.6-fold in Eag but 4.5-fold in DL26, the strain with the largest increase in resistance; moreover, complement-mediated opsonization by capsular antibody was greatly decreased. Thus, multiple mechanisms, capsule dependent as well as independent, appear to contribute to the serum factor-induced resistance of H. influenzae type b to antibody.

Acquired immunity toStreptococcus pneumoniae(pneumococcus) has long been assumed to depend on the presence of anticapsular antibodies. We found, however, that colonization with live pneumococci of serotypes 6B, 7F, or 14 protected mice against recolonization by any of the serotypes and that protection from acquisition of a heterologous or homologous strain did not depend on anticapsular antibody. Further, intranasal immunization by live pneumococcal colonization or by a killed, nonencapsulated whole-cell vaccine protected antibody-deficient mice against colonization, suggesting independence of antibodies to any pneumococcal antigens. Protection by intranasal immunization with whole-cell vaccine was completely abrogated in T cell-deficient mice, and in mice that were congenitally deficient in CD4+T cells or depleted of these cells at the time of challenge. In contrast, mice congenitally deficient in, or depleted of, CD8+T cells were fully protected. Protection in this model was observed beyond 2 months after immunization, arguing against innate or nonspecific immune mechanisms. Thus, we find that immunity to pneumococcal colonization can be induced in the absence of antibody, independent of the capsular type, and this protection requires the presence of CD4+T cells at the time of challenge.

SPECIFICITY PROBLEMS WITH THE ELISA FOR SEROTYPE-SPECIFIC STREPTOCOCCUS PNEUMONIAE (Spn) ANTIBODIES. † 1050

In healthy persons IgG antibodies to polysaccharides are predominantly of the IgG2 subclass.1 2 3 Selective deficiency of this subclass is associated with inability to produce antibodies to bacterial capsular polysaccharides, which confer protective immunity to encapsulated bacteria. However, the basis of the relation between antibody responses to capsular polysaccharide and the IgG2 subclass has not been defined. Some insight into that mechanism is offered by the present study of two children with selective IgG2 deficiency and a documented lack of antibody response to immunization with the capsular polysaccharide vaccine of Hemophilus influenzae b. Both patients were subsequently immunized . . .

Antibody to polyribophosphate, the capsular polysaccharide of Haemophilus influenzae type b, was measured in healthy ambulatory children by a radioactive antigen-binding assay. Titers fell from birth through nine months of age, then increased until six years, when they plateaued. Antibody activity was not correlated with the child's sex, ethnic status, or area of residence. Doses of 0.2-50 microgram of polyribophosphate given as single or booster doses had similar effects on antibody activity. Of 368 doses given to infants two to six months of age, 7% produced a significant antibody response; of 95 doses given to infants seven to 12 months old, 17% produced a response. The geometric mean titers of antibody resulting from immunization with polyribophosphate given at various times in relation to diphtheria-pertussistetanus vaccine did not differ significantly from one another or from titers observed in infants given only the latter vaccine. These data indicate that purified polyribophosphate will not provoke humoral immunity in young infants against H. influenzae type b and that it should no longer be considered as a candidate vaccine for this purpose.

Polyribophosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, is more effectively immunogenic when it is associated with the bacterium than when it is in the purified form that is being tested as a vaccine for humans. In an effort to analyze this difference, we isolated from H. influenzae type b a high-molecular-weight, soluble complex, in which PRP appears to be combined with protein (about 7% protein). The pyrogenicity and limulus lysate gelation activity of the complex suggest that a small amount of lipopolysaccharide also is present. The protein was resolved into five polypeptides by electrophoresis in polyacrylamide gel containing sodium dodecyl sulfate. In weanling rabbits, which do not respond to purified PRP, the complex induces high titers of antibody of PRP, in an anamnestic pattern. Bactericidal antibody to other bacterial components was also elicited. Equilibrium density gradient centrifugation of the complex indicated that most of the immunogenicity of PRP resides in the least dense fractions, which are high in protein, low in polysaccharide, and active in the limulus lysate test; denser fractions that react strongly with limulus lysate but are poor in protein were much less immunogenic.

