Determination of avidity of IgG against protein antigens from Streptococcus pneumoniae: assay development and preliminary application in clinical settings.

The measurement of antibody levels is a common test for the diagnosis of Streptococcus pneumoniae infection in research. However, the quality of antibody response, reflected by avidity, has not been adequately evaluated. We aimed to evaluate the role of avidity of IgG against eight pneumococcal proteins in etiologic diagnosis. Eight pneumococcal proteins (Ply, CbpA, PspA1 and 2, PcpA, PhtD, StkP-C, and PcsB-N) were used to develop a multiplex bead-based avidity immunoassay. The assay was tested for effects of the chaotropic agent, multiplexing, and repeatability. The developed assay was applied to paired samples from children with or without pneumococcal disease (n = 38 for each group), determined by either serology, polymerase chain reaction (PCR), or blood culture. We found a good correlation between singleplex and multiplex assays, with r ≥ 0.94.The assay was reproducible, with mean inter-assay variation ≤ 9% and intra-assay variation < 6%. Children with pneumococcal disease had lower median avidity indexes in the acute phase of disease for PspA1 and 2 (p = 0.042), PcpA (p = 0.002), PhtD (p = 0.014), and StkP-C (p < 0.001). When the use of IgG avidity as a diagnostic tool for pneumococcal infection was evaluated, the highest discriminative power was found for StkP-C, followed by PcpA (area under the curve [95% confidence interval, CI]: 0.868 [0.759-0.977] and 0.743 [0.607-879], respectively). The developed assay was robust and had no deleterious influence from multiplexing. Children with pneumococcal disease had lower median avidity against five pneumococcal proteins in the acute phase of disease compared to children without disease.

Detection of antibody responses against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis proteins in children with community-acquired pneumonia: effects of combining pneumococcal antigens, pre-existing antibody levels, sampling interval, age, and duration of illness.

We evaluated the effects of combining different numbers of pneumococcal antigens, pre-existing antibody levels, sampling interval, age, and duration of illness on the detection of IgG responses against eight Streptococcus pneumoniae proteins, three Haemophilus influenzae proteins, and five Moraxella catarrhalis proteins in 690 children aged <5 years with pneumonia. Serological tests were performed on acute and convalescent serum samples with a multiplexed bead-based immunoassay. The median sampling interval was 19 days, the median age was 26.7 months, and the median duration of illness was 5 days. The rate of antibody responses was 15.4 % for at least one pneumococcal antigen, 5.8 % for H. influenzae, and 2.3 % for M. catarrhalis. The rate of antibody responses against each pneumococcal antigen varied from 3.5 to 7.1 %. By multivariate analysis, pre-existing antibody levels showed a negative association with the detection of antibody responses against pneumococcal and H. influenzae antigens; the sampling interval was positively associated with the detection of antibody responses against pneumococcal and H. influenzae antigens. A sampling interval of 3 weeks was the optimal cut-off for the detection of antibody responses against pneumococcal and H. influenzae proteins. Duration of illness was negatively associated with antibody responses against PspA. Age did not influence antibody responses against the investigated antigens. In conclusion, serological assays using combinations of different pneumococcal proteins detect a higher rate of antibody responses against S. pneumoniae compared to assays using a single pneumococcal protein. Pre-existing antibody levels and sampling interval influence the detection of antibody responses against pneumococcal and H. influenzae proteins. These factors should be considered when determining pneumonia etiology by serological methods in children.

Specific antibody deficiency in children with recurrent respiratory infections: a controlled study with follow-up.

Specific antibody deficiency (SAD) to unconjugated pneumococcal vaccine (PPV) is an established primary B cell immunodeficiency. The occurrence and natural history of SAD in children is unclear. We conducted an observational study to identify SAD in children with recurrent respiratory infections. Ninety-nine children, mean age 5·9 (range 2-16) years, with recurrent or severe infections were vaccinated with PPV; serum antibody concentrations for serotypes 4, 6B, 9V, 14, 18C, 19F and 23F were measured before and 2 weeks after vaccination with enzyme immunoassay. The retrospective control group consisted of 89 healthy children matched for age and gender. No children had received previous conjugated pneumococcal vaccine (PCV) or PPV. The structured history of infectious diseases of all participants was collected. Ten of 91 (11%) children (eight excluded due to immunoglobulin G subclass deficiency) with recurrent respiratory infections had SAD. In the control group, three children (3%) responded inadequately to PPV (P = 0·05). Most children with SAD also had many other minor immune defects. After 0·5-5 years (medium 3·8), eight children with SAD were revaccinated with PPV; five responded adequately and three inadequately. Two SAD children were revaccinated with PCV, one developed an adequate and one an inadequate response. Two children with SAD received treatment with intravenous immunoglobulin; the remaining eight children recovered without replacement therapy during the follow-up. SAD is common in young children with recurrent respiratory infections, but it is often transient and resolves itself within a few years without specific treatment.

