Development and use of a serum bactericidal assay using pooled human complement to assess responses to a meningococcal group A conjugate vaccine in African toddlers.

A meningococcal group A polysaccharide (PS) conjugate vaccine (PsA-TT) has been developed for African countries affected by epidemic meningitis caused by Neisseria meningitidis. Complement-mediated serum bactericidal antibody (SBA) assays are used to assess protective immune responses to meningococcal vaccination. Human complement (hC') was used in early studies demonstrating antibody-mediated protection against disease, but it is difficult to obtain and standardize. We developed and evaluated a method for sourcing hC' and then used the SBA assay with hC' (hSBA) to measure bactericidal responses to PsA-TT vaccination in 12- to 23-month-old African children. Sera with active complement from 100 unvaccinated blood donors were tested for intrinsic bactericidal activity, SBA titer using rabbit complement (rSBA), and anti-group A PS antibody concentration. Performance criteria and pooling strategies were examined and then verified by comparisons of three independently prepared hC' lots in two laboratories. hSBA titers of clinical trial sera were then determined using this complement sourcing method. Two different functional antibody tests were necessary for screening hC'. hSBA titers determined using three independent lots of pooled hC' were within expected assay variation among lots and between laboratories. In African toddlers, PsA-TT elicited higher hSBA titers than meningococcal polysaccharide or Hib vaccines. PsA-TT immunization or PS challenge of PsA-TT-primed subjects resulted in vigorous hSBA memory responses, and titers persisted in boosted groups for over a year. Quantifying SBA using pooled hC' is feasible and showed that PsA-TT was highly immunogenic in African toddlers.

Anthrax vaccine-induced antibodies provide cross-species prediction of survival to aerosol challenge.

Because clinical trials to assess the efficacy of vaccines against anthrax are not ethical or feasible, licensure for new anthrax vaccines will likely involve the Food and Drug Administration's "Animal Rule," a set of regulations that allow approval of products based on efficacy data only in animals combined with immunogenicity and safety data in animals and humans. U.S. government-sponsored animal studies have shown anthrax vaccine efficacy in a variety of settings. We examined data from 21 of those studies to determine whether an immunological bridge based on lethal toxin neutralization activity assay (TNA) can predict survival against an inhalation anthrax challenge within and across species and genera. The 21 studies were classified into 11 different settings, each of which had the same animal species, vaccine type and formulation, vaccination schedule, time of TNA measurement, and challenge time. Logistic regression models determined the contribution of vaccine dilution dose and TNA on prediction of survival. For most settings, logistic models using only TNA explained more than 75% of the survival effect of the models with dose additionally included. Cross-species survival predictions using TNA were compared to the actual survival and shown to have good agreement (Cohen's κ ranged from 0.55 to 0.78). In one study design, cynomolgus macaque data predicted 78.6% survival in rhesus macaques (actual survival, 83.0%) and 72.6% in rabbits (actual survival, 64.6%). These data add support for the use of TNA as an immunological bridge between species to extrapolate data in animals to predict anthrax vaccine effectiveness in humans.

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.

Interlaboratory comparison of results of an anthrax lethal toxin neutralization assay for assessment of functional antibodies in multiple species.

The anthrax lethal toxin neutralization assay (TNA) will likely be used to correlate the protection offered by new anthrax vaccines in animal models to the immunogenicity that will be provided in humans. TNA data are being generated in several different laboratories to measure the immune responses in rabbits, nonhuman primates, and humans. In order to compare data among species and laboratories, a collaborative study was conducted in which 108 samples from the three species were analyzed in seven independent laboratories. Six of the seven laboratories had participated in an interlaboratory technology transfer of the TNA. Analysis of the titration curves generated by samples from each species indicated that the behaviors of the samples from all species were similar; the upper and lower asymptotes and the slopes of the curves were less than 30% divergent from those for human reference material. Dilutional linearity was consistent among samples from each species, with spike to effective dilution at 50% inhibition (ED(50)) slopes of less than 1.2 for all species. Agreement among the laboratories with consensus values was within 10% of the ED(50)s for all samples and within 7.5% of the quotients of the test sample ED(50) and the reference standard ED(50) (NF(50)s) for all samples. The relative standard deviations obtained when data from all laboratories and for all species were combined were 45% for the ED(50)s and 35% for the NF(50)s. These precision data suggest that the NF(50) readout may normalize the values generated by different laboratories. This study demonstrates that the TNA is a panspecies assay that can be performed in several different laboratories with a high degree of quantitative agreement and precision.

