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We investigated the anti-polysaccharide antibody responses in subjects with Down syndrome (DS) because DS subjects show decreased peripheral B-lymphocyte numbers in all age groups, and a clinical picture of recurrent respiratory tract infections and increased incidence of autoimmune diseases which is reminiscent of common variable immunodeficiency disorders (CVID)-like disease. We determined titers and opsonophagocytosis in response to conjugated and unconjugated pneumococcal serotypes in 18 DS subjects aged 6-24 years. The results show adequate serotype-specific antibody titers in response to all conjugated and almost all unconjugated serotypes used. Opsonophagocytosis activity as measured against pneumococcal serotypes 9N, 19F and 23F was also found to be intact. We conclude that DS subjects do not have a clear defect in their anti-polysaccharide antibody response., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: To describe the increase of invasive Haemophilus influenzae type b (Hib) infections in The Netherlands before and after the introduction of Hib vaccination in 1993, and to hypothesise about possible explanations., Design: Descriptive., Method: Data on the prevalence of invasive Hib infections, such as meningitis and epiglottitis, during 1990-2004 were obtained from The Netherlands Reference Laboratory for Bacterial Meningitis, which collects Hib isolates from spinal fluid and blood from across the country., Results: The incidence of invasive Hib infections decreased substantially for a few years after 1993. The total number of isolates was at a minimum in 1999 (n = 12) and increased to 49 in 2004. The annual number of patients with vaccine failure was 5 or less during 1995-2001, but was between 10 and 15 from 2002 onwards. A definite explanation for the increase in the incidence of invasive Hib infections cannot be given. Improbable causes are a surveillance artefact, an impaired response to the vaccine due to vaccination-scheme changes or interaction with other vaccines, or selection of Hib variants that are less sensitive to the vaccine-induced immunity. It most likely involves secondary vaccine failure: Hib carriership is decreased by mass vaccination, whereupon natural boosting occurs less frequently later in life. Subsequently, immunity decreases and susceptibility to invasive infection increases. Careful surveillance of invasive Hib infections in The Netherlands remains important.

The immune response to polysaccharides is initiated when polysaccharides bind complement factor C3d, and these polysaccharide-C3d complexes subsequently localize on splenic marginal zone B cells strongly expressing CD21 (complement receptor 2). Infants and children under the age of 2 years have low or absent expression of CD21 on their marginal zone B cells, and consequently do not adequately respond to polysaccharides. In contrast, polysaccharide-protein conjugate vaccines are able to induce antibodies at this young age. Conjugate vaccines apparently overcome the necessity for CD21-C3d interaction for an antipolysaccharide immune response. We demonstrate in a rat model that localization of pneumococcal polysaccharides on splenic marginal zone B cells indeed is complement dependent. We also show that pneumococcal conjugates do not specifically localize on splenic marginal zone B cells and that splenic localization of polysaccharide conjugates is independent of the presence of complement. Thus, the induction of antipolysaccharide antibodies by conjugate vaccines apparently can occur independently of CD21-C3d interaction. These basic findings may explain the effectiveness of conjugated vaccines in young children and may open the way for their application in other patient groups.

As a defective anti-polysaccharide response can exist in the absence of an immunoglobulin deficiency, a series of 26 patients with bronchiectasis of unknown aetiology was vaccinated with a 23-valent pneumococcal polysaccharide vaccine. All patients suffered from recurrent respiratory tract infections. When measuring total antibody levels to pneumococcal serotypes 3, 4 and 9, a normal polysaccharide antibody response was found in 22 patients. However, only 11 of these subjects showed a normal pneumococcal antibody response within the IgA and/or IgG2 subclass, and thus could be classified as true responders, while 15 patients did not respond in either the IgA class or in the IgG2 subclass. When analysing differences between the responder (n = 11) and nonresponder (n = 15) groups, the latter demonstrated higher frequencies of respiratory tract infections and more severe lung pathology, as revealed by the presence of more bronchi visualised in the peripheral third of the lung by high-resolution computed tomography scanning. Moreover, nonresponders needed extensive lung surgery more often in order to control their disease (number of resected segments eight versus five). In conclusion, an important fraction of patients presenting with idiopathic bronchiectasis is associated with a selective anti-polysaccharide response deficiency and this subgroup appears to represent a more severe clinical phenotype. Therefore, it can be regarded as a separate clinical entity with possible therapeutic targets. In order to identify IgA and IgG2 anti-polysaccharide nonresponders, all patients presenting with bronchiectasis of unknown aetiology should be immunised with a pneumococcal polysaccharide vaccine, and IgA and IgG2 isotype responses should be evaluated as well as the total antibody response.

