Avidin-biotin complex-based capture coating platform for universal Influenza virus immobilization and characterization

Influenza virus mutates quickly and unpredictably creating emerging pathogenic strains that are difficult to detect, diagnose, and characterize. Conventional tools to study and characterize virus, such as next generation sequencing, genome amplification (RT-PCR), and serological antibody testing, are not adequately suited to rapidly mutating pathogens like Influenza virus where the success of infection heavily depends on the phenotypic expression of surface glycoproteins. Bridging the gap between genome and pathogenic expression remains a challenge. Using sialic acid as a universal Influenza virus binding receptor, a novel virus avidin-biotin complex-based capture coating was developed and characterized that may be used to create future diagnostic and interrogation platforms for viable whole Influenza virus. First, fluorescent FITC probe studies were used to optimize coating component concentrations. Then atomic force microscopy (AFM) was used to profile the surface characteristics of the novel capture coating, acquire topographical imaging of Influenza particles immobilized by the coating, and calculate the capture efficiency of the coating (over 90%) for all four representative human Influenza virus strains tested.


Reviewers' Comments to the Authors:
Reviewer 1 1. Comment: What is the main purpose of this system? For the diagnosis of influenza viruses, there are several methods. Compare the current method with pervious approaches.
Author Response: Thank you for pointing out the weakness in our claims on originality and for giving us the opportunity to emphasize the novelty and purpose of this work, especially as it compares to current and previous approaches. We have addressed this concern on multiple fronts.
On the comparison to current diagnostic techniques and how our capture coating may integrate into a novel diagnostic set up, we have added clarifying language and strategic emphasis on the adaptable potential of this work to second half of the Introduction section, lines 70 to 117. These paragraphs detail the pros and cons of existing diagnostic techniques (PCR, ELISA, and glycan microarrays) and the progress, potential, and, up to this point, difficulty of molecular spectroscopy techniques that require universal Influenza virus immobilization. Studies using our novel bPEG2kSA capture coating in conjunction with nano-resolution vibrational spectroscopy to differentiate between Influenza virus strains are ongoing, but we felt are outside the scope of this stand-alone manuscript describing the capture coating itself. In addition, in the following paragraph lines 239 to 243, we reference the only previous study (Guo et. al 2018) we are aware of that uses a pegylated sialic aid conjugate for Influenza virus immobilization. We use this study as support for the receptor binding functionality of our bPEG2kSA end chain, though our study goals are different. Table 1, why is the fluorescence signal error data so high?

Comment: In
Author Response: We agree with your assessment that the standard deviation of our fluorescence experiments summarized in Table 1 is high and have addressed potential reasons with the addition of lines 206 to 209 in the revised manuscript: "The high standard deviation of these fluorescence tests, especially at the higher 100 µM bPEG2kSA concentration, may be due to steric hindrance effects caused by a high concentration of receptors. Steric hinderance may be why 10 µM of bPEG2kSA receptor significantly outperformed a higher concentration of 100 µM bPEG2kSA receptor." Reflection on this also motivated us to solidify a claim of significance with new statistical analysis (Welch's t-test, described in Methods section, lines 152 to 156), which required handling the statistic calculations with data from the lognormal distribution typical of fluorescence tagged data. This allowed us to claim the optimized bPEG2kSA concentration of 10 µM had "significantly higher fluorescent reading compared to the 0 µM bPEG2kSA controls (IAV H1N1 p =0.041 , IAV H3N2 p = 0.032, IBV Yamagata p < 0.001)." (revised manuscript lines 203-204) despite the high standard deviation of the data. Values that met the significance criteria are now marked with a "*" in Table 1 and S1 Fig. To reflect these changes, we report the data as geometric mean and standard deviation.
3. Comment: For the calculation of capturing efficiency, the related AFM data should be provided.
Author Response: Thank you for this observation that made us realize our mistake in erroneously omitting the proper standard deviation data in the "Mean Particle Count" column of Table 3 which is used to calculate the capture efficiency. This data has been added. Further improvements have been made to the AFM section of this manuscript in regard to additional analysis, characterization, and visualization of individual viruses and the capture coating. In the interest of brevity, please refer to our responses to the specific requests of Reviewer 2.
Reviewer 1, thank you again for your invaluable insight and questions pertaining to the originality and data behind this work. We hope we have satisfactorily addressed your concerns on the novelty of our bPEG2kSA and ABC-based capture coating and readily invite additional feedback as you see fit.  (Fig 2A), capture coating on sapphire substrate without virus (Fig 2B), and a cluster of Influenza A H3N2 virions immobilized by capture coating on sapphire substrate (Fig 2C), and a single Influenza A H3N2 virions immobilized by capture coating on sapphire substrate ( Fig  2D). The measured height of the individual virion, 36 nm, and diameter, 160 nm, agree well with the known size of Influenza virus given the relatively large diameter (~30 nm) of the AFM probe tip causing a broadening edge artefact in the diameter measurement. [37][38][39][40] ." In addition, we added a new column to Table 2 with the corresponding characterizing surface parameters for the individual virion in Fig 2D. We feel it is important to keep Fig 2C as it provides a wonderful visual of the tendril-like structure of the capture coating. However, now it is properly and clearly described as capturing a cluster of viruses as to not mislead the reader. Reviewer 2, thank you again for your insight and for challenging us to look closer at our AFM data. We believe these changes have improved our manuscript greatly and hope we have addressed your concerns aptly. We gladly invite additional feedback as needed.