We studied an immunogen consisting of oligosaccharides derived from Haemophilus influenzae type b capsular polysaccharide (PRP) coupled to CRM197, a nontoxic relative of diphtheria toxin. Subcutaneous injections were given to eight subjects at ages 2, 4, and 6 months, simultaneously with conventional diphtheria-tetanus-pertussis (DTP) vaccine. After the first immunization, total serum anti-PRP antibodies declined in all subjects, but increased in most after the second immunization and after the third in seven of seven subjects analyzed. In these seven infants, the geometric mean level at age 9 months (0.73 micrograms/ml) exceeded by at least 40 times the means of historical control groups given DTP only or DTP plus (uncoupled) PRP vaccine. An isotype-specific assay showed that IgM antibodies increased after the first immunization with the coupled vaccine in all eight infants. Against the background of declining maternal IgG antibody, elevations in IgG antibody were detected after the second or third immunization in six of the eight. These six at age 9 to 11 months were immunized with (uncoupled) PRP vaccine, and a "boost" in anti-PRP antibody, including an IgG component, was found.

Haemophilus influenzae type b (H.i.b) has been investigated with respect to phenotypic and genetic variations resulting in differential susceptibility to bactericidal antibody. Previous studies had shown that after growth in infected rats or in dialysate of rat serum, H.i.b strain Eag became more resistant to the bactericidal activity of antisomatic antibody. We now report that a similar phenotypic shift occurs when strain Eag is incubated with dialysate of human serum, that the increased resistance is to antibodies against determinants in the lipopolysaccharide not for the somatic antigens generally, and that most strains of H.i.b undergo the shift. To assess genetic differences in exposed antigens, a panel of 13 H.i.b isolates from cerebrospinal fluid were analyzed with cross-adsorbed antisera. Seven different patterns were found that could be accounted for through the variable expression of six antigens. These ranged from infrequent (found on 1:13 strains) to common (10:13 strains). At least four were somatic rather than capsular determinants; the most common (antigen 1) was contained in lipopolysaccharide. The epidemiologic relevance of the genetic variations was explored using pairs of isolates from two children who had had two documented infections with H.i.b. In both cases the isolates varied in somatic antigen expression. The strains from one patient differed in the expression of antigen 1. The isolates from the other were indistinguishable in sub-typing for the six classified antigens, but differed in the expression of an additional antigen identified by use of the patient's serum.

In human volunteers, single injections of purified polyribophosphate elicited antibodies detectable by passive hemagglutination and by serum bactericidal and opsonizing activities against viable Hemophilus influenzae, type b. All three activities rose by 2 wk to maximal levels, at which they remained for at least 6 months. Doses of 1 mug elicited antibody responses in nearly all recipients; higher doses of the antigen, however, produced larger increases in titer. Booster doses of 1 mug given at 6 months did not further increase the antibody titers. A tuberculin-like response was often observed at the site of injections given intradermally.

In radioantigen-binding assays for antibody to Haemophilus influenzae type b capsular polysaccharide, an apparent antibody concentration [( (["Ab"]) is assigned to test sera on the basis of binding equivalence to dilutions of standard adult antiserum. In childrens' sera, ["Ab"] can disagree between laboratories and between intralaboratory assays using different preparations of radioantigen. Immunochemical variables causing disagreement include the dilution of test serum at which ["Ab"] is assigned and the concentration and size distribution of radioantigen. Binding-dilution curves of many childrens' sera displayed shallower slopes than did the standard, thus ["Ab"] increased with dilution, particularly in low-binding sera. An increase in radioantigen concentration also raised ["Ab"]. Using larger-sized radioantigen raised the binding in standard and childrens' sera comparably and thus would not directly affect ["Ab"] but would raise ["Ab"] indirectly by increasing the serum dilution. These results are theoretically predicted if the affinity of childrens' antibodies are lower than that of the standard.

Haemophilus influenzae type b (Hib) capsular polysaccharide (PRP) was selectively hydrolyzed to reducing oligosaccharides, and the fraction containing 3-10 ribosylribitolphosphate repeating units (VS) was conjugated by reductive amination to diphtheria toxin (DTx), its nontoxic derivative CRM197 (Dcr), or diphtheria toxoid (DTd). Conjugate DTx-VS retained approximately 1% of native toxicity, which was eliminated by treatment with formalin. Immunization of rabbits with the conjugates elicited antibody (Ab) to PRP and to DTx but not to a model for the linkage determinant. Human adults given single subcutaneous injections had rises in serum Ab to PRP and in bactericidal activity in vitro; the Ab protected infant rats challenged with Hib. Adults had rises also in Ab to DTd, and these Ab protected rabbits against DTx. A series of two injections of the conjugates Dcr-VS and DTd-VS was tested in infants beginning at 19-23 mo of age. Rises in anti-PRP Ab after the primary resembled the rises after PRP vaccine. In contrast to PRP, the conjugates elicited large rises after the secondary vaccinations and a substantial IgG component. Development of bactericidal activity paralleled the rises in anti-PRP Ab. Secondary rises after Dcr-VS were higher than after DTd-VS. In infants 12-16 mo of age, Dcr-VS (but not DTd-VS) elicited strong primary and secondary Ab responses that included IgG and bactericidal activity. Both conjugates produced consistent rises in Ab to DTd.