Protection against infections with Streptococcus pneumoniae depends on the presence of antibodies against capsular polysaccharides that facilitate phagocytosis. Asplenic patients are at increased risk for pneumococcal infections, since both phagocytosis and the initiation of the antibody response to polysaccharides take place in the spleen. Therefore, vaccination with pneumococcal polysaccharide vaccines is recommended prior to splenectomy, which, as in the case of trauma, is not always feasible. We show that in rats, vaccination with a pneumococcal conjugate vaccine can induce good antibody responses even after splenectomy, particularly after a second dose. The spleen remains necessary for a fast, primary response to (blood-borne) polysaccharides, even when they are presented in a conjugated form. Coadministration of a conjugate vaccine with additional nonconjugated polysaccharides of other serotypes did not improve the response to the nonconjugated polysaccharides. We conclude that pneumococcal conjugate vaccines can be of value in protecting asplenic or hyposplenic patients against pneumococcal infections.

Infant vaccination with conjugated Haemophilus influenzae type b (Hib) vaccine is highly effective in protecting against invasive Hib infections, but vaccine failures do occur. Twenty-one vaccine failures are reported since the introduction of the Hib conjugate vaccine in The Netherlands. Of the 14 evaluable patients, 6 children showed no antibody response to Hib polysaccharide in convalescent-phase serum (immunoglobulin [Ig] G anti-Hib level <1.0 microg/mL), including 1 child with hypogammaglobulinemia and 1 child with IgG2 deficiency. After revaccination, almost all children developed anti-Hib antibodies. In case of Hib vaccine failure, case investigation should be performed, including measurement of serum Ig concentrations as well as specific anti-Hib antibodies. Invasive Hib disease after infant conjugate Hib vaccination may be the presentation of an underlying immunodeficiency, but more often, only a decreased antibody response to Hib is found; revaccination with conjugated Hib vaccine is advised.

Children with frequent recurrent episodes of otitis media may have a deficient IgG2 antibody response to polysaccharide antigens. Five otitis-prone children were vaccinated with heptavalent pneumococcal conjugate vaccine. While all had an IgG1 antibody response to all pneumococcal serotypes included in the conjugate vaccine, the IgG2 response, especially to serotypes 6B, 9V, 19F, and 23F, was poor. However, vaccination with a 23-valent polysaccharide vaccine 6 months after conjugate vaccination induced an 11.5- to 163-fold increase in IgG2 anti-polysaccharide antibody titers. Thus, an IgG2 polysaccharide antibody deficiency can be overcome by priming with a pneumococcal conjugate vaccine followed by a booster with a polyvalent polysaccharide vaccine.

Newborns and infants up to the age of 1.5-2 years of age are unable to produce antibodies to bacterial capsular polysaccharides. As a consequence, children up to the age of 2 years have an increased susceptibility for infections with encapsulated bacteria. Capsular polysaccharides are classified as so-called T cell independent type 2 antigens and induce IgG2 antibodies. The mechanism of B lymphocyte activation by polysaccharides differs from that of protein antigens and involves co-stimulation by CD21 (type 2 complement receptor). Reduced expression of CD21 on neonatal B lymphocytes can explain unresponsiveness to polysaccharides. Polysaccharide protein conjugates have the ability to overcome unresponsiveness to polysaccharides early in life. The response induced is predominant IgGl.

