ICS Assays Research Articles

Abstract CT107: Durable immunogenicity of ELI-002 2P in AMPLIFY-201: Lymph node targeted mKRAS-specific amphiphile vaccine in pancreatic and colorectal cancer

Abstract Background: Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are the second and third leading causes of cancer death in the United States. Kirsten rat sarcoma driver mutations (mKRAS) are prevalent in CRC (52%) and PDAC (93%), making them attractive targets for immunotherapy where therapeutic options are otherwise limited. ELI-002 2P immunotherapy enhances lymph node delivery and immune responses using Amphiphile (Amph) modification of vaccine components. 84% of patients (21/25) induced mKRAS-specific T-cells post-vaccination as assessed by direct ex vivo Fluorospot and/or ICS assays with an average 58-fold increase from baseline. A balanced CD4+ and CD8+ T cell response was observed in 59% of patients. Reductions in ctDNA or serum tumor biomarker antigen from baseline were observed in 84% of patients (21/25) and 24% (6/25) had complete biomarker clearance. The induction of strong mKRAS T cell responses correlated with reductions in tumor biomarker response/clearance and relapse-free survival (RFS) in patients. Methods: ELI-002-001 is a first-in-human Phase 1 trial (NCT04853017) of ELI-002 2P cancer vaccine as adjuvant treatment for patients with high relapse-risk mKRAS+ PDAC and CRC. ELI-002 2P consists of 2 Amph-modified mKRAS peptide antigens, Amph-G12D and Amph-G12R (Amph-Peptides 2P), and an Amph-modified immune-stimulatory oligonucleotide adjuvant (Amph-CpG-7909). 25 patients received ELI-002 2P at 1.4 mg of Amph-Peptides 2P and Amph-CpG-7909 at 5 dose levels; 0.1, 0.5, 2.5, 5, and 10 mg. Peripheral blood and circulating tumor DNA (ctDNA) or serum tumor antigen were collected longitudinally to assess T cell immunogenicity and reductions in clinical biomarkers. Results: A majority of patients who received the booster immunizations maintained or increased mKRAS-specific T cell responses relative to baseline. A high frequency of polyfunctional cells secreting IFNγ, TNFα, IL-2, and/or Granzyme B was observed. CD4+ T regulatory cells were not induced after ELI-002 2P immunization. Assessment of the breadth of responses to 7 different KRAS mutations revealed broad cross-reactivity to KRAS mutants, including non-immunizing epitopes, and no responses to WT. Additional phenotypic and functional qualities were assessed. Conclusions: ELI-002 is an off-the-shelf vaccine targeting common KRAS tumor mutations demonstrating several key advantages: lymph node-targeted vaccine design, high immunogenicity, with balanced CD4+ and CD8+ T cell responses, HLA-agnostic activity, and targeting of vaccine antigens critical for tumor survival. A randomized Phase 2 clinical trial (NCT05726864) investigating a 7-peptide formulation (G12D, R, V, A, C, S, G13D) is underway. Citation Format: James R. Perry, Haley VanWyk, Amy M. Tavares, Thian Kheoh, Esther Welkowsky, Christopher M. Haqq, Peter C. DeMuth, Lisa K. McNeil. Durable immunogenicity of ELI-002 2P in AMPLIFY-201: Lymph node targeted mKRAS-specific amphiphile vaccine in pancreatic and colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT107.

An Evaluation of the Appropriateness of Prescribing Ultrasensitive Cardiac Troponin I in the Emergency Department of the Mohammed V Military Training Hospital in Rabat

In emergency medicine, Troponin Ic is a useful diagnostic and prognostic biomarker in the management of ischemic cardiac pathologies. However, its prescription often exceeds international recommendations. The aim of this study is to evaluate practices concerning the prescription of Troponin assays in the emergency department of the Mohammed V Military Training Hospital in Rabat. This is a prospective study. Was included in this study, every patient admitted to the emergency department of the Mohammed V Military Training Hospital in Rabat and having at least one troponin assay during the period from December 25, 2020 to January 12, 2021. The assay was performed using the "ARCHITECT STAT Highly sensitive troponin-I" method. 135 patients for whom have prescribed a Troponin assay during the period of study. A total of 219 assays were performed. The mean age was 64 years, with a predominance of males. The main reasons for admission were chest pain (29.6%), dyspnea (26.6%), neurological disorders (16.3%) and respiratory distress (10.3%). A high Troponin Ic value during the first determination was observed in almost 37% of cases. In 63% of cases, the value was negative. 53.3% of prescriptions were considered as irrelevant according to pre-established criteria. A repeated Troponin Ic assays were performed in 31% (n=127) of cases. They were deemed relevant in 25% of cases. In 6% of patients, the appropriateness of repeat assays was not demonstrated. Compliance with good prescribing practices for Troponin significantly reduces the number of unjustified samples, with a real economic impact.

