Assignment of Immunoglobulin G1 and G2 Concentrations to Pneumococcal Capsular Polysaccharides 3, 6B, 14, 19F, and 23F in Pneumococcal Reference Serum 89-SF

We describe standardization of an enzyme-linked immunosorbent assay (ELISA) for measuring immunoglobulin G1 (IgG1) and IgG2 concentrations of antibodies to pneumococcal capsular polysaccharide (Pnc PS). The ELISA uses a human pneumococcal reference serum pool, lot 89-SF, as a reference. IgG1 and IgG2 concentrations were assigned to antibodies to Pnc PS serotypes 3, 6B, 14, 19F, and 23F in 89-SF by ELISA using affinity-purified monoisotypic IgG1 and IgG2 preparations. The sum of IgG1 and IgG2 concentrations in 89-SF agrees well with the previously assigned IgG concentrations. The IgG1 and IgG2 values in 89-SF were used to measure antibodies to Pnc PS 6B, 14, and 23F in adult pre- and postimmunization sera and the sum of IgG1 and IgG2 concentrations correlated well with the IgG values. Furthermore, the IgG2/IgG1 ratio did not affect the detection of IgG1, the isotype usually represented by a lower concentration.     

免疫比浊法检测梅毒螺旋体抗体的方法评价

目的评价免疫比浊法在梅毒螺旋体抗体检测中的临床应用价值。方法分析免疫比浊法的精密度、线性、稳定性、干扰因素、携带污染指标,并与化学发光微粒子免疫分析法(CMIA)进行阳性率比较。结果免疫比浊法批内CV为1.64%~2.22%、批间CV为3.57%~3.74%;黄疸、脂血及溶血对本方法无明显干扰;阳性率均为16.8%,差异无统计学意义(x^2=0,P>0.05)。结论免疫比浊法检测结果与CMIA法检测结果相关性良好,可以满足临床诊断需要。     

Association of Serotype-Specific Antibody Concentrations and Functional Antibody Titers with Subsequent Pneumococcal Carriage in Toddlers Immunized with a 9-Valent Pneumococcal Conjugate Vaccine

Association of pneumococcal nasopharyngeal carriage with the concentration and opsonophagocytic activity (OPA) of serum serotype-specific antibodies was determined for toddlers 1 month after immunization with a 9-valent pneumococcal conjugate vaccine. Higher anti-serotype 14 and anti-serotype 19F IgG and anti-serotype 14 IgM correlated with a lowered probability of pneumococcal acquisition. Postvaccination OPA did not correlate with pneumococcal carriage.     

The Human in Vitro Anti-type 4 Pneumococcal Polysaccharide Antibody Response is Regulated by Suppressor T Cells

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.     

Evaluation of the Specificity of Pneumococcal Polysaccharide Enzyme-Linked Immunosorbent Assay and the Effect of Serum Adsorption Based on Standard Pneumococcal Serogroup- or Serotype-Specific Rabbit Antisera

