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

Background

Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2^c,d,e,f,g,h) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182–207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.

Methods

We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.

Findings

Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115–119. Using a 9 Å resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.

Conclusions

The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.     

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.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ?-propionolactone (?-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.     

Development of pan-specific antibody against trimethyllysine for protein research

Background  

Trimethylation of the Nε-lysine residues in a protein is one of the most important events of posttranslational modifications. Simple methods for rapid detection and isolation of the Nε-trimethylated protein species are needed. This report introduces a novel method to prepare the affinity purified antibody specific for the Nε-trimethylated lysine (tMeK). The applications of the purified antibody are also reported in this paper.     

Arboviral Etiologies of Acute Febrile Illnesses in Western South America, 2000–2007

Background

Arthropod-borne viruses (arboviruses) are among the most common agents of human febrile illness worldwide and the most important emerging pathogens, causing multiple notable epidemics of human disease over recent decades. Despite the public health relevance, little is know about the geographic distribution, relative impact, and risk factors for arbovirus infection in many regions of the world. Our objectives were to describe the arboviruses associated with acute undifferentiated febrile illness in participating clinics in four countries in South America and to provide detailed epidemiological analysis of arbovirus infection in Iquitos, Peru, where more extensive monitoring was conducted.

Methodology/Findings

A clinic-based syndromic surveillance system was implemented in 13 locations in Ecuador, Peru, Bolivia, and Paraguay. Serum samples and demographic information were collected from febrile participants reporting to local health clinics or hospitals. Acute-phase sera were tested for viral infection by immunofluorescence assay or RT-PCR, while acute- and convalescent-phase sera were tested for pathogen-specific IgM by ELISA. Between May 2000 and December 2007, 20,880 participants were included in the study, with evidence for recent arbovirus infection detected for 6,793 (32.5%). Dengue viruses (Flavivirus) were the most common arbovirus infections, totaling 26.0% of febrile episodes, with DENV-3 as the most common serotype. Alphavirus (Venezuelan equine encephalitis virus [VEEV] and Mayaro virus [MAYV]) and Orthobunyavirus (Oropouche virus [OROV], Group C viruses, and Guaroa virus) infections were both observed in approximately 3% of febrile episodes. In Iquitos, risk factors for VEEV and MAYV infection included being male and reporting to a rural (vs urban) clinic. In contrast, OROV infection was similar between sexes and type of clinic.

Conclusions/Significance

Our data provide a better understanding of the geographic range of arboviruses in South America and highlight the diversity of pathogens in circulation. These arboviruses are currently significant causes of human illness in endemic regions but also have potential for further expansion. Our data provide a basis for analyzing changes in their ecology and epidemiology.     

Analysis of microRNAs induced by Venezuelan equine encephalitis virus infection in mouse brain

MicroRNAs (miRNA) are small RNA (∼22nts) molecules that are expressed endogenously in cells and play an important role in regulating gene expression. Recent studies have shown that cellular miRNA plays a very important role in the pathogenesis of viral infection. Venezuelan equine encephalitis virus (VEEV) is an RNA virus and is a member of the genus Alphavirus in the family Togaviridae. VEEV is infectious in aerosol form and is a potential biothreat agent. In this study, we report for the first time that VEEV infection in mice brain causes modulation of miRNA expression. Pathway analyses showed that majority of these miRNAs are involved in the neuronal development and function. Target gene prediction of the modulated miRNAs correlates with our recently reported mRNA expression in VEEV infected mice brain.     

Intravenous administration of anti-vascular endothelial growth factor humanized monoclonal antibody bevacizumab improves articular cartilage repair

Introduction  

In this study, we investigate the efficacy of repairing an osteochondral defect in rabbit knee joints by administering bevacizumab, a humanized monoclonal anti-vascular endothelial growth factor (VEGF) antibody.     

The Use of NanoTrap Particles as a Sample Enrichment Method to Enhance the Detection of Rift Valley Fever Virus

Background

Rift Valley Fever Virus (RVFV) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. The current state of diagnosis has led to misdiagnosis early on in infection. Here we describe the use of a novel sample preparation technology, NanoTrap particles, to enhance the detection of RVFV. Previous studies demonstrated that NanoTrap particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. Here we extend these findings by demonstrating the capture and enrichment of viruses.