In a prospective study, serological responses and opsonic activity towards Streptococcus pneumoniae were measured in 60 HIV-infected patients and 25 controls after the administration of the 23-valent pneumococcal vaccine (PneumovaxR). Serum samples were collected before vaccination and at weeks 1, 2, 4, and 12 after vaccination and were tested for the presence of antibodies against a mixture of capsular polysaccharide antigens (pool) and against type 3 and type 4 antigens (PS3 and PS4), using an ELISA. A serological response was defined as a two-fold or greater increase in serum titer after vaccination. Opsonophagocytosis was measured in patients with a definite response against PS3. Generally, prevaccination antipneumococcal antibody titers were clearly higher in HIV-infected patients than in healthy controls. After vaccination, antipool antibody responses were found in 76% of vaccinated patients; 24% of the patients were non-responders. In patients with more than 0.300 x 10(9) CD4 + cells per liter the percentage of responders was 94%; in patients with fewer than 0.300 x 10(9) CD4 + cells per liter this percentage was 68% (P < 0.05). The antipool response in control subjects was 92%. A serological response to PS3 and PS4 was found in 29% and 49% of the patients, respectively, and was correlated with CD4 + cell count. In controls, these percentages were 48% and 92%, respectively. In 30% of responding patients, antibody titers dropped already to prevaccination levels by week 12 after vaccination. Opsonophagocytosis was not significantly improved by vaccination, probably because of a relatively high preexisting opsonic activity. Although prevaccination conditions may have had an important influence on the study outcome, the results are not in favor of a significant beneficial effect of vaccination with PneumovaxR on antibody formation in HIV-infected patients. This raises further questions as to the relevance of pneumococcal vaccination in this population.

Anti-group B streptococcal type III polysaccharide-specific human B cells from the peripheral circulation can be activated and detected in an in vitro culture system. It is possible to detect both IgM- and IgG- producing cells from both seropositive and seronegative donors. The specificity of the response was demonstrated by inhibition with excess liquid phase antigen and the use of related but antigenically distinct control antigens. The response was absent without the addition of T cells, optimal at 10% and 25% T cells respectively for IgM- and IgG-secreting cells, and undetectable using 50% T cells. The optimal antigen concentration for in vitro B-cell activation is 2.5 x 10(-4) micrograms/ml. Cells from 5 of 6 seropositive donors and 3 of 7 seronegative donors produced specific IgM antibody after culture with antigen. We conclude that the control of the human antibody response to the group B streptococcal type III polysaccharide is influenced by T cells. The response seen in the culture system may be of value in assessing future vaccine candidates designed to prevent neonatal infections.

Background: Recurrent pyogenic infections are known to occur in patients with an impaired response to polysaccharide antigens. We investigated the occurrence of deficient responses to pneumococcal capsular polysaccharides in patients with recurrent respiratory tract and recurrent systemic infections., Methods: Forty-five patients, 1.7 to 17.1 years of age, were immunized with 23-valent pneumococcal polysaccharide vaccine. Antibody levels to seven pneumococcal serotypes (3, 4, 6A, 9N, 14, 19F, 23F) were determined by ELISA before and after immunization. In addition, patients received a booster immunization with diphtheria toxoid, tetanus toxoid, and poliomyelitis virus vaccine., Results: Thirty-five patients had normal serum immunoglobulin levels. Five of these patients (14%) had low antipneumococcal preimmunization antibody levels and failed to respond to pneumococcal vaccination, whereas the response to booster immunization with protein antigens was appropriate. Three patients were younger than 3 years old, and one had a family history of IgG2 deficiency. Low IgG developed in a fifth patient during follow-up. Ten patients had a humoral immunodeficiency. Seven of these patients failed to respond to pneumococcal vaccination., Conclusions: We conclude that a defective immune response to polysaccharide antigens in patients requires long-term follow-up to distinguish transient maturational delay from a persistent selective impaired response to polysaccharide antigens, which on occasion may precede the development of humoral immunodeficiency disease.

