In addition, it might not be feasible to store plates for batch analysis

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In addition, it might not be feasible to store plates for batch analysis. In summary, we have established a new reporter gene-based RSV neutralization assay using rA2-Rluc (RSV-A2 strain) that is simple, rapid, high throughput, and less labor intensive than the traditional RSV-PRNT. titers for these human sera. This newly developed simple, high throughput, RSV-Luc-NeuT could be easily automated and applied in measurement of RSV neutralization titers in large vaccine trials. Keywords:Respiratory Syncytial Virus, RSV, Neutralization, Antibody, Luciferase, Assay, Rapid, F protein, G protein, PRNT, RSV-Luc-NeuT, Vaccine, Infection == 1. Introduction == Human Respiratory Syncytial Virus (RSV) is the leading cause of severe respiratory tract disease in the pediatric populations [1], and contributes significantly to the burden of disease in the elderly/frail and immunocompromised individuals [2]. Vaccine development Rabbit Polyclonal to A4GNT was greatly hampered by the negative outcome of a clinical trial with formalin-inactivated RSV (FI-RSV) vaccine in the 1960s that did not confer long-term protection and was associated with enhanced respiratory disease and death in infants exposed to natural RSV infection following vaccination [3-5]. Neutralizing antibodies play a major role in protection against RSV infection and disease. RSV F and G proteins are the major surface viral proteins demonstrated to induce neutralizing antibodies that confer protection in animal models [6]. Furthermore, high titers of RSV specific serum neutralizing antibodies correlate with protection against RSV challenge in adult volunteers [7], and with a lower risk of RSV infection in children and the elderly [8-10]. Passively-acquired maternal anti-RSV antibodies with neutralizing activity also provide protection against lower respiratory tract infection for the first few months of life [8,9]; likewise, passively administered human immunoglobulin containing anti-RSV neutralizing antibodies protected high-risk infants from lower respiratory tract infection that led to approval of Respigam (human immunoglobulin, intravenous) for the prevention of hospitalization due to RSV disease [11,12]. Similarly, a humanized anti-RSV-F monoclonal antibody with neutralizing activity was shown to provide significant protection against RSV disease in high-risk infants under 2 years of age in controlled clinical trials [13,14]. Renewed efforts are underway to develop a safe and effective RSV vaccine. Several vaccine prototypes have entered clinical trials [15,16]. The standard assay currently used to measure RSV neutralization is the plaque reduction neutralization test (RSV-PRNT) [17,18]. However, the RSV-PRNT is labor intensive, time-consuming (56 days), and not readily adaptable to a high throughput technology. Most importantly, the RSV PRNT is inherently variable and difficult to validate. In earlier studies in our laboratory, the level of variability for three operators was tested using RSV Lot 1 (at 1% IgG) in 27 independent RSV-PRNT assays on Hep-2 cell monolayers in the presence of 5% GPC. Plaque counts for the virus control (no antibody) for these assays ranged from 29 pfu/well to 51 pfu/well with a mean of 43 pfu/well. 50% endpoint titers ranged from 1:298 to 1 1:2427 (~8-fold difference between the lowest and highest titer) with a GMT SD of 1090 430 and a coefficient of variation of SMER-3 43%. In this study, we describe the development of a simple, sensitive, fast, high-throughput reporter gene-based RSV SMER-3 microneutralization assay usingRenillaluciferase-expressing RSV-A2 (rA2-Rluc) virus. We demonstrate that the RSV-Luciferase Neutralization Test (RSV-Luc-NeuT) is highly specific and sensitive for detecting both anti-F and anti-G RSV neutralizing antibodies. RSV-Luc-NeuT is more rapid (18 h) and has the potential for a higher throughput when compared with the traditional RSV-PRNT. Importantly, side-by-side comparison of monoclonal and polyclonal antibodies from animals and humans demonstrated a strong correlation between the inhibition seen using the RSV-Luc-NeuT and virus-neutralization quantified using RSV-PRNT. == 2. Materials and methods == == 2.1. Cells and viruses == A549 cells (CCL-185) and Vero cells (CCL-81) were obtained from ATCC (Manassas, VA, USA). Recombinant RSV expressingRenillaluciferase (rA2-Rluc) was generated by cloning a cassette consisting of theRenillaluciferase (Rluc) ORF-NS1 gene end signal-NS1 gene start signal into an antigenomic cDNA (D53) of RSV at the position of the NS1 ATG. This encodes Rluc SMER-3 at the first position in the genome, preserving the RSV sequence from the leader through the NS1 5 UTR (Fig. 1A). The Rluc containing D53 was then used to recover recombinant RSV in BSR-T7 cells as described [19]. Working virus stocks SMER-3 of rA2-Rluc were grown in A549 cells. For traditional RSV-PRNT assay, RSV strain A2, the kind gift of Drs. Robert Chanock and Brian Murphy, was plaque purified three times and then amplified in HEp-2 cell monolayers in the presence of EMEM containing 1% FBS, glutamine, penicillin, streptomycin and SMER-3 amphotericin B as.

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