Proficiency tests to evaluate the impact on assay outcomes of harmonized influenza-specific Intracellular Cytokine Staining (ICS) and IFN-É£ Enzyme-Linked ImmunoSpot (ELISpot) protocols.

The magnitude and quality of cell-mediated immune responses elicited by natural infection or vaccination are commonly measured by Interferon-É£ (IFN-É£) Enzyme-Linked ImmunoSpot (ELISpot) and Intracellular Cytokine Staining (ICS). To date, laboratories apply a variety of in-house procedures which leads to diverging results, complicates interlaboratory comparisons and hampers vaccine evaluations. During the FLUCOP project, efforts have been made to develop harmonized Standard Operating Procedures (SOPs) for influenza-specific IFN-É£ ELISpot and ICS assays. Exploratory pilot studies provided information about the interlaboratory variation before harmonization efforts were initiated. Here we report the results of two proficiency tests organized to evaluate the impact of the harmonization effort on assay results and the performance of participating FLUCOP partners. The introduction of the IFN-É£ ELISpot SOP reduced variation of both background and stimulated responses. Post-harmonization background responses were all lower than an arbitrary threshold of 50 SFU/million cells. When stimulated with A/California and B/Phuket, a statistically significant reduction in variation (p < 0.0001) was observed and CV values were strongly reduced, from 148% to 77% for A/California and from 126% to 73% for B/Phuket. The harmonizing effect of applying an ICS SOP was also confirmed by an increased homogeneity of data obtained by the individual labs. The application of acceptance criteria on cell viability and background responses further enhanced the data homogeneity. Finally, as the same set of samples was analyzed by both the IFN-É£ ELISpot and the ICS assays, a method comparison was performed. A clear correlation between the two methods was observed, but they cannot be considered interchangeable. In conclusion, proficiency tests show that a limited harmonization effort consisting of the introduction of SOPs and the use of the same in vitro stimulating antigens leads to a reduction of the interlaboratory variation of IFN-É£ ELISpot data and demonstrate that substantial improvements for the ICS assay are achieved as comparable laboratory datasets could be generated. Additional steps to further reduce the interlaboratory variation of ICS data can consist of standardized gating templates and detailed data reporting instructions as well as further efforts to harmonize reagent and instrument use.

Harmonization and qualification of intracellular cytokine staining to measure influenza-specific CD4+ T cell immunity within the FLUCOP consortium.

Despite the knowledge that cell-mediated immunity (CMI) contributes to the reduction of severe influenza infection, transmission, and disease outcome, the correlates of protection for cell-mediated immunity remain still unclear. Therefore, measuring the magnitude and quality of influenza-specific T cell responses in a harmonized way is of utmost importance to improve characterisation of vaccine-induced immunity across different clinical trials. The present study, conducted as part of the FLUCOP project, describes the development of a consensus protocol for the intracellular cytokine staining (ICS) assay, in order to reduce inter-laboratory variability, and its qualification. In order to develop a consensus protocol, the study was divided into different stages. Firstly, two pilot studies evaluated critical parameters in the analytical (read-outs) and post-analytical (gating strategies and data analysis) methods applied by eight different laboratories within the FLUCOP consortium. The methods were then harmonized by fixing the critical parameters and the subsequent consensus protocol was then qualified by one FLUCOP member. The antigen-specific cell population was defined as polypositive CD4+ T cells (i.e. positive for at least two markers among CD40L/IFNγ/IL2/TNFα), which was shown to be the most sensitive and specific read-out. The qualification of this consensus protocol showed that the quantification of polypositive CD4+ T cells was precise, linear and accurate, and sensitive with a lower limit of quantification of 0.0335% antigen-specific polypositive CD4+ T cells. In conclusion, we provide the description of a harmonized ICS assay, which permits quantitative and qualitative evaluation of influenza vaccine-induced T cell responses. Application of this harmonized assay may allow for future comparisons of T cell responses to different influenza vaccines. It may facilitate future assessments of potential correlates of protection with the promise of application across other pathogens.

