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Reductive amination coupling an aldehyde-containing polysaccharide, generated by periodate oxidation, with the amino groups in protein has been widely used in the synthesis of glycoconjugate vaccines. The conjugation is often achieved under slightly basic conditions via a Schiff's base intermediate followed by its reduction with sodium cyanoborohydride. We observed that oxidized capsular polysaccharides such as Streptococcus pneumoniae type 6B (Pn-6B) and Haemophilus influenzae type a (HiA) underwent significant degradation during the conjugation in slightly basic media leading to sub-optimal glycoconjugates. Further study on oxidized Pn-3, Pn-6A, Pn-6C, Pn-2 polysaccharides and dextran provided evidence that the degradation is a result of base-catalysed β-elimination. In contrast to HiA, Pn-2, Pn-3, Pn-6B polysaccharides and dextran, oxidized Pn-6A and Pn-6C polysaccharides were stable under basic conditions due to lack of the leaving group at the β-position of the aldehyde. By performing conjugation of oxidized polysaccharides to bovine serum albumin (BSA) in phosphate buffer at pH 6.0, 6.8, 7.2 and 8.0, we concluded that the reductive amination proceeds best in slightly acidic media, particularly with those β-elimination susceptible polysaccharides., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

After the introduction of the glycoconjugate vaccine based upon the capsular polysaccharide ofHaemophilus influenzaetype b in the mid 1980s there was a remarkable decrease in the number of invasive cases reported for this organism. Since the 1990s several groups have observed the emergence ofHaemophilus influenzaetype a (Hia), especially in indigenous communities in the northern regions of Canada and Alaska, to a stage where a solution is warranted to prevent further unnecessary deaths due to this pathogen. A glycoconjugate vaccine solution based upon the type a capsular polysaccharide (CPS) was investigated pre-clinically in an effort to illustrate the proof of concept for this approach. In this study we describe the growth of Hia and the isolation, purification and conjugation of the CPS to several carrier proteins. The resulting glycoconjugates were immunised in mice and rabbits provoking sera that facilitated bactericidal killing against all type a strains that we tested. This study has illustrated the pre-clinical proof of concept of a glycoconjugate vaccine based on the CPS of Hia asa solution to this emerging disease., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

The introduction of vaccines containing the capsular polysaccharides of N. meningitidis, S. pneumonia, and H. influenzae type b has driven a significant reduction in cases of disease caused by these bacteria. The polysaccharide-specific antibody responses following vaccination are well characterized, however less is known about the B cells underlying this response. Here, we summarize the plasma cell (PC) and memory B cell (BMEM) responses following plain polysaccharide and protein-polysaccharide conjugate vaccination, drawing together studies covering a range of vaccines and age groups. These studies show that infant primary PC and BMEM responses to polysaccharide-conjugate vaccines are low in relation to older age groups but are significantly higher following booster doses. PC kinetics have generally been found to follow a similar pattern irrespective of vaccine type or age group, whereas divergent BMEM responses have been reported following plain polysaccharide and conjugate vaccination. A degree of correlation between early BMEM responses and maintenance of protective antibody levels has been identified in some studies, but the relationship between the 2 remains unclear. Identification of the B cell subsets involved and the mechanisms by which they are induced may provide a better understanding of the role of B cells in maintaining protective immunity through vaccination.

The polysaccharide-encapsulated bacteria, Haemophilus influenzae type b, Neisseria meningitidis and Streptococcus pneumoniae are important causes of invasive bacterial infection in childhood, accounting for most of the cases of bacterial pneumonia and meningitis worldwide. Protein-polysaccharide conjugate vaccines have been developed over the last 20 years and have proven very effective in controlling these infections. Although studies have consistently shown that herd immunity is critical for population protection, long-term individual protection against polysaccharide-encapsulated bacteria appears to depend on persisting antibody and, perhaps to a lesser extent, immunological memory. However, some studies have reported that the concentration of serum antibody and vaccine effectiveness are not sustained after infant immunization, despite persistence of immunological memory. In this article, we detail the mechanisms of protection against invasion by encapsulated bacteria, describe the age-dependent B-cell and antibody responses to protein-polysaccharide conjugate vaccines and propose strategies to guarantee protection during periods of increased disease burden.

This review focuses on the immune response to non-conjugated and conjugated polysaccharide vaccines derived from encapsulated pathogens, such as Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis. Special attention is paid to a number of side effects observed following the use of some of these vaccines. For example, we discuss the long-lasting specific refractoriness induced by unconjugated polysaccharides, and the absence of an effective immune response in adults vaccinated with some conjugated vaccines. We argue that studies performed in the mouse model can help to understand those paradoxical effects observed in humans, and the mechanisms underlying such processes.

Objective: Before 1991, Alaska Native children experienced one of the highest rates of invasive Haemophilus influenzae type b disease. H influenzae type b vaccine has led to a near-elimination of invasive H influenzae type b disease in the United States. We describe challenges encountered in controlling H influenzae type b disease in Alaska and update the current status of H influenzae disease and carriage in Alaska as lessons to other populations., Patients and Methods: We reviewed data from statewide H influenzae disease surveillance conducted during 1980-2004. Vaccine coverage data were based on audits from tribal facilities and the National Immunization Survey. H influenzae type b colonization data were based on 6 carriage studies., Results: After universal infant vaccination in 1991, H influenzae type b disease among Alaska Native and non-Native children < 5 years of age decreased by 94% and 96%, respectively. After a 1996 change in H influenzae type b vaccine from polyribosylribitol phosphate-outer membrane protein conjugate vaccine to H influenzae type b oligosaccharide-CRM197 vaccine, the incidence of H influenzae type b disease increased in rural Alaska Natives from 19.8 to 91.1 cases per 100000 per year < 5 years of age. During 2001-2004, with use of polyribosylribitol phosphate-outer membrane protein conjugate vaccine, the rate of H influenzae type b disease in Alaska Native and non-Native children aged < 5 years decreased to 5.4 and 0 per 100000 per year, respectively. In postvaccine studies, H influenzae type b carriage has decreased in Alaska Native children < 5 years of age., Conclusions: H influenzae type b vaccination has resulted in a dramatic decrease in invasive H influenzae type b disease in Alaska; however, despite high rates of H influenzae type b vaccine coverage, H influenzae type b disease rates among rural Alaska Native children < 5 years of age remain higher than the rates among non-Native Alaska and other US children. Equity in disease rates may not be achieved in indigenous populations with the current vaccines unless other environmental and household factors contributing to disease transmission are addressed.