In a study of the issue of revaccination with Haemophilus influenzae b polysaccharide vaccine, 36 children who had been vaccinated with polysaccharide vaccine at age 18 to 20 months were stratified according to their 3-week postvaccination anticapsular antibody concentration determined by radioantigen binding assay, then sequentially assigned to one of three groups so as to have comparable distributions of peak antibody concentrations. These children were revaccinated with polysaccharide vaccine at 24 to 27, 30 to 33, or 36 to 39 months of age. The post-reimmunization geometric mean antibody concentrations in these three groups were 3.1, 3.0 and 7.8 micrograms/ml, respectively, and the percentages rising to greater than 1 microgram/ml were 75, 80 and 93%. The geometric means and antibody response rates were nearly identical to age-matched control groups not previously vaccinated. Thus there was no evidence of priming (memory induction) or of tolerance induction by the early (18 months old) primary vaccination.

In systemic infections caused by Hemophilus influenzae, type b, the capsular polysaccharide, polyribophosphate, is released into the circulation. Polyribophosphate was quantitated in serial serum and cerebrospinal fluid samples from 45 children with H. influenzae, type b meningitis by means of a radiolabeled antigen-binding inhibition assay. Polyribophosphate was regularly found in acute serum and cerebrospinal fluid samples and could be detected in unbound form for periods of 1-30 days after initiation of effective therapy. Complexes of polyribophosphate dissociable with acid and pepsin were detected in serum samples from 17 patients, in one case for a period of 145 days after hospitalization. Polyribophosphate levels and patterns of clearance were studied in relation to hospital course and antibody response. Patients with prolonged antigenemia had protracted fevers and severe neurological symptoms during hospitalization, frequently with focal complications.Antipolyribophosphate antibody responses were detected during the first 100 days of convalescence by radioimmunoassay in 79% of the patients studied, including 60% of the children 1 yr or less in age. The intensity of antibody response although clearly related to the age of the patient, was more reliably predicted by the efficiency of antigen clearance. Antibody responses were uniformly of low magnitude in patients with prolonged antigenemia, irrespective of age. Paients who failed to develop antibody to polyribophosphate after meningitis also exhibited impaired antigen clearance. These studies suggest that mechanisms necessary for clearance of polyribophosphate may influence the development and intensity of the humoral immune response and raise the possibility of developmental deficiencies in the clearance system in infants and children.

The role of antibodies to capsular and somatic antigens in the clearance of Haemophilus influenzae was investigated by active and passive immunization. The clearance index (k) and the proportion of strain b organisms cleared 30 min after intravenous administration (deltaY30) were greater in eight-week-old actively immunized rats (k = 0.693, deltaY30 = 4.07) than in nonimmune animals (k = 0.075, deltaY30 = 0.95)(P less than 0.025 for all); however, clearance correlated imprecisely with titers of bactericidal or anticapsular antibody. In three-week-old rats, intranasal immunization with strain b or U significantly increased (P less than 0.005) the rate of clearance of strains b and U. Passive immunization with antibodies to somatic or capsular antigens significantly increased the rate of clearance (P less than 0.001) and the proportion of bacteria cleared (P less than 0.05) with all test strains. The increased clearance associated with passive immunization correlated with increased splenic uptake of 32P-labeled H. influenzae (r = 0.83, P less than 0.025). Analysis of the disappearance of viable organisms and bacterial 32P suggested that bacteriolysis of H. influenzae did not occur during clearance of the bacteremia. Either antibody to capsular antigen or antibody to somatic antigen, administered or evoked in rats, accelerates intravenous clearance of H. influenzae by promotion of reticuloendothelial phagocytosis.

Both gonococci and Haemophilus influenzae type b (Hib) in response to incubation with low-molecular-weight components of blood are converted to a relative resistance to killing by antibody and complement in vitro. Recently, cytidine 5′-mono-phospho-N-acetylneuraminic acid (a sialyl donor present in mammalian tissues) was found to convert gonococci. Here we report that this compound was inactive in Hib. In further contrast, the Hib-converting activity of serum was not retained by 500-Mr filters, not sensitive to pH 2, and not active in the presence of growth media. Thus the phenotypic resistances in Hib and gonococci appear to have a different metabolic basis. The low-Mr serum factors active in Hib remain to be identified. Filtrates of human nasopharyngeal washes were also active, further suggesting that the converted form resembles the phenotype of Hib in vivo.

