Comparison of Different Cell Substrates on the Measurement of Human Influenza Virus Neutralizing Antibodies

Abstract

Eight cell lines were systematically compared for their permissivity to primary infection, replication, and spread of seven human influenza viruses. Cell lines were of human origin (Caco-2, A549, HEp-2, and NCI-H292), monkey (Vero, LLC-MK2), mink (Mv1 Lu), and canine (MDCK). The influenza viruses included seasonal types and subtypes and a pandemic virus. The MDCK, Caco-2, and Mv1 Lu cells were subsequently compared for their capacity to report neutralization titers at day one, three and six post-infection. A gradient of sensitivity to primary infection across the eight cell lines was observed. Relative to MDCK cells, Mv1 Lu reported higher titers and the remaining six cell lines reported lower titers. The replication and spread of the seven influenza viruses in the eight cell substrates was determined using hemagglutinin expression, cytopathic effect, and neuraminidase activity. Virus growth was generally concordant with primary infection, with a gradient in virus replication and spread. However, Mv1 Lu cells poorly supported virus growth, despite a higher sensitivity to primary infection. Comparison of MDCK, Caco-2, and Mv1 Lu in neutralization assays using defined animal antiserum confirmed MDCK cells as the preferred cell substrate for influenza virus testing. The results observed for neutralization at one day post-infection showed MDCK cells were similar (<1 log2 lower) or superior (>1 log2 higher) for all seven viruses. Relative to Caco-2 and Mv1 Lu cells, MDCK generally reported the highest titers at three and six days post-infection for the type A viruses and lower titers for the type B viruses and the pandemic H9N2 virus. The reduction in B virus titer was attributed to the complete growth of type B viruses in MDCK cells before day three post-infection, resulting in the systematic underestimation of neutralization titers. This phenomenon was also observed with Caco-2 cells.

Mechanism and Significance of Cell Type-Dependent Neutralization of Flaviviruses

“…The extent of virion maturation is one factor shown to govern how many antibodies bind the virion at a given concentration of antibody (25). In several instances, the results of neutralization assays have been shown to vary when the assays are performed on multiple cellular substrates (26)(27)(28)(29)(30)(31)(32). Interest in cell type-dependent patterns of neutralization is heightened by results of the first large-scale phase IIb clinical study of a tetravalent DENV vaccine (33).…”

Cell culture-based influenza vaccines: A necessary and indispensable investment for the future

“…89 MDCK cells may also be better suited for neutralization assays. 92 Second, MDCK cells have been shown to be the most suitable for large-scale production of influenza virus. 89,93,94 Head-to-head comparison in laboratory scale bioreactors showed that MDCK cells yielded more virus than did Vero cells.…”

“…148 Cytopathic effects can be observed earlier in these cells, and titres of influenza A viruses are higher in these cells than in MDCK cells. 92,[148][149][150] However, further characterization and clinical studies would be required before they could be adopted as cell substrates for influenza vaccine production.…”

“…Another issue is the fact that the reference reagents used for quality control and potency assays were largely produced using ECE-grown virus, and there is evidence that reactivity could be broader when MDCK-grown virus is used. 46,55,92 However, this could be overcome by shifting to producing reagents using cell culture-derived virus. It is also possible that some virus strains may not grow well in certain cell types, and several cell lines or clones may need to be tested for initial expansion of the vaccine virus.…”

“…177,178 Other issues with cell culture-derived vaccines include lower levels of neuraminidase in the virion compared to virus grown in ECE, 46 and differential glycosylation patterns. 92,152,176,177 Although these may influence immunogenicity in animal models, 179 overwhelming evidence from clinical studies in humans suggests that there is no significant difference in immunogenicity. However, this has not been supported with a detailed characterization of how these differences affect the various components of the immune response.…”