Immunization against pneumococcal disease in HIV-infected patients: conjugate versus polysaccharide vaccine before or after reconstitution of the immune system (CTN-147).

To investigate whether patients should be immunized immediately or delay immunization until after reconstitution of the immune system and whether a conjugate or polysaccharide vaccine results in a better immunologic response. Seventy-nine patients were randomly assigned, utilizing a two by two factorial design to receive immediate immunization or delay immunization. Baseline characteristics were similar for the four arms: 78% men, median age 41 years, median time since HIV diagnosis 0.3 years, median CD4 60 cells/mm(3) and median HIV viral load 5.02 log copies/mL. Results in favour of delayed immunization were observed for those serotypes showing a response. The proportional odds ratios for delayed versus immediate immunization were 0.341 (P = 0.04) and 0.204 (P = 0.004) at months 6 and 12, respectively. No differences in immunological response were observed between the two individual vaccines for the shared serotypes studied. HIV-infected adults produced a higher immunological response to pneumococcal vaccine after reconstitution of the immune system.

Antibody response to Haemophilus influenzae type-b conjugate vaccine in children and young adults with congenital asplenia or after undergoing splenectomy.

Absence of the spleen constitutes a risk of infection caused by encapsulated bacteria. The aim of our study was to determine the immune response to Haemophilus influenzae type-b (Hib) conjugate vaccine (HibCV) in asplenic individuals, considering the cause of asplenia, the age when splenectomy was carried out, and previous Hib vaccinations. Twenty asplenic patients, aged five to 25 years, were immunized with a single dose of HibCV. The specific antibody concentrations against HibCV were measured by enzyme-linked immunosorbent assay. Before vaccinations, the geometric mean antibody concentration (GMC) had an average value of 3.21 µg/ml and was comparable for all of the patients, regardless of the causes of asplenia. After vaccinations, the GMC was significantly higher, with an average of 6.78 µg/ml. Further, 4.5 years after vaccinations, the GMC was comparable to that of previously unvaccinated children. Moreover, 17/20 patients had GMC ≥ 1.0 µg/ml, which included all of the children with congenital asplenia, children splenectomized before the age of six years, and only 57% of children splenectomized after that age. HibCV gives asplenic patients long-term protection. Hence, HibCV should be administered regardless of previous vaccinations and time from splenectomy, even if antibody evaluation is not available.

Immune response to the 7-valent pneumococcal conjugate vaccine in 30 asplenic children.

The aim of the study was to determine the concentration of pneumococcal antibodies after a dose of 7-valent pneumococcal conjugate vaccine (PCV7) in 30 asplenic children between 4 months and 19 years of age. Fifteen children had received pneumococcal polysaccharide vaccine (PPV) approximately 5 years prior to vaccination with PCV7. The antibody concentrations against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were measured by ELISA before and after the PCV7 vaccination. Before vaccination with PCV7, the antibody concentrations were similar in children who had or had not received PPV previously. A dose of PCV7 stimulated a good immune response in asplenic patients. Prior immunization with PPV did not affect the antibody concentration after the vaccination with PCV7. In conclusion, asplenic children vaccinated with PPV may need revaccination with PPV earlier than the recommended 3-5 years after the first dose. PCV7 induces a satisfactory immune response in asplenic patients and should be considered as an alternative vaccine in that patient group.

A randomized study on donor immunization with tetanus-diphtheria, Haemophilus influenzae type b and inactivated poliovirus vaccines to improve the recipient responses to the same vaccines after allogeneic bone marrow transplantation.

The HLA-identical sibling donors of 111 bone marrow transplantation (BMT) recipients were randomised to receive or not to receive tetanus-diphtheria (T-d), Haemophilus influenzae type b (Hib), and inactivated poliovirus (IPV) vaccines 2-10 weeks before BM harvest. Fifty-three (DV+ group) recipients received the graft from a vaccinated donor and 58 (DV- group) from an unvaccinated donor. All recipients were vaccinated with the T-d, Hib and IPV vaccines at 3, 6 and 12 months after BMT. Diphtheria and Hib antibody concentrations were consistently higher in the DV+ than in the DV- group from 6 months post transplantation onwards. The differences were significant at 6 and 13 months for diphtheria and at 12 months for Hib antibody concentrations. Tetanus, PV1, PV2 and PV3 antibody levels were similar in both groups. Patients transplanted from donors with high tetanus, diphtheria and Hib antibody concentrations had higher respective antibody concentrations after BMT than those transplanted from donors with low antibody concentrations. Especially patients whose donors have low-specific antibody concentrations may benefit from donor vaccination with protein and conjugate vaccines.