The immunoglobulins involved in the human serologic response to polyribophosphate (PRP) 4 , the capsular antigen of Hemophilus influenzae type b, have been studied. After immunization with 50 μg of PRP, serum demonstrated primary antigen-binding capacity and a classic precipitin curve. Per unit weight, the IgM from this serum was 100-fold more active than the IgG in PRP-specific hemagglutination and 2-fold greater in complement-dependent bactericidal activity. Specific anti-PRP antibody separated from the IgG by immunoabsorption was active in both assays; of the IgG subgroups, only IgG1 was detected in this preparation. The high salt extract from a PRP precipitate of hyperimmune serum, however, contained IgG2 and IgG1 in the ratio of 1.6:1. Phagocytosis of H. influenzae b in vitro was enhanced by heated serum, IgG, and the anti-PRP IgG1 preparation but not by IgM; this activity was approximately doubled by the addition of complement. Inhibitor studies indicated that C 3 peptidase was active in this system.

Humoral immunity to Hemophilus influenzae, type b was studied in normal human adults by means of assays for serum bactericidal and opsonizing activities against the organism and for passive hemagglutinating activity using erythrocytes sensitized with polyribophosphate, the type-specific capsular antigen. Hemagglutinating activity was detectable in about 60% of the 114 sera tested. Serum bactericidal and opsonizing activities were found in all sera tested; the levels in some sera, however, were quite low. The antibacterial activities were due not only to antibodies directed against the polyribophosphate capsule but also to antibodies that appear to be directed against somatic antigens. Type b strains differed in their susceptibility to the antisomatic antibodies of particular sera but were uniformly sensitive to anticapsular antibody.

These studies were performed to assess the utility of the baboon as a nonhuman primate model to evaluate vaccines for use in humans. Specifically, we examined the antibody response of baboons immunized during the third trimester of pregnancy with Haemophilus influenzae type b (Hib) polyribosylribitol phosphate (PRP) conjugate and unconjugated polysaccharide vaccines. Some of the vaccinated mothers failed to respond to a single immunization with unconjugated Hib PRP. Specific Hib PRP immunoglobulin G (IgG) but not IgM antibodies cross the baboon placenta and are detected in the offspring. Higher-titer baboon anti-Hib PRP did not express two previously defined cross-reactive human anti-Hib PRP idiotypes and was biased towards lambda light-chain expression. Spectrotype analysis indicated that baboon anti-Hib PRP was restricted in heterogeneity and oligoclonal.

Ab to PRP are protective against systemic infections by Hib, but infants under 18 mo usually have an inadequate Ab response to PRP vaccine. To circumvent this unresponsiveness, we conjugated oligosaccharides of PRP to a protein carrier, CRM197, a nontoxic version of diphtheria toxin. Infants were injected with the conjugate at ages 2, 4, and 6 mo. At age 9 mo the geometric mean titer (GMT) of anti-PRP Ab in a group of 5 such infants was 2.0 μg/ml; the response included IgG Ab. In an unvaccinated control group of 13 infants the GMT was 0.015 μg/ml, with no IgG included. Both groups were then vaccinated with PRP vaccine, and the anti-PRP Ab was measured 1 mo later. In the control group, 3/13 infants had a rise, and the GMT rose to 0.024 μg/ml. In the group primed with conjugate, 4/5 had a rise, and the GMT rose to 7.2 μg/ml. Thus the conjugate is able not only to raise the Ab to high levels, but also to prime for an enhanced response to the polymer. It seems likely that such a response would occur also when the infant encounters Hib. Further, boosting with PRP after priming with conjugate may maintain the Ab at an elevated level.

Top of pageAbstract Vaccines composed of Haemophilus influenzae b capsular polysaccharide (PRP) oligomers coupled to diphtheria toxoid (DTd), (DTd-ol), have been shown to induce antibodies (Ab) to PRP after repetitive immunization (imm) of infants at ages at which there is no response to PRP vaccine. To further examine the immunogenicity of DTd-ol, a 3-yr-old child with the Wiskott-Aldrich syndrome (WA) and a 7-yr-old child with isolated IgG2 subclass deficiency (G2−) who had both failed to produce Ab following two imms with purified PRP were then imm 3X with DTd-ol. Anti-PRP Ab, μg/ml, increased as follows: Both children produced a normal Ab titer to DTd after the first imm. The anti-PRP Ab isotype induced by DTd-ol was almost exclusively the IgG isotype in WA and both IgG and IgM isotypes in G2−. Over 90% of the anti-PRP Ab of the IgG isotype was of the IgG1 subclass in both WA and G2−, but IgG2 subclass Ab did increase after imm in both WA and G2− and was not detected preimm. The Ab light chain was predominately kappa type. Analytical isoelectric focusing patterns of Ab showed a restricted number of IgG Ab clonotypes. These results suggest that the poor response to polysaccharides in some immunodeficiency diseases can be bypassed by imm with oligosaccharide-protein conjugate vaccines and offer insight into the response of young infants to DTd-ol.