Phytochemical, antioxidant, and anticancer assessments of Atriplex halimus extracts: In silico and in vitro studies

In recent years, the global health community has become increasingly interested in harnessing the therapeutic potential of traditional plants. This study focuses on Atriplex halimus L., a halophytic plant known as Mediterranean saltbush or "Lgtef", traditionally heralded for its medicinal properties. Our primary objective was to scientifically validate its traditional use by examining the antioxidant and anticancer activities of two extracts: aqueous (AHAE) and hydroethanolic (AHHE) derived from its leaves. Both extracts demonstrated significant antioxidant capabilities when assessed through DPPH, BC, ABTS, IC, and TAC assays. Notably, AHHE exhibited exceptional potency, with a DPPH IC50 value of 0.59 ± 0.12 mg/mL. In anticancer evaluations against HepG2 and MDA-MB-463 cell lines, AHHE again stood out, indicating cytotoxic activity with IC50 values of 31.22 ± 3.19 µg/mL and 48.22 ± 2.03 µg/mL, respectively. These in vitro findings were bolstered by computational analyses, which confirmed the antioxidant and anticancer potential, highlighting AHHE's superior efficacy.In summary, our research affirms A. halimus as a rich source of natural bioactive agents, underscoring its traditional medicinal significance. By bridging indigenous knowledge with scientific validation, this study offers a promising avenue for future research into plant-derived remedies, especially against the backdrop of rising global cancer rates and challenges with synthetic drugs. Our findings underscore the therapeutic promise of A. halimus, emphasizing its potential as a cornerstone in life and health sciences.

Letermovir conversion after valganciclovir treatment in cytomegalovirus high‐risk abdominal solid organ transplant recipients may promote development of cytomegalovirus‐specific cell mediated immunity

To evaluate the association of conversion from valganciclovir to letermovir on cytomegalovirus-specific cellular immunity. Adult patients were included if they received a kidney or liver transplant between 8/1/2018-12/31/20, developed symptomatic, high-level CMV viremia and were converted to letermovir 480mg daily as monotherapy after treatment with ganciclovir-derivatives for a minimum of 4weeks and had subsequent CMV cell-mediated immunity (CMI) testing via ICS assay by flow cytometry (Viracor Eurofins T Cell Immunity Panel). Seven patients met inclusion criteria; 87.5% were male and recipients of a kidney transplant. All patients were CMV high risk (D+/R-). Mean time from transplant to CMV disease was 200±91 days. Peak viral load (VL) during CMV treatment was 540,341±391,211 IU/mL. Patients received a mean of 30±24weeks (range: 4-78weeks) of therapy with ganciclovir-derivatives at induction doses prior to letermovir introduction. The median absolute lymphocyte count (ALC) at letermovir initiation was 400/μL (IQR 575) and the median VL was 51.6 (range: ND-490) IU/mL. Most patients (n=5/7, 71.4%) experienced an increase in VL 1 and/or 2weeks after conversion to letermovir. All patients had positive CMI per ICS assay after conversion. Patients received a mean of 10.3±6.9weeks of letermovir prior to having a positive result. Median ALC at positivity was 900/μL. Immunosuppression was not further reduced from initiation of letermovir to demonstration of CMV CMI. No patient had progressive replication or breakthrough disease while maintained on letermovir and three patients (42.9%) underwent antiviral withdrawal without recurrence at the last follow-up. In this case series of abdominal transplant recipients with severe or persistent CMV infection, patients developed CMV-specific CMI after conversion to letermovir monotherapy. These data suggest that using letermovir in place of valganciclovir for secondary prophylaxis may address the lack of efficacy previously seen with this approach, as well as the issues that plague antiviral withdrawal with systematic monitoring. Future prospective studies are needed to evaluate this effect in a more controlled research environment with serial CMI testing to elucidate the optimal duration of letermovir when used in this way.

Can the Wondfo® SARS-CoV-2 IgM/IgG antibodies be used as a rapid diagnostic test?