Results

Screening of NanoTrap particles indicated that one particle, NT53, was the most efficient at RVFV capture as demonstrated by both qRT-PCR and plaque assays. Importantly, NT53 capture of RVFV resulted in greater than 100-fold enrichment from low viral titers when other diagnostics assays may produce false negatives. NT53 was also capable of capturing and enhancing RVFV detection from serum samples. RVFV that was inactivated through either detergent or heat treatment was still found bound to NT53, indicating the ability to use NanoTrap particles for viral capture prior to transport to a BSL-2 environment. Furthermore, both NP-40-lysed virus and purified RVFV RNA were bound by NT53. Importantly, NT53 protected viral RNA from RNase A degradation, which was not observed with other commercially available beads. Incubation of RVFV samples with NT53 also resulted in increased viral stability as demonstrated through preservation of infectivity at elevated temperatures. Finally, NanoTrap particles were capable of capturing VEEV and HIV, demonstrating the broad applicability of NanoTrap particles for viral diagnostics.

Conclusion

This study demonstrates NanoTrap particles are capable of capturing, enriching, and protecting RVFV virions. Furthermore, the use of NanoTrap particles can be extended to a variety of viruses, including VEEV and HIV.     

Signal stability of Cy3 and Cy5 on antibody microarrays

Background  

The antibody microarray technique is a newly emerging proteomics tool for differential protein expression analyses that uses fluorescent dyes Cy 3 and Cy 5. Environmental factors, such as light exposure, can affect the signal intensity of fluorescent dyes on microarray slides thus, it is logical to scan microarray slides immediately after the final wash and drying processes. However, no research data are available concerning time-dependent changes of fluorescent signals on antibody microarray slides to this date. In the present study, microarray slides were preserved at -20°C after regular microarray experiments and were rescanned at day 10, 20 and 30 to evaluate change in signal intensity.     

Correlation of antinuclear antibody and anti-double-stranded DNA antibody with clinical response to infliximab in patients with rheumatoid arthritis: a retrospective clinical study

Introduction  

The induction of antinuclear antibodies (ANAs) or anti-double-stranded (ds) -DNA antibodies (Abs) after infliximab (IFX) therapy in rheumatoid arthritis (RA) is a well-known phenomenon, but the correlation of such Abs with the clinical response to IFX has not yet been determined. The aims of this retrospective observational study were to examine the prevalence of positive ANA and anti-ds-DNA Abs before and after IFX therapy in patients with RA and to investigate whether an increased titer of such Abs is associated with the clinical efficacy of IFX.     

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

Background  

Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.     

Characterization of single chain antibody targets through yeast two hybrid

Background  

Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.     

Alternating evolutionary pressure in a genetic algorithm facilitates protein model selection

Background  

Automatic protein modelling pipelines are becoming ever more accurate; this has come hand in hand with an increasingly complicated interplay between all components involved. Nevertheless, there are still potential improvements to be made in template selection, refinement and protein model selection.     

Microbiological quality of drinking water from dispensers in Italy

Background  

Water coolers are popular in office buildings and commercial stores and the quality of this source of drinking water has the potential to cause waterborne outbreaks, especially in sensitive and immunocompromised subjects. The aim of this study was to determine the quality of water plumbed in coolers from commercial stores in comparison with tap water in Italy.     

Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail

Background  

High purity of tumour samples is a necessity for accurate genetic and expression analysis and is usually achieved by positive selection in chronic lymphocytic leukaemia (CLL).     

Antimicrobial activity of copper surfaces against suspensions of <Emphasis Type="Italic">Salmonella enterica</Emphasis> and <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Salmonella enterica and Campylobacter jejuni are amongst the more prevalent bacterial pathogens that cause foodborne diseases. These microorganisms are common contaminants of poultry and poultry products. This study was aimed to evaluate the antibacterial activity of metallic copper surfaces on these important enteropathogens, and to determine the potential acquisition of copper by food exposed to this metal.     

An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody

Background  

Phage-display panning is an integral part of biomedical research. Regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. Here, we produce an efficient antigen-specific single chain fragment variable (scFv) antibody by using a target-related molecule that favored selection ofrecombinant antibodies.     

ElliPro: a new structure-based tool for the prediction of antibody epitopes

Background  

Reliable prediction of antibody, or B-cell, epitopes remains challenging yet highly desirable for the design of vaccines and immunodiagnostics. A correlation between antigenicity, solvent accessibility, and flexibility in proteins was demonstrated. Subsequently, Thornton and colleagues proposed a method for identifying continuous epitopes in the protein regions protruding from the protein's globular surface. The aim of this work was to implement that method as a web-tool and evaluate its performance on discontinuous epitopes known from the structures of antibody-protein complexes.     

Contemporary epidemiology of gout in the UK general population

Introduction  

The objective of this study was to investigate the contemporary incidence of gout, examine potential risk factors, and evaluate specific gout treatment patterns in the general population.