Mixture model analysis for establishing a diagnostic cut-off point for pertussis antibody levels.

Previous studies of pertussis (whooping cough) that have derived diagnostic cut-off points for pertussis antibody levels have assumed a single distribution for antibody levels and have used small sample sizes. In a recent study of 5409 serum samples from the Third National Health and Nutrition Examination Survey (NHANES III), a finite mixture model was developed to examine the distribution of immunoglobulin G (IgG) antibody levels against pertussis toxin (PT), an antigen specific to the Bordetella pertussis bacterium. The mixture model identified three component populations with antibody levels greater than the quantitative assay's lower limit of quantitation (LLQ) and included a point distribution located at or below the LLQ to account for the excess number of antibody values that fell below the LLQ. The mixture model analysis accounted for the NHANES III design. A cut-off point for anti-PT IgG levels was chosen to have a 99 per cent model specificity based on the two overlapping normal distributions assumed for the two component populations with the highest antibody levels. This cut-off point may have a higher diagnostic sensitivity for acute B. pertussis infection than other cut-off points derived by assuming a single distribution for antibody levels.

por Variable-region typing by DNA probe hybridization is broadly applicable to epidemiologic studies of Neisseria gonorrhoeae.

The porin gene (porB) of Neisseria gonorrhoeae encodes the major outer membrane protein identified as PI or Por. To examine the utility of por variable-region (VR) typing, porB from 206 isolates was characterized by using oligonucleotide probes in a checkerboard hybridization assay that identifies the sequence types of five VRs of both PIA and PIB porB alleles. The strains represented temporally and geographically distinct isolates, isolates from a large cluster, epidemiologically linked partner isolates, and a collection of strains from disseminated gonococcal infections. By using rigorous epidemiologic criteria for transmission of infection between sex partners, por VR typing was more discriminatory than serovar typing in classifying isolates from both members of 43 epidemiologically linked pairs: 39 of 43 pairs were classified as coinciding by por VR typing compared to 43 of 43 by serovar determination (P = 0.058). porB sequence data confirmed the accuracy of the por VR method. Relationships between VR type and serovar typing monoclonal antibodies were observed for all six PIB and three of six PIA antibodies. por VR typing is a molecular tool that appears to have broad applicability. This method can be adapted to a wide range of technologies from simple hybridization to microarray and may allow for typing from noncultured clinical specimens.

Genetic typing of the porin protein of Neisseria gonorrhoeae from clinical noncultured samples for strain characterization and identification of mixed gonococcal infections.

Molecular methods that characterize the Neisseria gonorrhoeae porin protein Por are needed to study gonococcal pathogenesis in the natural host and to classify strains from direct clinical samples used with nucleic acid amplification-based tests. We have defined the capabilities of por variable region (VR) typing and determined suitable conditions to apply the method to direct clinical specimens. Nested PCR from spiked urine samples detected 1 to 10 copies of template DNA; freezing spiked whole urine greatly reduced the ability to amplify porB. In a laboratory model of mixed gonococcal infections, the por type of one strain could be determined in the presence of a 100-fold excess of another. por VR typing was used to examine clinical samples from women enrolled in studies conducted in Baltimore, Md., and Madagascar. por type was determined from 100% of paired cervical swab and wick samples from 20 culture-positive women from Baltimore; results for eight individuals (40%) suggested infection with more than one strain. In frozen urine samples from Madagascar, porB was amplified and typed from 60 of 126 samples from ligase chain reaction (LCR)-positive women and 3 samples from LCR-negative women. The por VR types of 13 samples (21%) suggested the presence of more than one gonococcal strain. Five por types, identified in >45% of women with typed samples, were common to both geographic areas. Molecular typing is an important adjunct to nucleic acid amplification-based diagnostics. Methods that utilize direct clinical samples and can identify mixed infections may contribute significantly to studies of host immunity, gonococcal epidemiology, and pathogenesis.

Establishment of diagnostic cutoff points for levels of serum antibodies to pertussis toxin, filamentous hemagglutinin, and fimbriae in adolescents and adults in the United States.