Humoral and cellular immunogenicity and safety following a booster dose of a tetravalent dengue vaccine 5+ years after completion of the primary series in Singapore: 2-year follow-up of a randomized phase II, placebo-controlled trial.

The tetravalent dengue vaccine (CYD-TDV) is approved for use as a 3-dose series for the prevention of dengue in seropositive individuals ≥9 years. A randomized, placebo-controlled, phase II study of a booster dose of CYD-TDV in individuals who completed the 3-dose schedule >5 years previously (NCT02824198), demonstrated that a booster restored neutralizing antibody titers to post-dose 3 levels. We present additional immunogenicity assessments up to 24 months post-booster, and B- and T-cell responses in a participant subset. Participants aged 9-45 years that had received all three doses of CYD-TDV were randomized 3:1 to receive a booster dose of CYD-TDV (n = 89) or placebo (n = 29). Neutralizing antibody levels at Months 1, 6, 12, and 24 post-booster were assessed by plaque reduction neutralization test. In a subset, B-cell responses were assessed by a fluorescent immunospot assay, and T-cells analyzed by flow cytometry at Days 0, 7, 12, Months 1 and 12. We observed an increase of antibody titers Month 1 post-booster, then a gradual decline to Month 24. In the CYD-TDV booster group, an increase in plasmablasts was seen at Day 7 declining by Day 14, an increase in memory B-cells was observed at Day 28 with no persistence at Month 12. CYD-TDV booster recalled a CD8+ T-cell response, dominated by IFN-γ secretion, which decreased 12 months post-booster. This study showed a short-term increase in antibody titers and then gradual decrease following CYD-TDV booster injection >5 years after primary immunization, and the presence of memory B-cells activated following the booster, but with low persistence.

Prevention of post-operative pediatric tracheotomy wounds: A multidisciplinary team approach.

OBJECTIVE: Tracheotomy-related pressure wounds have been reported as high as 29%. All advanced stage (stage 3 or 4) wounds are reported by hospitals, and CMS will no longer reimburse healthcare costs to manage them. We present the results of an intensive, multidisciplinary wound prevention strategy starting in the operating room at the time of tracheotomy placement. METHODS: Prospective analysis of a tracheostomy wound care protocol at an academic, tertiary-care pediatric hospital from September 2012 to February 2016. Participants include all patients having undergone tracheostomy placement followed by protocoled daily dressing changes until the first tracheostomy tube change by team consisting a senior level otolaryngology resident or pediatric otolaryngology fellow, certified wound care specialist, respiratory therapist, and bedside nurse. RESULTS: Post-operative tracheostomy-related wound data from 3 years prior to clinical intervention was obtained and compared to the intervention cohort. From March 2010-August 2012, 161 tracheotomy procedures were performed with 36 (22.4%) subsequent pressure wounds; 31% of these were stage 3 or 4 wounds. After multidisciplinary protocol implementation, there have been 121 additional tracheotomy procedures with reduction to 12 (9.9%) total tracheotomy-related wounds (p = 0.0064) and none (0%) were stage 3 or 4 (p = 0.0014). All stage 1 and 2 wounds were identified early, underwent appropriate intervention, and avoided further progression. CONCLUSIONS: This protocol was successful in the elimination of all tracheostomy-related advanced stage, hospital-acquired pressure injuries and led to prompt identification of early stage wounds post-operatively.

Persistence of Th1/Tc1 responses one year after tetravalent dengue vaccination in adults and adolescents in Singapore.

