Utilization of serologic assays to support efficacy of vaccines in nonclinical and clinical trials: meeting at the crossroads.

In May 2009 the National Institute of Allergy and Infectious Diseases hosted a workshop on serologic assays that support vaccine efficacy evaluations. The meeting promoted exchange of ideas among investigators from varying disciplines who are working on anti-infectious agent vaccines at different stages of development. The presentations and discussions at the workshop illustrated the challenges common across various pathogens with recurring themes: (1) A thorough understanding of the science regarding the pathogen and the host response to disease and immunization is fundamental to assay selection. (2) The intended use of the immunoassay data must be clearly defined to ensure appropriate specificity, accuracy, and precision; a laboratory must also commit resources to assure data quality and reliability. (3) During vaccine development, an immunoassay may evolve with respect to quality, purpose, and degree of standardization, and, in some cases, must be changed or replaced as data are accumulated. (4) Collaboration on standardized reagents and methods, harmonization efforts, and multidisciplinary teams facilitate consistent generation of quality data. This report provides guidance for effective development and utilization of immunoassays based on the lessons learned from currently licensed vaccines. Investigators are encouraged to create additional opportunities for scientific exchange, noting that the discussed themes are relevant for immunoassays used for other purposes such as therapeutics and diagnostics.

Approach to validating an opsonophagocytic assay for Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) polysaccharide serotype-specific antibodies that have opsonophagocytic activity are considered a primary mechanism of host defense against pneumococcal disease. In vitro opsonophagocytic assays (OPAs) with antibody and complement to mediate opsonophagocytic killing of bacteria have been designed and developed as an adjunct to the standardized serum immunoglobulin G antipneumococcal capsular polysaccharide enzyme immunoassay to assess the effectiveness of pneumococcal vaccines. OPA presents challenges for assay standardization and assay precision due to the multiple biologically active and labile components involved in the assay, including human polymorphonuclear leukocytes or cultured effector cells, bacteria, and complement. Control of these biologically labile components is critical for consistent assay performance. An approach to validating the performance of the assay in accordance with International Conference for Harmonization guidelines, including its specificity, intermediate precision, accuracy, linearity, and robustness, is presented. Furthermore, we established parameters for universal reagents and standardization of the use of these reagents to ensure the interlaboratory reproducibility and validation of new methodologies.

Assignment of weight-based immunoglobulin G1 (IgG1) and IgG2 units in antipneumococcal reference serum lot 89-S(F) for pneumococcal polysaccharide serotypes 1, 4, 5, 7F, 9V, and 18C.

Weight-based assignments for immunoglobulin G1 (IgG1) and IgG2 subclass antibodies to Streptococcus pneumoniae capsular polysaccharides (PnPs) in antipneumococcal standard reference serum lot 89-S (lot 89-S), also known as lot 89-SF, have been determined for serotypes 1, 4, 5, 7F, 9V, and 18C. This extends the usefulness of lot 89-S beyond the IgG1 and IgG2 subclass assignments for serotypes 3, 6B, 14, 19F, and 23F made previously (A. Soininen, H. Kayhty, I. Seppala, and T. Wuorimaa, Clin. Diagn. Lab. Immunol. 5:561-566, 1998) to cover 11 major serotypes associated with the highest percentage of pneumococcal disease worldwide. A method of equivalence of absorbances in enzyme immunosorbent assays was used to determine the IgG1 and IgG2 antibody concentrations for the additional serotypes in lot 89-S, based on the subclass values previously assigned for PnPs serotypes 6B, 14, and 23F. This cross-standardization method assures consistency with previous antibody assignments in that reference serum. The newly assigned subclass values for serotype 9V, and previously assigned values for serotype 14, were used to quantitate PnPs antibodies in sera from adult and pediatric subjects immunized with a pneumococcal conjugate vaccine. There was a predominance of IgG1 anti-PnPs antibodies in pediatric sera and IgG2 anti-PnPs antibodies in the adult sera. The IgG1 and IgG2 subclass assignments for the 11 PnPs serotypes in antipneumococcal standard reference serum lot 89-S are useful for quantitating and characterizing immune responses to pneumococcal infection and vaccination regimens.

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.

Long-term antibody levels and booster responses in South African children immunized with nonavalent pneumococcal conjugate vaccine.

Children who had initially received three doses of either a nonavalent pneumococcal conjugate vaccine containing serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F or placebo at 6, 10, and 14 weeks of age were bled at 9 and 18 months for determination of antibody concentrations. The children were then randomized to receive a booster dose of either the 9-valent pneumococcal conjugate vaccine or a 23-valent polysaccharide vaccine and antibody levels determined 1 month later. At 9 months, the geometric mean concentrations (GMCs) were significantly higher for all vaccine serotypes in vaccinated children compared with controls (means varied from 0.49 microg/ml for serotype 4 to 2.37 microg/ml for serotype 14). At 18 months, antibody concentrations remained significantly higher in vaccinated children (means varied from 0.19 microg/ml for serotype 4 to 1.1 microg/ml for serotype 14). In children who had received conjugate vaccine in infancy, the conjugate vaccine at 18 months produced a significant booster response for serotypes 1, 6B, 14, 19F, and 23F (means varied from 2.74 microg/ml for serotype 19F to 15.52 microg/ml for serotype 6B) and produced a comparable response to a first dose of conjugate at this age for serotypes 4, 5, 9V, and 18C. Boosting at 18 months with polysaccharide vaccine produced higher antibody concentrations to all serotypes in children who had previously received conjugate vaccine compared to children who had not received the conjugate vaccine in infancy. In conclusion, the 9-valent pneumococcal conjugate vaccine given in infancy elicits significant and long-lasting antibody responses which can be boosted with either the conjugate or polysaccharide vaccines.