Worldwide, Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality, especially in infants and elderly people. Pneumococcal capsular polysaccharides are well characterized, and more than 90 different serotypes have been identified. Serotype-specific antibodies against the capsular polysaccharide are produced during infection. Detection of antibodies against pneumococci by enzyme-linked immunosorbent assay (ELISA) is performed according to WHO guidelines, using antigens provided by ATCC. However, testing the ELISA for specificity is challenging due to the difficulty in obtaining human naïve serum with pneumococcal antibodies as well as human serum with antibodies against a single serotype. The application of well-defined serotype-specific sera produced in animals to evaluate the specificity of the ATCC antigens and the effect of adsorption with cell wall and 22F polysaccharides has not been performed before, to our knowledge. In this study, the specificity of ATCC antigens (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) was tested by using commercial serotype-, serogroup-, and pool-specific pneumococcal rabbit antisera.Worldwide, Streptococcus pneumoniae (pneumococcus) is a significant cause of morbidity and mortality, especially in infants and elderly people. Pneumococcal infections range from mild upper respiratory tract diseases and otitis media to pneumonia, bacteremia, and meningitis (4). Pneumococcal strains possess a polysaccharide capsule, and more than 90 different varieties (serotypes) have been identified (11, 18). Serotype-specific antibodies against the capsular polysaccharides provide protection against the corresponding serotypes. Four vaccines have been developed, two of which are currently used, including a 23-valent polysaccharide (Pneumovax) for the elderly and for children over the age of 2 years and a 7-valent protein-conjugated vaccine (Prevenar) for children under 2 years of age (9). In 2009, a PCV-10 vaccine (PhiD-CV; GlaxoSmithKline) and a PCV-13 vaccine (Wyeth) are expected to be licensed and used (9).Detection of antibodies against pneumococci is performed by an enzyme-linked immunosorbent assay (ELISA) according to WHO guidelines. Antigens are provided by ATCC, with the exact compositions being unknown (16; www.vaccine.uab.edu).The ATCC antigens used in the WHO ELISA are believed to be serotype specific; however, very few studies have been performed to investigate the actual specificity of the antigens (15, 17). These studies indicate that cross-reactions between serotypes occur, resulting in diagnostic challenges. Furthermore, the antibody responses in human sera seem to differ between immunized and naturally infected subjects (15, 17). Adsorbing the human sera by using pneumococcal cell wall polysaccharides (C-Ps) and 22F polysaccharide was shown to improve the specificity of the WHO ELISA (3). However, due to the widespread colonization/infection of humans by different pneumococcal serotypes, it is uncertain if individuals have been exposed to the bacterium and to what extent. Thus, it is almost impossible to obtain a human serum naïve of pneumococcal antibodies as well as human serum with antibodies against a single serotype. The use of alternative, well-defined serotype-specific sera produced in animals to evaluate the specificity of the ATCC antigens and the effect of adsorption with C-Ps and 22F polysaccharide has not been tested previously, to our knowledge. Thus, the specificity of the ATCC antigens was evaluated in this study by using commercial pneumococcal rabbit antisera specific to capsular polysaccharides (13). Furthermore, the effect of serum adsorption with C-Ps and/or 22F polysaccharide on the pneumococcal titer was determined.     

Antibody Response to Pneumococcal Polysaccharide Vaccine in Young, Adult and Old Mice

The anti-pneumococcal antibody response was studied in young (5-week-old) and adult (10-week-old) BALB/c and CBA/J mice and in adult (9–10-week-old) and old (12-, 18- and 24-month-old) AB6F₁ and B6D2F₁ mice after s.c. immunization with a 23-valent pneumococcal polysaccharide vaccine. Both young and adult mice showed a significant IgM antibody response to the vaccine 6 days after immunization with 111 /ig antigen. There were significant immune responses to serotypes 1, 2, 4 and 7F in contrast to small responses to serotypes 14, 19F and 23F after immunization with the vaccine. One month after immunization, there were only marginal differences in IgM anti-pneumococcal antibody levels to the vaccine (anti-PPS) between immunized and unimmunized BALB/c mice, whereas in CBA/J mice the anti-PPS remained higher in immunized than in unimmunized mice. Immunization of old mice induced a significant IgM antibody response 6 days after immunization, but the anti-PPS thereafter decreased rapidly towards preimmunization values in AB6F₁ mice. A significant IgG anti-PPS was not detected in any of the mice studied. The IgA anti-PPS tended to vary over time with no consistent pattern. It is important to carefully consider age and strain of the mice used when studying the immune response to pneumococcal polysaccharide antigens.     

Assignment of Weight-Based Antibody Units for Seven Additional Serotypes to a Human Pneumococcal Standard Reference Serum, 007sp

