Although the microneutralization (MN) assay has been shown to be more sensitive than the hemagglutination inhibition (HAI) assay for the measurement of humoral immunity against influenza viruses, further evidence relating MN titres to protective efficacy against infection is needed. Serum antibodies against seasonal H1N1 and H3N2 influenza were measured in children and adolescents (n = 656) by MN and hemagglutination inhibition (HAI) assays. Compared to HAI, the MN assay is more sensitive in detecting serum antibodies and estimates of protective effectiveness against PCR-confirmed infection were higher for both subtypes. Given our findings, the MN assay warrants further consideration as a formal tool for the routine evaluation of vaccine-induced antibody responses.

Standardization of the hemagglutination inhibition (HAI) assay for influenza serology is challenging. Poor reproducibility of HAI results from one laboratory to another is widely cited, limiting comparisons between candidate vaccines in different clinical trials and posing challenges for licensing authorities. In this study, we standardized HAI assay materials, methods, and interpretive criteria across five geographically dispersed laboratories of a multidisciplinary influenza research network and then evaluated intralaboratory and interlaboratory variations in HAI titers by repeatedly testing standardized panels of human serum samples. Duplicate precision and reproducibility from comparisons between assays within laboratories were 99.8% (99.2% to 100%) and 98.0% (93.3% to 100%), respectively. The results for 98.9% (95% to 100%) of the samples were within 2-fold of all-laboratory consensus titers, and the results for 94.3% (85% to 100%) of the samples were within 2-fold of our reference laboratory data. Low-titer samples showed the greatest variability in comparisons between assays and between sites. Classification of seroprotection (titer > 40) was accurate in 93.6% or 89.5% of cases in comparison to the consensus or reference laboratory classification, respectively. This study showed that with carefully chosen standardization processes, high reproducibility of HAI results between laboratories is indeed achievable.T he hemagglutination inhibition (HAI) assay is the primary method for determining quantitative antibody titers for influenza virus and is widely used both for licensure of vaccines and for seroepidemiologic studies examining protection in populations (1-3). The assay relies on the ability of the hemagglutinin protein on the surface of influenza virus to bind to sialic acids on the surface of red blood cells (RBCs) (4). If the patient's serum contains antibodies that block viral attachment, this interaction is inhibited. Direct comparison of results between studies has been problematic, as the reproducibility of HAI assays between laboratories has historically been poor (5-12). These studies have shown that HAI titers reported for identical specimens in different laboratories can vary as much as 80-fold or 128-fold (9, 11), with the geometric coefficient of variation (GCV) as high as 803% (5). This variability in results may relate to differences in biological reagents, protocols, and personnel training. The use of international standards (IS) may reduce interlaboratory variability (6, 11, 13), but such reagents currently exist only for influenza A H1N1 and H5N1 clade 1 viruses (14, 15). The need for standardization of HAI assays and other laboratory methods (e.g., microneutralization [MN] assays) has been highlighted as a priority of CONSISE, the Consortium for the Standardization of Influenza Seroepidemiology (16,17). CONSISE collaborators have recently published data showing that a standardized MN protocol improves the comparability of serologic results between laboratories (18); however, the conso...

During the 2009–2010 influenza pandemic, an adjuvanted, dose-sparing vaccine was recommended for most Canadians. We hypothesize that differences exist in the responses to AS03-adjuvanted, low antigen (Ag) dose versus unadjuvanted, full-dose vaccines. We investigated the relationship between Ag dose and the oil-in-water emulsion Adjuvant System AS03. BALB/c mice received two IM doses of AS03A or AS03B with exaggerated dilutions of A/Uruguay/716/2007 H3N2 split virion vaccine Ag. Immune responses were assessed 3 weeks after the booster. Unadjuvanted “high” (3 μg) and low-dose (0.03–0.003 μg) vaccines generated similar serum antibody titers and cytokine secretion patterns in restimulated splenocytes. Compared to unadjuvanted “high-dose” vaccination, both AS03A and AS03B-adjuvanted low-dose vaccines tended to elicit higher serum antibody titers, broader induction of cytokine secretion and generated more influenza-specific antibody secreting cells and cytokine-secreting CD4 and CD8 T cells in splenocytes. We show that varying Ag and/or AS03 dose in this influenza vaccination mouse model can strongly influence both the magnitude and pattern of the immune response elicited. These findings are highly relevant given the likelihood of expanded use of adjuvanted, dose-sparing vaccines and raise questions about the use of “standard” doses of vaccines in pre-clinical vaccine studies.