Aim: To investigate the possible relationship between serum levels and avidities of antibodies against tetanus toxoid (TT) and Haemophilus influenzae type b (Hib) in children that were vaccinated after treatment for childhood acute lymphoblastic leukaemia (ALL)., Methods: The study groups were 31 paediatric patients with ALL and 18 healthy controls. All subjects were vaccinated with TT and a protein conjugated Hib vaccine. Antibody levels were analysed at three time points: At diagnosis of ALL, after cessation of treatment before vaccination and three weeks after vaccination. Avidity was measured twice, with a thiocyanate elution assay, at diagnosis of ALL and three weeks after vaccination., Results: There was a correlation between level and avidity of tetanus antibodies after vaccination (r(s) = 0.59, P < 0.001). In the standard-risk and intermediate-risk ALL groups, all patients responded with protective levels of tetanus antibodies with normal avidity. In the high-risk ALL group 7/9 patients had subprotective levels of tetanus antibodies after vaccination and concomitantly the lowest avidity, implying poor protection against tetanus. No patients were found with low levels and low avidity of anti-Hib IgG, and 29/31 patients had full protection after a single dose of conjugated Hib-vaccine., Conclusion: The vaccination strategy after childhood ALL must be different for low-risk and high-risk ALL groups, since the high-risk group fail to elicit a recall response to tetanus.

Passive transfer of a pediatric human serum pool generated against polysaccharide-protein D conjugate vaccines conferred approximately 34% protection against development of ascending NTHI-induced OM when used in a chinchilla viral-bacterial co-infection model. These data are in line with results obtained using a similar 11-valent-protein D conjugate vaccine in a pediatric clinical trial, wherein a vaccine efficacy of 35.6% was shown against acute OM episodes caused by NTHI. These observations strongly support the chinchilla passive transfer-superinfection model as one that could predict clinical trials outcomes for vaccines to prevent NTHI-induced OM.

Aims: (1) To determine the causes of meningitis in children immunized with Hib vaccine, presenting without a non-blanching rash; (2) to review the use of dexamethasone in this group., Method: Retrospective review of all children with more then 10 white cells/mm(3) in their cerebrospinal fluid (CSF), admitted between January 1998 and August 2002. Children were excluded if they had a non-blanching rash on admission or if their discharge diagnosis was not meningitis. Local guidelines recommended dexamethasone to be given before antibiotics for children with meningitis and no rash., Results: One hundred and eight children were identified. Causes of proven meningitis were: viral 41 (enterovirus 40), bacterial 22. CSF culture or PCR was the only diagnostic test in 31 children. Dexamethasone was given to 16 children. Length of admission was shorter in children with viral compared with bacterial meningitis (4 vs 8 days; P < 0.0001)., Summary: Viral meningitis is the commonest cause of meningitis without rash. Enteroviral PCR was the most useful test and needs to be widely available. Confirmation of enteroviral meningitis allowed early discharge. Few children were given dexamethasone, but only 5/108 may have benefited., Conclusions: The most common cause of meningitis without a rash in British children is enterovirus. The use of dexamethasone in children with meningitis without a rash should be reconsidered or, at least, individualised.

Introduction: Haemophilus influenzae type b (Hib) persists as a major cause of pediatric meningitis and pneumonia in developing countries in which Hib conjugate vaccines are not used. Demonstration of decreases in severe Hib disease after countries introduce Hib conjugate vaccine will help justify the resources necessary to purchase and provide the vaccine. Because surveillance for culture-confirmed Hib meningitis is not available in many countries, alternative means to measure the impact of Hib conjugate vaccine would be useful., Methods: Laboratory records from the years before and after introduction of the Hib conjugate vaccine were reviewed at 4 hospitals, 2 in Argentina and 2 in South Africa. Potential indicators of bacterial meningitis including cerebrospinal fluid (CSF) culture, white blood cell count, appearance, protein and glucose were recorded., Results: After introduction of Hib conjugate vaccine, culture-confirmed Hib meningitis declined significantly at 3 of 4 hospitals (2 in Argentina and 1 in South Africa). In the same 3 hospitals, there was a significant decline after vaccine introduction in some of the following CSF indicators of bacterial meningitis: proportion of CSF specimens with white blood cell count > or = 100 x 10(6)/L, 500 x 10(6)/L and 1,000 x 10(6)/L; glucose <40 mg/dL; protein >100 mg/dL; and turbid appearance., Conclusions: Culture-confirmed Hib meningitis declined at 3 of the 4 hospitals after Hib vaccine introduction. Surrogate indicators of bacterial meningitis also declined and might be useful measures of Hib conjugate vaccine impact at hospitals where capacity to culture Hib is not available.

The ability of 2 mutants of heat-labile Escherichia coli enterotoxin (LTK63 and LTR72) to enhance the immunogenicity of 2 protein polysaccharide conjugate vaccines, Neisseria meningitidis group C (MenC) and Haemophilus influenzae type B (Hib), both of which are conjugated to the nontoxic mutant of diphtheria toxin (CRM197), after intranasal (inl) immunization in mice was evaluated. In addition, the question of whether combining both vaccines in a single formulation with heat-labile E. coli enterotoxin mutants reduced the response to either vaccine was investigated. The results showed that potent serum antibody responses against MenC and Hib could be elicited by inl immunization in combination with the mucosal adjuvants. Moreover, IgA mucosal responses were induced only in animals immunized through the inl route. Finally, the coadministration of 2 conjugate vaccines simultaneously did not adversely affect the responses against either. These studies support the rationale for developing mucosal vaccines, based on combining protein polysaccharide conjugates with heat-labile E. coli enterotoxin mutants, for infants and young children.