The pneumococcal enzyme-linked immunosorbent assay (ELISA) reference standard serum, lot 89SF, has been in use since 1990 and was replaced in 2013 with a new reference standard, 007sp, that is projected to be available for the next 25 years. 007sp was generated under an FDA-approved clinical protocol; 278 adult volunteers were immunized with the 23-valent unconjugated polysaccharide vaccine Pneumovax II, and a unit of blood was obtained twice from each immunized subject within 120 days following immunization. Pooled serum was prepared from the plasma of 262 subjects, filled at 6 ml per vial, and lyophilized. Five independent laboratories participated in bridging the serotype-specific IgG assignments for 89SF to the new reference standard, 007sp, to establish equivalent reference values for 13 pneumococcal capsular serotypes (1,3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) by using the WHO reference ELISA. In a second study involving three laboratories, a similar protocol was used to assign weight-based IgG concentrations in micrograms per ml to 007sp of seven serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) also present in the 23-valent pneumococcal unconjugated polysaccharide vaccine. In addition, the IgG assignments for a 12-member WHO quality control (QC) serum panel were also extended to cover these seven serotypes. Agreement was excellent, with a concordance correlation coefficient (r_c) of >0.996 when each laboratory was compared to the assigned values for the 12 WHO QC serum samples. There are four remaining pneumococcal serotypes (2, 9N, 17F, and 20) found in Pneumovax II for which IgG assignments exist for 89SF and remain to be bridged.     

Specificity of the Antibody Response to the Pneumococcal Polysaccharide and Conjugate Vaccines in Human Immunodeficiency Virus-Infected Adults

Nonspecific antibodies, which are thought to be nonprotective, have been shown to contribute a substantial proportion of the measured concentration in the standardized immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for pneumococcal polysaccharide capsular antibodies. The presence of such antibodies in human immunodeficiency virus (HIV)-infected persons has not been evaluated. The amount of nonspecific antibodies is proportional to the reduction in IgG antibody concentration that occurs with serum absorption with the heterologous polysaccharide 22F. We measured the amount of nonspecific antibodies before and after vaccination with the pneumococcal conjugate vaccine (PCV; n = 33) or the pneumococcal polysaccharide vaccine (PPV; n = 34) in HIV-infected adults with CD4 counts of ≥200 cells/mm³. Blood was drawn before and 2 months after vaccination. For prevaccination sera, we found a substantial amount of nonspecific antibodies for serotypes 4, 6B, 9V, and 23F (23 to 47% of measured IgG concentration), but not for serotype 14. There tended to be proportionately less nonspecific antibodies in postvaccine sera than prevaccine sera for PCV, but not for PPV. Subjects with a low HIV viral load (≤400 copies/ml) had proportionately more nonspecific antibodies than those with higher viral load before and after both vaccines. After 22F absorption, the geometric mean concentrations of antibodies were significantly higher post-PCV than post-PPV for the high viral load group for all five serotypes, but for no serotypes in the low viral load group. These findings confirm that absorption with a heterologous pneumococcal polysaccharide (e.g., 22F) is necessary to remove nonspecific antibodies in a standardized IgG ELISA for pneumococcal capsular antibodies in HIV-infected adults.     

The Immune Response to Pneumococcal Polysaccharide Type III is Followed by Increased Production of an Antibody Antifactor

The study shows that serum from pneumococcal polysaccharide type III (SSSIII)-immunized and nonimmunized mice that has been absorbed until devoid of anti-SSSIII antibodies inhibits anti-SSSIII plaque-forming cells (PFC) but not antisheep red blood PFC in antibody plaque assay. A large fraction of the inhibitions activity can be absorbed by and eluted from anti-SSSIII-Sepharose, but not normal mouse serum-Sepharose. It has low or no affinity for protein A and concanavalin A. The titre of the inhibitor increases with repeated immunizations and inhibits the anti-SSSIII PFC of rats or mice of different allotypes. On the basis of these characteristics, I classify the inhibitor as an anti-idiotypic nonimmunoglobulin factor directed against a common determinant of anti-SSSIII antibodies of mouse and rat, with an immuno-regulatory function.     

IgG Subclass Antibody Responses to Pneumococcal Polysaccharide Vaccine in Splenectomized, Otherwise Normal, Individuals

Subclasses of IgG antibodies to pneumococcal polysaccharide serotype antigens 4, 6A, and 23F were measured before and 4 weeks after vaccination with pneumococcal vaccine in young individuals splenectomized because of trauma and in a control group. An ELISA technique was applied. IgG2 anti-pneumococcal antibodies predominated before vaccination, especially against serotypes 4 and 6A. The youngest individuals in the splenectomy group tended to have lower IgG2 anti-pneumococcal antibody levels than the older ones. Vaccination induced antibodies of the IgG1 and IgG2 subclasses, and in some individuals also of the IgG4 subclass. Splenectomy does not seem to influence the IgG subclass pattern of antipneumococcal antibodies.     