Theoretic coverage of heptavalent pneumococcal conjugate vaccine in the prevention of community-acquired pneumonia in children in Italy.

In order to evaluate the theoretic coverage of the heptavalent conjugate vaccine against community-acquired pneumonia (CAP) during the first years of life, the anti-capsular IgG antibodies to the nine more common pneumococcal serotypes (1, 4, 5, 6B, 9V, 14, 18C, 19F, 23F) were quantitated in 196 affected children aged 2-5 years by means of enzyme-linked immunosorbent assays of acute and convalescent serum samples. Acute Streptococcus pneumoniae infection associated with the nine tested serotypes was diagnosed in 57 children (29.1%). Serological data indicated that 16.8% of CAPs were caused by serotypes included in the heptavalent conjugate vaccine. This study confirms the significant role of S. pneumoniae in pediatric CAP, and highlights the importance of pneumococcal conjugate vaccines in preventing it during the first years of life.

Immunogenicity after one, two or three doses and impact on the antibody response to coadministered antigens of a nonavalent pneumococcal conjugate vaccine in infants of Soweto, South Africa.

BACKGROUND: Children <6 months of age are at increased risk of pneumococcal disease. The early immunogenicity of conjugate vaccines therefore may be important to prevent disease in young children. OBJECTIVES: To determine the immunogenicity of a nonavalent pneumococcal conjugate vaccine after one dose, two doses and three doses and its impact on the antibody response to coadministered antigens. METHODS: A total of 500 infants from Soweto were immunized at 6, 10 and 14 weeks of age with either placebo (n = 250) or 9-valent pneumococcal conjugate vaccine (n = 250) containing serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F conjugated to CRM(197) mutant diphtheria protein. Blood was taken for determination of serotype-specific IgG before the first dose and 1 month after each dose. RESULTS: Before the first dose at 6 weeks of age >80% of infants had >0.15 microg/ml antibody to six of the nine antigens, >70% to serotypes 18C and 23F and >50% to serotype 4. Geometric mean concentrations (GMCs) after one dose ranged from 0.27 microg/ml for serotype 23F to 2.98 microg/ml for serotype 1; >90% of infants had serotype-specific antibody >0.15 microg/ml except for serotypes 23F (70%) and 6B (80%). After two doses GMCs ranged from 1.14 microg/ml for serotype 23F to 5.68 microg/ml for serotype 1; >95% of infants had serotype-specific antibody >0.15 microg/ml and >75% had >0.5 microg/ml for all nine serotypes. GMCs after three doses ranged from 2.73 microg/ml for serotype 23F to 6.18 microg/ml for serotype 5; >98% of infants had serotype-specific antibody >0.15 microg/ml and >92% had >0.5 microg/ml for all nine serotypes. Antibody concentrations after three doses were significantly higher to Haemophilus influenzae type b-polyribosylribitol phosphate vaccine in children who received pneumococcal conjugate vaccine, but they had lower antibodies to pertussis toxin than controls. CONCLUSIONS: A single dose of this pneumococcal conjugate vaccine produces a potentially protective antibody response to most serotypes in the majority of children in this population.

A glycoconjugate vaccine for Neisseria meningitidis induces antibodies in human infants that afford protection against meningococcal bacteremia in a neonate rat challenge model.

The functional activities of serum samples from human infants immunized with a glycoconjugate vaccine for Neisseria meningitidis serogroup C were assessed in a complement-mediated antibody-dependent serum bactericidal assay (SBA) and in a neonate rat model of protection from bacteremia. Selective serum samples from individual human infants were combined to make a panel of 11 serum pools to obtain a sufficient volume for testing. Each pool was assayed (i) for the anti-N. meningitidis serogroup C capsular polysaccharide (PS) immunoglobulin G (IgG) concentration as determined by reactivity in a direct-binding enzyme-linked immunosorbent assay, (ii) for bactericidal activity against N. meningitidis serogroup C strain C11, and (iii) for the ability to reduce bacteremia after passive transfer into a neonate rat model. Representative serum samples from infants who were not previously immunized with any N. meningitidis serogroup C vaccine served as a negative control. The prepared serum pools ranged in antibody concentration from 0.18 to 17.31 micro g of IgG specific for N. meningitidis serogroup C PS per ml. For this serum panel, a direct relationship between concentrations of anti-N. meningitidis serogroup C PS-specific IgG and serum SBA titers (r = 0.9960) was observed. Passive transfer to neonate rats demonstrated the ability of postimmunization serum samples to significantly reduce (> or =2-log(10) reduction compared to control animals) the level of bacteremia following a challenge. Of 79 neonate rats that received > or =0.031 micro g of human infant anti-N. meningitidis serogroup C PS IgG, 75 (94.9%) had a > or =2-log(10) reduction in bacteremia, whereas of the animals that received <0.031 micro g of antigen-specific IgG, 10.3% (4 of 39 rats) showed a > or =2-log(10) reduction in bacteremia. It was concluded that the anti-N. meningitidis serogroup C PS IgG antibody induced by this glycoconjugate vaccine had in vitro functional activity (as determined by a SBA) and also afforded protection against meningococcal bacteremia in an animal model.