Molecular mimicry is one of the most important pathogenic factor of microorganism and is defined as a structural similarity of microbial molecules to host tissue contributing to the pathogenicity. Mimicry can be observed at the molecular, serological and functional level. In the review the infectious diseases have been discussed where the mimicry phenomenon may occur, and also autoimmune disease where due to the molecular mimicry bacterial structures are potent to induce adverse immune reactions. The cross-reacting molecules mimicking the host structures comprise colominic acid, sialic acid containing capsular polysaccharides of Streptococcus group B, phosphocholine containing antigen, lipopolysaccharides of Campylobacter jejuni contributing in induction of Guillain-Barré syndrome or Lewis antigen containing lipopolysaccharides of Helicobacter pylori inducing gut carcinoma. Knowledge on the phenomenon of molecular mimicry is important when new conjugate vaccine has to be constructed, because great care should be paid not to induce autoantibodies with synthetic immunogen. Investigation of microbial factors reveal that many autoimmune diseases are of infection etiology.

Concentrations of serum anti-Haemophilus influenzae type b (anti-Hib) capsular polysaccharide (CPS) >/=0.15 and >/=1.0 microgram/mL are widely used as surrogates for protection against invasive Hib disease. However, the relationship between serum anti-Hib CPS following immunization and protection against colonization is not known, making it difficult to evaluate new Hib vaccines or combination vaccines. In the Dominican Republic, nasopharyngeal swabs were collected from 546 9-month-old infants who had received Hib conjugate vaccine at ages 2, 4, and 6 months and from 600 unvaccinated infants of the same age. The prevalence of Hib colonization was lower among vaccinated infants than among unvaccinated infants (0.9% vs. 2.3%). Among vaccinated infants, protection against colonization was significantly correlated with anti-Hib CPS concentrations >/=5 microgram/mL 1 month following the third dose of vaccine. These results suggest that the concentration of serum anti-Hib CPS needed for protection against colonization is greater than that needed for protection for invasive disease.

We describe the use of Nuclear Magnetic Resonance (NMR) spectroscopy to control the identity of purified bulk capsular polysaccharide [called poly(ribosylribitolphosphate) or PRP] from Haemophilus influenzae type b (Hib), and derivatised forms, used in the production of Hib polysaccharide-protein conjugate vaccines. We describe the approaches we have developed to validate this test., (Copyright 2000 The International Association for Biologicals.)

Background: A current debate is whether the immunologic priming of infants with Haemophilus influenzae type b (Hib) conjugate vaccines would be protective in the absence of circulating antibody to the capsular polysaccharide (PS). Data from the prevaccine era on the PS antibody responses of older children to Hib meningitis may be informative on this issue., Methods: PS antibody was assayed by radioantigen binding in sera taken in the first month postadmission in 47 children ages 2 to 136 months with culture-proved Hib meningitis., Results: Sera obtained on admission had very low antibody concentrations, and the subsequent response during convalescence was age-dependent. The major finding is that some patients, including 10 of 11 children older than 2 years, had substantial antibody elevations within a few days of admission, increases resembling the response to PS vaccine in infants primed with PS-protein conjugate vaccines., Conclusions: In this group of patients with Hib meningitis, natural priming did not prevent infection. Hib may have the ability to invade despite the capacity for a vigorous antibody response.

This study compared Hib antibody responses to a single lot of PRP-OMPC vaccine given at 2, 4 and 12 months to 57 Aboriginal infants in rural areas of the Northern Territory and 56 Caucasian infants in Sydney, Australia. The Aboriginal infants had lower levels of antibody in cord blood (P>0.05), which were significantly lower (P<0.02) by 2 months of age. Antibody responses to one or two doses of vaccine, measured at 4 and 12 months of age, were similar but the geometric mean titre following the booster dose in Aboriginal infants was significantly lower (1.98 vs. 6.04 mcg/ml, P = 0.002). Low preimmunisation antibody is consistent with the early onset of Hib disease in Aboriginal infants before immunisation. Lower responses to boosting could correlate with persistence of Hib colonisation in indigenous populations.

The joining of polysaccharide antigens to various proteins can result in increased immunogenicity of vaccines composed of such antigens. This article discusses conjugated polysaccharide vaccines for Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitis. Increased availability and use of such vaccines may result in the ability to give more effective vaccines earlier in life, further reducing the incidence of diseases caused by these organisms.

Acute otitis media (AOM) is a common infectious disease in children. Some children experience recurrent episodes of AOM. Recent investigations demonstrate antigen-specific immunological deficiencies in children prone to AOM. In the present study, the immune responses to non-typeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae (S. pneumoniae) were further investigated in otitis-prone children and normal children. Forty-eight percent of otitis-prone children exhibited reduced IgG2 levels to S. pneumoniae and 55% exhibited reduced IgG levels to NTHi. These data suggest that otitis proneness appears to be related to numerous immunological derangements. Pathogen-specific antibodies are a reliable measure of otitis proneness.

We measured the levels of serum IgG antibodies to CD outer membrane protein of Moraxella catarrhalis, P6 outer membrane protein of non-typeable Haemophilus influenzae and capsular polysaccharides of Streptococcus pneumoniae in 168 children with otitis media with effusion (OME) who were followed prospectively, using ELISA. Serum IgG antibodies to CD, P6 and pneumococcal capsular polysaccharides were detected in all samples. The anti-pneumococcal polysaccharides antibody level was highest, followed by the anti-P6 antibody level and anti-CD antibody was lowest (median:interquartile ranges were 45.9:19.1-100 microg/ml, 15.6:9.70-23.2 microg/ml and 1.06:0.73-1.87 microg/ml, respectively). In children aged 0-6 years, there were positive correlations among the antibody levels (anti-CD vs anti-P6, r=0.325, p <0.001; anti-CD vs anti-polysaccharide, r=0.397, p <0.0001; anti-P6 vs anti-polysaccharide, r=0.175, p=0.057). However, no relationship was seen in children aged 7-15 years. Children were classified according to severity of OME during the 1-year follow-up. In children aged 0-6 years, the severity of OME correlated inversely with the levels of anti-CD antibody (r=-.23, p=0.012), of anti-P6 antibody (r=-0.292, p=0.0015), and of anti-pneumococcal polysaccharides antibody (r=-0.25, p=0.0064). However, no correlation was found between antibody levels and severity of OME in children aged 7-15 years. These data suggest that persistence and/or recurrence of OME may be due to an insufficient serum antibody response to middle ear pathogens in young children.