A number of studies have indicated that the complement receptor type 2 (CR2), which is the receptor for C3d, a degradation fragment of the complement component C3, regulates B lymphocyte activation and growth. Early reports have described that C3 regulates T cell-dependent (TD) antibody responses. The involvement of CR2 in the antibody response to T cell-independent type 2(TI-2) antigens was investigated because neonatal B cells, which are unresponsive to TI-2 antigens both in vivo and in vitro, express a significantly decreased level of CR2 as compared to B cells of adult donors. We utilized type 4 pneumococcal polysaccharide (PS4) as a model TI-2 antigen. In order to study the relationship between CR2 and the response to PS4, B cells were costimulated with PS4 and monoclonal antibodies (MAb) to CR2. HB5 and OKB7 anti-CR2 monoclonal antibodies enhanced the in vitro response of adult B cells to PS4, as measured in a PS4-specific spot-forming cell assay. Neonatal B cells could only be induced to respond to PS4 using high concentrations of OKB7 anti-CR2 MAb. The 8-mercaptoguanosine (8MGuo), an agent that can overcome the in vitro unresponsiveness to PS4 of neonatal B cells, increased CR2 expression on adult and neonatal B cells. Furthermore, 8MGuo synergizes strongly with anti-CR2 antibodies in augmenting the anti-PS4 antibody response. Data presented in this report provide evidence of CR2 involvement in the antibody response to PS4 and that the neonatal B cell unresponsiveness to TI-2 antigens may be due to the decreased expression of CR2.

Synthetically prepared ribosylribitol phosphate dimer and trimer analogous to fragments of the capsular polysaccharide of Haemophilus influenzae type b, containing either an amino- or a (masked) thiol-functionalized spacer, were conjugated to protein by two different methods. The thiol-containing carbohydrates were conjugated to tetanus toxoid or H. influenzae outer membrane protein using N-succinimidyl 3-(2-pyridyldithio)propionate. Glutaric dialdehyde was used to conjugate the amino-containing carbohydrates with tetanus toxoid. All conjugates were able to bind antibodies raised against the native bacterial polysaccharide, as determined by competition ELISA. The glutaric dialdehyde conjugate prepared from ribosylribitol phosphate trimer and tetanus toxoid induced a strong polysaccharide-specific antibody response in mice and rabbits.

An international collaborative study was conducted at ten sites to examine the performance of enzyme immunoassays (EIAs) for the quantitation of IgG1, IgG2, IgG3, IgG4 and total IgG anti-Haemophilus influenzae type b (Hib) capsular polysaccharide in human serum. All groups used the same reagents: microtiter plates coated with polyribosylribitol phosphate (PRP) conjugated to poly-L-lysine (PLL), reference, control and test human sera, biotin-conjugated International Union of Immunological Societies (IUIS)-documented monoclonal anti-human IgG1-4 and IgG Pan detection antibodies, avidin-peroxidase and TMB substrate. Initial mixing of soluble PRP antigen or an equal volume of buffer with the 20 test sera prior to analysis confirmed PRP antigen specificity in all five EIAs with greater than 80% competitive inhibition at most sites. Positive correlation between the total IgG anti-Hib and sum of IgG1-4 anti-Hib was demonstrated (r2 = 0.99, Y = 1.13X -0.15). Good agreement was shown between the total IgG anti-Hib as measured by EIA and the total Hib-specific antibodies measured by the current radiolabeled antigen binding assay (r2 = 0.97, Y = 4.6X -5.8). Assay parallelism was demonstrated with an average interdilutional %CV of 22% and parallel dose-response curve slopes. The interdilutional %CVs were calculated as an average per sample of the variation of microgram/ml (corrected for dilution) at different dilutions per laboratory for all participating sites. The interlaboratory variation was the only performance parameter studied that exceeded the target level of 35% CV in all IgG1-4 and total IgG anti-Hib assays. IgG subclass distributions in the test sera demonstrated a predominance of IgG1 anti-Hib in the pediatric serum pools and IgG2 anti-Hib in the adult sera, with low but detectable levels of IgG3 and IgG4 anti-Hib in each group.

Polyribosilribitolphosphate (PRP), the capsular polysaccharide from Haemophilus influenzae type b, is a T-cell-independent type 2 antigen. In vitro culture of adult peripheral blood T cells with 15 micrograms/ml PRP leads to induction of interleukin-2 receptor (IL-2R) expression on up to 10% of T cells. These cells are CD4+ and carry the alpha beta T-cell receptor. PRP, at concentrations above 1-5 micrograms/ml, can also induce in vitro proliferation of both adult and neonatal T cells. We conclude that PRP acts as a human T-cell mitogen. The in vitro proliferative response as well as IL-2R expression was studied in T cells derived from adults after vaccination with native PRP, with PRP conjugated to a carrier protein, or with diphtheria toxoid. Vaccination with conjugated PRP decreased the doses of PRP required for in vitro induction of IL-2R expression and T-cell proliferation. This indicates that vaccination with PRP conjugated to a carrier protein improves the in vitro T-cell response to PRP activation.

