Development of a safe and convenient neutralization assay for rapid screening of influenza HA-specific neutralizing monoclonal antibodies

Abstract

The worldwide outbreak of the swine-origin 2009 H1N1 influenza A virus (IAV) and an increasing number of influenza cases caused by a highly pathogenic avian influenza (HPAI) H5N1 have accelerated the need to develop vaccines and antiviral agents against IAVs. Among various antivirals, neutralizing monoclonal antibodies (mAbs) are considered important passive therapeutics having an immediate effect against viral pathogens. Here we report a pseudovirus neutralization assay for rapid screening of neutralizing mAbs targeting hemagglutinin (HA) of H5N1 and H1N1 IAV. In this study, we generated six pseudoviruses with an HIV-1 backbone, respectively, expressing HA of four clades of H5N1 IAV and the 2009 epidemic H1N1 IAV. The resulting pseudoviruses were able to infect a variety of human and non-human cells, with 293T cells from human kidney as the most susceptible target cells. Using the established pseudovirus neutralization assay, we showed that three of ten selected mAbs specific to HA could potently neutralize infection of a pseudovirus bearing HA from the homologous IAV A/VietNam/1194/2004(H5N1) strain. This was highly consistent with the result of a microneutralization assay testing the same strain of a live IAV. Since the pseudovirus neutralization assay does not involve an infectious virus and can be performed without the requirement of a biosafety-3 laboratory, it may be applied for safe and rapid screening of neutralizing mAbs and antiviral agents targeting HA of IAVs.

Fig. 1

Western blot analysis of the generated HA pseudoviruses. Pseudoviruses (50 ng/ml p24) were, respectively, detected by mAbs (1:1000) specific for HIV-1 p24 (A) and IAV HA (#8, B).

Fig. 2

Infectivity and host cell tropism of HA pseudoviruses. (A) Comparison of infection rates of pseudoviruses with and without exogenous NA in 293T cells. * indicates P < 0.05 when comparing each pseudovirus with and without NA. (B) Detection of cell tropism of HA pseudoviruses in MDCK, A549, Vero, CHO-K1 and 293T cells. For each HA pseudovirus, * indicates P < 0.05 when comparing the infectivity in 293T cells and other cells. VSV-G pseudovirus was used as the positive control, and Env− pseudovirus and cells only (mock) were used as the negative control. Pseudoviruses (50 ng/ml p24) were used for infection of cells, and the data are expressed as the Mean RLU ± SD of 3 parallel wells in 96-well culture plates. The experiment was repeated three times and similar results were obtained.

Fig. 3

Antibody responses and neutralizing titer detection of mAbs targeting HA of A/VietNam/1194/2004(H5N1) by pseudovirus neutralization assay. (A) ELISA detection of IgG response of 20 screened mAbs specific to HA. The data are expressed as absorbance at 450 nm (A450). (B) Pseudovirus neutralization detection of HA-specific mAbs against infection of HA pseudovirus in 293T cells. Ten mAbs with the highest antibody titer were selected for screening neutralizing activity. 33G4 mAb and VSV-G were used as negative mAb and pseudovirus controls, respectively. The data are presented as Mean NT₅₀ ± SD of 3 parallel wells of each dilution in 96-well culture plates. The dotted line indicates the detection limit. The experiment was repeated three times and similar results were obtained.