Branda T. Hu

Approach to Validating an Opsonophagocytic Assay for Streptococcus pneumoniae

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

ABSTRACT 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 standards 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.

Correlates of Protection against Influenza in the Elderly: Results from an Influenza Vaccine Efficacy Trial

ABSTRACT Although a number of studies have investigated and quantified immune correlates of protection against influenza in adults and children, data on immune protection in the elderly are sparse. A recent vaccine efficacy trial comparing standard-dose with high-dose inactivated influenza vaccine in persons 65 years of age and older provided the opportunity to examine the relationship between values of three immunologic assays and protection against community-acquired A/H3N2 influenza illness. The high-dose vaccine induced significantly higher antibody titers than the standard-dose vaccine for all assays. For the hemagglutination inhibition assay, a titer of 40 was found to correspond with 50% protection when the assay virus was antigenically well matched to the circulating virus—the same titer as is generally recognized for 50% protection in younger adults. A dramatically higher titer was required for 50% protection when the assay virus was a poor match to the circulating virus. With the well-matched virus, some protection was seen at the lowest titers; with the poorly matched virus, high levels of protection were not achieved even at the highest titers. Strong associations were also seen between virus neutralization test titers and protection, but reliable estimates for 50% protection were not obtained. An association was seen between titers of an enzyme-linked lectin assay for antineuraminidase N2 antibodies and protection; in particular, the proportion of treatment effect explained by assay titer in models that included both this assay and one of the other assays was consistently higher than in models that included either assay alone. (This study has been registered at ClinicalTrials.gov under registration no. NCT01427309.)

1059Expanded immunogenicity of high-dose inactivated influenza vaccine compared to standard-dose inactivated influenza vaccine in older adults

Background: previous studies have demonstrated the superior immunogenicity of high-dose inactivated influenza vaccine (IIV-HD) in older adults compared to standard-dose inactivated influenza vaccine (IIV-SD), as measured by hemagglutination inhibition (HAI) antibody titers against egg-propagated vaccine antigens. The 2012-2013 northern hemisphere influenza season was characterized by high H3N2 activity and by mismatch between predominant circulating strains and egg-propagated vaccines. There is growing interest in evaluating influenza vaccine immune responses beyond the traditional HAI assay. Methods: samples collected as part of a randomized controlled trial (NCT01427309) evaluating the efficacy of IIV-HD vs IIV-SD in adults ≥65 years were available for testing; one third of trial participants provided post-vaccination sera. This sub-study utilized a case-cohort design, in which 675 representative samples collected during the 2012-2013 season of the study (from individuals who either developed polymerase chain reaction/culture confirmed H3N2 influenza illness [N=123] or belonged to a random subset of 10% of non-cases [N=552]) were selected for expanded testing. Expanded immunogenicity was assessed with an HAI assay using an MDCK cell-propagated A/ Victoria/361 (H3N2) antigen, a viral neutralization assay (NT) using both eggand cell-propagated A/Victoria/361 (H3N2) antigens, and an enzyme-linked lectin assay (ELLA) for anti-neuraminidase (N2) antibodies. Geometric mean titers (GMT) were estimated for IIV-HD and IIV-SD recipients using weighted averages and were compared as GMT ratios (IIV-HD/IIV-SD). Results: samples from 318 IIV-HD and 357 IIV-SD recipients were assayed. The GMT ratios (and 95% Confidence Intervals) were 1.48 (1.26; 1.73), 1.53 (1.29; 1.82), 1.75 (1.43; 2.15), and 1.42 (1.23; 1.65) for cell-propagated HAI, egg-propagated NT, cell-propagated NT, and N2-ELLA, respectively. The GMT ratio for the full immunogenicity subset (2879 IIV-HD and 2872 IIV-SD recipients) assessed by HAI assay using egg-propagated A/Victoria/361 was 1.82 (1.71; 1.94). Conclusion: IIV-HD is associated with significantly improved immunogenicity compared to IIV-SD in older adults as assessed using a range of different assays. BACKGROUND • Adults ≥65 years of age are particularly vulnerable to influenza-associated complications, accounting for most seasonal influenza-related hospitalizations and deaths1,2 • The high burden of influenza in this population persists despite documented improvements in vaccination rates3 • An unmet medical need is therefore recognized for improved influenza vaccines in adults 65 years of age and older4,5 • Previous studies have demonstrated the superior immunogenicity of high-dose inactivated influenza vaccine (IIV-HD) in older adults compared to standard-dose inactivated influenza vaccine (IIV-SD), as measured by hemagglutination inhibition (HAI) antibody titers against egg-propagated vaccine antigens6,7,8 • A recently completed randomized, controlled efficacy study demonstrated that IIV-HD was 24.2% (95% confidence interval [CI], 9.7%–36.5%) more efficacious than a IIV-SD in preventing laboratory-confirmed symptomatic influenza in adults 65 years of age and older9 • The second year of the efficacy study (2012-2013 northern hemisphere influenza season) was characterized by high H3N2 activity and by mismatch between predominant circulating strains and egg-propagated vaccines such as the ones used in the study. In contrast, cell-propagated vaccine antigens were considered well-matched to circulating H3N2 viruses10,11 • Consistent with previous studies, the immunogenicity of IIV-HD was significantly improved compared to IIV-SD as determined by the standard HAI assay using egg-propagated vaccine antigens in the full Year 2 immunogenicity subset (2879 IIV-HD and 2872 IIV-SD recipients), with a corresponding GMT ratio of 1.82 (95% CI 1.71; 1.94)9 Figure 1. HAI (Egg-propagated) G M T 0 100 200 300 400 500 600 IIV-HD IIV-SD IIV: inactivated influenza vaccine; HD: high-dose; SD: standard-dose GMT: Geometric Mean Titers; HAI: Hemagglutination inhibition; Error bars represent 95% confidence intervals • There is growing interest in evaluating influenza vaccine immune responses beyond the traditional HAI assay.12 OBJECTIVES • To compare the immunogenicity of IIV-HD vs IIV-SD in adults ≥65 years of age against the A/Victoria/361/2011 (H3N2) influenza virus using methods other than the HAI assay based on egg-propagated antigens.