Abs using the kappaII-A2 V gene segment predominate the human Ab repertoire to the Haemophilus influenzae b (Hib) polysaccharide (PS). All A2 anti-Hib PS Abs sequenced to date possess a 10-amino acid L chain complementarity-determining region-3 (CDR-3) having an insertional arginine (Arg) at position 95a, the V-J junction. These findings suggest an essential requirement for this conserved Arg residue in determining Hib PS-binding affinity. We examined this requirement by performing chain recombination experiments in which a series of A2 L chains, differing at position 95a, were combined individually with an Fd region known to generate a Hib PS-combining site when paired with an A2-Arg(95a)-Jkappa1 V region. Hib PS binding of the recombinant Fabs was evaluated quantitatively using a radioantigen-binding assay. Fabs having A2 L chains with either Arg or lysine in position 95a in combination with Jkappa1 gave equivalent and strongest binding to Hib PS. Fabs having A2-Jkappa1 L chains with either tyrosine, glycine, alanine, leucine, serine, or threonine in position 95a, or having an A2-Arg(95a)-Jkappa3 L chain, gave intermediate binding. Fabs having A2-Jkappa1 L chains with glutamate or aspartate at 95a or with no junctional residue showed little or no Hib PS binding. These results demonstrate the importance of L chain junctional residue, as well as Jkappa usage and CDR-3 length, in determining Hib PS-binding affinity. Contrary to expectation, an Arg junctional residue is not essential for generating either high or intermediate affinity-binding sites.

Objective: To study the kinetics of Haemophilus influenzae type b (Hib)-specific antibody in infants born to mothers immunized with an Hib polysaccharide or one of two Hib conjugate vaccines., Study Design: Serum antibody to the polyribosylribitol (PRP) moiety of Hib was measured by radioimmunoassay and enzyme-linked immunosorbent assay at birth and at 2 and 6 months of age in infants born to women immunized with Hib polysaccharide or conjugate vaccine (PRP-D and HbOC). A subset of infants > or = 6 months of age was immunized with Hib conjugate vaccine after licensure of this vaccine for infants. A comparison group of 18 infants born to unimmunized women received the same Hib conjugate vaccine on a similar schedule., Results: Total PRP antibody concentrations were 1.50, 14.4 and 20.4 microg/ml in 2-month-old infants born to mothers immunized with polysaccharide, PRP-D and HbOC vaccines, respectively, and 2.54, 1.35 and 2.46 microg/ml in 6-month-old infants. Infants born to mothers immunized with polysaccharide vaccine had significantly less PRP antibody at 2 months of age but similar antibody concentrations at 6 months of age. Persistence or increases in total PRP antibody during 6 months were noted in 21 of 47 (44.6%) study infants. A subset of study and comparison infants was immunized with a mean of 2.6 doses of Hib vaccines between 6 months and 2 years of age, and all infants had total PRP antibody concentrations > or = 0.15 microg/ml., Conclusion: Conjugate Hib vaccines administered during the last trimester of pregnancy resulted in significantly higher PRP antibody titers in infants at birth and 2 months of age than did polysaccharide vaccine. A subset of infants born to immunized mothers was subsequently immunized with Hib conjugate vaccine and had antibody concentrations similar to those in infants born to nonimmunized women.

Objective: To assess the immunogenicity in Indian infants to Haemophilus influenzae b oligosaccharide conjugate vaccine (HbOC)., Design: Prospective multicenter study., Setting: Pediatric Out Patient Department of general hospitals in Pune and Mumbai., Subjects: 124 full term healthy infants brought for routine DPT/OPV immunization., Methods: Infants were administered 3 doses of 0.5 ml of HbOC, on the same day as their DPT/OPV immunization, injected intramuscularly on the limb opposite to that where DPT vaccine was administered. Data on local reactions and general symptoms was collected for three days after every dose. The children had their blood collected for assay of anti PRP (polyribosil ribitol phosphate) antibody titers, along with the first injection and one month after the third injection. One hundred and three infants completed the study protocol with two blood collections., Results: The initial geometric mean titers (GMT) of 0.124 mcg/ml rose by 37 times to 4.552 mcg/ml. Ninety eight children (95.1%) had a final titer of > or = 0.15 mcg/ml, the minimum level associated with protection, and 77 children (74.8%) had a final level of > or = 1.0 mcg/ml, a level associated with long term protection., Conclusion: HbOC is immunogenic in Indian infants when used as per the locally recommended DPT/OPV immunization schedule.

We have measured antibodies to pneumococcal and Haemophilus polysaccharides in a prospective study of 450 children aged 2-16 years with otitis media requiring grommets (ear tubes). Pneumococcal antibody levels were significantly higher in the 2-6 year (P < 0.004) and 7-10 year (P < 0.04) study groups in comparison with age-matched controls. There was no difference in Haemophilus antibody levels between the study and control group children for the age groups 2-6 years and 11-16 years. Haemophilus antibody levels were significantly lower in the 7-10 year (P < 0.003) group in comparison with age-matched controls. Eighty-eight out of 450 (19.6%) children had pneumococcal antibody levels below the 25th percentile. Nineteen out of 88 (21.6%) children with pneumococcal antibody levels below the 25th centile were test immunized with 23 valent Pneumococcal polysaccharide and unconjugated Haemophilus type b capsular polysaccharide. Of these 19 children (aged 4-11 years), five mounted suboptimal responses to both polysaccharide antigens, whilst one child failed to respond to Haemophilus polysaccharide alone. There was no significant difference in the prevalence of IgG subclass deficiency between the normal responders and poor responders to immunization (P = 0.12). We found no evidence of specific polysaccharide antibody deficiency in the vast majority of the 450 children studied. However, the significance of poor antibody responses to test immunization in a small minority of children with otitis media is unclear. Long-term follow up of these children is required to determine whether poor immunization responses herald the development of frank antibody deficiency.

