Abstract
A novel electronic cell sensor array technology, the real-time cell analysis (RTCA) system, was developed to monitor cell events. Unlike the conventional methods labeling the target cells with fluorescence, luminescence, or light absorption, the RTCA system allows for label-free detection of cell processes directly without the incorporation of labels. Here, we used this new format to measure the cytopathic effect (CPE) of the 2009 influenza A (H1N1) virus and the efficacy of neutralizing antibodies in human sera to this virus. The real-time dynamic monitoring of CPE was performed on MDCK cell cultures infected with the H1N1 virus, ranging from 5.50×102 to 5.50×107 copies/mL. The resulting CPE kinetic curves were automatically recorded and were both time and viral load dependent. The CPE kinetics were also distinguishable between different H1N1 stains, as the onset of CPE induced by the A/Shanghai/37T/2009 H1N1 virus was earlier than that of the A/Shanghai/143T/2009 H1N1 virus. Furthermore, inhibition of H1N1 virus-induced CPE in the presence of human specific anti-sera was detected and quantified using the RTCA system. Antibody titers determined using this new neutralization test correlated well with those obtained independently via the standard hemagglutination inhibition test. Taken together, this new CPE assay format provided label-free and high-throughput measurement of viral growth and the effect of neutralizing antibodies, illustrating its potential in influenza vaccine studies.
Highlights
With the 2009 global outbreak of pandemic influenza, large-scale vaccination against the new strain of influenza A virus subtype H1N1 was employed to prevent its spread in China, and numerous clinical trials were initiated to estimate the antibody responses elicited with the 2009 influenza A (H1N1) virus vaccine [1,2,3]
The absorbance values representing the viable cell population were decreased due to cytopathogenic effect (CPE). These results demonstrated the accurate monitoring of H1N1-induced CPE using the real-time cell analysis (RTCA) system
To evaluate seasonal or pandemic candidate vaccines, a sensitive, specific, and reproducible serological method is necessary to measure the antibody response elicited with a candidate vaccine against wildtype viruses
Summary
With the 2009 global outbreak of pandemic influenza, large-scale vaccination against the new strain of influenza A virus subtype H1N1 was employed to prevent its spread in China, and numerous clinical trials were initiated to estimate the antibody responses elicited with the 2009 influenza A (H1N1) virus vaccine [1,2,3]. The traditional measurement of influenza virus neutralizing antibodies involves hemagglutination, immunological procedures, or various methods for cytopathogenic effect (CPE) identification, such as microscopic, colorimetric, and fluorometric assays [4,5,6,7]. Due to the the novel electronic cell sensor array technology, the RTCA system can provide dynamic monitoring of most of the cells present in the wells without the label molecules By this means, a wide range of noninvasive, label-free cell-based assays can be performed, such as monitoring of cytotoxicity and cell death, stem cell proliferation, RNA interference, virus-mediated cytopathogenicity, etc. As a proof of concept, this format was used to measure neutralizing antibodies in pre- and post-immune serum from 21 healthy individuals who had received the 2009 H1N1 vaccination; the data were compared with those obtained by hemagglutination inhibition tests
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