To characterize the cell mediated immunity (CMI) induced by the investigational CYD tetravalent dengue vaccine (TDV), we developed a whole-blood, intracellular cytokine staining (ICS) assay and a multiplex assay, each requiring 3 mL of blood. We assessed CMI before and 28 d after a first and third injection of CYD-TDV and one year after the third injection in a subset of 80 adolescents and adults enrolled in a phase II trial in Singapore (ClinicalTrial.gov NCT NCT00880893). CD4/IFNγ/TNFα responses specific to dengue NS3 were detected before vaccination. Vaccination induced YF-17D-NS3-specific CD8/IFNγ responses, without significant TNFα, and a CYD-specific Th1/Tc1 cellular response in all participants, which was characterized by predominant IFNγ secretion compared with TNFα, associated with low level IL-13 secretion in multiplex analysis of peripheral blood mononuclear cells (PBMC) supernatants after restimulation with each the CYD vaccine viruses. Responses were directed mainly against CYD-4 after the first vaccination, and were more balanced against all four serotypes after the third vaccination. The same qualitative profile was observed one year after the third vaccination, with approximately 2-fold lower NS3-specific responses, and 3-fold lower serotype-specific cellular responses. These findings confirm previous observations regarding both the nature and specificity of cellular responses induced by CYD-TDV, and for the first time demonstrate the persistence of cellular responses after one year. We also established the feasibility of analyzing CMI with small blood samples, allowing such analysis to be considered for pediatric trials.

Cell-mediated immunity induced by chimeric tetravalent dengue vaccine in naive or flavivirus-primed subjects.

Three independent, phase 1 clinical trials were conducted in Australia and in USA to assess the safety and immunogenicity of sanofi pasteur dengue vaccine candidates. In this context, Dengue 1-4 and Yellow Fever 17D-204 (YF 17D)-specific CD4 and CD8 cellular responses induced by tetravalent chimeric dengue vaccines (CYD) were analyzed in flavivirus-naive or flavivirus-immune patients. Tetravalent CYD vaccine did not trigger detectable changes in serum pro-inflammatory cytokines, whatever the vaccinees immune status, while inducing significant YF 17D NS3-specific CD8 responses and dengue serotype-specific T helper responses. These responses were dominated by serotype 4 in naive individuals, but a booster vaccination (dose #2) performed 4 months following dose #1 broadened serotype-specific responses. A similar, broader response was seen after primary tetravalent immunization in subjects with pre-existing dengue 1 or 2 immunity caused by prior monovalent live-attenuated dengue vaccination. In all three trials, the profile of induced response was similar, whatever the subjects' immune status, i.e. an absence of Th2 response, and an IFN-gamma/TNF-alpha ratio dominated by IFN-gamma, for both CD4 and CD8 responses. Our results also showed an absence of cross-reactivity between YF 17D or Dengue NS3-specific CD8 responses, and allowed the identification of 3 new CD8 epitopes in the YF 17D NS3 antigen. These data are consistent with the previously demonstrated excellent safety of these dengue vaccines in flavivirus-naive and primed individuals.

Evaluation by flow cytometry of antibody-dependent enhancement (ADE) of dengue infection by sera from Thai children immunized with a live-attenuated tetravalent dengue vaccine.

Sera from Thai children immunized with a live-attenuated tetravalent dengue virus vaccine or from naturally infected age-matched site-control subjects were examined for immune enhancement capacity by a highly reproducible flow cytometric assay in Fc receptor-bearing K562 human cells. None of the sera under study corresponded to cases of severe dengue disease. In parallel assays employing each dengue virus serotype, we found no or only minimal antibody-dependent enhancement (ADE) when sera from vaccinated or control subjects were used at a low serum dilution [1/12] that approximated the in vivo condition. Among sera that exhibited homotypic neutralizing antibody activity against DV1-3, the level correlated with absence of ADE or infection with the respective serotype. Similarly, a broad heterotypic neutralizing antibody response that included all four serotypes was linked to complete absence of K562 cell infection. In contrast, at higher serum dilutions a correlation between breadth of antibody response and heightened immune enhancement emerged, a pattern identical to that observed among control subjects. These findings support the use of live dengue vaccines and protocols that induce broad serotype-specific neutralizing antibody responses, but they also suggest that clinically relevant immune enhancement may not be likely if this is not uniformly achieved after the first immunization.