To determine whether the human antibody (Ab) repertoire to the Haemophilus influenzae type b capsular polysaccharide (Hib PS) could be studied at the molecular level with phage display technology, we constructed a phage Fab library by using peripheral blood from a vaccinated adult. Phage were selected based on Hib PS binding. Two distinct Hib PS-specific phage clones were identified whose Fab fragments used the same V(H) region paired with two different V(L) regions. The V(L) regions were derived from two independent rearrangements of the A2c gene with Jkappa1, and both contained a nontemplated arginine codon at the V-Jkappa junction. The two A2 V gene segments differed from the A2c germ line sequence in 0 and 5 bases. The V(H) region consisted of the V(H)26 gene segment having 98% identity to the germline nucleotide sequence, a D region of 9 bases, and J(H)4b1. Usage of V(H)26 in combination with A2 V regions containing a junctional arginine is a predominant configuration of naturally occurring Hib PS-specific Abs. Liquid- and solid-phase assays showed that phage-derived Fab reacted with Hib PS and expressed HibId-1, an idiotype associated with the kappaII-A2 V region. These findings extend the database of V region polymorphisms that can contribute to the Hib PS repertoire and demonstrate that Hib PS-specific Fab fragments isolated from combinatorial phage libraries use V gene combinations which mirror the natural repertoire.

Background: Recurrent bacterial sepsis is common in pediatric HIV infection and immunization against Haemophilus influenzae type b (Hib) is recommended. Long term persistence of anti-Hib antibody and the need for, or timing of, a booster dose has not been adequately studied., Methods: Immunogenicity during a 12-month period following immunization with Hib-tetanus conjugate vaccine (ACT-HIB; Merieux) was evaluated in 48 vertically HIV-infected children and 36 uninfected children, born to HIV-positive mothers. A titer of anti-Hib polysaccharide antibody of > or = 0.15 microgram/ml was considered to indicate short term and > or = 1 microgram/ml long term protection., Results: At 1 month postvaccination 36 (100%) uninfected and 42 (88%) HIV-infected children achieved titers of > or = 1 microgram/ml. However, by 1 year titers had dropped below this value in 18 (43%) infected compared with only 4 (11%) uninfected children (chi square, 9.7; P = 0.002). Although the rate of fall of antibody titer was greater in uninfected than in infected children, this was no longer the case after adjustment for the 1-month postimmunization titer. The rate of antibody titer decline was not significantly related to HIV disease status or to either the age-related CD4 count at the time of immunization or the change in age-adjusted CD4 count during the 12 months after immunization., Conclusions: Not only was the initial antibody response to Hib conjugate vaccine decreased in children with HIV infection and AIDS but also 1 year later only 57% of the initial responders had persisting titers above the level associated with long term protection. The need for reimmunization of children with HIV infection against Hib requires further evaluation.

In this study we compared natural vs. induced Haemophilus influenzae type b (Hib) anti-capsular polyribosylribitol phosphate (PRP) antibody response in a low socioeconomic population. One hundred twenty five 2-month-old children received the complete HbOC vaccine immunization scheme and a booster dose at 15 months of age. One hundred twenty five non-immunized children served as the control group. Serum Hib anti-PRP antibody titers were determined by ELISA in all children. We found at the end of the primary immunization scheme an antibody concentration of 27.28 micrograms/ml in the immunized group vs. 7.48 micrograms/ml in the control group. The antibody response was mainly of the IgG1 class in both groups. After the booster dose the antibody concentration was 30.14 g/ml in the vaccinated group vs. 6.06 micrograms/ml in the control group (p < 0.01). Ninety nine percent of immunized and non-immunized infants had titers greater than 1 microgram/ml. These results confirm that immunization with the HbOC vaccine induces an important increase in anti-PRP specific antibody titer, but they also demonstrate that natural exposure induces responses higher than those referred as protective (1 microgram/ml).

The mechanism of antibody-mediated reduction of Haemophilus influenzae type b (Hib) carriage was studied in the infant rat colonization model. Monoclonal Hib polysaccharide (PS) antibody (MAb) given intranasally or intraperitoneally and human secretory anti-Hib PS IgA given intranasally inhibited colonization by Hib during the entire follow-up period (2-48 h after challenge) but did not affect colonization by Hi, a noncapsulated variant of Hib. F(ab')2 fragments, prepared from the MAb or from human serum anti-Hib IgG reduced Hib colonization as efficiently as the uncleaved molecules. Complement depletion by cobra venom treatment had no effect on the antibody-mediated reduction of Hib colonization. These results indicate that Fc-mediated activities of immunoglobulins are not essential in the reduction of Hib colonization. Instead, antibodies to Hib most likely reduce colonization by a direct effect on growth of the bacteria or their adherence to the nasopharyngeal mucosa.

Restricted V region diversity is a key feature of Abs to many haptens and simple polysaccharides. Two possible mechanisms exist: 1) selection of many clonally unrelated B cells using very similar or identical VDJ and VJ rearrangements; and 2) selection of a heavily expanded progeny of few virgin B cells. How many virgin B cells eventually give rise to the total Ab response to a simple Ag is a fundamental immunologic question. In this report, we address this question in human adults by analyzing the rearranged VkappaJkappa genes of B cells responding to a single dose of the capsular polysaccharide of Haemophilus influenzae type b coupled to tetanus toxoid. We combined affinity purification of circulating vaccine-induced Ab-secreting cells with PCR amplification of cDNA followed by cloning and sequencing. Forty-eight and 42 kappa VJ gene transcripts were analyzed from two adults, respectively. Both individuals used extremely restricted repertoires with >90% of the cells using a single Vkappa gene rearranged to a single Jkappa gene. Despite the fact that the Ab responses engaged high numbers of Ab-secreting cells, analysis of the many shared, somatically acquired mutations showed that the majority of the cells originated from a common virgin B cell. Kinetic considerations implied that an extremely selected population of hypermutated memory B cells must have existed in these individuals before the first systemic immunization with the Ag. A possible role for the mucosal immune system in the priming and selection of these cells is proposed.