Background: An outbreak of novel coronavirus (SARS-CoV-2) disease (COVID-19) has rapidly spread worldwide. The aim of this study was to evaluate and validate the performance of the Wondfo® lateral-flow immunochromatographic assay that detect SARS-CoV-2- IgG, IgM antibodies (Wondfo® IC), using the results obtained by the fluorescence immunoassay test as reference diagnostic. Material and methods: 97 serum specimens collected and analyzed by four independent laboratories of Sergipe/Brazil was used for validated the Wondfo® SARS-CoV-2 IgM/IgG antibodies test. The COVID-19 positive serum specimens were determined by fluorescence immunoassay technique, used as reference standard. Results: An overall of 97 serum specimens show 39 (39/97) SARS-CoV-2 IgG positive specimens, 33 (33/97) SARS-CoV-2 IgM positive specimen and 25 non-reagent specimens (25/97). However, the Wondfo® IC assay detected only 9 (9/97) IgM/IgG positive specimen and 25 (25/97) no-reagent specimen. A weak correlation was found between the outcomes of the Wondfo® IC assay and fluorescence test. The accuracy between the two tests was 32.08%. The sensitivity, specificity, positive predictive value, and negative predictive value of Wondfo® IC assay were of 11.12%, 100%, 100% and 25.27%, respectively. Moreover, no false positive sample was determinate, whereas 88.89% of false negative results were found. Conclusion: The Wondfo® IC test failed in providing a quick, valid, and reliable results and appears not to be a good alternative for clinical use in detecting pandemic coronavirus. However, if the limitations of the rapid test are known, some correction factors can be used in order to adjust the epidemiological data.

Serological and Molecular Diagnosis of Bovine Brucellosis in Menoufia Province

Brucellosis represents a serious problem in cattle relaying huge economic losses. Brucella meletensis on of brucella strains that can infect human which its usual host is sheep, might isolated from cattle. The aim of present study was detection of brucellosis among cattle population in Menoufia governorate, Egypt and analyze risk factors. Serologically brucella were detected with RBPT. Evaluation of the specificity and sensitivity of RBPT, IC assay and cELISA against CFT test were done. Molecular detection of brucella using PCR and bacteriological isolation followed by phenotypic and molecular typing of the isolated bacteria. The overall prevalence using RBT was 1.44%. Holstein Friesian cattle have highest prevalence (1.57%) while native cattle were (1.28%). Cattle over 3 years old have the highest seroprevalence (2.77%). The positive result for RBPT, cELISA, IC Assay and CFT were 91%, 60%, 91% and 88% respectively after examination 100 serum sample. Brucella melitensis biovar 3 was isolated from the tissue specimens (uterus and/or lymph nodes). PCR targeting (Immuno-dominant antigen, gene bp26) generated product of 450 bp from (16/20) tissues specimens. These findings assisting in future planning pragmatic control strategies against bovine brucellosis in Egypt and for herd health fertility.

Distinct Phenotypes of Islet Antigen-Specific CD4+ T Cells among the Three Subtypes of Type 1 Diabetes

