Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling

Abstract

Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and beta-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and beta-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.

FIG. 1.

Hemagglutination titers (left) and NA activities (right) of human influenza A(H3N2) virus and avian influenza A(H7N3) virus after treatment with heat (A), β-PL (B), Triton X-100 (C), or formalin (D). Controls were subjected to the same procedures as the treatment specimens except for the treatment itself: they were heated at 22°C and had no β-PL, Triton X-100, or formalin added. Hemagglutination titers and NA activities after Triton X-100 treatment were determined after the removal of the detergent. Hemagglutination titers of <2 are expressed as 1.

FIG. 2.

Hemagglutination titers (left) and NA activities (right) of seasonal A(H1N1) and pandemic A(H1N1)v viruses after inactivation using Triton X-100 (A and B) or formalin (C and D). The left bars show the results for the original stock viruses, and the right bars show the results for the viruses after inactivation. Hemagglutination titers and NA activities after Triton X-100 treatment were determined after the removal of the detergent. Hemagglutination titers of <2 are expressed as 1. NA activities of live A(H1N1)v viruses were not determined and are therefore not shown for these viruses in panels B and D.

FIG. 3.

Results obtained using inactivated influenza viruses in NA inhibition assays. (A) The NA activity increases after 1% Triton X-100 treatment but is subsequently reduced by column-based detergent removal. Lines connect results from experiments with the same virus strain. (B) Effects of 1% Triton X-100 and formalin treatments on the performance of the MUNANA NA inhibition assay with oseltamivir-sensitive and -resistant influenza viruses. Results obtained after column-based detergent removal (Triton + column) are indicated. RFU, relative fluorescence units. (C) IC₅₀ values obtained with the MUNANA assay (for live and Triton X-100-treated virus samples, virus samples treated with Triton X-100 and subjected to detergent removal, and formalin-treated virus samples) and the NA-Star assay (for live and Triton X-100-treated virus samples and virus samples treated with Triton X-100 and subjected to detergent removal) for NA inhibition with oseltamivir-sensitive and -resistant seasonal influenza A(H1N1) viruses. (D) NAI susceptibilities of 12 1% Triton X-100-treated influenza A(H1N1)v virus isolates as measured with the MUNANA and NA-Star NA inhibition assays.

FIG. 4.

HI titers of four ferret antisera with live or formalin-treated influenza A(H1N1) viruses. Only one of the A(H1N1)v pandemic strains (A/California/04/09) showed some cross-reactivity with ferret antisera against recent seasonal A(H1N1) influenza viruses. Formalin-treated A/Netherlands/602/09 and A/Paris/2590/09 viruses had too-low hemagglutination titers to be included in the HI assay (Fig. 2C). HI titers of <20 are not shown.

FIG. 5.

T-cell response kinetics after PBMC stimulation with live or formalin-treated influenza virus A/Wisconsin/67/05 (H3N2). (A) Percentages of CD4⁺ CD107a⁺ T cells; (B) percentages of CD8⁺ CD107a⁺ T cells; (C) percentages of CD4⁺ IL-2⁺ T cells; (D) percentages of CD8⁺ IL-2⁺ T cells. Mock, culture supernatant from uninfected cells; *, P < 0.05 for live and formalin-inactivated A(H3N2) virus samples compared to mock-treated samples.

FIG. 6.

Detection of T-cell responses against live or formalin-treated A/New Caledonia/20/99 (H1N1) virus demonstrates significant T-cell epitope antigenicity 72 h poststimulation. (A) Percentages of CD4⁺ CD107a⁺ T cells (white bars) and CD8⁺ CD107a⁺ T cells (black bars); (B) percentages of CD4⁺ IL-2⁺ T cells (white bars) and CD8⁺ IL-2⁺ T cells (black bars). SEB, positive control using S. aureus enterotoxin B; mock, culture supernatant from uninfected cells. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 for live and formalin-inactivated A(H1N1) virus samples compared to mock-treated samples.

FIG. 7.

Detection of T-cell responses against formalin-treated pandemic influenza A/Paris/2590/09 (H1N1)v virus demonstrates significant T-cell epitope antigenicity. (A) Percentages of CD4⁺ CD107a⁺ T cells; (B) percentages of CD8⁺ CD107a⁺ T cells; (C) percentages of CD4⁺ IL-2⁺ T cells; (D) percentages of CD8⁺ IL-2⁺ T cells. SEB, positive control using S. aureus enterotoxin B; mock, culture supernatant from uninfected cells.