Analysis of the Thermal and pH Stability of Human Respiratory Syncytial Virus

Abstract

Respiratory syncytial virus (RSV) was studied as a function of pH (3-8) and temperature (10-85 degrees C) by fluorescence, circular dichroism, and high-resolution second-derivative absorbance spectroscopies, as well as dynamic light scattering and optical density as a measurement of viral aggregation. The results indicate that the secondary, tertiary, and quaternary structures of RSV are both pH and temperature labile. Derivative ultraviolet absorbance and fluorescence spectroscopy (intrinsic and extrinsic) analyses suggest that the stability of tertiary structure of RSV proteins is maximized near neutral pH. In agreement with these results, the secondary structure of RSV polypeptides seems to be more stable at pH 7-8, as evaluated by circular dichroism spectroscopy. The integrity of the viral particles studied by turbidity and dynamic light scattering also revealed that RSV is more thermally stable near neutral pH and particularly prone to aggregation below pH 6. By combination of the spectroscopic data employing a multidimensional eigenvector phase space approach, an empirical phase diagram for RSV was constructed. The pharmaceutical utility of this approach and the optimal formulation conditions are discussed.

High-throughput screening of stabilizers for Respiratory Syncytial Virus: Identification of stabilizers and their effects on the conformational thermostability of viral particles

“…24 We have also observed that a common mechanism of the destabilization of RSV is pH and temperature induced particle aggregation. 24 In this work, an empirical phase diagram was created employing measurement of the virus by a variety of techniques as a function of pH and temperature. The resultant colored diagram displayed clearly delineated empirical phase boundaries that indicated conditions under which the structure of the virus was altered (i.e., degraded).…”

“…RSV samples were further purified by a 30-60% w/v sucrose gradient made in NTE buffer (10 mM Tris-HCl, 140 mM NaCl, 1 mM EDTA) pH 7.4, and concentrated by centrifugation in a 1.45 M sucrose cushion as previously reported. 24,25 Virus titration was performed by an ELISA sandwich using the method of Malley et al as previously reported. 24,26 As determined by ELISA, stock suspensions of purified RSV contained approximately 10 5 to 10 6 PFU/mL.…”

“…24,25 Virus titration was performed by an ELISA sandwich using the method of Malley et al as previously reported. 24,26 As determined by ELISA, stock suspensions of purified RSV contained approximately 10 5 to 10 6 PFU/mL. Stock viral suspensions of about 1 mL were dialyzed at 4˚C against 2 L of 20 mM citrate/phosphate (CP) buffer pH 6.0 using Slide-a-Lyzer ® dialysis cassettes, 10,000 MWCO (Pierce, Rockford, IL).…”

“…The pH and temperature conditions under which RSV showed intrinsic marginal stability were selected based on aggregation traces and the phase boundaries observed in the previously published pH/temperature empirical phase diagram (EPD) for RSV. 24 Using this approach, the pH/temperature regions under which the degradation pathways of RSV can be accelerated are easily identified as an abrupt color change in the EPD. 24 The nature of the physical changes observed and the adaptability of the assay to commercial 96 -well microtiter plate readers dictate the type of test to be used for the high-throughput screening.…”

“…23 In this context, we have recently shown that the thermal stability of RSV is maximized at neutral pH. 24 We have also observed that a common mechanism of the destabilization of RSV is pH and temperature induced particle aggregation. 24 In this work, an empirical phase diagram was created employing measurement of the virus by a variety of techniques as a function of pH and temperature.…”