Subclass distribution of natural salivary IgA antibodies against pneumococcal capsular polysaccharide of type 14 and pneumococcal surface adhesin A (PsaA) in children.

A number of studies have shown that the ratio of IgA1 and IgA2 subclasses in secretions can depend upon the nature of the antigen inducing their production. In order to evaluate the effect of the nature of the antigen on the subclass distribution of the naturally occurring salivary IgA antibodies against Streptococcus pneumoniae, we used enzyme immunoassay to measure the levels of natural IgA, IgA1 and IgA2 antibodies to pneumococcal capsular polysaccharide type 14 (PS14) and pneumococcal surface adhesin A (PsaA) in saliva of children during their first 2 years of life. The sum of anti-PS14 and anti-PsaA IgA1 and IgA2 correlated significantly with the antigen-specific total IgA, which showed that IgA1 and IgA2 add up to IgA. IgA1 was the predominant subclass for both antigens. The median of anti-PS14 and anti-PsaA IgA1 was higher than that of IgA2, and the antigen-specific IgA1 was found in a larger proportion of samples than IgA2. The ratio of IgA1 to IgA2 (IgA1/IgA2 ratio) was lower for anti-PS14 than for anti-PsaA, suggesting that the PS antigen induced more IgA2 than the protein antigen. The possible impact of the IgA subclass distribution on protection of mucosal surfaces by natural or vaccine-induced antibodies needs to be determined.

Maternal immunization with pneumococcal polysaccharide vaccine in the Philippines.

A randomized, controlled study was conducted to evaluate the immunogenicity and reactogenicity of the 23-valent pneumococcal (Pnc) polysaccharide (PS) vaccine among pregnant women and to ascertain the transfer of anti-Pnc antibody (Ab) from mother to infant. One hundred and sixty women received either one dose of Pnc PS vaccine, Haemophilus influenzae type b conjugate vaccine and tetanus toxoid (TT) (Pnc vaccine group, N=106) or TT only (control group, N=54). Sera were obtained from all mothers prior to vaccination and 4 weeks after from the vaccinated group. Cord blood was obtained in 75% of deliveries. Anti-Pnc Ab for serotypes 1, 5, 6B, 14, 18C and 19F was determined using enzyme immunoassay. The Pnc vaccine and control groups were comparable in terms of age, parity, gravidity, prior doses of TT, and pre-vaccination geometric mean concentration (GMC in microg/ml) of anti-Pnc Ab. Between 66 and 87% of the mothers had type-specific Ab prior to vaccination. There was a significant rise in anti-Pnc Ab (varying from 3.3- to 9.1-fold for the individual serotypes) between the pre and post-vaccination samples. Adverse reactions were mild and required no treatment. The level of anti-Pnc Ab in cord blood was significantly lower in the control group compared to the Pnc vaccine group. Vaccination of pregnant women with Pnc Ps vaccine induces good immune response and Ab can be transferred to their infants via cord blood thus providing enhanced protection.

Effects of sample collection and storage methods on antipneumococcal immunoglobulin A in saliva.

Saliva contains components of both the mucosal and systemic immune systems. Variable flow rates, immunoglobulin proteases, and variation in collection and storage methods all introduce differences in the estimated concentrations of antibodies. We evaluated the effect of four collection methods and three storage protocols on the concentrations of immunoglobulin A (IgA) antibodies to pneumococcal capsular antigens 1, 5, 6B, and 14 and to pneumococcal surface adhesin A (PsaA) in saliva. Specimens were collected from 30 healthy Kenyan adults by collecting drool, by pipette suction, and with two commercial kits, OraSure and Oracol. Aliquots from each specimen were snap-frozen with glycerol in liquid nitrogen or stored for 4 to 8 h at +4 degrees C either with or without the addition of protease enzyme inhibitors prior to storage at -70 degrees C. Anticapsular IgA concentrations were not significantly different with different collection methods, but snap-freezing the specimens in liquid nitrogen led to concentrations 41 to 47% higher than those of specimens stored by the other methods (P < 0.0005).