Objective: To evaluate two- or three-dose "mixed" regimens of Haemophilus influenzae type b conjugate vaccines in the priming series., Design: Two randomized clinical trials with 140 and 181 infants, respectively., Setting: Private practices in New Orleans and Chicago., Methods: In trial I, infants received one of four regimens. Two were recommended regimens for polyribosylribitol phosphate (PRP)-meningococcal protein conjugate (M) and PRP-tetanus toxoid conjugate (T). Two mixed regimens consisted of M at 2 months followed by two doses of T or PRP-diphtheria toxoid conjugate (D) at 4 and 6 months. Trial II consisted of three groups. Two were recommended regimens for M and T. The third was a two-dose mixed regimen consisting of M at 2 months and T at 4 months. Parents were interviewed and instructed to record side effects after each vaccination. Serum was assayed for H influenzae type b anticapsular antibody (anti-PRP)., Results: Minor differences in safety profiles likely reflected alpha error. In trial I, M (lot 0884T, one of several known to have had decreased immunogenicity), probably primed for substantial increase in serum antibody when D or T was given at 4 and 6 months. In trial II, infants who received the two-dose mixed regimen (M from immunogenic lot 0116W at 2 months and T at 4 months) had a significantly higher mean area under the curve than recipients of the three-dose TTT regimen when antibody concentration was plotted against age, although the geometric mean anti-PRP antibody concentration for the MT-recipients was significantly lower at 7 months., Conclusions: M used in trial I may have primed infants despite poor immunogenicity. The two-dose mixed regimen (MT-) in trial II produced a mean anti-PRP antibody concentration with higher sustained anti-PRP concentrations from 2 to 7 months, as judged by the area under the curve, but a lower mean anti-PRP antibody concentration at 7 months.

The development of Haemophilus influenzae type b (Hib) conjugate vaccines has led to the virtual elimination of systemic infections caused by that pathogen, has provided insights into the pathogenesis of and immunity to other capsulated bacteria, and has contributed to the development of new vaccines. Meningitis, a common and serious infection of children, and other infections caused by Hib have been virtually eliminated in countries that have achieved widespread vaccination with Hib conjugates, including the United States, Canada, the United Kingdom, Iceland, Scandinavia, France, and Germany. Hib conjugates have also been shown to be highly effective in developing countries. The principles derived from the use of these vaccines, along with studies of other capsulated pathogens, should allow the rapid inclusion of new polysaccharide-based conjugates into routine vaccination schedules of infants, and should help to realize further reductions in serious systemic infectious diseases.

Immunization of mothers during pregnancy may be an effective means of providing protection to infants during the first months of life against many pathogens. Previous studies have identified factors that influence the transfer of immunoglobulin across the placenta, including the time of vaccination during pregnancy and isotypes of specific immunoglobulins. By studying antibodies to Haemophilus influenzae type b polysaccharide (Hib-PS) in 26 pairs of maternal-cord sera obtained from unimmunized healthy women and 22 pairs of maternal-cord sera from women immunized with one of three different Hib vaccines, we have found that the immunoglobulin transfer is also dependent on the V region of antibodies. Anti-Hib-PS derived from the V kappa II gene "A2" was transferred about ten times more efficiently to the fetus than other anti-Hib-PS antibodies (20% vs 1-2%). It was found that antibodies derived from the A2 V kappa gene are primarily IgG whereas other antibodies are preferentially associated with the IgM isotype. The potential association between the antibody V region with preferential placental transfer should be considered for future studies involving maternal immunization.

Objective: To evaluate the response of human immunodeficiency virus (HIV)-infected and -exposed infants to the primary series and booster dose of Haemophilus influenzae type b (Hib) conjugate vaccine., Design: Retrospective study., Patients and Setting: The HIV-exposed and -infected infants who were attending the Special Immunology Family Clinic at The Children's Hospital of Philadelphia (Pa)., Main Outcome Measures: Geometric mean antibody titers (GMTs) to Hib polyribosyl ribitol phosphate capsular antigen were assessed after the primary series and again after the 15-month booster doses. In addition, the percentages of patients who responded with polyribosyl ribitol phosphate antibody levels greater than both 0.15 and 1.0 mg/L were compared between groups., Results: After the 3-dose primary series, the GMTs were lower in the HIV-infected infants compared with those in the HIV-exposed, uninfected infants (0.86 vs 2.30, P = .02). Forty-six percent of the HIV-infected infants mounted a response ( > 1.0 mg/L) compared with that in 79% of the HIV-exposed infants (P = .05). Among the HIV-infected infants, there was no difference in the GMTs based on CD4+ cell counts or HIV-related symptoms. After the 15-month booster dose, the GMTs were not significantly different in the HIV-infected and -exposed infants. As a group, the HIV-infected infants responded to the booster dose with a 2-fold increase in the GMTs, and significantly more of these infants had antibody concentrations above 1.0 mg/L compared with their response to the primary series (62% vs 38%, P = .02)., Conclusions: Most of the HIV-infected infants responded to the primary series of Hib conjugate vaccine with antibody concentrations greater than 0.15 mg/L, but the GMTs were significantly lower than those in the uninfected infants. The primary series of Hib conjugate vaccine appeared to be capable of inducing specific immunologic memory in the HIV-infected infants. The HIV-infected infants had a significant response to a booster dose of Hib conjugate vaccine, as measured by using the GMTs and the percentage of infants with antibody concentrations greater than 1.0 mg/L. The duration of protective titers will need to be followed in this population of patients who are at a high risk for serious bacterial disease.

A new ELISA technique using Nunc CovaLink NH microtiter plates has been developed to measure anticapsular polysaccharide specific antibodies. Capsular polysaccharide (PS) of Haemophilus influenzae type b (PRP) and pneumococcal antigens types 3, 6, 8, 14, 19, 23 were immobilized on CovaLink NH. These are modified plates with secondary amino groups bound to their surface which, in the presence of a water-soluble carbodiimide as coupling reagent, facilitate the direct binding of polysaccharides. We compared the binding characteristics of PS antigens to CovaLink NH and a conventional polystyrene ELISA plate. Checkerboard titration of PS antigens between 0.04-30 micrograms/ml clearly demonstrated that with Covalink NH optimal binding of a pooled serum from immunized donors was achieved for all PS antigens tested at a concentration of 1 microgram/ml, while binding of PS to the conventional plate was rather poor even at concentrations of 30 micrograms/ml. The CVs for the ELISA ranged from 1.1 to 2.8% for intra-assay comparisons and from 3.6 to 7.3% for inter-assay comparisons. In addition, when PRP-IgG antibodies were determined with the CovaLink NH ELISA and compared with the Farr assay an acceptable correlation ( r = 0.89, p < 0.0001) was obtained. The technique described provides a simple and sensitive tool for evaluating specific immunity to PS antigens.

