Single radial haemolysis compared to haemagglutinin inhibition and microneutralization as a correlate of protection against influenza A H3N2 in children and adolescents.

BACKGROUND: Serum 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. OBJECTIVES: We 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. METHODS: Of 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. RESULTS: The 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.

Low hemagglutinin antigen dose influenza vaccines adjuvanted with AS03 alter the long-term immune responses in BALB/c mice.

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 µg hemagglutinin (HA)/dose) or low-dose (LD) formulations (0.03 µg or 0.003 µg HA) with AS03 and were followed to 34 weeks post-boost (pb). We examined serologic responses, spleen and bone marrow (BM) HA-specific antibody-secreting cells (ASCs) by ELISpot, influenza-specific cytokine/chemokine production in re-stimulated splenocytes by multiplex ELISA, and antigen-specific CD4+ T cells that express cytokines (IL-2, IFNγ, TNFα 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 +AS03 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 LD+AS03 groups generated broader, more durable responses at later timepoints. Finally, the 0.03 µg HA+AS03 group generated the greatest number of antigen-specific CD4+ T cells and the highest percentage of poly-functional 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.

Standardization of Hemagglutination Inhibition Assay for Influenza Serology Allows for High Reproducibility between Laboratories.

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.

Correction: Microneutralization Assay Titres Correlate with Protection against Seasonal Influenza H1N1 and H3N2 in Children.

[This corrects the article DOI: 10.1371/journal.pone.0131531.].

AS03-Adjuvanted, Very-Low-Dose Influenza Vaccines Induce Distinctive Immune Responses Compared to Unadjuvanted High-Dose Vaccines in BALB/c Mice.

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.

Microneutralization assay titres correlate with protection against seasonal influenza H1N1 and H3N2 in children.

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.

Unusual patterns of IgG avidity in some young children following two doses of the adjuvanted pandemic H1N1 (2009) influenza virus vaccine.

During the 2009-2010 H1N1 influenza pandemic, an adjuvanted monovalent vaccine containing ∼25% of the normal antigen dose and AS03 adjuvant was widely used in Canada. This vaccine was found to be well-tolerated and immunogenic in young children (D. W. Scheifele et al., Pediatr. Infect. Dis. J. 30:402-407, 2011). We report here additional analyses to further characterize the humoral response to this vaccine. We measured standard hemagglutination inhibition (HAI) and microneutralization (MN) titers, as well as influenza virus-specific IgG avidity and subclass distribution by enzyme-linked immunosorbent assay in 73 subjects. Sera were collected before (day 0) and 3 weeks after each dose of vaccine (days 21 and 42). Most children (55/73) had undetectable HAI and MN titers at day 0 (presumed to be antigen naive) and mounted good responses at days 21 and 42. The majority of these children (43/55) had the expected pattern of an increasing IgG avidity index (AI) after each dose of vaccine (not detected [ND], 0.30, and 2.97 at days 0, 21, and 42, respectively). The avidity responses in the remaining children (12/55) were quite different, with AIs increasing abruptly after the first dose and then declining after the second dose of vaccine (ND, 8.83, and 7.15, respectively). These children also had higher concentrations of influenza virus-specific IgG1 and IgG3 antibodies at day 21. Although the antibody titers were similar, some antigen-naive children demonstrated an unusual pattern of avidity maturation after two immunizations with AS03-adjuvanted, low-dose influenza virus vaccine. These data suggest the presence of subtle differences in the quality of the antibodies produced by some subjects in response to this vaccine.

Intranasal proteosome-based respiratory syncytial virus (RSV) vaccines protect BALB/c mice against challenge without eosinophilia or enhanced pathology.

A safe and effective vaccine against respiratory syncytial virus (RSV) is still unavailable. Proteosome-based adjuvants are derived from the outer membrane proteins (OMP) of Neisseria species and are potent inducers of both mucosal and systemic immunity in humans and animals. Candidate RSV subunit vaccines comprising enriched RSV proteins (eRSV) formulated with proteosomes alone or with LPS (Protollin) were produced. Administered intranasally in BALB/c mice, both vaccines elicited long-lasting systemic and mucosal RSV-specific antibodies and fully protected against challenge. In vitro restimulation of lymphocytes from the Protollin-eRSV immunized mice with F (MHC-I) and G (MHC-II) peptides elicited F peptide-specific CD8(+) T cells and supernatant IFNgamma, TNFalpha, IL-2 and IL-10 while the formalin-inactivated RSV (FI-RSV) vaccine elicited predominantly IL-5. Pulmonary eosinophilia did not develop following immunization with either proteosome-based vaccine following challenge compared to mice immunized with FI-RSV. Proteosome-based eRSV vaccines can therefore protect against RSV challenge in mice without increasing the risk of pulmonary immunopathologic responses.

A novel intranasal Protollin-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice.

Protollin-MV is a vaccine produced by mixing split measles virus (MV) antigen with the novel adjuvant Protollin (Neisseria meningitidis outer membrane proteins non-covalently complexed with Shigella flexneri 2a lipopolysaccharide). Intranasal immunization of mice with two or three doses of Protollin-MV induces both serum IgG and mucosal IgA with strong neutralizing activity. There is a dose-dependent shift towards lower IgG1:IgG2a ratios and MV-specific IFNgamma production in splenocytes. Intranasal Protollin-MV can therefore induce systemic and mucosal neutralizing antibody responses as well as elicit a balanced TH1/TH2-type response.

Differential regulation of PHEX expression in bone and parathyroid gland by chronic renal insufficiency and 1,25-dihydroxyvitamin D3.

Mutations in the PHEX gene are responsible for X-linked hypophosphatemia, a renal phosphate-wasting disorder associated with defective skeletal mineralization. PHEX is predominantly expressed in bones and teeth and in the parathyroid gland of patients with chronic renal failure and tertiary hyperparathyroidism. The purpose of the present study was to examine the effects of renal insufficiency and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the regulation of PHEX expression in rat tibia and parathyroid gland. In rats fed a high-phosphate (Pi) diet, nephrectomy elicited a significant increase in the serum parathyroid hormone (PTH) concentration that was associated with a significant increase in the abundance of PHEX mRNA and protein in the tibia and a significant increase in PHEX mRNA in the parathyroid gland. In contrast, 1,25(OH)2D3 administration to intact rats fed a control diet elicited a significant decrease in the serum PTH concentration that was accompanied by a significant decrease in PHEX mRNA and protein abundance in the tibia and a significant decrease in PHEX mRNA in the parathyroid gland. In addition, the increases in serum PTH levels and PHEX mRNA in the tibia and parathyroid gland in nephrectomized rats fed a high-Pi diet were blunted by 1,25(OH)2D3. Serum PTH concentration was positively and significantly correlated with tibial PHEX mRNA and protein abundance. In summary, we demonstrate that PHEX expression in the tibia and parathyroid gland is increased by chronic renal insufficiency and decreased by 1,25(OH)2D3 administration and suggest that PTH status may play an important role in mediating these changes in PHEX expression.