Ab to PRP are protective against Hib systemic infections, but infants of age

Antisera to the pilus of a single strain of Hib (C54) were made by hyperimmunization with a pilus-enriched (p+) variant of C54 followed by absorption against a variant of C54 that was identical in encapsulation, outer membrane protein composition, and lipopolysaccharide subtype but lacked pili (p-). 4 different antisera were prepared (2 in different species of mice and 2 in guinea pigs). All 4 antisera agglutinated (aggl) the homologous C54 p+ but did not aggl C54 p-. C54 causes hemagglutination (HA) of human RBCs and adheres well to human epithelial cells (EC) (4.8 bacteria/EC) whereas the p- variant is HA- and adheres poorly (0.2/EC). Addition of antisera resulted in complete inhibition of HA and >60% reduction in C54 bacterial adherence. 9 strains isolated from CSF, blood, and mucosal surfaces were pilus-enriched and studied for cross-reactivity with the pilus of C54. 5 were HA+ with human RBCs only (like C54 and 80% of all Hib isolates), 2 were HA+ with human and rat RBCs (like 12% of all Hib) and 2 were HA+ with all animal and human RBCs tested (like 8% of all Hib). Despite these HA species differences (the only type of heterogeneity found to date) the 4 antisera aggl all 9 strains when p+ but not when p-. Similarly, the antisera inhibited HA of human RBCs and adherence to EC. Thus, pili from different strains of Hib share antigenic determinant(s), and common antipilus antibodies can produce the biologically relevant effect of inhibiting bacterial adherence.

ANTIBODY (Ab) TO THE CAPSULAR POLYSACCHARIDE (PRP) OF HAEMOPHILUS INFLUENZAE B IN 2 TO 6-MO-OLD INFANTS VACCINATED WITH PRP OLIGOSACCHARIDES CONJUGATED TO THE DIPHTHERIA PROTEIN CRM 197

Immune responses to Hemophilus influenzae, type b, polyribophosphate (PRP) were studied in 4 infants vaccinated at 6 months with 3 injections of PRP spaced 2 to 3 weeks apart. The injections totaled 1.6ugm of PRP mixed with 0.2ugm of cellular protein. Antibody to PRP was assayed by the Farr technique. Cellular responses were evaluated by incubating peripheral blood lymphocytes with several dilutions of pure PRP or PRP with protein and assaying for either protein or DNA synthesis. Generally significant increases in antibody to PRP were not seen as a result of this vaccination. No pre or post-vaccination lymphocyte cultures displayed increased DNA synthesis to the various dilutions and preparations of PRP tested. On the other hand, post-vaccination lymphocyte cultures from 2 infants displayed increased protein synthesis to pure PRP and one of these infants reacted similarly to PRP-protein. A third infant had significant pre-vaccination stimulation of protein synthesis and remained so following vaccination. The significance of cellular immune responses to PRP is unknown, however since antibody to PRP is not normally generated in the young infant, evaluation of both humoral and cellular immune responses may reveal additional mechanisms of immunity to infection with H. influenzae.

Prior to a planned conception, 24 women were immunized with purified HIB capsular polysaccharide to attempt to extend their offspring's period of passive antibody protection against systemic HIB disease. All women had an antibody rise. Geometric mean titer prior to immunization was 1.2 μg/ml and at 1, 7-10, and 19-27 months after immunization was 35 μg/ml, 23 μg/ml, and 16 μg/ml respectively as measured by radioantigen binding. Most post-immunization antibody was of the IgG class as determined by an enzyme-linked immunosorbent assay. However, there was a wide range of post-immunization titers (2.7-310 μg/ml at 7-10 months post-immunization). The low magnitude antibody responders also had low IgG antibody levels that were not boostable. The offspring of 6 women, born 11½ to 17½ months after immunization, had antibody titers markedly higher than those of age-matched infants of unvaccinated women. Their titers persisted above the estimated protective level through 6-12 months of age. Four infants, all born to low-responder women, had titers similar to natural antibody titers. These results suggest that the interval of passive antibody protection can be extended. This approach may be especially useful in populations highly susceptible to HIB and may be applicable to the prevention of other childhood and neonatal infections.