The broad use of conjugated vaccines against Haemophilus influenzae type b may select for strains to which the polysaccharide vaccine does not provide immunity. We analyzed 392 consecutive H. influenzae isolates from Swiss children 0 to 16 years of age with invasive disease during the years 1986 to 1993. Bacterial strains were characterized by serotyping, capsular genotyping, outer membrane protein (OMP) subtyping, and ribotyping. Of 392 strains, 372 were serotype b, 1 was serotype a, 3 were serotype f, and 16 were nontypeable H. influenzae. After the introduction of Haemophilus conjugate vaccines in 1990, there was a relative increase of nontypeable strains from 3 to 6.6% (P = 0.27). Of the type b strains, 281 (75.5%) had the same OMP subtype and ribotype pattern. This clone predominated in the pre- and postvaccine periods. After the year 1990, the proportions of OMP subtype 1c and OMP subtype 3 tended to increase. Isolates from previously vaccinated (n = 10) and nonvaccinated patients did not differ in their subtype distributions. We conclude that the administration of conjugated vaccines decreased invasive disease caused by the most prevalent H. influenzae type b clone. However, further surveillance of circulating H. influenzae strains during the period of vaccination is indicated.

Background: Haemophilus influenzae type b (Hib) conjugate vaccines have demonstrated an impressive impact in diminishing Hib disease in industrialized countries. However, their high cost prompts nonindustrialized countries to corroborate their effectiveness under local conditions before considering their programmatic implementation. Such a postlicensure evaluation of vaccine effectiveness was undertaken in Chile., Methods: After its licensure in Chile polyribosylribitol phosphate-tetanus toxoid protein conjugate vaccine (PRP-T), combined with diphtheria-tetanus-pertussis vaccine, was introduced into the Expanded Program on Immunization schedules in 36 health centers throughout metropolitan Santiago for 12 months, whereas 35 similar health centers administered only diphtheria-tetanus toxoid-pertussis vaccine. Bacteriologic surveillance data for invasive Hib cases collected over the ensuing 30 months were analyzed., Results: In an intent-to-vaccinate (effectiveness) analysis, PRP-T provided 90.2% protection (95% confidence interval, 74.5 to 100%) against invasive Hib disease (40 vs. 4 cases, P < 0.001). Vaccine effectiveness was 91.3% against meningitis (22 vs. 2 cases) and 80% against pneumonia and empyema (10 vs. 2 cases, P = 0.039). Vaccine efficacy among infants who received all three doses of PRP-T was 91.7% (95% confidence interval, 64.8 to 100%)., Conclusions: Programmatic use of Hib conjugate vaccine administered in combination with diphtheria-tetanus-pertussis vaccine constitutes a highly effective and practical intervention in Chile, a newly industrializing country.

Since immunization of infants with conjugated Haemophilus influenzae type b (Hib) capsular polysaccharide (PS) vaccines results in a reduction of colonization, we determined the inhibitory effect of anti-Hib PS on two steps in the colonization, i.e., adherence of H. influenzae to nasopharyngeal epithelium and bacterial growth. Monoclonal antibody (MAb) E117-5 specific for Hib PS inhibited at a concentration of at least 80 microg/ml in vitro the adherence of Hib strain 770235f+b+ to oropharyngeal epithelial cells by 50% (P <, 0.02), but this MAb and sera from children immunized with Hib PS conjugate vaccine (n = 10) were not inhibitory in final dilutions containing up to 20 microg of anti-Hib PS per ml. The growth of Hib strain 770235f+b+ did completely stop in the presence of 5 microg of anti-Hib PS MAb E117-5 per ml and human sera with an anti-Hib PS concentration of 2 microg/ml or more, in contrast to the growth of the nonencapsular variant strain 770235f+b0.

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.

Immunogenicity of one dose of Haemophilus influenzae type b (Hib) conjugate vaccine in infancy and its ability to induce immunologic memory was studied in infants immunized at 4 and 14 months with either PRP-OMP (Hib polysaccharide conjugated with Neisseria meningitidis group B outer membrane protein complex) or PRP-T (Hib polysaccharide-tetanus toxoid conjugate) and compared with three doses of the same vaccines at 4, 6, and 14 months. Each group received diphtheriatetanus-pertusis vaccine at 3, 4, and 5 months of age. At 7 months of age, both vaccines were immunogenic after one dose, even though higher antibody concentrations were achieved after two doses. A booster dose given at 14 months resulted in a high antibody concentration and a strongly IgG-dominated isotype distribution, speaking for a secondary-type response in all groups, including those who had received only one dose in infancy. Subsequent persistence of antibodies suggestive of full protection for up to 36 months was similar in all groups.

Haemophilus influenzae type b (Hib) is an important pathogen for young children, and children can be protected with antibodies (Abs) to Hib polysaccharide (PS) capsule, a linear polymer of ribosyl ribitol phosphate. The structure of anti-Hib-PS Abs has been well characterized at the molecular level; about two-thirds of anti-Hib-PS Abs use a V kappa gene named A2, and the remaining anti-Hib-PS Abs use one of many other VL genes. In order to understand the structural basis for the variability in the function of these Abs, we prepared 18 clonally pure Abs from adults and studied their affinity, avidity, bactericidal potency in vitro, and ability to reduce bacteremia in newborn rats. Affinities and avidities were determined as the inverse of the concentrations of short (3 repeating units) and long (20 repeating units) ligands which could bind 50% of anti-Hib-PS Ab in solution, respectively. No significant correlations between the protection of newborn rats and affinity (r = 0.02) or avidity (r = 0.16) were observed. The amount of Ab required to kill 50% of bacteria in vitro decreased with avidity (r = -0.32), as expected. However, Abs with high affinity were unexpectedly found to have less bactericidal activity (r = 0.38). This suggests that avidity may be a better predictor of Ab function than affinity. Affinity and avidity results were negatively correlated (r = 0.76, P = 0.0022), and Abs that had A2 V kappa gene products had higher avidity (P < 0.05) and lower affinity (P = 0.06) than Abs that had other VL genes. A possible explanation of these observations is that the epitope for Abs with the A2 gene is within the Hib-PS chain itself, whereas the epitope for Abs with a non-A2 gene is the terminus of Hib-PS.