A Gene of the Immunoglobulin H-Chain Cluster Controls the Murine Antibody Response to Pneumococcal Polysaccharide Type 14

Mice of various strains were immunized with pneumococcal polysaccharide type 14 (pneumo-14), and their anti-pneumo-14 antibodies were measured by the Farr test. Mice of strains BALB/c, ST/b, NZB and CBA (Ig allotypes a, e or j) had 300-1700 ng of antibody nitrogen per millilitre of serum on day 7. The corresponding values for C57BL/Ka, RF or AKR mice (allotypes b, c or d) were 40-300 ng/ml. Two families of congenic strains were tested, one with the C57BL and the other with the BALB/c background genome. Their response was either high or low depending on the VH genes, and other gene loci had little effect on the concentration of anti-pneumo-14 antibodies.     

Kinetics of IgM and IgA Antibody Response to 23-Valent Pneumococcal Polysaccharide Vaccination in Healthy Subjects

Purpose

A poor antibody response of IgM and IgA antibodies upon vaccination with pneumococcal polysaccharides (PnPS) is discussed as independent risk factors for bronchiectasis in patients with antibody deficiency syndrome (ADS) receiving immunoglobulin replacement therapy. However, the kinetics of the specific IgM and IgA response to vaccination with multivalent pneumococcal polysaccharides requires a more detailed knowledge. In this study we aimed i) to develop a standardised multivalent PnPS-IgM and IgA-ELISA, and ii) to compare the sensitivity of the multivalent to the serotype specific antibody response, and iii) to determine the kinetics of the anti-PnPS IgM and IgA antibodies in healthy subjects.

Methods

We immunised n?=?20 healthy adults with a 23-valent PnPS vaccine (Pneumovax®). The kinetics of the 23-valent antibody response was assessed for 1 year with newly developed ELISAs for IgM and IgA isotypes, along with serotype specific responses.

Results

The IgA and IgM antibody response peaked at 2 and 3 weeks, respectively. IgM antibody levels remained at a plateau (above 80 % of peak response) for 3 months. After one year, specific antibody levels were still at about 30 % of the peak response. The 23-valent antibody response yielded significantly higher responder rates than assessment of single serotypes.

Conclusion

Testing the IgM and IgA immune response to polysaccharide vaccination with a multivalent PnPS ELISA may be a feasible tool for assessment of the immune function in patient groups who receive IgG replacement therapy.     

Evaluation of Pneumococcal Polysaccharide Immunoassays Using a 22F Adsorption Step with Serum Samples from Infants Vaccinated with Conjugate Vaccines