Haemophilus influenzae type b and cross-reactive antigens in natural Hib infection dynamics; modelling in two populations.

Natural immunity to Haemophilus influenzae type b (Hib) invasive disease is based on antibodies arising in response to encounters with Hib or cross-reactive (CR) bacteria. The relative importance of Hib and CR contacts is unknown. We applied a statistical model to estimate the total rate of immunizing infections of Hib and CR prior to wide-scale vaccinations in Finland and the UK. The average rates of these contacts were 0.7 and 1.2 per year per child in Finland and the UK, respectively. Using a rough estimate of 0.1 Hib acquisitions per year per child in the UK based on carriage rates, the proportion of Hib among all immunizing contacts was in the order of 10%, suggesting that CR bacteria have a major role. In general, varying frequency of CR contacts may explain some differences in the pre-vaccination incidence and age-distribution of invasive disease in different countries.

Current state of pneumococcal vaccines.

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and acute otitis media in children and adults worldwide. According to World Health Organization estimates, at least 1 million children under 5 years of age die each year from pneumococcal pneumonia. The emergence of resistant strains necessitates the development of an effective vaccine with a large serotype coverage. The 11 most common serotypes cause 72-83% of all serious pneumococcal diseases worldwide. Currently marketed 23-valent pneumococcal polysaccharide vaccine provides large serotype coverage and offers a less expensive option. However, it is efficacious only in adults but not in infants. Conjugate vaccines offer a solution by generating immunological memory already at early age. A recently licensed 7-valent conjugate vaccine is immunogenic and efficacious in infants. Its serotype coverage might be sufficient in Europe and North America, but not in Africa, Asia and Oceania. A need exists to develop pneumococcal vaccines with lower cost and larger serotype coverage. Several 11-valent pneumococcal conjugate vaccines are being evaluated in phase I-III trials. This study reviews the current state of pneumococcal problem and pneumococcal vaccines in clinical use.

Bridging phase 2 and phase 3 pneumococcal immunologic data for future combination vaccines.

Pneumococcal conjugate vaccines (PncCs) will be introduced into childhood vaccination programs now that the first PncC has been licensed for use. The next generation of PncCs and possible combination vaccines containing PncC will most probably be approved on the basis of phase 2 immunogenicity and safety data. PncCs are combination vaccines that include, at present, 7-11 components. Immune response to different components may vary. Furthermore, there seem to be population-based differences in immune response. Whether these differences are due to the other vaccines that are given simultaneously or due to the genetic background remains to be seen. Immune response can be evaluated by determining both the quantity and the quality of antibodies after vaccination. However, data are still missing on the minimal protective immune response and serologic correlates or surrogates of protection.

Avidity and subclasses of IgG after immunization of infants with an 11-valent pneumococcal conjugate vaccine with or without aluminum adjuvant.

Finnish and Israeli infants received an 11-valent mixed-carrier pneumococcal conjugate vaccine with or without aluminum adjuvant at 2, 4, 6, and 12 months of age. The relative avidity of serotype 1-, 5-, 6B-, 14-, 19F-, and 23F-specific IgG antibodies in serum obtained at 7, 12, and 13 months of age was measured by EIA, using thiocyanate as a chaotropic agent. For all serotypes, except 14, avidity increased between the ages of 7 and 12 months. After boosting at 12 months, avidity further increased for all serotypes. The adjuvant improved antibody avidity against serotype 5. The IgG antibodies produced were mainly IgG1 subclass, although some infants also produced IgG2 after boosting. In conclusion, the immunization of infants with this 11-valent pneumococcal conjugate vaccine increased avidity of IgG, suggesting successful immunologic priming.

Booster response to the tetanus and diphtheria toxoid carriers of 11-valent pneumococcal conjugate vaccine in adults and toddlers.

We measured the tetanus and diphtheria antitoxin responses after administration of one dose of a mixed carrier (tetanus and diphtheria toxoids) 11-valent pneumococcal conjugate vaccine (PncDT) in 20 Finnish adults (mean age 26.1 years) and 20 Finnish (mean age 23.2 months) and 23 Israeli (mean age 18.5 months) toddlers. The vaccinees had previously been immunised with multiple doses of vaccines containing diphtheria and tetanus toxoids. A double-antigen ELISA was used to measure the antitoxin concentrations. PncDT induced significant booster responses in both adults and toddlers to the tetanus and the diphtheria carrier proteins. Thus, the effect on the tetanus and diphtheria immunity of multivalent conjugate vaccines containing tetanus and diphtheria toxoids as carriers needs to be evaluated before such vaccines are routinely implemented.