Objective: To evaluate the effectiveness in low birth weight (LBW) infants of the currently recommended immunization schedule for conjugated Haemophilus influenzae type b (HIB) vaccine., Methods: We quantified antibody responses in 36 preterm infants with a mean birth weight of 1060 g and a mean gestational age of 28 weeks. Infants were immunized with 0.5 mL of HIB vaccine at 2 and 4 months' postnatal age. Specific HIB antibodies were quantified on cord blood, immediately before each immunization and 2 months after the last immunization., Results: Even though the geometric mean titers increased significantly during the study period, they were still markedly lower than values reported in term infants. After the second immunization, only 24 infants (67%) attained antibody concentrations of more than 0.25 micrograms/mL, defined as seropositivity. Also, only 53% of infants achieved antibody concentrations of more than 1.0 micrograms/mL compared with 92% as reported in term infants. Stepwise logistic regression identified gestational age of 27 weeks or less and the amount of intravenous immunoglobulin received as the significant variables influencing the antibody response after the first immunization. The incidence of side effects was negligible., Conclusions: We conclude that LBW infants, and especially those born at 27 or less weeks' gestation, do not respond as effectively to the HIB vaccine. We speculate that reevaluation of the current immunization schedule may be required for very LBW infants.

In 1992 it was decided to implement vaccination against Haemophilus influenzae type b (Hib) in the Norwegian vaccination programme. The chosen vaccine consists of Hib-polysaccharide conjugated to tetanus toxoid. To suit the Norwegian vaccination schedule, Hib-vaccine was given together with vaccine against diphtheria, tetanus and pertussis (DTP) at three, five and ten months of age. Since all earlier studies with the Hib-vaccine have used other time schedules, a pilot study was conducted to test antibody response and side effects when using the Norwegian schedule. 44 infants were vaccinated with Hib-vaccine and DTP at three, five and ten months of age. Blood samples showed that 95% had protective antibody titer against Hib after two doses, 100% after three. The response to the other antigens was adequate. There were only minor side effects. After introduction of the Hib-vaccine the incidence of invasive Hib-infection in children below three years of age has decreased considerably.

We investigated the relationship between the form of the Haemophilus influenzae type B (Hib) polysaccharide (PS)-protein conjugate vaccine, Id expression, and Ab quality. Two post-vaccination pools were prepared from sera of infants vaccinated with either Hib PS oligomers coupled to CRM197, a mutant diphtheria toxin (HbOC), or with higher m.w. Hib PS coupled to an outer membrane protein complex of Neisseria meningitidis group B (Hib-OMP). The mean anti-Hib PS Ab avidity of the serum pool from the infants vaccinated with HbOC was threefold higher than that of the pool from infants vaccinated with Hib-OMP. Using sequential immunoabsorption, three IgG1 idiotypically-defined anti-Hib PS fractions were isolated from each of the serum pools: Hibld-1, Hibld-2, and a Hibld-1/-2-depleted population, designated Hibld-0. Hibld-1 and Hibld-2 are idiotypic markers for anti-Hib PS Abs expressing kappa II-A2 and lambda VII V regions, respectively. Hibld-1 anti-Hib PS Abs had significantly higher avidity, 2- to 19-fold higher in vitro bactericidal activity, and were more protective against Hib bacteremia in infant rats, than the respective Hibld-2 Abs isolated from each of the pools. Comparing the two vaccines, Hibld-1 anti-Hib PS Abs elicited by HbOC had significantly higher avidity and 10-fold higher bactericidal and rat protective activity than the Hibld-1 Abs elicited by Hib-OMP. These findings demonstrate that the molecular form of the Hib PS immunogen dictates both V region usage and quality of Ab function.

IgG and IgA antibodies to Haemophilus influenzae type b (Hib) capsular polysaccharide (PS) were measured in saliva of 7- to 19-month-old children after vaccination with 2 or 3 doses of Hib conjugate vaccine. Both the concentration and the prevalence of these antibodies were higher after 3 than after 2 doses of vaccine. The presence and concentration of IgG Hib PS antibodies in saliva correlated with their concentration in serum. Fifty-four percent (20 of 37) of the children with serum IgG concentrations higher than 30 micrograms/ml had detectable salivary IgG, whereas the 21 children with a low serum IgG concentration (< 3 micrograms/ml) had no IgG in saliva. The IgA anti-Hib PS in saliva was mainly secretory and was found in saliva of 29% (19 of 65) of the children who had no detectable serum IgA anti-Hib PS. The results suggest that the IgG anti-Hib PS in saliva was derived from serum, whereas the IgA antibodies were locally produced. The Hib PS antibodies on mucosa are suggested to be responsible for the reduction of Hib carriage observed among children vaccinated with Haemophilus type b conjugates.

In order to define abnormalities of humoral immunity which determine susceptibility to respiratory tract infections in IgA-deficient adults, serum IgG subclass concentrations, and serum concentrations of pneumococcal antibodies and Haemophilus influenzae type B (Hib) antibodies sera from IgA-deficient adults with and without susceptibility to respiratory tract infections were compared. Infection susceptibility was not related to the degree of IgA deficiency, but was related to deficiency of IgG4 and, to a lesser extent, IgG2, as well as to low basal serum concentrations of pneumococcal polysaccharide antibodies. The combination of IgG2 and/or IgG4 deficiency and a non-protective basal serum concentration of antibody against two or more pneumococcal polysaccharides was present in the serum of six of 12 (50%) patients with severe infections, but only one of 44 (2%) patients without infections. Furthermore, the preservation of antibody responses against the most immunogenic pneumococcal polysaccharide type 3, but not against the less immunogenic types 7F, 9N and 14, in patients with severe infections suggested that abnormalities of pneumococcal polysaccharide antibody responses might include defects of affinity maturation.