The history of the pneumococcal polysaccharide enzyme-linked immunosorbent assay (ELISA) is characterized by a continuous search for increased specificity. A third-generation ELISA that uses 22F polysaccharide inhibition has increased the specificity of the assay, particularly at low antibody concentrations. The present work compared various 22F ELISAs and non-22F ELISAs. The comparisons involved three different laboratories, including a WHO reference laboratory, and included sera from subjects from different geographic areas immunized with different pneumococcal conjugate vaccines, including the licensed 7-valent Prevenar vaccine and the 10-valent Synflorix vaccine. All comparisons led to the same conclusion that the threshold defined as 0.35 μg/ml for the WHO non-22F ELISA is lower when any 22F ELISA is used. The use of highly purified polysaccharides for coating further improved the specificity of the assay. In conclusion, we confirm that the 22F ELISA can be recommended as a reference method for the determination of antibodies against pneumococcal polysaccharides.It has been amply demonstrated that protection against the various manifestations of pneumococcal disease is mediated by antibodies induced to the type-specific pneumococcal polysaccharides (PSs) (10, 19). The biological function of these antibodies is to bind onto the surface of the pneumococcal cell and in so doing induce complement activation, leading to the uptake and killing of the pneumococcal cells by human phagocytic cells, particularly polymorphonuclear leukocytes (26). Such antibodies are referred to as being opsonic, and the whole process of uptake and killing is referred to as opsonophagocytosis. With the development of new pneumococcal conjugate vaccines (PCVs), it is critical that highly specific immunoassays be used to measure the vaccine-induced antibodies that are associated with protective immunity. This implies that the widely used anti-PS enzyme-linked immunosorbent assay (ELISA) should selectively measure those antibodies with opsonic activity. For that purpose, several improvements have been made to the initial ELISA, principally through the recognition of the role played by the antibodies directed against pneumococcal determinants other than the PSs.One of these determinants is the common cell wall polysaccharide (CWPS). CWPS is covalently linked to the serotype-specific capsular PS through as yet to be determined linkages (4, 9). Thus, when the pneumococcal serotype PS is purified, the CWPS is copurified. The PSs distributed by the American Type Culture Collection (ATCC) to laboratories that perform ELISA for the quantitation of antipneumococcal antibodies are the 23 PSs included in the 23-valent PS vaccine manufactured by Merck (Whitehouse Station, NJ) and contain CWPS as a contaminant.The preadsorption of postimmunization sera from adults and children with CWPS alone may not be sufficient for the measurement of antibody concentrations that are predictive of vaccine efficacy (24, 31), the reason being that there is a poor correlation between the antibody concentration and opsonic activity (coefficient of correlation [r] = about 0.5 rather than 0.8 or higher). Yu et al. reported in 1999 (31) that many heterologous pneumococcal PSs reduced the level of antibody binding of sera from PS vaccine-immunized adults to the serotype 6B PS over that removed by CWPS alone. They concluded that the antibodies removed by the heterologous pneumococcal PSs were against a novel epitope not found on CWPS. Importantly, they found that these antibodies against novel epitopes were not opsonic. They went on to suggest that the specificity of the pneumococcal PS ELISA could be improved by preadsorption with an unrelated pneumococcal PS, in addition to the CWPS. Soininen at al. (24) examined PSs obtained from the ATCC and from three manufacturers. They observed that the amount of cross-reactive antibody removed by preadsorption with heterologous pneumococcal PS in sera from both adults and infants differed depending upon the manufacturer of the purified pneumococcal PS used in the ELISA. This is in line with the observation that ATCC PS batches have varing degrees of contaminants (30). Xu et al. reported that CWPS is present in two forms: one form is bound and the other form is unbound to the capsular PS (29). Thus, it would be expected that those methods of PS purification that disassociate unbound CWPS would yield purer capsular PS.From the observations made above, Concepcion and Frasch recommended that each serum be doubly adsorbed: with CWPS, as previously recommended for the second-generation pneumococcal PS ELISA, and with a heterologous pneumococcal serotype PS (2). They recommended that the 22F PS be used for the second adsorption, because the 22F PS was not being considered for use in any of the pneumococcal conjugate vaccines and it was available from the ATCC. The use of 22F as the second adsorbent has now been evaluated in a number of laboratories (5, 7, 21, 23) and is included in the WHO-recommended third-generation pneumococcal ELISA (25).Some countries that have introduced the pneumococcal conjugate vaccine into their routine immunization schedules had instituted a reduced, accelerated schedule. For example, the United Kingdom has instituted two primary doses at 2 and 4 months of age and a booster at about 12 months of age. As noted above, adults have rather high levels of IgG to CWPS, and maternal antibodies will be more evident in the infant when a compressed or shorter immunization schedule is used. This means that the added 22F adsorption will be required for the measurement of serotype-specific antibodies after primary immunization when such schedules are used.Recommendations for the serological criteria to be considered for the licensure of new pneumococcal conjugate vaccines for use in infants were given as an appendix in volume 927 of the WHO Technical Report Series, published in 2005 (28). The serological criteria included the demonstration of noninferiority against a registered vaccine, with the primary end point being the IgG antibody concentration, as measured by ELISA, in sera from infants collected 4 weeks after a three-dose primary series, with a single threshold of IgG antibody concentration of 0.35 μg/ml for all serotypes. Of note, the threshold value was defined by using pooled immunogenicity and efficacy data from three different efficacy trials with invasive disease end points. The reference value was first proposed to be 0.20 μg/ml, on the basis of a vaccine efficacy trial conducted in Northern California (1, 8). When pooled data from three different efficacy trials with invasive disease end points performed in Northern California, in South Africa, and among Navajo Indians were taken into account (1, 8, 11, 14), an antibody concentration of 0.35 μg/ml, aggregated across the seven serotypes, was determined. However, the WHO agreed that alternative methods may be used for the validation of new conjugate vaccine formulations, and this may eventually lead to the establishment of an alternative threshold (28), but no guidance for bridging was provided. It was further agreed that other clinical end points (e.g., pneumonia or otitis media) would not be taken into account. Nevertheless, this reference threshold value, determined by using data from an ELISA without 22F adsorption, does not necessarily predict protection in an individual subject. A possible correlation between antibody concentrations in the range of 0.20 to 0.35, as measured by ELISA, and a titer of 1:8, as measured by an opsonophagocytosis assay (OPA), was mentioned. At last, the demonstration of the functional capacity of the antibody through determination of opsonophagocytic activity, as measured by OPA after a three-dose priming series, was defined as an important secondary end point. These recommendations published in 2005 have recently been confirmed in a 2009 WHO publication, based upon a consensus meeting held in July 2008 in Ottawa, Ontario, Canada (3).There is an important inconsistency in the 2003 WHO recommendations for serological criteria. The WHO-recommended third-generation ELISA, approved by WHO in 2000 and set up in two WHO reference laboratories, includes the routine use of the 22F adsorbent, as described by Wernette et al. (25). However, as noted above, the recommended threshold concentration was derived without the use of the 22F adsorbent.While the 22F adsorbent did not affect the antibody concentrations in sera from infants in the Northern California efficacy trial (20), 22F adsorption reduced the antibody concentrations over 30%, on average, in vaccinated children in the South African trial (13). In the case in which a reduced primary immunization schedule is used, it will be important to remove maternal unspecific nonopsonizing antibody. Henckaerts et al. (5) made observations comparable to those derived from the South African study.In order to further investigate the impact of 22F adsorption and improved ELISA coating conditions, a series of comparison ELISA measurements was carried out. These involved three different laboratories and used serum samples from pediatric subjects after immunization with different PCVs, including the Prevenar and Synflorix vaccines.     