BackgroundSerum antibodies are often used as correlates of protection for influenza. Three commonly used serological assays for detecting influenza‐specific serum antibodies are single radial haemolysis (SRH), haemagglutinin inhibition (HAI) and microneutralization (MN). However, here are limited data on SRH as well as HAI and MN as correlates of protection against influenza in children and adolescents. There are also limited data that compare SRH to HAI and MN.ObjectivesWe sought primarily to understand how SRH titres correlate to protection against influenza infection in children and adolescents. We also compare SRH to HAI and MN.MethodsOf 732 healthy Hutterite children and adolescents aged between 3 and 15 years were enrolled from Saskatchewan and Alberta, Canada, in the 2008‐2009 flu season. Blood samples were drawn from participants at baseline and between 3 and 5 weeks post‐vaccination. Serum antibodies against seasonal H3N2 influenza were measured by SRH, HAI and MN assays.ResultsThe estimates of protective efficacy fluctuated when the cut‐off SRH values increased. The correlation between HAI and SRH titres was 0.53 (P<.01); between MN and SRH 0.82 (P<.01); and between HAI and MN 0.50 (P<.01). Sixteen per cent of participants had SRH titres below the detection limit, compared to 7% and 34% for the MN and HAI assays.Conclusions SRH had the worst correlation with protection against seasonal H3N2 in children and adolescents compared to MN and HAI. SRH, HAI and MN titres were significantly correlated with each other. SRH was less sensitive than MN but more sensitive than HAI.

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We investigated the long-term immune profiles of dose-sparing, AS03-adjuvanted vaccines compared to a traditional high-dose, unadjuvated influenza vaccine formulation. BALB/c mice received 2 IM injections of influenza A/Uruguay/716/2007 (H3N2) split vaccine antigen: high-dose (HD) (3 mg hemagglutinin (HA)/ dose) or low-dose (LD) formulations (0.03 mg or 0.003 mg HA) with AS03 and were followed to 34 weeks post-boost (pb). We examined serologic responses, spleen and bone marrow (BM) HA-specific antibodysecreting cells (ASCs) by ELISpot, influenza-specific cytokine/chemokine production in re-stimulated splenocytes by multiplex ELISA, and antigen-specific CD4C T cells that express cytokines (IL-2, IFNg, TNFa and IL-5) by flow cytometry. All formulations elicited robust serum antibody titers that persisted for at least 34 weeks. The number of antigen-specific ASCs in the spleen and BM were higher in the 2 LD CAS03 groups, but despite having fewer ASCs, the average spot size in the HD-unadjuvanted group was larger at later time-points, suggesting greater antibody production per cell. Striking differences in the long-term profiles induced by the different vaccine formulations may contribute to these different ASC profiles. The HD-unadjuvanted vaccine elicited strong Th2 cytokines during the first 6 weeks pb but LDCAS03 groups generated broader, more durable responses at later timepoints. Finally, the 0.03 mg HACAS03 group generated the greatest number of antigen-specific CD4C T cells and the highest percentage of polyfunctional cells that expressed 2 or more cytokines. Although all of the tested vaccines induced durable antibody responses, we show that different vaccine formulations (dose-sparing, adjuvant) generate distinct long-term immune profiles. Furthermore, our data suggest that the different profiles may be generated through unique mechanisms.