Mohamed R. Hamed

Human Blood Autoantibodies in the Detection of Colorectal Cancer.

Colorectal cancer (CRC) is the second most common malignancy in the western world. Early detection and diagnosis of all cancer types is vital to improved prognosis by enabling early treatment when tumours should be both resectable and curable. Sera from 3 different cohorts; 42 sera (21 CRC and 21 matched controls) from New York, USA, 200 sera from Pittsburgh, USA (100 CRC and 100 controls) and 20 sera from Dundee, UK (10 CRC and 10 controls) were tested against a panel of multiple tumour-associated antigens (TAAs) using an optimised multiplex microarray system. TAA specific IgG responses were interpolated against the internal IgG standard curve for each sample. Individual TAA specific responses were examined in each cohort to determine cutoffs for a robust initial scoring method to establish sensitivity and specificity. Sensitivity and specificity of combinations of TAAs provided good discrimination between cancer-positive and normal serum. The overall sensitivity and specificity of the sample sets tested against a panel of 32 TAAs were 61.1% and 80.9% respectively for 6 antigens; p53, AFP, K RAS, Annexin, RAF1 and NY-CO16. Furthermore, the observed sensitivity in Pittsburgh sample set in different clinical stages of CRC; stage I (n = 19), stage II (n = 40), stage III (n = 34) and stage IV (n = 6) was similar (73.6%, 75.0%, 73.5% and 83.3%, respectively), with similar levels of sensitivity for right and left sided CRC. We identified an antigen panel of sufficient sensitivity and specificity for early detection of CRC, based upon serum profiling of autoantibody response using a robust multiplex antigen microarray technology. This opens the possibility of a blood test for screening and detection of early colorectal cancer. However this panel will require further validation studies before they can be proposed for clinical practice.

Protective antibodies against Clostridium difficile are present in intravenous immunoglobulin and are retained in humans following its administration

The prevalence of serum antibodies against Clostridium difficile (CD) toxins A and B in healthy populations have prompted interest in evaluating the therapeutic activity of intravenous immunoglobulin (IVIg) in individuals experiencing severe or recurrent C. difficile infection (CDI). Despite some promising case reports, a definitive clinical role for IVIg in CDI remains unclear. Contradictory results may be attributed to a lack of consensus regarding optimal dose, timing of administration and patient selection as well as variability in specific antibody content between commercial preparations. The purpose of this study was to investigate retrospectively the efficacy of three commercial preparations of IVIg for treating severe or recurrent CDI. In subsequent mechanistic studies using protein microarray and toxin neutralization assays, all IVIg preparations were analysed for specific binding and neutralizing antibodies (NAb) to CD antigens in vitro and the presence of anti‐toxin NAbs in vivo following IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multi‐isotype specific antibodies to a 7‐plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of anti‐CD antibodies and/or anti‐toxin neutralizing capacities prior to fractionation.

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

ABSTRACT Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost.

High prevalence of subclass-specific binding and neutralizing antibodies against clostridium difficile toxins in adult cystic fibrosis sera: Possible mode of immunoprotection against symptomatic c. difficile infection

Objectives Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. Methods Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. Conclusion A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera monitoring of anti-C. difficile toxin antibody titers may be of clinical value.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A signalome screening approach in the autoinflammatory disease TNF receptor associated periodic syndrome (TRAPS) highlights the anti-inflammatory properties of drugs for repurposing

&NA; TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease caused by mutations in TNF Receptor 1 (TNFR1). Current therapies for TRAPS are limited and do not target the pro‐inflammatory signalling pathways that are central to the disease mechanism. Our aim was to identify drugs for repurposing as anti‐inflammatories based on their ability to down‐regulate molecules associated with inflammatory signalling pathways that are activated in TRAPS. This was achieved using rigorously optimized, high through‐put cell culture and reverse phase protein microarray systems to screen compounds for their effects on the TRAPS‐associated inflammatory signalome. 1360 approved, publically available, pharmacologically active substances were investigated for their effects on 40 signalling molecules associated with pro‐inflammatory signalling pathways that are constitutively upregulated in TRAPS. The drugs were screened at four 10‐fold concentrations on cell lines expressing both wild‐type (WT) TNFR1 and TRAPS‐associated C33Y mutant TNFR1, or WT TNFR1 alone; signalling molecule levels were then determined in cell lysates by the reverse‐phase protein microarray. A novel mathematical methodology was developed to rank the compounds for their ability to reduce the expression of signalling molecules in the C33Y‐TNFR1 transfectants towards the level seen in the WT‐TNFR1 transfectants. Seven high‐ranking drugs were selected and tested by RPPA for effects on the same 40 signalling molecules in lysates of peripheral blood mononuclear cells (PBMCs) from C33Y‐TRAPS patients compared to PBMCs from normal controls. The fluoroquinolone antibiotic lomefloxacin, as well as others from this class of compounds, showed the most significant effects on multiple pro‐inflammatory signalling pathways that are constitutively activated in TRAPS; lomefloxacin dose‐dependently significantly reduced expression of 7/40 signalling molecules across the Jak/Stat, MAPK, NF‐&kgr;B and PI3K/AKT pathways. This study demonstrates the power of signalome screening for identifying candidates for drug repurposing. Graphical abstract Figure. No caption available.