Serum Opsonic Activity in Infants with Sickle-Cell Disease Immunized with Pneumococcal Polysaccharide Protein Conjugate Vaccine

Pneumococcal infections are an important cause of morbidity and mortality in children with sickle-cell disease (SCD). Pneumococcal conjugate vaccines (PCVs) are immunogenic in healthy infants <2 years of age but have not been evaluated in young children with SCD. Infants with SCD were immunized with a 7-valent PCV (Wyeth-Lederle Vaccines & Pediatrics) at 2, 4, and 6 months of age. A booster dose of 23-valent pneumococcal polysaccharide vaccine (PPV; Pnu-Immune) was administered at 24 months of age. Antipneumococcal type 6B and 14 serum opsonic activity was measured to assess the biologic function of the antibody. Following the administration of three doses of PCV, opsonic activity against serotype 6B increased from 4.8% at 2 months to 33.5% at 7 months, with a subsequent decline to 8.1% at 12 months and 7.5% at 24 months and with an increase to 30.7% at 25 months after administration of a booster dose of PPV. Similar trends were seen with serotype 14 (opsonic activities were 9.4% at 2 months, 24.9% at 7 months, 16.5% at 12 months, and 12.6% at 24 months, and the opsonic activity was 27.3% 1 month after the administration of PPV). Serum opsonic activity correlated with antibody levels for both serotypes. PCV induces serum opsonic activity in infants with SCD. Antipneumococcal serum opsonic activity correlates with antibody levels.     

Use and Clinical Interpretation of Pneumococcal Antibody Measurements in the Evaluation of Humoral Immune Function

Pneumococcal vaccination is a commonly used technique for assessing the humoral immune status of a patient suspected of having immunodeficiency. Interpretation of what constitutes an adequate response, however, can be challenging. This is due to the complexity of the data generated from serotype-specific assays, historical variations in the assays used to measure pneumococcal antibodies, and varying recommendations on the relevant cut points that define response. In this review, we summarize the historical evolution of assays used for this purpose and discuss the analytical considerations that have influenced published data. We also examine current clinical recommendations for defining an adequate response to vaccination, with a particular focus on the interpretation of serotype-specific data generated by multiplex assays.     