We have prepared conjugates of pneumococcal type 4 polysaccharides (PS4) or oligosaccharides to tetanus toxoid using the carbodiimide method. The use of a spacer, 6-aminohexanoic acid, resulted in higher incorporation of carrier protein. Conjugates contained up to 10% free polysaccharide, but no free protein. In general, polysaccharide conjugates induced higher anti-PS4 IgG antibody titers than oligosaccharide conjugates. Conjugates with the highest amount of incorporated protein were the most immunogenic. The response to conjugated PS4 does show characteristics of a T cell-dependent antibody response, in terms of both isotype distribution and induction of immunological memory. Repeated immunization with high doses of PS4TT conjugate resulted in a virtually negative anti-PS4 IgG response, suggestive of the induction of high dose tolerance.

Previous studies with saccharide-protein conjugates have demonstrated that antibody responses to the saccharide can be improved by the preexistence of carrier immunity. Here we report that prior exposure to the carrier protein can either enhance or suppress antibody response to polysaccharides administered in saccharide-protein conjugates. A dose-dependent role for carrier priming in the antisaccharide antibody response to three saccharide-protein conjugate vaccines, i.e., a Streptococcus pneumoniae type 4 polysaccharide-tetanus toxoid (TT) conjugate (PS4TT), a Neisseria meningitidis group C polysaccharide-TT conjugate (MenCTT), and a N. meningitidis group C oligosaccharide-diphtheria mutant toxin conjugate (MenCCRM), was investigated. The results showed that an increase in the antipolysaccharide antibody response could be obtained for both PS4TT and MenCTT but not for MenCCRM with low-dose carrier priming (0.025 to 0.25 microgram). However, suppression of the antipolysaccharide antibody response was observed with the PS4TT and MenCTT vaccines with high-dose (25-micrograms) carrier priming. There was no suppression effect with MenCCRM. The increase in the antipolysaccharide antibody response was shown to be restricted to the immunoglobulin G1 (IgG1) subclass, whereas suppression with high-dose carrier priming affected all antipolysaccharide subclass antibodies induced by PS4TT (IgG1, IgG2b, and IgG3) and only two of the four subclass antibodies induced by MenCTT (IgG2a and IgG2b). The increase in the antipolysaccharide antibody response was also present at the antipolysaccharide IgM antibody level but was not observed at the anti-carrier IgG antibody level.

In order to study the regulation of the human B-cell response to T-cell independent type 2 (TI-2) antigens, we analysed the role of T cells in the in vitro antibody response to type 4 pneumococcal polysaccharide (PS4). We found that T cells can positively regulate the in vitro antibody response to PS4 when they are added into an in vitro B-cell culture system. In addition we demonstrated that T cells exert a negatively regulating activity. We found that T cells, when cultured for 24 h in the presence of high concentrations of PS4, can suppress the anti-PS4 antibody response. This down-regulation of the anti-PS4 B-cell response is shown to be antigen specific and MHC-restricted. Furthermore, PS4-specific T-cell mediated suppression appears to be radioresistant and confined to the T-cell preparations enriched for CD8+ cells. The results show that analogous to results obtained in mice, human T cells are able to exert a regulatory control of the antibody response to TI-2 antigens.