Type 1 diabetes (T1D) is classified into three subtypes in Japan based on the manner of disease progression: acute onset (AT1D), slowly progressive (SP1D), and fulminant (FT1D). While cellular immunity is considered to play a major role in pancreatic β cell destruction in T1D, the phenotypes of autoreactive T cells in each subtype remain largely unknown. In this study, we performed an integrated assay that permits the determination of ex-vivo cellular immune responses and T cell repertoires specific for islet antigens. Briefly, peripheral blood mononuclear cells obtained from 20 AT1D, 17 SP1D, 18 FT1D patients, and 17 nondiabetics (ND) were stimulated for 48 h with GAD65, preproinsulin (PPI), IGRP, and ZnT8 peptide clusters, after which cytokine/chemokine secretion was measured (ex-vivo assay). Subsequently, peptide-reactive T cells were further expanded with IL-2 to identify CD4+ T cell subsets based on intracellular cytokine staining (ICS assay). In the ex-vivo assay, GAD65-specific IL-6 and IP-10 responses, and PPI-specific IP-10 responses were significantly upregulated in AT1D as compared to ND. Furthermore, GAD65-specific IL-1β and G-CSF, and PPI-specific G-CSF responses were significantly upregulated in FT1D when compared to ND. The ICS assay further revealed that GAD65- and PPI-specific CD4+ T cells were skewed towards Th1 cells in AT1D (P = 0.007 and P = 0.004, respectively), whereas GAD65- and IGRP-specific Th2 cells were more prevalent in SP1D (P = 0.01 and P = 0.03, respectively) than in ND. Interestingly, GAD65-specific Th1 cells were more abundant in SP1D with HLA-DR9 than in those without DR9 (P = 0.03). Of note, FT1D displayed significantly less Tr1 cells specific for all four antigens and GAD65-specific Th2 cells than ND. In conclusion, the phenotypes of islet antigen-specific CD4+ T cells differ among the three T1D subtypes. These distinct phenotypes of pathogenic T cells may be associated with the manner of progression of β cell destruction. Disclosure D. Chujo: Research Support; Self; Mitsubishi Tanabe Pharma Corporation, Sanofi K.K., Kyowa Hakko Kirin Co., Ltd.. A. Kawabe: None. N. Takahashi: None. M. Matsushita: None. C. Tsutsumi: Research Support; Self; MSD K.K.. F. Haseda: None. A. Imagawa: Research Support; Self; Kowa Pharmaceutical, Sanwa Kagaku Kenkyusho Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Novo Nordisk A/S, Eli Lilly and Company, MSD K.K., Mitsubishi Tanabe Pharma Corporation, Shionogi & Co., Ltd., Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Sanofi K.K., Novo Nordisk A/S, Mitsubishi Tanabe Pharma Corporation, Daiichi Sankyo Company, Limited, Nipro, Eli Lilly and Company, AstraZeneca, Nippon Boehringer Ingelheim Co. Ltd., Taisho Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Terumo Medical Corporation. T. Hanafusa: Speaker's Bureau; Self; Novo Nordisk Inc.. K. Yagi: None. M. Shimoda: None. K. Ueki: Speaker's Bureau; Self; Daiichi Sankyo Company, Limited, Eli Lilly and Company, Boehringer Ingelheim Pharmaceuticals, Inc., MSD K.K., Novo Nordisk Inc., Mitsubishi Tanabe Pharma Corporation, Kyowa Hakko Kirin Co., Ltd., Takeda. Research Support; Self; Takeda, Astellas, Novo Nordisk Inc.. H. Kajio: None.

Validation of an automated gating pipeline for ICS assays in vaccine trials

Validation of an automated gating pipeline for ICS assays in vaccine trials

Development and evaluation of a broad-specific immunochromatographic assay for screening of both tadalafil and its analogues in functional foods

ABSTRACTIllegal additives of tadalafil and related analogues in functional foods threaten public health. To generate broad-specific antibodies against both tadalafil and its analogues, tadalafil was activated through an oximation reaction for synthesis of conjugates, leaving the common structure of tadalafil analogues exposed as a major antigenic site. The desired antisera showed satisfied specificities to tadalafil and its major analogues with immunochromatographic (IC50) values ranging from 16.1 to 29.5 ng mL−1 in a referring indirect competitive enzyme-linked immunosorbent assay. The optimized IC assay showed detection thresholds ranging in 10∼20 μg g–1 for tadalafil and major analogues when detected fortified herbal samples in a dilution ratio of 1:103. Sixty herbal food supplements were screened and five were found to be positive using IC strip. It was confirmed by UPLC-PDA-MS/MS that positive samples contain target illegal additives in levels of 10∼20 mg g−1 (1%∼2%). In this range of containing percentage, sensitivity of the IC strip is adequate to screen tadalafil and its potential analogues in herbal products.

Development of IFN-γ secretory ELISPOT based assay for screening of ADCC responses

Antibody dependent cell mediated cytotoxicity has been established as one of the important protective immune mechanisms against HIV making it essential to evaluate it while testing immunogenicity of emerging vaccine candidates. IFN-γ secretory ELISPOT assay, widely used for evaluation of CTL response in HIV vaccine trials, was adapted for measuring ADCC responses and the results were compared with the standard ICS based assays. IFN-γ responses elicited by plasma samples of 23 HIV infected individuals against Env and Gag peptides using granulocytes as antigen presenting cells were assessed by both the methods. Supernatants of the activated cells in ELISPOT assay were also assessed for cytokine/chemokine estimation. ELISPOT assays detected significantly more ADCC responders against HIV-Env and Gag peptide pools than ICS assay. The magnitude of IFN-γ response in both the assay correlated significantly (p=0.002). NK cells were found to be the predominant cell type secreting IFN-γ in the assay. Although IFN-γ and IL-6 levels were significantly higher in supernatants of Env peptides stimulated cells, IP-10 and MCP-1α levels were found to be more against Gag peptides. Thus, IFN-γ secretory ELISPOT assay was found to be more sensitive in detecting ADCC responders than ICS assay making it a valuable tool for screening of ADCC responses in future vaccine trials. Differences in cytokine pattern of Env versus Gag stimulated cells warrants a need for investigating their role in protection against HIV infection.

