A novel platform for virus-like particle-display of flaviviral envelope domain III: induction of Dengue and West Nile virus neutralizing antibodies.

CD16-RIgE is a chimeric human membrane glycoprotein consisting of the CD16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE (RIgE). Coexpression of CD16-RIgE and HIV-1 Pr55Gag polyprotein precursor (Pr55GagHIV) in insect cells resulted in the incorporation of CD16-RIgE glycoprotein into the envelope of extracellular virus-like particles (VLPs), a phenomenon known as pseudotyping. Taking advantage of this property, we replaced the CD16 ectodomain of CD16-RIgE by the envelope glycoprotein domain III (DIII) of dengue virus serotype 1 (DENV1) or West Nile virus Kunjin (WNVKun). The two resulting chimeric proteins, DIII-DENV1-RIgE and DIII-WNVKun-RIgE, were addressed to the plasma membrane, exposed at the surface of human and insect cells, and incorporated into extracellular VLPs when coexpressed with Pr55GagHIV in insect cells. The DIII domains were accessible at the surface of retroviral VLPs, as shown by their reactivity with specific antibodies, and notably antibodies from patient sera. The DIII-RIgE proteins were found to be incorporated in VLPs made of SIV, MLV, or chimeric MLV-HIV Gag precursors, indicating that DIII-RIgE could pseudotype a wide variety of retroviral VLPs. VLP-displayed DIII were capable of inducing specific neutralizing antibodies against DENV and WNV in mice. Although the neutralization response was modest, our data confirmed the capability of DIII to induce a flavivirus neutralization response, and suggested that our VLP-displayed CD16-RIgE-based platform could be developed as a vaccine vector against different flaviviruses and other viral pathogens.

Membrane-based electrochemical nanobiosensor for the detection of virus.

A membrane-based electrochemical nanobiosensor sensitive toward whole viral particles is fabricated by forming a submicrometer thick nanoporous alumina membrane over a platinum disk electrode. Antibody probe molecules are physically adsorbed onto the walls of the membrane nanochannels. The sensing signal is based on the monitoring of the electrode's Faradaic current response toward ferrocenemethanol, which is extremely sensitive to the formation of immunocomplex within the nanoporous membrane. This nanobiosensor is demonstrated for the sensing of West Nile virus protein domain III (WNV-DIII) and the inactivated West Nile viral particle, using anti-WNV-DIII immunoglobulin M (IgM) as the biorecognition probe. The detection of the viral protein and the particle are logarithmically linear up to 53 pg mL(-1) (R(2) = 0.99) and 50 viral particles per 100 mL (R(2) = 0.93) in pH 7, with extremely low detection limits of 4 pg mL(-1) and ca. 2 viral particles per 100 mL, comparable to sensitivities of polymerase chain reaction techniques. The relative standard deviation (RSD) of whole viral particle detection in whole blood serum is 6.9%. In addition, the simple nanobiosensor construction procedure, minimal sample preparation, and short detection time of 30 min are highly attractive properties and demonstrate that the detection of a wide range of proteins and viruses can be achieved.