Numerous reports have documented that serologic methods are much more sensitive than culture for the diagnosis of pertussis in adolescents and adults. However, a standardized serologic test for pertussis is not routinely available to most clinicians, and the serologic test levels or cutoff points correlated with diseases have not been determined. The goal of the present study was to examine the distribution of immunoglobulin G (IgG) levels against three Bordetella pertussis antigens (pertussis toxin [PT], filamentous hemagglutinin [FHA], and fimbria types 2 and 3 [FIM]) and to determine population-based antibody levels for the purpose of establishing such diagnostic cutoff points. Enzyme-linked immunosorbent assays (ELISAs) were performed with sera from >6,000 U.S. residents aged 6 to 49 years who participated in the Third National Health and Nutrition Examination Survey. Mixture models were developed to identify hypothesized exposure groups and establish diagnostic cutoffs. Quantifiable (>20 ELISA units/ml [EU]) anti-FHA and anti-FIM IgG antibodies were common (65 and 62% of individuals, respectively), but quantifiable anti-PT IgG antibodies were less frequent (16%). Given the distributions of antibody levels, an anti-PT IgG level of > or =94 EU was proposed as the diagnostic cutoff point. Application of this cutoff point to culture-confirmed illness in a prior study investigating cough illness yielded a high diagnostic sensitivity (80%) and specificity (93%). A standardized ELISA for anti-PT IgG with a single serum sample appears to be useful for the identification of recent B. pertussis infection in adolescents and adults with cough illness. The PT cutoff point will be further evaluated in prospective studies of confirmed B. pertussis infection.

Porin variation among clinical isolates of Neisseria gonorrhoeae over a 10-year period, as determined by Por variable region typing.

The Neisseria gonorrhoeae porin protein (Por) is a potential vaccine target and is the antigenic determinant for serovar typing. Two classes of Por, PIA and PIB, and antigenically distinct variants within each class result from sequence variations in the por gene variable regions (VRs) encoding surface-exposed loops. Oligonucleotide probes to 5 VRs of each class were used in checkerboard hybridizations to type 282 clinical gonococcal isolates selected from strains collected over the course of 10 years. PIA strains (n=63) showed limited por diversity, with 90% having 1 of 4 por types. PIB strains (n=219) were more diverse, although several common por types were identified that persisted over time. Variation within individual VRs was found to be limited. The present study provides information about the diversity of Por in strains circulating in a single geographic region over time, illustrates the utility of a novel por typing method, and has implications for vaccine development.

Use of coefficient of variation in assessing variability of quantitative assays.

We have derived the mathematical relationship between the coefficient of variation associated with repeated measurements from quantitative assays and the expected fraction of pairs of those measurements that differ by at least some given factor, i.e., the expected frequency of disparate results that are due to assay variability rather than true differences. Knowledge of this frequency helps determine what magnitudes of differences can be expected by chance alone when the particular coefficient of variation is in effect. This frequency is an operational index of variability in the sense that it indicates the probability of observing a particular disparity between two measurements under the assumption that they measure the same quantity. Thus the frequency or probability becomes the basis for assessing if an assay is sufficiently precise. This assessment also provides a standard for determining if two assay results for the same subject, separated by an intervention such as vaccination or infection, differ by more than expected from the variation of the assay, thus indicating an intervention effect. Data from an international collaborative study are used to illustrate the application of this proposed interpretation of the coefficient of variation, and they also provide support for the assumptions used in the mathematical derivation.

Effect of O acetylation of Neisseria meningitidis serogroup A capsular polysaccharide on development of functional immune responses.

The importance of O-acetyl groups to the immunogenicity of Neisseria meningitidis serogroup A polysaccharide (PS) was examined in studies using human sera and mouse immunization. In 17 of 18 postimmunization human sera, inhibition enzyme-linked immunosorbent assay indicated that the majority of antibodies binding to serogroup A PS were specific for epitopes involving O-acetyl groups. Studies with mice also showed an essential role for O-acetyl groups, where serum bactericidal titers following immunization with de-O-acetylated (de-O-Ac) conjugate vaccine were at least 32-fold lower than those following immunization with O-Ac PS-conjugate vaccine and 4-fold lower than those following immunization with native capsular PS. Inhibition studies using native and de-O-Ac PS confirmed the specificity of murine antibodies to native PS. The dramatic reduction in immunogenicity associated with removal of O-acetyl groups indicates that O acetylation is essential to the immunogenic epitopes of serogroup A PS. Since levels of bactericidal antibodies are correlated with protection against disease, O-acetyl groups appear to be important in protection.