Differential kinetics of CD4+ and CD8+ T cell responses: implications for the assessment of immune responses

Abstract Intracellular cytokine staining (ICS) to detect cellular immune responses to infection or vaccination has become routine. Laboratories develop and optimize ICS assays independently and there are many variations of the assay in use today. However, little is known regarding the underlying biology that results in the variable outcomes following assay optimization. The results from the different assays can range from highly similar (slight changes in magnitude) to incredibly different, such as large changes in magnitude or a change in the responding cell types detected. These large differences prompted our investigation into the underlying reasons for the discrepancies in reported data for clinical trials. Here, we used leukapheresis samples from CMV-reactive volunteers to examine viability over time, the effect of costimulation, and response kinetics. The data show that golgi transport inhibitors diminish cell viability after 12 hours of stimulation, emphasizing the importance of viability markers particularly during long stimulations. Costimulatory antibodies increase responses but at the expense of sensitivity, given the increase in background. Interestingly, we found that CD4+ and CD8+ T cells exhibit differential kinetics in their response to CMVpp65 peptides. CD8+ T cells were largely exhausted by 6 hours following the initiation of stimulation, while CD4+ T cells continue to make cytokines well past 20 hours of stimulation. These data have implications on the design of ICS assays and the interpretation of data generated by ICS. Furthermore, the data provide a basis for understanding the differences in ICS assays and provide insight into the kinetics of CD4+ and CD8+ T cell responses.

A novel DNA vaccine against Mycobacterium tuberculosis infection (VAC4P.1109)

Abstract Background: Mycobacterium tuberculosis (M.tb.) pandemic continues to grow together with serious issues of increasing drug resistance, HIV co-infection and mortality. Since the existing BCG vaccine does not provide significant protection in adults, novel protective strategies should be explored with urgency. Methods: DNA vaccines that express TB antigens Ag85B, ESAT-6, Rv2660c and fusion BER, were investigated in BALB/c mice intramuscularly with electroporation (EP). Immunogenicity and efficacy assessments of these DNA vaccines were then determined before or after aerosol challenge of 100CFU M.tb H37Rv strain. Results: Compared with BCG and other DNA/EP vaccines, pBER/EP induced high frequency of Ag85B-specific CD8+ T cells (up to 41%) in tetramer assay. Moreover, pBER/EP also elicited significantly higher frequencies of antigen-specific CD4+ T cells by ELIspot and ICS assays. Surprisingly, these T cell responses were also significantly higher than those induced by a heterologous DNA/EP prime and vaccinia-85B boost regimen. Critically, pBER/EP conferred significantly better protection against M.tb H37Rv challenge when compared to other DNA/EP vaccines, with lower CFU count in the lungs (p<0.01) and the absence of lung pathology. Conclusions: pBER/EP is a promising DNA vaccine candidate that does not have major issues of safety and pre-existing anti-vector immunity (e.g. vaccinia vector). Our findings warrant further investigation of pBER/EP for TB prevention and immunotherapy.

Multi-well microelectrode array recordings detect neuroactivity of ToxCast compounds

Spontaneous activity in neuronal cultures on microelectrode arrays (MEAs) is sensitive to effects of drugs, chemicals, and particles. Multi-well MEA (mwMEA) systems have increased throughput of MEAs, enabling their use for chemical screening. The present experiments examined a subset of EPA's ToxCast compounds for effects on spontaneous neuronal activity in primary cortical cultures using 48-well MEA plates. A first cohort of 68 compounds was selected from the ToxCast Phase I and II libraries; 37 were positive in one or more of 20 individual ToxCast Novascreen assays related to ion channels (NVS_IC), with the remainder selected based on known neuroactivity. A second cohort of 25 compounds was then tested with 20 originating from the ToxCast Phase I and II libraries (not hits in NVS_IC assays) and 5 known negatives from commercial vendors. Baseline activity (1h) was recorded prior to exposing the networks to compounds for 1h, and the weighted mean firing rate (wMFR) was determined in the absence and presence of each compound. Compounds that altered activity by greater than the weighted change of DMSO-treated wells plus 2SD were considered “hits”. Of the first set of 68 compounds, 54 altered wMFR by more than the threshold, while in the second set, 13/25 compounds were hits. MEAs detected 30 of 37 (81.1%) compounds that were hits in NVS_IC assays, as well as detected known neurotoxicants that were negative in NVS_IC assays, primarily pyrethroids and GABAA receptor antagonists. Conversely, wMFR of cortical neuronal networks on MEAs was insensitive to nicotinic compounds, as only one neonicotinoid was detected by MEAs; this accounts for the bulk of non-concordant compounds between MEA and NVS_IC assays. These data demonstrate that mwMEAs can be used to screen chemicals efficiently for potential neurotoxicity, and that the results are concordant with predictions from ToxCast NVS_IC assays for interactions with ion channels.

