Bart Rombaut

Potential Use of Antiviral Agents in Polio Eradication

In 1988, the World Health Assembly launched the Global Polio Eradication Initiative, which aimed to use large-scale vaccination with the oral vaccine to eradicate polio worldwide by the year 2000. Although important progress has been made, polio remains endemic in several countries. Also, the current control measures will likely be inadequate to deal with problems that may arise in the postpolio era. A panel convoked by the National Research Council concluded that the use of antiviral drugs may be essential in the polio eradication strategy. We here report on a comparative study of the antipoliovirus activity of a selection of molecules that have previously been reported to be inhibitors of picornavirus replication and discuss their potential use, alone or in combination, for the treatment or prophylaxis of poliovirus infection.

Hybridoma antibodies to poliovirus N and H antigen

SummaryA set of 13 hybridoma antibodies to type 1 poliovirus has been studied with regard to neutralization and binding to N antigen, H antigen and capsid proteins. Two hybridomas produced heterogeneous antibodies, even after repeated attempts to reclone them.

A simple quantitative protein A micro-immunoprecipitation method; assay of antibodies to the N and H antigens of poliovirus.

Staphylococcus aureus (Cowan strain I) was used to absorb immune complexes from antiserum to poliovirus to which labeled N or H poliovirus antigens had been added, and the radioactivity in the pelleted organisms and in the supernatant was measured. Excellent agreement was obtained between values calculated separately from the pellet and supernatant readings, validating the use of supernatant measurements from a microtitration plate method.

A pH-dependent antigenic conversion of empty capsids of poliovirus studied with the aid of monoclonal antibodies to N and H antigen

Abstract The antigenic specificity and sedimentation coefficient of poliovirus empty capsids prepared at 4° from infected HeLa cells differ according to the pH of the extraction buffer: at pH 6.0 these particles are N antigenic (65 S), but at pH 8.5 they are H (80 S). N (65 S) particles are rapidly converted to H (80 S) at pH 8.5, 4°. The time course and the influence of temperature and pH on this conversion is studied. The N antigen associated with 65 S particles is more sensitive to alkaline pH than that of 160 S particles.

A comparison of WIN 51711 and R 78206 as stabilizers of poliovirus virions and procapsids

The thermal denaturation of poliovirus virions and procapsids in the 42 to 48 degrees C range was studied using N- and H-specific monoclonal antibodies. The half-life of the N antigen of Mahoney and Sabin 1 virions was extended 50- to 250-fold by either 10 microM WIN 51711 or R 78206. The minimum concentrations required for full stabilization at 46 degrees C (1.0 microM for WIN 51711, 0.2 microM for R 78206) were independent of the strain or serotype of the virus; 30 to 60 molecules of stabilizer per virion were required for full protection. R 78206 was the most efficient stabilizer of Mahoney procapsids; the half-life of the N-specific epitopes of these particles at 44 degrees C was extended from less than 1 min to 1 day.

Epitope evolution in poliovirus maturation

SummaryEight monoclonal antibodies specific for native (N) poliovirus antigen all recognized empty capsids extracted from infected HeLa cells; only three of them recognized 14S particles. The facts point to the existence of two different epitopes N1 and N2, only one of which (N1) is present on 14S particles (these observations confirm and extend findings byEminiet al). Another epitope of 14S particles is recognized by antibodies to heated (H) poliovirus antigen.Infected cell extract-catalyzed polymerization of 14S particles yielded mainly empty capsids carrying the N1 and N2, but no H epitopes.

Microfluidics in macro-biomolecules analysis: macro inside in a nano world

Use of microfluidic devices in the life sciences and medicine has created the possibility of performing investigations at the molecular level. Moreover, microfluidic devices are also part of the technological framework that has enabled a new type of scientific information to be revealed, i.e. that based on intensive screening of complete sets of gene and protein sequences. A deeper bioanalytical perspective may provide quantitative and qualitative tools, enabling study of various diseases and, eventually, may offer support for the development of accurate and reliable methods for clinical assessment. This would open the way to molecule-based diagnostics, i.e. establish accurate diagnosis and disease prognosis based on identification and/or quantification of biomacromolecules, for example proteins or nucleic acids. Finally, the development of disposable and portable devices for molecule-based diagnosis would provide the perfect translation of the science behind life-science research into practical applications dedicated to patients and health practitioners. This review provides an analytical perspective of the impact of microfluidics on the detection and characterization of bio-macromolecules involved in pathological processes. The main features of molecule-based diagnostics and the specific requirements for the diagnostic devices are discussed. Further, the techniques currently used for testing bio-macromolecules for potential diagnostic purposes are identified, emphasizing the newest developments. Subsequently, the challenges of this type of application and the status of commercially available devices are highlighted, and future trends are noted.

Cell-free synthesis of poliovirus: 14S subunits are the key intermediates in the encapsidation of poliovirus RNA.

In a cell-free system of uninfected HeLa cells, programmed with poliovirus RNA, extraneous radiolabelled 14S subunits assembled with endogenous 14S subunits and interacted with newly synthesized RNA to form virions (160S). This result suggests that 14S subunits are the key intermediates in the encapsidation of poliovirus RNA.

In vitro antiviral activity of single domain antibody fragments against poliovirus.

VHHs or Nanobodies are single-domain antigen-binding fragments derived from heavy chain antibodies found in camelids. It has already been shown that complex protein mixtures and even whole organisms elicit good immune responses in camelids; therefore we hypothesized that VHHs selected from a dromedary immunized with poliovirus type 1 might inhibit the in vitro replication of poliovirus through binding to essential biological sites on the viral capsid. In this study, we aimed to determine whether VHHs inhibit wild-type and vaccine strains of poliovirus type 1. Interestingly, VHHs showed a potent antipolio activity with EC50 values in the low nanomolar range. Moreover, these antibody fragments completely blocked viral multiplication at higher concentrations. Remarkably, no (immune) escape variants against some of these VHHs could be generated. In conclusion, VHHs fulfil several in vitro requirements to be assigned as potential antiviral compounds for further development of an anti-poliovirus drugs.

Stabilization by Host Cell Components and Mg2+ of the Neutralization Epitopes of Poliovirus

When type 1 poliovirions were extracted from infected HeLa cells and heated at 56 degrees C in the presence of 1 mM-Mg2+, RNA-free, pluri-antigenic particles were formed. These particles possessed neutralization epitopes as well as epitopes associated with H antigen and with the isolated capsid protein VP3. The heating caused the formation of exclusively H antigenic particles when the Mg2+ concentration was 10(-4) M or lower, or when the virus was further purified in order to remove remaining cellular components. The ability of purified virus to form pluri-antigenic particles upon heating could be restored by incubation at 37 degrees C with infected cell extract.