Pre-vaccination (Day 1) and post-vaccination (Day 22) geometric mean titre against a) A/H1N1, b) A/H3N2 and c) B viral strains in subjects without pre-vaccination immunoprotection

<p><b>Copyright information:</b></p><p>Taken from "Safety and immunogenicity of an MF59™-adjuvanted subunit influenza vaccine in elderly Chinese subjects"</p><p>http://www.immunityageing.com/content/5/1/2</p><p>Immunity & ageing : I & A 2008;5():2-2.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2291031.</p><p></p

Geometric mean titre ratios (Day22:Day1) in all subjects in the Phase II/III trial

<p><b>Copyright information:</b></p><p>Taken from "Safety and immunogenicity of an MF59™-adjuvanted subunit influenza vaccine in elderly Chinese subjects"</p><p>http://www.immunityageing.com/content/5/1/2</p><p>Immunity & ageing : I & A 2008;5():2-2.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2291031.</p><p></p

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

<div><p>The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen's neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.</p></div

Calculated specificity, sensitivity, and positive and negative predictive values for the PVLA in comparison to the CPE.

<p>PPV: Positive predictive value; NPV: Negative predictive value.</p

Comparison between CPE and PVLA measurements.

<p>(A) Correlation analysis was performed for 180 double-positive EV71-NtAb samples tested by both the CPE and PVLA methods. The solid line represents the linear regression fitting of the data. The result demonstrated that there was a good relativity between the two assays (r = 0.91, p<0.0001). (B) A Bland-Altman difference plot of EV71-NtAb measurements was constructed using the results of the CPE and PVLA methods. The x-axis corresponds to the average (log₁₀) concentration of EV71-NTAb determined by CPE or PVLA, and the y-axis is a measure of the difference between the concentrations as determined by CPE and PVLA. The solid line represents the mean value, while dashed lines represent the 95% confidence limits. The results indicated that the new method has a high-degree of agreement with the “gold standard” assay. Only four values fell outside ±2 standard deviations (−1.003 to 0.809log₁₀).</p

Optimization of assay parameters.

<p>(<b>A</b>) <b>Determination of RD cell density.</b> RD cells (5×10⁴ cells per well) were incubated in a 96-well plate for 12 h with various dilutions of pseudovirus (from 2×10³ to 2×10⁷ copies per well) in a final volume of 200 μl per well. The data showed that the RLUs decreased with pseudotype in a dose dependent manner. The variation coefficient was lower than 5% for 5×10⁴ and 1×10⁵ cells per well at all the doses of pseudovirus. However, when the RD cell density was 2×10⁴ cells per well, the variation coefficient of RLU was higher than 15% with lower pseudovirus dosage. So the 5×10⁴ cells per well was chosen as the optimized RD cell density. (<b>B</b>) <b>Evaluation of luciferase activity at various incubation times.</b> RD cells (5×10⁴ cells per well) were incubated with 10⁶ copies pseudovirus per well in a 96-well plate for various time points, from 0.5 to 24 h, and each luciferase reading shown is the average of eight replicates. The results indicated that luciferase activity increased gradually with the extension of the incubation over time and reached a plateau at 12 h after incubation. therefore, 12 h was chosen as the optimized incubation time. (<b>C</b>) <b>Infection curve of EV71(FY)-Luc.</b> RD cells were incubated at 35°C together with a range of EV71 pseudovirus concentrations in a final volume of 200 μl per well in 96-well plates. Luciferase activity was measured 12 h post-infection. Each data point represents the average of eight replicates. Linear regression analysis was conducted using Graph Pad Prism. The results showed that when the dose of pseudovirus were within the range of 1×10⁴–10⁷ copies per well, there was a strong correlation between the viral dose and luciferase activity.</p

Contingency table between PVLA and the CPE assay using different cutoffs^a.

a<p>n = 326.</p

Study of the Integrated Immune Response Induced by an Inactivated EV71 Vaccine

<div><p>Enterovirus 71 (EV71), a major causative agent of hand-foot-and-mouth disease (HFMD), causes outbreaks among children in the Asia-Pacific region. A vaccine is urgently needed. Based on successful pre-clinical work, phase I and II clinical trials of an inactivated EV71 vaccine, which included the participants of 288 and 660 respectively, have been conducted. In the present study, the immune response and the correlated modulation of gene expression in the peripheral blood mononuclear cells (PBMCs) of 30 infants (6 to 11 months) immunized with this vaccine or placebo and consented to join this study in the phase II clinical trial were analyzed. The results showed significantly greater neutralizing antibody and specific T cell responses in vaccine group after two inoculations on days 0 and 28. Additionally, more than 600 functional genes that were up- or down-regulated in PBMCs were identified by the microarray assay, and these genes included 68 genes associated with the immune response in vaccine group. These results emphasize the gene expression profile of the immune system in response to an inactivated EV71 vaccine in humans and confirmed that such an immune response was generated as the result of the positive mobilization of the immune system. Furthermore, the immune response was not accompanied by the development of a remarkable inflammatory response.</p> <p> <em>Clinical Trial Registration: NCT01391494 and NCT01512706.</em></p> </div

Confirmation of the genes modulated in vaccinated subjects by real-time RT-PCR.

<p>Real-time RT-PCR analyses of 30 selected genes of PBMCs from the vaccinated infants (Vac, n = 20) at day 28 after vaccine booster. The <i>y</i>-axis indicates the relative quantity of the specific mRNA in the samples compared with the pre-vaccinated samples. The results are normalised to the level of endogenous GAPDH. The error bars indicate the SD of the relative quantities.</p

Immune responses were induced by the inactivated EV71 vaccine.

<p>The humoral (a) and cellular immune responses (b) against EV71 antigen were induced by the inactivated EV71 vaccine in volunteers (n = 20 for Vaccinated and n = 10 for Placebo). (a) The neutralizing antibodies targeting the EV71 antigen in the serum of experimental subjects. The geometric mean titers (GMTs) of neutralizing antibodies to EV71 were measured by a neutralization test as described in the Methods. **, P values of vaccinated group vs baseline value (at 0 day before primary immunization) and vs placebo group were <0.01. (b) The specific T cell response to EV71 antigenic peptides (sequences described in result section), as determined by an Elispot assay. PBMCs from the experimental subjects were incubated in the presence of 10 µM peptides, as described in the Methods section. The values are expressed as the mean±S.D. (n = 20 for Vaccinated and n = 10 for Placebo). **, P values of vaccinated group vs baseline value (at 0 day before primary immunization) and vs placebo group were <0.01. (c) The inactivated EV71 vaccination induces multiple arms of the innate and adaptive responses. Heat map of significantly modulated genes associated with immune responses against the EV71 antigen were analyzed on vaccinated cohort (Vac, n = 20) compared placebo cohort (Pla, n = 10). The listed genes were fallen under different functional categories, which included classification associated with activation of T cell, B cell, innate response adaptive response and MHC respectively. The values are shown in log₂ scale.</p