Identification of minimal human MHC-restricted CD8+ T-cell epitopes within the Plasmodium falciparum circumsporozoite protein (CSP)

BackgroundPlasmodium falciparum circumsporozoite protein (CSP) is a leading malaria vaccine candidate antigen, known to elicit protective antibody responses in humans (RTS,S vaccine). Recently, a DNA prime / adenovirus (Ad) vector boost vaccine encoding CSP and a second P. falciparum antigen, apical membrane antigen-1, also elicited sterile protection, but in this case associated with interferon gamma ELISpot and CD8+ T cell but not antibody responses. The finding that CSP delivered by an appropriate vaccine platform likely elicits protective cell-mediated immunity provided a rationale for identifying class I-restricted epitopes within this leading vaccine candidate antigen.MethodsLimited samples of peripheral blood mononuclear cells from clinical trials of the Ad vaccine were used to identify CD8+ T cell epitopes within pools of overlapping 15mer peptides spanning portions of CSP that stimulated recall responses. Computerized algorithms (NetMHC) predicted 17 minimal class I-restricted 9-10mer epitopes within fifteen 15mers positive in ELISpot assay using PBMC from 10 HLA-matched study subjects. Four additional epitopes were subsequently predicted using NetMHC, matched to other study subjects without initial 15mer ELISpot screening. Nine of the putative epitopes were synthesized and tested by ELISpot assay, and six of these nine were further tested for CD8+ T cell responses by ELISpot CD4+ and CD8+ T cell-depletion and flow cytometry assays for evidence of CD8+ T cell dependence.ResultsEach of the nine putative epitopes, all sequence-conserved, recalled responses from HLA-matched CSP-immunized research subjects. Four shorter sequences contained within these sequences were identified using NetMHC predictions and may have contributed to recall responses. Five (9-10mer) epitopes were confirmed to be targets of CD8+ T cell responses using ELISpot depletion and ICS assays. Two 9mers among these nine epitopes were each restricted by two HLA supertypes (A01/B07; A01A24/A24) and one 9mer was restricted by three HLA supertypes (A01A24/A24/B27) indicating that some CSP class I-restricted epitopes, like DR epitopes, may be HLA-promiscuous.ConclusionsThis study identified nine and confirmed five novel class I epitopes restricted by six HLA supertypes, suggesting that an adenovirus-vectored CSP vaccine would be immunogenic and potentially protective in genetically diverse populations.

Exogenous, TAP‐independent lysosomal presentation of a respiratory syncytial virus CTL epitope

Correction to: Immunology and Cell Biology (2012) 90, 978–982; doi:10.1038/icb.2012.43; published online 28 August 2012 Regrettably, the name of the virus in Figure 1 corrupted on upload. It reads ‘vvFsig?’ but should read ‘vvFsig−’. The correct Figure 1 appears below. The typesetter would like to apologise for their error. Proteasome-independent and TAP-independent presentation of epitope F249-258 follows a lysosomal exogenous pathway. (a) rVACV encoding different forms of RSV F glycoprotein used in this study were based on the Western Reserve (WR) wild-type vaccinia strain. Virus vvF encodes the wild-type F protein of RSV. Hydrophobic regions are signal peptide (sp), fusion peptide (fp) and transmembrane region (tm). Processing by furin-like proteases in the ectodomain (arrows) yields the F1 and F2 chains. Virus vvFsig− encodes a cytosolic form of the F protein lacking the signal peptide. The CTL epitope (▴) presented by Kd assessed in this study is F249-258 (TYMLTNSELL) and is resistant to brefeldin A (BFA) and to lactacystin (LC). (b) CV-1 cells were infected for 24 h with the indicated virus at the indicated m.o.i., p.f.u. counted and cells stained with anti-RSV F antibody. For UVi, an estimated m.o.i.⩽10−7 was calculated. T2/Kd cells were infected for 5 h with indicated rVACV (m.o.i. 5) or with their UVi counterparts (m.o.i.⩽10−5). Background staining by cells infected with WR is shown in grey. (c–f) T2/Kd cells were infected with rVACV at a m.o.i. of 3–15 (light grey bars) or with the same volume of UVi non-infectious virus, equivalent to a m.o.i.⩽10−5 (dark grey bars). ICS assays were performed by stimulating CTL F/F249-258 monospecific for epitope F249-258 with the indicated fixed targets in the presence of brefeldin A to intracellularly accumulate IFNγ produced upon CTL activation. Represented data are the percentages of total CD8+ lymphocytes that expressed IFNγ or the percentage of inhibition of CTL activation. When the mean is represented, the s.d. is shown by error bars. Lack of side effects by inhibitors was controlled by measuring CTL activation by limiting concentrations of exogenous peptide in the presence of inhibitors, as well as their effect on rVACV protein expression. (c) Data are the mean of at least five independent experiments. DC, dendritic cells. (d) Cells were infected with the indicated virus in presence of protein-synthesis inhibitor cycloheximide (CH). Data in the left panel are the mean of one to five independent experiments. A representative experiment is shown in the right panel. (e) Cells were incubated with vvF, UVi vvF or parental strain WR at 37 °C for the indicated times. As control, CTL activation induced by cells incubated with synthetic peptide just before fixation and washing is shown (x). Results from a representative experiment are shown. (f) Cells were infected with vvFsig− in the presence of inhibitors NH4Cl (lysosomotropic agent), Leup (inhibitor of lysosomal serine and cysteine proteases) or 3-methyladenine (3MA) (autophagy inhibitor). Percentage activated CTLs in the absence of inhibitors ranged from 11 to 38. Data are from one to two independent experiments.