Priming of Immunological Memory by Pneumococcal Conjugate Vaccine in Children Unresponsive to 23-Valent Polysaccharide Pneumococcal Vaccine

Pneumococcal polysaccharide vaccine (PPV) is of limited immunogenicity in infants and immunocompromised patients. Our prospective randomized controlled trial investigated whether priming with pneumococcal conjugate vaccine (PCV) induced specific immunological memory in previously nonresponders to PPV. Of a total of 33 children (2 to 18 years) with polysaccharide-specific immunodeficiency (PSI), group A (n = 16) received two doses of 7-valent PCV in a 4- to 6-week interval, and a booster dose of 23-valent PPV after one year. Group B (n = 17) received two doses of PPV in a 1-year interval exclusively. Specific antibody concentrations for serotypes 4, 5, 6B, 9V, 14, 18C, 19F, and 23F were determined (enzyme-linked immunosorbent assay) before and at 7 and 28 days after administration of the PPV booster and compared to an opsonophagocytosis assay. Of group A, 64 to 100% had antibody concentrations of ≥1 μg/ml on day 28 after the booster versus 25 to 94% of group B. Group A had significantly higher antibody concentrations for all PCV-containing serotypes already on day 7, indicating early memory response. Antibody concentrations were in accordance with functional opsonic activity, although opsonic titers varied among individuals. Pneumococcal vaccination was well tolerated. The incidence of airway infections was reduced after priming with PCV (10/year for group A versus 15/year for group B). Following a PPV booster, even patients primarily not responding to PPV showed a rapid and more pronounced memory response after priming with PCV.     

Assignment of Weight-Based Antibody Units for 13 Serotypes to a Human Antipneumococcal Standard Reference Serum, Lot 89-S(F)

Weight-based immunoglobulin G (IgG), IgM, IgA, and total Ig antibody assignments were made to human antipneumococcal standard reference serum lot 89-S, also known as lot 89-SF, for Streptococcus pneumoniae capsular polysaccharide (PnPs) serotypes 2, 6A, 8, 9N, 10A, 11A, 12F, 15B, 19A, 17F, 20, 22F, and 33F, as well as for C-polysaccharide (C-Ps), extending the standard's usefulness for pneumococcal vaccine evaluation beyond the original serotype 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F assignments (S. A. Quataert, C. S. Kirch, L. J. Quackenbush Wiedl, D. C. Phipps, S. Strohmeyer, C. O. Cimino, J. Skuse, and D. V. Madore, Clin. Diagn. Lab. Immunol. 2:590-597, 1995). The additional 14 assignments were determined using an equivalence of absorbance method with an anti-PnPs serotype 6B reference enzyme-linked immunosorbent assay (EIA). To assure accuracy, anti-PnPs EIA for serotype 14 antibodies, a previously assigned serotype, was performed concurrently. This method assures consistency of the new microgram-per-microliter assignments with previous antiserotype assignments to lot 89-S. The sum of the experimentally derived isotype assignments for anti-PnPs serotypes in lot 89-S agrees well with the separately determined total Ig assignment for each serotype. The lot 89-S assignments for serotypes 1, 5, 6B, 14, 18C, 19F, and 23F were used for pneumococcal conjugate vaccine clinical trial evaluation and to generate data in efficacy trials where serological correlates for protection have been proposed. The assignment of antibody concentrations to additional pneumococcal serotypes in this reference reagent facilitates the consistent and accurate comparison of serum antibody concentrations across clinical trials.     

Anti-ENA Antibody in Serum Determined by ELISA-Technique

An enzyme linked immune sorbent assay, ELISA, for determination of serum antibodies against two different extractable nuclear antigens, the RNAse sensitive ribonucleoprotein (RNP), and the RNAse resistant Sm-antigen is described. The technical variables are systematically evaluated and a recommended procedure selected and described.