High Prevalence of Subclass-Specific Binding and Neutralising Antibodies Against Clostridium Difficile Toxins in Adult Cystic Fibrosis Sera: Possible Mode of Protection Against Symptomatic Clostridium Difficile Infection

Objectives: Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. Methods: Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results: Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. Conclusion: A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera monitoring of anti-C. difficile toxin antibody titers may be of clinical value.

Generation and Characterisation of HCV Genotype 1 Viruses Most Recent Common Ancestor

Background: Hepatitis C virus (HCV) causes acute and chronic liver diseases in humans. Its two envelope glycoproteins, E1 and E2, interact with host cell receptors and provide a target for neutralising antibodies. Past vaccine studies using unmodified E2 proteins have failed to convincingly generate broadly neutralising antibody responses. Objectives: This study sought to generate and evaluate an immune-focused, vaccine candidate for HCV. Methodology: A synthetic construct based on most recent common ancestral sequence (MRCA) of HCV genotype 1 viruses was generated using sequences available from the Los Alamos HCV database [720 sequences (360 subtype 1a and 360 subtype 1b sequences)], after exclusion of epidemiologically-related sequences. Soluble E2 (sE2) proteins were generated by stably transfected S2 cells and purified using Strep- tag purification and size exclusion chromatography. The MRCA construct was subsequently interrogated using a linear (AP33) and conformational (1:7) monoclonal antibodies directed at E2. A full length E1E2 construct was used for production of HCV pseudoparticles (HCVpp). The infectivity of the HCVpp was measured in the presence of monoclonal antibodies; AP33 1:7 and AR3A. Results: Monomeric proteins of the MRCA generated using a Drosophila expression system were conformationally intact when examined by the monoclonal antibody 1:7 that targets the conformational epitope on E2 responsible for interaction with the CD81 receptors. The full length MRCA E1E2 construct showed functionality in the HCV pseudo-particle (HCVpp) system. The MRCA HCVpp construct was susceptible to neutralisation by AP33, 1:7 and AR3A, in dose- dependant manner. Conclusion: This study demonstrates the generation of a functional construct that could be used as a vaccine candidate in a potential vaccine approach to minimise the problem of genetic diversity between the vaccine construct and contemporary viruses.

PTU-263 Profiling humoral immune responses to clostridium difficile-specific antigens by protein microarray

Introduction The main objective of this study was to develop, validate and implement a novel microarray platform to enable the simultaneous quantification of systemic IgG immune responses to a 7-plex panel of highly purified C. difficile-specific virulence factors, including whole toxins A and B (toxinotype 0, strain VPI10463, ribotype 087), recombinant fragments of toxin A and B, PCR ribotype-specific surface layer proteins (001, 002, 027) and control proteins (tetanus and candida). We evaluated the performance of the microarray technique against tradional ELISA. Method Microbial antigens were diluted to 200 μg/ml in printing buffer in a 384-well plate and spotted in quadruplicate onto poly-L-Lysine-coated slides using a Biorobotics MicroGridII arrayer. The slides were blocked with 5% BSA diluted in PBS-Tween-20 wash buffer. After washing, all slides were incubated with sera diluted 1:500 in antibody diluent. Following washing, slides were incubated with biotinylated anti-human IgG diluted 1:20,000, washed and then incubated with Streptavidin Cy5 diluted 1:2,000 in 5% BSA. After final washes, slides were dried by centrifugation and scanned using a GenePix 4200AL scanner. The resultant TIFF images were processed with Axon GenePix Pro-6 microarray image analysis software. Protein signals were finally determined with background subtraction using RPPanalyzer, a module within the R statistical language on CRAN (http://vran.r-project.org). Kruskall Wallis was used with Dunn’s post test. Correlation was evaluated using Spearman rank correlation coefficient. P values of < 0.05 were considered statistically significant. Results A total of 327 serum samples from patients with C. difficile infection (CDI), cystic fibrosis (CF) and healthy controls (HC) were tested by microarray for the presence of specific IgG antibody. The microarray assay fell within acceptable limits of precision for both intra- and inter-assay variability for all antigens tested (<10% CV). Additionally, there was significant correlation (p < 0.0001) between antibody detection levels using the microarray system and the ELISA format. Conclusion This is the first reported use of protein microarray to assess serum immunoreactivities to C. difficile-specific antigens. Our results suggest that the microarray platform allows accurate, precise and reproducible specific antibody quantifcation. Additional modifications of this approach could be used to simultaneously study multiple isotype specificities to several C. difficilestrain-specific antigens for large collections of patient sera. Disclosure of interest O. Negm: None Declared, M. Hamed: None Declared, E. Abbott: None Declared, P. Tighe: None Declared, C. Shone: None Declared, C. Loscher: None Declared, L. Edwards: None Declared, M. Wilcox Grant/ Research Support from: Multiple therapeutic and diagnostic companies, Consultant for: Multiple therapeutic and diagnostic companies, T. Monaghan: None Declared.