Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains

<p>Abstract</p> <p>Background</p> <p>There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV), as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology.</p> <p>Results</p> <p>In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv) isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge.</p> <p>Conclusion</p> <p>A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans.</p> <p>Crown Copyright © 2009</p

Intraductal fully covered self-expanding metal stents in the management of post-liver transplant anastomotic strictures: a UK wide experience.

Background: Fully covered intraductal self-expanding metal stents (IDSEMS) have been well described in the management of post-liver transplant (LT) anastomotic strictures (ASs). Their antimigration waists and intraductal nature make them suited for deployment across the biliary anastomosis. Objectives: We conducted a multicentre study to analyse their use and efficacy in the management of AS. Design: This was a retrospective, multicentre observational study across nine tertiary centres in the United Kingdom. Methods: Consecutive patients who underwent endoscopic retrograde cholangiopancreatography with IDSEMS insertion were analysed retrospectively. Recorded variables included patient demographics, procedural characteristics, response to therapy and follow-up data. Results: In all, 162 patients (100 males, 62%) underwent 176 episodes of IDSEMS insertion for AS. Aetiology of liver disease in this cohort included hepatocellular carcinoma (n = 35, 22%), followed by alcohol-related liver disease (n = 29, 18%), non-alcoholic steatohepatitis (n = 20, 12%), primary biliary cholangitis (n = 15, 9%), acute liver failure (n = 13, 8%), viral hepatitis (n = 13, 8%) and autoimmune hepatitis (n = 12, 7%). Early AS occurred in 25 (15%) cases, delayed in 32 (20%) cases and late in 95 (59%) cases. Age at transplant was 54 years (range, 12-74), and stent duration was 15 weeks (range, 3 days-78 weeks). In total, 131 (81%) had complete resolution of stricture at endoscopic re-evaluation. Stricture recurrence was observed in 13 (10%) cases, with a median of 19 weeks (range, 4-88 weeks) after stent removal. At removal, there were 21 (12%) adverse events, 5 (3%) episodes of cholangitis and 2 (1%) of pancreatitis. In 11 (6%) cases, the removal wires unravelled, and 3 (2%) stents migrated. All were removed endoscopically. Conclusion: IDSEMS appears to be safe and highly efficacious in the management of post-LT AS, with low rates of AS recurrence

Isolation and Characterisation of a Human-Like Antibody Fragment (scFv) That Inactivates VEEV In Vitro and In Vivo

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required

The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity

Although the murine immune response to Venezuelan equine encephalitis virus (VEEV) is well-characterized, little is known about the human antibody response to VEEV. In this study we used phage display technology to isolate a panel of 11 VEEV-specfic Fabs from two human donors. Seven E2-specific and four E1-specific Fabs were identified and mapped to five E2 epitopes and three E1 epitopes. Two neutralizing Fabs were isolated, E2-specific F5 and E1-specific L1A7, although the neutralizing capacity of L1A7 was 300-fold lower than F5. F5 Fab was expressed as a complete IgG1 molecule, F5 native (n) IgG. Neutralization-escape VEEV variants for F5 nIgG were isolated and their structural genes were sequenced to determine the theoretical binding site of F5. Based on this sequence analysis as well as the ability of F5 to neutralize four neutralization-escape variants of anti-VEEV murine monoclonal antibodies (mapped to E2 amino acids 182–207), a unique neutralization domain on E2 was identified and mapped to E2 amino acids 115–119

Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage

DNA vaccination elicits humoral and cellular immune responses and has been shown to confer protection against several viral, bacterial, and parasitic pathogens. Here we report that optimized codon usage of an injected DNA sequence considerably increases both humoral and cellular immune responses. We recently generated a synthetic human immunodeficiency virus type 1 gp120 sequence in which most wild-type codons were replaced with codons from highly expressed human genes (syngp120). In vitro expression of syngp120 is considerably increased in comparison to that of the respective wild-type sequence. In BALB/c mice, DNA immunization with syngp120 resulted in significantly increased antibody titers and cytotoxic T-lymphocyte reactivity, suggesting a direct correlation between expression levels and the immune response. Moreover, syngp120 is characterized by rev-independent expression and a low risk of recombination with viral sequences. Thus, synthetic genes with optimized codon usage represent a novel strategy to increase the efficacy and safety of DNA vaccination

DNA Vaccines against Dengue Virus Type 2 Based on Truncate Envelope Protein or Its Domain III

Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2

Scientific assessment of the use of sugars as cigarette tobacco ingredients: A review of published and other publicly available studies

Sugars, such as sucrose or invert sugar, have been used as tobacco ingredients in American-blend cigarettes to replenish the sugars lost during curing of the Burley component of the blended tobacco in order to maintain a balanced flavor. Chemical-analytical studies of the mainstream smoke of research cigarettes with various sugar application levels revealed that most of the smoke constituents determined did not show any sugar-related changes in yields (per mg nicotine), while ten constituents were found to either increase (formaldehyde, acrolein, 2-butanone, isoprene, benzene, toluene, benzo[k]fluoranthene) or decrease (4-aminobiphenyl, N-nitrosodimethylamine, N-nitrosonornicotine) in a statistically significant manner with increasing sugar application levels. Such constituent yields were modeled into constituent uptake distributions using simulations of nicotine uptake distributions generated on the basis of published nicotine biomonitoring data, which were multiplied by the constituent/nicotine ratios determined in the current analysis. These simulations revealed extensive overlaps for the constituent uptake distributions with and without sugar application. Moreover, the differences in smoke composition did not lead to relevant changes in the activity in in vitro or in vivo assays. The potential impact of using sugars as tobacco ingredients was further assessed in an indirect manner by comparing published data from markets with predominantly American-blend or Virginia-type (no added sugars) cigarettes. No relevant difference was found between these markets for smoking prevalence, intensity, some markers of dependence, nicotine uptake, or mortality from smoking-related lung cancer and chronic obstructive pulmonary disease. In conclusion, thorough examination of the data available suggests that the use of sugars as ingredients in cigarette tobacco does not increase the inherent risk and harm of cigarette smoking