A number of pneumococcal type 4 poly- and oligosaccharide tetanus toxoid conjugates were prepared using identical conjugation methods. Purified conjugates with similar m.w. were injected in mice; polysaccharide conjugates (PS4TT) were more immunogenic than oligosaccharide conjugates (OS4TT). Polysaccharide conjugates with a PS4:TT ratio less than 1 (w/w) appeared to be the most immunogenic conjugates. This was observed in both NIH (outbred) and BALB/c (inbred) strains of mice. Oligosaccharide tetanus toxoid conjugates required w/w ratios of greater than 1 to acquire optimal immunogenicity. Oligosaccharides (12 repeating units) of pneumococcal type 4, obtained by periodate cleavage, appeared to retain full antigenicity as measured by competition ELISA. Both PS4TT and OS4TT conjugates induced an antibody response with the characteristics of a T cell-dependent type of immune response. An anti-PS4 IgG and IgM booster effect could be demonstrated for all conjugates. The IgG subclass response induced by PS4TT and OS4TT conjugates is primarily IgG1 but IgG3 is also detectable. However, the amounts of anti-PS4 IgG3 differed for the various conjugates. We conclude that the immune response induced by pneumococcal type 4 saccharide tetanus toxoid conjugates can be manipulated by variation of the saccharide:protein ratio and the saccharide chain length, whereas keeping the m.w. of such conjugates at constant values.

The immunoregulatory function of the complement system has been the focus of many investigations. In particular, fragments of complement factor C3 have been shown to play a role in B-lymphocyte activation and proliferation, lymphokine production, and the generation of in vitro antibody production. Purified pneumococcal polysaccharides (PS) can induce direct activation of C3 via the alternative pathway. Using sera of C1q-deficient patients and healthy subjects, we demonstrated that C3d, a split product of C3 that is generated after degradation of iC3b, can be bound to PS antigens. The binding of C3d to PS can occur in the absence of specific antibodies. Subsequently, we showed that PS complexed with C3d can be recognized by complement receptor type 2 that is expressed on B cells. Treatment of B cells with a monoclonal antibody recognizing the C3d-binding site of complement receptor type 2 reduces the binding of PS-C3d to the cells. In addition, we showed that PS4 complexed with C3d exerted an increased immunogenicity compared with free PS4. Our results show that the complement system plays a role in the activation of PS-specific B cells, carrying membrane receptors for C3d. Consequently, the complement system plays a regulatory role in the antibody response to T-cell-independent type 2 antigens such as PS.

In order to gain insight into the relatively late appearance in ontogeny of responsiveness to T-cell independent (TI) antigens, the in vitro human B-cell response to type 4 pneumococcal polysaccharides (PS4, a human TI-2 antigen) was studied. B cells obtained from adults differentiate into anti-PS4 antibody forming cells upon culturing with PS4, but neonatal B cells obtained from cord blood fail to respond. The culture system used does, however, allow the differentiation of B cells reactive with TD antigens, for example ovalbumin. In order to evaluate the concept that in man the anti-PS4 response is derived from a particular B-cell subset we separated adult B cells on the basis of expression of the determinant recognized by the monoclonal antibody FMC7. The anti-PS4 response is found mainly, but not exclusively, in the FMC7+ subset. The selective unresponsiveness of neonatal B cells to TI-2 antigens is not, however, due to the absence of FMC7+ B cells because, unlike adult blood B cells of which about 50% are FMC7+, 100% of neonatal B cells are FMC7+.

We have shown previously that pneumococcal polysaccharides behave as human T cell-independent type 2 Ag. When cultured in vitro with type 4 pneumococcal polysaccharides (PS4), human neonatal B cells do not or only marginally respond. Limiting dilution analysis of neonatal B cells polyclonally activated by a combination of phorbol esters, calcium ionophore, and T cells and T cell factors, however, showed that Ag-reactive B cells are present in cord blood. The frequency of anti-PS4 reactive B cells in cord blood is comparable with that of adult peripheral blood. In order to obtain more insight into the activation requirements of these PS4-reactive neonatal B cells, 8-mercaptoguanosine (8MGuo) was added to in vitro cultures. Addition of 0.5 to 1.0 mM 8MGuo resulted in a 3- to 10-fold amplification of the anti-PS4 response. the effect of 8 MGuo was most prominent when added 3 days after initiation of the culture. Based on kinetic studies, we propose that in vitro activated cells are target for 8MGuo. These data further indicate that neonatal unresponsiveness to polysaccharide Ag is not due to the physical absence of Ag-reactive B cells but more likely to be the consequence of different activation requirements as compared with adult B cells.