Production, Application, and Preliminary Validation of Influenza Pseudovirus Detecting H5-Neutralizing Antibodies in Vaccinated Chicken Serum

Abstract

Background: Highly pathogenic avian influenza virus (H5N1) is a highly contagious and deadly pathogen among birds, but it can also be transmitted to humans. The surface glycoprotein, hemagglutinin (HA), is the most important antigenic determinant against which immune responses are elicited during infection or following vaccination. The virus neutralization test (VNT) is considered the gold standard method for detecting and measuring antibody levels; however, it requires the use of live virus. An alternative approach for neutralization assays involves the use of pseudoviruses, which are non-replicative particles expressing HA. Objectives: This study aimed to evaluate the suitability of the constructed pseudovirus for assessing virus-neutralizing antibodies. Methods: A pseudovirus harboring H5 HA was constructed using retroviral vectors. Following transfection of HEK293T cells with pcDNA-HA, pLOX, and psPAX plasmids, pseudovirus production and titration were evaluated by measuring P24 protein, and the amount of green fluorescent protein (GFP) expression was detected by flow cytometry. The functionality and specificity of the H5 pseudovirus were assessed by VNT. Results: Chicken sera containing H5 antibodies effectively neutralized the pseudovirus in a titer-dependent manner. The results of the neutralization test and the hemagglutination inhibition (HI) test were consistent. The cross-reactivity analysis of H5 pseudovirus and human influenza viruses with chicken and human sera highlighted the specificity of the H5 pseudoviruses. Conclusions: The VNTs based on pseudoviruses expressing H5 on their surface are recognized as reliable and safe alternatives for detecting neutralizing antibodies to avian influenza viruses in routine biosafety level 2 (BSL-2) laboratories.