Development of a novel simian adenovirus 24 based vaccine vector

Methods Neutralizing antibodies against sAd24 were determined using a panel of 106 rhesus macaque sera and 128 human sera from Rwanda and South Africa using a luciferasebased adenovirus neutralization assay. The immunogenicity of a single dose of 10E7, 10E8 or 10E9 virus particles of sAd24-SIV Gag based vector was determined in C57BL/6 mice. SIV-Gag-specific immune responses were assessed by Db/AL11 tetramer binding assays, IFN-g ELISPOT assays and ICS assays.

Optimization and qualification of an 8-color intracellular cytokine staining assay for quantifying T cell responses in rhesus macaques for pre-clinical vaccine studies

Vaccination and SIV challenge of macaque species is the best animal model for evaluating candidate HIV vaccines in pre-clinical studies. As such, robust assays optimized for use in nonhuman primates are necessary for reliable ex vivo measurement of immune responses and identification of potential immune correlates of protection. We optimized and qualified an 8-color intracellular cytokine staining assay for the measurement of IFNγ, IL-2, and TNF from viable CD4 and CD8 T cells from cryopreserved rhesus macaque PBMC stimulated with peptides. After optimization, five laboratories tested assay performance using the same reagents and PBMC samples; similar results were obtained despite the use of flow cytometers with different configurations. The 8-color assay was then subjected to a pre-qualification study to quantify specificity and precision. These data were used to set positivity thresholds and to design the qualification protocol. Upon completion of the qualification study, the assay was shown to be highly reproducible with low inter-aliquot, inter-day, and inter-operator variability according to the qualification criteria with an overall variability of 20–40% for each outcome measurement. Thus, the 8-color ICS assay was formally qualified according to the ICH guidelines Q2 (R1) for specificity and precision indicating that it is considered a standardized/robust assay acceptable for use in pre-clinical trial immunogenicity testing.

Evaluation of Antioxidant Properties and Mineral Composition of Purslane (Portulaca oleracea L.) at Different Growth Stages

The main objective of this research was to appraise the changes in mineral content and antioxidant attributes of Portulaca oleracea over different growth stages. The antioxidant activity was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) assays. The iodine titration method was used to determine the ascorbic acid content (AAC). DPPH scavenging (IC50) capacity ranged from 1.30 ± 0.04 to 1.71 ± 0.04 mg/mL, while the ascorbic acid equivalent antioxidant activity (AEAC) values were 229.5 ± 7.9 to 319.3 ± 8.7 mg AA/100 g, total phenol content (TPC) varied from 174.5 ± 8.5 to 348.5 ± 7.9 mg GAE/100 g. AAC 60.5 ± 2.1 to 86.5 ± 3.9 mg/100 g and FRAP 1.8 ± 0.1 to 4.3 ± 0.1 mg GAE/g. There was good correlation between the results of TPC and AEAC, and between IC50 and FRAP assays (r2 > 0.9). The concentrations of Ca, Mg, K, Fe and Zn increased with plant maturity. Calcium (Ca) was negatively correlated with sodium (Na) and chloride (Cl), but positively correlated with magnesium (Mg), potassium (K), iron (Fe) and zinc (Zn). Portulaca olerecea cultivars could be used as a source of minerals and antioxidants, especially for functional food and nutraceutical applications.