Serum and salivary anti-capsular antibodies in infants and children vaccinated with octavalent pneumococcal conjugate vaccines, PncD and PncT.

We studied the immunogenicity of two octavalent pneumococcal (Pnc) conjugate vaccines; Pnc polysaccharides (PS) of serotypes 3, 4, 6B, 9V, 14, 18C, 19F, and 23F were conjugated to diphtheria or tetanus toxoid (PncD and PncT, respectively). Fifty healthy Finnish infants were vaccinated at the ages of 2, 4, 6, and 15 months with either PncD or PncT. Serum IgG antibodies to the vaccine serotypes were analysed by enzyme-linked immunosorbent assay (EIA). All eight PSs induced a significant antibody response both after the primary series and after the booster. Response to PncD was higher for PSs 3, 9V, 14 and 18C and to PncT for serotype 4. Salivary IgA and IgG anti-Pnc antibodies were measured for serotypes 4, 6B, 9V, 14, 18C, and 19F. Mucosal antibodies were found rarely after the primary series but in a greater proportion after the booster. In conclusion, both vaccines were immunogenic.

Haemophilus influenzae type b serology in childhood leukaemia: a case-control study.

Antibody to Haemophilus influenzae type b (Hib) polysaccharide (PRP) was measured in 42 children with acute lymphoblastic leukaemia (ALL) and 42 non-leukaemic hospital controls. Modelling anti-PRP concentrations as a function of age revealed that the slopes of the trend lines differed significantly between cases and controls (P = 0.05); anti-PRP concentrations were lower among younger cases, and higher among older cases, than among controls of the same ages.

Immunogenicity and reactogenicity of diphtheria, tetanus and pertussis toxoids combined with inactivated polio vaccine, when administered concomitantly with or as a diluent for a Hib conjugate vaccine.

In an open trial, 400 infants were randomized to vaccination with a combined diphtheria-tetanus-pertussis-inactivated polio vaccine (DTaP-IPV) either mixed with a Haemophilus influenzae type b (Hib) tetanus toxoid conjugate immediately before injection (DTaP-IPV/Hib (mix)) or given concurrently with the Hib conjugate at separate injection sites (DTaP-IPV+Hib (sep)). The pertussis component consisted of pertussis toxoid alone. The vaccines were given intramuscularly at 3, 5 and 12 months of age. No vaccine-related serious adverse events occurred. Local reactions were evaluated from diary cards completed by the parents. Infants who received DTaP-IPV/Hib (mix) experienced fewer local reactions. Sera were obtained 28-45 days after the second and third vaccinations. Total Hib capsular antibodies were similar in the two groups after the second injection but lower in the group receiving DTaP-IPV/Hib (mix) than in the group receiving DTaP-IPV+Hib (sep) after the third injection (geometric mean 6.1 vs 10.4 microg/ml). Mixing of the vaccines also led to somewhat lower diphtheria toxin antibodies (5.9 vs. 7.7 IU/ml after the third injection) while tetanus antibodies were higher (3.9 vs. 2.5 IU/ml after the third injection). Antibodies against pertussis toxin and the three polio virus types were similar in the two groups. The moderate impairment of the Hib antibody response caused by mixing of the Hib conjugate with aluminium adsorbed DTaP may be due to physicochemical interference but is probably of little clinical importance because of the ability of the Hib conjugates to induce an immunologic memory.

Cross-reactivity of antibodies to type 6B and 6A polysaccharides of Streptococcus pneumoniae, evoked by pneumococcal conjugate vaccines, in infants.

Streptococcus pneumoniae serotypes 6B and 6A are important causes of infections, yet only 6B is included in current vaccines. It is, therefore, crucial to evaluate whether functional antibodies are produced against both types after vaccination. Concentration and opsonophagocytic activity (OPA) of antibodies to 6B and 6A polysaccharides were determined in serum samples from infants immunized with 3 different pneumococcal conjugate vaccines containing serotype 6B. In all vaccine groups, a significant correlation was found between the anti-6B and -6A antibody concentration and OPA. However, OPA to the vaccine serotype was detectable more commonly than OPA to the cross-reactive type. Furthermore, 5%-15% of the serum samples showed high OPA against the 6B but not the 6A strain. On average, 2-6 times more anti-6B antibodies were needed for 50% opsonophagocytic killing of the type 6A than the type 6B strain. Although pneumococcal type 6B conjugate vaccines elicit antibodies that cross-react with type 6A, not all anti-6B antibodies are functionally cross-reactive.