Mo1121 Novel Influenza (H1N1) Infection Could Cause Gastrointestinal Illness, but Not Significantly - Result of Epidemiological Survey and Antiviral Treatment Influences

[Background] There was pandemic infection of the novel influenza (H1N1) all over the world in the last winter (2009-2010 ). We have cared the novel influenza patients including adults and children in the department of General and Intensive Care Medicine. We present this study referring to gastrointestinal illness in H1NI during the last winter, because there have been very few reports about it associated with the novel influenza. [Method] Persons with fever and/or some influenza-like symptoms during 6 months from July to March 2010, were studied. Novel influenza was diagnosed by detection of viral antigen using immunochromatography assay (IC). We used two commercialized rapid IC kits (QuicNaviFluTM, OTSUKA PHARMACEUTICAL Co., LTD, Tokyo, Japan and Capilia FLU A+BTM, TAUNS LABORATORIES INC. Numazu, Japan ) for the diagnosis of novel influenza (H1N1). These kits are recommended as an authoritative rapid test for H1NI and widely indicated in Japan. We decided it H1NI when the rapid IC assay indicated type A infection. To understand gastrointestinal symptoms (diarrhea, vomiting, and abdominal pain ), all patients were classified into 4 groups; A group is composed of persons with positive influenza A check (medicated with an antiviral agent ), B group with negative influenza A check, C group without influenza check and medicated with oseltamivir, and D group without influenza and medicated with zanamivir. [Result] H1NI was diagnosed in 1567 out of 6859 suspicious patients by rapid IC assay. Gastrointestinal symptom were observed; 41 diarrhea (2.6%), 94 vomiting(6.0%), 37 abdominal pain (2.4%) respectively. Antiviral medication for treatment of H1NI infection was indicated to 3132 patients (1954 children and 1178 adults). There was no significant difference between Group A and Group B about gastrointestinal symptoms both in children and adults. No difference was found between group C and Group D about gastrointestinal symptom. There was no death due to H1NI through out the period in our department. [Conclusion] Nausea and vomiting were most frequently observed in children and adolescences with H1NI, followed by abdominal pain and diarrhea. They were not significant and further examination and treatment were not essential. Antiviral treatment ( oseltamivir and zanamivir ) could cause vomiting in only 0.5%, less than reported previously.

Discovery and development of telaprevir: an NS3-4A protease inhibitor for treating genotype 1 chronic hepatitis C virus

Infection with hepatitis C virus (HCV) is a major medical problem with over 170 million people infected worldwide. Substantial morbidity and mortality are associated with hepatic manifestations (cirrhosis and hepatocellular carcinoma), which develop with increasing frequency in people infected with HCV for more than 20 years. Less well known is the burden of HCV disease associated with extrahepatic manifestations (diabetes, B-cell proliferative disorders, depression, cognitive disorders, arthritis and Sjögren's syndrome). For patients infected with genotype 1 HCV, treatment with polyethylene glycol decorated interferon (peginterferon) α and ribavirin (PR) is associated with a low (40-50%) success rate, substantial treatment-limiting side effects and a long (48-week) duration of treatment. In the past 15 years, major scientific advances have enabled the development of new classes of HCV therapy, the direct-acting antiviral agents, also known as specifically targeted antiviral therapy for hepatitis C (STAT-C). In combination with PR, the HCV NS3-4A protease inhibitor telaprevir has recently been approved for treatment of genotype 1 chronic HCV in the United States, Canada, European Union and Japan. Compared with PR, telaprevir combination therapy offers significantly improved viral cure rates and the possibility of shortened treatment duration for diverse patient populations. Developers of innovative drugs have to blaze a new path with few validated sign posts to guide the way. Indeed, telaprevir's development was once put on hold because of its performance in a standard IC(50) assay. Data from new hypotheses and novel experiments were required to justify further investment and reduce risk that the drug might fail in the clinic. In addition, the poor drug-like properties of telaprevir were a formidable hurdle, which the manufacturing and formulation teams had to overcome to make the drug. Finally, novel clinical trial designs were developed to improve efficacy and shorten treatment in parallel instead of sequentially. Lessons learned from the development of telaprevir suggest that makers of innovative medicines cannot rely solely on traditional drug discovery metrics, but must develop innovative, scientifically guided pathways for success.